Patent application title: MUTATED PROTOPORPHYRINOGEN IX OXIDASE (PPX) GENES
Inventors:
Gregory F.w. Gocal (San Diego, CA, US)
Peter R. Beetham (Carlsbad, CA, US)
Peter R. Beetham (Carlsbad, CA, US)
Aura De Schopke (San Diego, CA, US)
Sarah Dumm (San Diego, CA, US)
James Pearce (La Jolla, CA, US)
Christian Schopke (San Diego, CA, US)
Keith A. Walker (San Diego, CA, US)
Keith A. Walker (San Diego, CA, US)
IPC8 Class: AC12N504FI
USPC Class:
435410
Class name: Chemistry: molecular biology and microbiology plant cell or cell line, per se (e.g., transgenic, mutant, etc.); composition thereof; process of propagating, maintaining, or preserving plant cell or cell line; process of isolating or separating a plant cell or cell line; process of regenerating plant cells into tissue, plant part, or plant, per se, where no genotypic change occurs; medium therefore
Publication date: 2012-05-17
Patent application number: 20120122223
Abstract:
Provided are compositions and methods relating to gene and/or protein
mutations in transgenic or non-transgenic plants. In certain embodiments,
the disclosure relates to mutations in the protoporphyrinogen IX (PPX)
gene. In some embodiments the disclosure relates to plants that are
herbicide resistant.Claims:
1. A non-transgenic plant cell comprising a mutated protoporphyrinogen IX
oxidase (PPX) gene, wherein said gene encodes a protein comprising a
mutation at one or more amino acid positions corresponding to a position
selected from the group consisting of 52, 85, 105, 111, 130, 139, 143,
144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219,
220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 305,
311, 316, 318, 332, 343, 354, 357, 359, 360, 366, 393, 403, 424, 426,
430, 438, 440, 444, 455, 457, 470, 478, 483, 484, 485, 487, 490, 503, 508
and 525 of SEQ ID NO: 1 or wherein said gene encodes a protein comprising
a mutation at one or more amino acid positions corresponding to a
position selected from the group consisting of 58, 64, 74, 84, 93, 97,
98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195,
214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396,
404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 500
of SEQ ID NO: 9.
2.-62. (canceled)
Description:
CROSS REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This application claims priority to PCT/US2011/046330, filed Aug. 2, 2011, which claims priority under 35 U.S.C. ยง119(e) to U.S. Application Ser. No. 61/370,436, filed Aug. 3, 2010, each of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] This disclosure relates, at least in part, to gene and/or protein mutations in plants.
BACKGROUND OF THE INVENTION
[0003] The following description is provided simply as an aid in understanding the invention and is not admitted to describe or constitute prior art.
[0004] Examples of certain mutations in the PPX genes of plants have been reported. For example, U.S. Pat. No. 5,767,373 discloses "eukaryotic DNA sequences coding for native protoporphyrinogen oxidase (protox) or modified forms of the enzyme which are herbicide tolerant;" U.S. Pat. No. 6,282,837 discloses "eukaryotic DNA sequences coding for native protoporphyrinogen oxidase (protox) or modified forms of the enzyme which are herbicide tolerant and a method for controlling weeds using plants having altered protox activity which confers tolerance to herbicides;" U.S. Pat. No. 6,308,458 discloses "methods for controlling the growth of undesired vegetation comprising applying an effective amount of a protox-inhibiting herbicide to a population of transgenic plants or plant seed transformed with a DNA sequence coding for a modified protox enzyme that is tolerant to a protox-inhibiting herbicide or to the locus where a population of the transgenic plants or plant seeds is cultivated;" U.S. Pat. No. 6,905,852 discloses "[a] protoporphyrinogen oxidase tolerant to photobleaching herbicide and derivatives thereof, comprising a polypeptide having the amino acid sequence represented by SEQ ID No. 2 [a PPX protein] or mutated peptides derived therefrom by deletion, addition, substitution, etc. of one or more amino acids in the above amino acid sequence and having an activity substantially equivalent to that of the protoporphyrinogen oxidase;" US patent No. discloses "methods to confer resistance to protoporphyrinogen-inhibiting herbicides onto crop plants. Resistance is conferred by genetically engineering the plants to express cloned DNA encoding a protoporphyrinogen oxidase resistant to porphyric herbicides;" US Patent Application Publication No. 20020086395 discloses "[a]method for evaluating the ability of a compound to inhibit the protoporphyrinogen oxidase activity, which comprises the steps of: (1) culturing a transformant expressing a protoporphyrinogen oxidase gene present in a DNA fragment in a medium containing substantially no protoheme compounds in each comparative system of the presence and absence of a test compound to measure a growth rate of the transformant under each condition, said transformant being resulted from a host cell deficient in the growing ability based on the protoporphyrinogen oxidase activity transformed with the DNA fragment in which a promoter functionable in the host cell and a protoporphyrinogen oxidase gene are operatively linked, and (2) determining the ability of the compound to inhibit the protoporphyrinogen oxidase activity by comparing the growth rates; and the like;" Patzoldt W L, et al., PNAS USA 103:12329-34 (2006) discloses a "3-bp deletion corresponding to the G210 codon" of PPX; and Li X, et al., Plant Physiology 133:736-47 (2003) discloses "isolation of plant protoporphyrinogen oxidase (PPO) genes and the isolation of herbicide-resistant mutants." The terms PPO and PPX are used interchangeably herein.
SUMMARY OF THE INVENTION
[0005] The present disclosure relates, at least, in part to methods and compositions relating to gene and protein mutations in plants. In some aspects and embodiments, the present disclosure may also relate to compositions and methods for producing herbicide-resistant plants. The present disclosure methods and compositions relate, at least in part to mutations in a protoporphyrinogen IX oxidase (PPX) gene.
[0006] In one aspect, there is provided a plant or a plant cell including a mutated PPX gene. In certain embodiments, the mutated PPX gene encodes a mutated PPX protein. In certain embodiments, a plant having a plant cell that includes a mutated PPX gene may be herbicide-resistant; for example, resistant to a PPX-inhibiting herbicide. In certain embodiments, the plant or the plant cell is non-transgenic. In certain embodiments, the plant or the plant cell is transgenic. The disclosure also provides recombinant vectors including such mutated PPX genes, as well as transgenic plants containing such mutated PPX genes.
[0007] As used herein, the term "PPX gene" refers to a DNA sequence capable of generating a PPX polypeptide that shares homology and/or amino acid identity with amino acid sequence SEQ ID NO: 1, and/or encodes a protein that demonstrates PPX activity. In certain embodiments, the PPX gene has 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or 100% identity to a specific PPX gene; for example, the mitochondrial Russet Burbank PPX genes, for example, StmPPX1 or StmPPX2; or for example, a plastidal Russet Burbank PPX gene, for example, StcPPX1. In certain embodiments, the PPX gene has 60%; 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or 100% identity to a sequence selected from the sequences in FIGS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41 43 and 45. In some embodiments, a PPX gene is a mitochondrial PPX gene; for example, StmPPX1 or StmPPX2. In some embodiments, a PPX gene is a plastidal PPX gene; for example, StcPPX1. In some embodiments, a PPX gene is a mitochondrial PPX gene allele; for example, StmPPX2.1 or StmPPX2.2. In some embodiments, a PPX gene is a plastidal PPX gene allele; for example, StcPPX1 or StcPPX1.1. In some plants, such as water hemp, the protein product of a single PPX gene is both mitochondrial and plastidal as disclosed in Patzoldt W L, et al., PNAS USA 103:12329-34 (2006).
[0008] As used herein, the term "mutation" refers to at least a single nucleotide variation in a nucleic acid sequence and/or a single amino acid variation in a polypeptide relative to the normal sequence or wild-type sequence or a reference sequence, e.g., SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments a mutation refers to at least a single nucleotide variation in a nucleic acid sequence and/or a single amino acid variation in a polypeptide relative to a nucleotide or amino acid sequence of a PPX protein that is not herbicide resistant. In certain embodiments, a mutation may include a substitution, a deletion, an inversion or an insertion. In some embodiments, a substitution, deletion, insertion, or inversion may include a variation at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides. In some embodiments, a substitution, deletion, insertion, or inversion may include a variation at 1, 2, 3, 4, 5, 6, 7 or 8 amino acid positions. The term "nucleic acid" or "nucleic acid sequence" refers to an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, which may be single or double stranded, and represent the sense or antisense strand. A nucleic acid may include DNA or RNA, and may be of natural or synthetic origin. For example, a nucleic acid may include mRNA or cDNA. Nucleic acid may include nucleic acid that has been amplified (e.g., using polymerase chain reaction). The convention "NTwt###NTmut" is used to indicate a mutation that results in the wild-type nucleotide NTwt at position ### in the nucleic acid being replaced with mutant NTmut. The single letter code for nucleotides is as described in the U.S. Patent Office Manual of Patent Examining Procedure, section 2422, table 1. In this regard, the nucleotide designation "R" means purine such as guanine or adenine, "Y" means pyrimidine such as cytosine or thymine (uracil if RNA); "M" means adenine or cytosine; "K" means guanine or thymine; and "W" means adenine or thymine.
[0009] As used herein, the term "mutated PPX gene" refers to a PPX gene having one or more mutations at nucleotide positions relative to a reference PPX nucleic acid sequence. In certain embodiments a mutated PPX gene has one or more mutations relative to a corresponding wild type PPX sequence. As used herein, the term "wild-type" may be used to designate the standard allele at a locus, or the allele having the highest frequency in a particular population. In some instances, wild-type allele may be represented by a particular amino acid or nucleic acid sequence. For example, a wild-type potato plastidal PPX protein may be represented by SEQ ID NO: 7. For example, a wild-type potato mitochondrial PPX protein may be represented by SEQ ID NO: 9. In some embodiments a mutated PPX gene has one or more mutations relative to a reference PPX nucleic acid sequence, for example SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 43 or 45 or at homologous positions of paralogs thereof. In some embodiments, the mutated PPX gene is modified with at least one mutation. In other embodiments, the mutated PPX gene is modified with at least two mutations. In other embodiments, the mutated PPX gene is modified with at least three mutations. In some embodiments, a mutated PPX gene encodes a mutated PPX protein. In some embodiments, a mutated PPX gene includes two or more nucleic acid sequence mutations selected from Tables 2, 3a and 3b. In some embodiments, a mutated PPX gene encodes one or more mutated mitochondrial PPX proteins. In other embodiments, a mutated PPX gene encodes one or more mutated plastidal PPX proteins. In some embodiments, a mutated PPX gene is a mutated mitochondrial PPX gene; for example, mutated StmPPX1. In some embodiments, a mutated PPX gene is a mutated mitochondrial PPX gene; for example, mutated StmPPX2. In some embodiments, a mutated PPX gene is a mutated plastidal PPX gene; for example, mutated StcPPX1. In some embodiments, a mutated PPX gene is a mutated mitochondrial PPX gene allele; for example, mutated StmPPX2.1 or mutated StmPPX2.2. In some embodiments, a mutated PPX gene is a mutated plastidal PPX gene allele; for example, mutated StcPPX1 or mutated StcPPX1.1. In some embodiments, there is at least one mutation in a plastid PPX gene and at least one mutation in a mitochondrial PPX gene. In some embodiments, one or more mutations in a PPX gene leads to herbicide resistance; for example, resistance to a PPX-inhibiting herbicide. In some embodiments, the mutated PPX gene encodes a mutated PPX protein that has increased resistance to one or more herbicides as compared to a reference PPX protein.
[0010] In some embodiments, the mutations in a mutated PPX gene encodes a protein having a combination of two or more mutations. In certain embodiments, at least one mutation is in the plastid PPX gene and at least one mutation is in a mitochondrial PPX gene. In certain embodiments, the combinations are selected from Tables 4a and 4b. In some embodiments, the mutations in a mutated PPX gene encode a protein having a combination of three or more mutations; for example, combinations selected from Tables 4a and 4b. In some embodiments, the at least one mutation in the plastidal PPX gene and the at least one mutation in the mitochondrial PPX gene are at the same corresponding position. In other embodiments, the at least one mutation in the plastid PPX gene and the at least one mutation in the mitochondrial PPX gene are at different corresponding positions.
[0011] As used herein, the term "PPX protein" refers to a protein that has homology and/or amino acid identity to a PPX protein of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42 or 44 and/or demonstrates PPX activity. In certain embodiments, the PPX protein has 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or 100% identity to a specific PPX protein, such as for example, the mitochondrial Russet Burbank PPX protein or the plastidal Russet Burbank PPX proteins. In certain embodiments, the PPX protein has 70%; 75%; 80%; 85%; 90%; 95%; 96%; 97%; 98%; 99%; or 100% identity to a sequence selected from the sequences in FIG. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42 or 44.
[0012] As used herein, the term "mutated PPX protein" refers to a PPX protein having one or more mutations at positions of amino acids relative to a reference PPX amino acid sequence, or at homologous positions of paralogs thereof. In some embodiments, a mutated PPX protein has one or more mutations relative to a reference PPX amino acid sequence, for example, a reference PPX amino acid sequence having SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42 or 44, or portions thereof. In certain embodiments a mutated PPX protein has one or more mutations relative to a corresponding wild type protein. In some embodiments a mutated PPX protein has one or more mutations relative to a corresponding protein that is not herbicide resistant. In some embodiments, the PPX protein is modified with at least one mutation. In other embodiments, the PPX protein is modified with at least two mutations. In other embodiments, the PPX protein is modified with at least three mutations. In some embodiments, one or more mitochondrial PPX proteins are mutated. In other embodiments, one or more plastidal PPX proteins are mutated. In yet another embodiment one or more mitochondrial PPX proteins and one or more plastidal PPX proteins are mutated. In some embodiments, the term mutated PPX protein refers to a PPX protein that has increased resistance to one or more herbicides as compared to a reference protein.
[0013] In some embodiments, a mutated PPX protein includes a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of 52, 85, 105, 111, 130, 139, 143, 144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219, 220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 305, 311, 316, 318, 332, 343, 354, 357, 359, 360, 366, 393, 403, 424, 426, 430, 438, 440, 444, 455, 457, 470, 478, 483, 484, 485, 487, 490, 503, 508 and 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 500 of SEQ ID NO: 9. In some embodiments, a plant or plant cell may include a mutated protoporphyrinogen IX oxidase (PPX) gene wherein the gene encodes a protein including a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of G52, N85, N105, E111, G130, D139, P143, R144, F145, L147, F165, L167, I170, A180, P185, E192, S193, R199, V206, E212, Y219, A220, G221, L226, M228, K229, A230, K237, S244, R256, R257, K270, P271, Q272, S305, E311, T316, T318, S332, S343, A354, L357, K359, L360, A366, L393, L403, L424, Y426, S430, K438, E440, V444, L455, K457, V470, F478, F483, D484, I485, D487, K490, L503, V508 and I525 of SEQ ID NO: 1. In some embodiments, a plant or plant cell may include a mutated protoporphyrinogen IX oxidase (PPX) gene wherein the gene encodes a protein including a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of D58, E64, G74, G84, L93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, N195, P214, I215, K229, K230, C271, D274, F283, A292, S296, C307, N324, D330, S396, A404, R406, K410, L421, A423, C434, D447, S448, V449, D451, D454, Y465, K470 and T500 of SEQ ID NO: 9. In some embodiments, a PPX protein is a paralog of Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato plastidal PPX protein) and the PPX protein may have an N at the position corresponding to position 52 of SEQ ID NO:1, wherein the N is substituted with an amino acid other than an N; a K at the position corresponding to position 272 of SEQ ID NO:1, wherein the K is substituted with an amino acid other than a K; an S at the position corresponding to position 359 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S; and/or an S at the position corresponding to position 525 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S. In some embodiments, a mutated PPX protein includes two or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of G52, N85, N105, E111, G130, D139, P143, R144, F145, L147, F165, L167, I170, A180, P185, E192, S193, R199, V206, E212, Y219, A220, G221, L226, M228, K229, A230, K237, S244, R256, R257, K270, P271, Q272, S305, E311, T316, T318, S332, S343, A354, L357, K359, L360, A366, L393, L403, L424, Y426, S430, K438, E440, V444, L455, K457, V470, F478, F483, D484, I485, D487, K490, L503, V508, and I525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes two or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of D58, E64, G74, G84, L93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, N195, P214, I215, K229, K230, C271, D274, F283, A292, S296, C307, N324, D330, S396, A404, R406, K410, L421, A423, C434, D447, S448, V449, D451, D454, Y465, K470 and T500 of SEQ ID NO: 9. In some embodiments, a PPX protein is a paralog of Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato plastidal PPX protein) and the PPX protein has two or more mutations and has one or more of: (1) an N at the position corresponding to position 52 of SEQ ID NO:1, wherein the N is substituted with an amino acid other than an N; (2) a K at the position corresponding to position 272 of SEQ ID NO:1, wherein the K is substituted with an amino acid other than a K; (3) an S at the position corresponding to position 359 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S; and/or (4) an S at the position corresponding to position 525 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S. In some embodiments, a mutated PPX protein includes three or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of G52, N85, N105, E111, G130, D139, P143, R144, F145, L147, F165, L167, I170, A180, P185, E192, S193, R199, V206, E212, Y219, A220, G221, L226, M228, K229, A230, K237, S244, R256, R257, K270, P271, Q272, S305, E311, T316, T318, S332, S343, A354, L357, K359, L360, A366, L393, L403, L424, Y426, S430, K438, E440, V444, L455, K457, V470, F478, F483, D484, I485, D487, K490, L503, V508 and I525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes three or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of D58, E64, G74, G84, L93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, N195, P214, I215, K229, K230, C271, D274, F283, A292, S296, C307, N324, D330, S396, A404, R406, K410, L421, A423, C434, D447, S448, V449, D451, D454, Y465, K470 and T500 of SEQ ID NO: 9. In some embodiments, a PPX protein is a paralog of Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato PPX protein) and the PPX protein has three or more mutations and has one or more of: (1) an N at the position corresponding to position 52 of SEQ ID NO:1, wherein the N is substituted with an amino acid other than an N; (2) a K at the position corresponding to position 272 of SEQ ID NO:1, wherein the K is substituted with an amino acid other than a K; (3) an S at the position corresponding to position 359 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S; and/or (4) an S at the position corresponding to position 525 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S.
[0014] In conjunction with the various aspects, embodiments, compositions and methods disclosed herein, a mutated PPX protein includes one or more amino acid mutations selected from Tables 1, 2, 3a, 3b, 4a, 4b, 8a-f, 9a-d and 10. In some embodiments, a mutated PPX protein includes two or more amino acid mutations selected from Tables 1, 2, 3a, 3b, 4a, 4b, 8a-f, 9a-d and 10. In some embodiments, a mutated PPX protein includes three or more amino acid mutations selected from Tables 1, 2, 3a, 3b, 4a, 4b, 8a-f, 9a-d and 10. In some embodiments, a mutated PPX protein includes one or more nucleic acid sequence mutations selected from Tables 2, 3a and 3b. In some embodiments, the one or more mutations in a mutated PPX protein includes one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of a glycine to lysine at a position corresponding to position 52 of SEQ ID NO: 1; an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1; a glutamic acid to valine at a position corresponding to position 111 of SEQ ID NO: 1; a glycine to asparagine at a position corresponding to position 130 of SEQ ID NO: 1; an aspartic acid to histidine at a position corresponding to position 139 of SEQ ID NO: 1; a proline to arginine at a position corresponding to position 143 of SEQ ID NO: 1; an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1; an arginine to leucine at a position corresponding to position 144 of SEQ ID NO: 1; an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1; a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1; a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1; a leucine to valine at a position corresponding to position 147 of SEQ ID NO: 1; a phenylalanine to asparagine at a position corresponding to position 165 of SEQ ID NO: 1; an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1; a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 1; a proline to histidine at a position corresponding to position 185 of SEQ ID NO: 1; a proline to tyrosine at a position corresponding to position 185 of SEQ ID NO: 1; a glutamic acid to aspartic acid at a position corresponding to position 192 of SEQ ID NO: 1; a glutamic acid to lysine at a position corresponding to position 192 of SEQ ID NO: 1; a serine to threonine at a position corresponding to position 193 of SEQ ID NO: 1; an arginine to leucine at a position corresponding to position 199 of SEQ ID NO: 1; a valine to phenylalanine at a position corresponding to position 206 of SEQ ID NO: 1; a tyrosine to serine at a position corresponding to position 219 of SEQ ID NO: 1; an alanine to cysteine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to leucine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to valine at a position corresponding to position 220 of SEQ ID NO: 1; a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1; a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1; a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 1; an alanine to phenylalanine at a position corresponding to position 230 of SEQ ID NO: 1; a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1; a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1; an arginine to histidine at a position corresponding to position 256 of SEQ ID NO: 1; an arginine to serine at a position corresponding to position 256 of SEQ ID NO: 1; a lysine to glutamic acid at a position corresponding to position 270 of SEQ ID NO: 1; a lysine to glutamine at a position corresponding to position 270 of SEQ ID NO: 1; a proline to arginine at a position corresponding to position 271 of SEQ ID NO: 1; a glutamine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1; a serine to leucine at a position corresponding to position 305 of SEQ ID NO: 1; a glutamic acid arginine at a position corresponding to position 311 of SEQ ID NO: 1; a threonine to glycine at a position corresponding to position 316 of SEQ ID NO: 1; a threonine to glycine at a position corresponding to position 318 of SEQ ID NO: 1; a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1; a leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 1; a lysine to arginine at a position corresponding to position 359 of SEQ ID NO: 1; a lysine to threonine at a position corresponding to position 359 of SEQ ID NO: 1; a leucine to aspartic acid at a position corresponding to position 360 of SEQ ID NO: 1; a leucine to lysine at a position corresponding to position 360 of SEQ ID NO: 1; an alanine to glutamic acid at a position corresponding to position 366 of SEQ ID NO: 1; a leucine to methionine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 403 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1; a tyrosine to cysteine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to valine at a position corresponding to position 426 of SEQ ID NO: 1; a serine to leucine at a position corresponding to position 430 of SEQ ID NO: 1; a lysine to serine at a position corresponding to position 438 of SEQ ID NO: 1; a glutamic acid to lysine at a position corresponding to position 440 of SEQ ID NO: 1; a valine to isoleucine at a position corresponding to position 444 of SEQ ID NO: 1; a leucine to valine at a position corresponding to position 455 of SEQ ID NO: 1; a lysine to valine at a position corresponding to position 457 of SEQ ID NO: 1; a valine to serine at a position corresponding to position 470 of SEQ ID NO: 1; a valine to tyrosine at a position corresponding to position 470 of SEQ ID NO: 1; a phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO: 1; a phenylalanine to glycine at a position corresponding to position 483 of SEQ ID NO: 1; an aspartic acid to alanine at a position corresponding to position 484 of SEQ ID NO: 1; an isoleucine to glutamic acid at a position corresponding to position 485 of SEQ ID NO: 1; an aspartic acid to glycine at a position corresponding to position 487 of SEQ ID NO: 1; a lysine to asparagine at a position corresponding to position 490 of SEQ ID NO: 1; a leucine to phenylalanine at a position corresponding to position 503 of SEQ ID NO: 1; a valine to threonine at a position corresponding to position 508 of SEQ ID NO: 1; and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a PPX protein is a paralog of Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato PPX protein) and the PPX protein may have an N at the position corresponding to position 52 of SEQ ID NO:1, wherein the N is substituted with an amino acid other than an N; a K at the position corresponding to position 272 of SEQ ID NO:1, wherein the K is substituted with an amino acid other than a K; an S at the position corresponding to position 359 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S; and/or an S at the position corresponding to position 525 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S. In such embodiments, the one or more mutations in a mutated PPX protein includes one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of an asparagine to lysine at a position corresponding to position 52 of SEQ ID NO: 1; an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1; an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1; an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1; a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1; a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1; an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1; a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 1; a proline to histidine at a position corresponding to position 185 of SEQ ID NO: 1; an alanine to cysteine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to leucine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to valine at a position corresponding to position 220 of SEQ ID NO: 1; a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1; a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1; a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1; a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1; a lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1; a serine to leucine at a position corresponding to position 305 of SEQ ID NO: 1; a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1; a leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 1; a serine to arginine at a position corresponding to position 359 of SEQ ID NO: 1; a serine to threonine at a position corresponding to position 359 of SEQ ID NO: 1; a leucine to methionine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 403 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1; a tyrosine to cysteine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to valine at a position corresponding to position 426 of SEQ ID NO: 1; a phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO: 1; an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1; an aspartic acid to asparagine at a position corresponding to position 58 of SEQ ID NO: 9; a glutamic acid to valine at a position corresponding to position 64 of SEQ ID NO: 9; a glycine to cysteine at a position corresponding to position 74 of SEQ ID NO: 9; a glycine to asparagine at a position corresponding to position 84 of SEQ ID NO: 9; a leucine to histidine at a position corresponding to position 93 of SEQ ID NO: 9; a lysine to arginine at a position corresponding to position 97 of SEQ ID NO: 9; an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; an arginine to leucine at a position corresponding to position 98 of SEQ ID NO:9; an alanine to valine at a position corresponding to position 101 of SEQ ID NO: 9; a serine to asparagine at a position corresponding to position 119 of SEQ ID NO: 9; a phenylalanine to leucine at a position corresponding to position 121 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; an asparagine to tyrosine at a position corresponding to position 139 of SEQ ID NO: 9; an asparagine to arginine at a position corresponding to position 139 of SEQ ID NO: 9; an asparagine to histidine at a position corresponding to position 139 of SEQ ID NO: 9; a glutamic acid to aspartic acid at a position corresponding to position 150 of SEQ ID NO: 9; a glutamic acid to lysine at a position corresponding to position 150 of SEQ ID NO: 9; a serine to threonine at a position corresponding to position 151 of SEQ ID NO: 9; a glutamine to leucine at a position corresponding to position 157 of SEQ ID NO: 9; a valine to phenylalanine at a position corresponding to position 164 of SEQ ID NO: 9; a valine to alanine at a position corresponding to position 164 of SEQ ID NO: 9; an aspartic acid to glutamic acid at a position corresponding to position 170 of SEQ ID NO: 9; a cysteine to serine at a position corresponding to position 177 of SEQ ID NO: 9; a histidine to glutamine at a position corresponding to position 187 of SEQ ID NO: 9; a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9; an asparagine to lysine at a position corresponding to position 195 of SEQ ID NO: 9; a proline to serine at a position corresponding to position 214 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; an isoleucine to serine at a position corresponding to position 215 of SEQ ID NO: 9; an isoleucine to histidine at a position corresponding to position 215 of SEQ ID NO: 9; a lysine to glutamic acid at a position corresponding to position 229 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9; a lysine to arginine at a position corresponding to position 230 of SEQ ID NO: 9; a cysteine to arginine at a position corresponding to position 271 of SEQ ID NO: 9; an aspartic acid to glycine at a position corresponding to position 274 of SEQ ID NO: 9; a phenylalanine to glycine at a position corresponding to position 283 of SEQ ID NO: 9; an alanine to glycine at a position corresponding to position 292 of SEQ ID NO: 9; a serine to leucine at a position corresponding to position 296 of SEQ ID NO: 9; a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9; an asparagine to aspartic acid at a position corresponding to position 324 of SEQ ID NO: 9; an asparagine to lysine at a position corresponding to position 324 of SEQ ID NO: 9; an aspartic acid to glutamic acid at a position corresponding to position 330 of SEQ ID NO: 9; a serine to leucine at a position corresponding to position 396 of SEQ ID NO: 9; an alanine to serine at a position corresponding to position 404 of SEQ ID NO: 9; an arginine to lysine at a position corresponding to position 406 of SEQ ID NO: 9; a lysine to isoleucine at a position corresponding to position 410 of SEQ ID NO: 9; a leucine to valine at a position corresponding to position 421 of SEQ ID NO: 9; an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9; a cysteine to serine at a position corresponding to position 434 of SEQ ID NO: 9; a cysteine to tyrosine at a position corresponding to position 434 of SEQ ID NO: 9; an aspartic acid to glycine at a position corresponding to position 447 of SEQ ID NO: 9; a serine to alanine at a position corresponding to position 448 of SEQ ID NO: 9; a valine to glutamic acid at a position corresponding to position 449 of SEQ ID NO: 9; an aspartic acid to glycine at a position corresponding to position 451 of SEQ ID NO: 9; an aspartic acid to asparagine at a position corresponding to position 454 of SEQ ID NO: 9; a tyrosine to phenylalanine at a position corresponding to position 465 of SEQ ID NO: 9; a lysine to threonine at a position corresponding to position 470 of SEQ ID NO: 9; and a threonine to serine at a position corresponding to position 500 of SEQ ID NO: 9.
[0015] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, In certain embodiments, a mutated PPX protein may include a combination of mutations; for example a combination of mutations selected from Tables 4a and 4b. In some embodiments, the mutated PPX protein includes a combination of two or more mutations; for example, combinations selected from Tables 4a and 4b. In some embodiments, the mutated PPX protein includes a combination of three or more mutations; for example, combinations selected from Tables 4a and 4b. In some embodiments, the combination of mutations in a mutated PPX gene encode a protein having a mutation at a position corresponding to Y426 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: N85, R144, F145, A180, A220, L226, and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations in a mutated PPX gene encode a protein having a mutation at a position corresponding to L393 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: R144, F145, A220, S224 and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to L403 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: F145, A220 and L226 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to R144 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: G52, N85, A220, S244, L226, M228, K272, S332, L393, L424, Y426 and I525 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to N85 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: R144, F145, A180, A220, L226, M228, and Q272 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to L424 of SEQ ID NO: 1 and a mutation at the amino acid position corresponding to a position selected from the group consisting of: R144, F145, A220, L226 and L393 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to I525 of SEQ ID NO: 1 and a mutation at the amino acid position corresponding to a position N85, F144, F145, A180, L226 and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to R144 of SEQ ID NO: 1 and a mutation at the amino acid position corresponding to a position A220 of SEQ ID NO: 1. In some embodiments, a PPX protein is a paralog of Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato PPX protein) and the PPX protein may have an N at the position corresponding to position 52 of SEQ ID NO:1, wherein the N is substituted with an amino acid other than an N; a K at the position corresponding to position 272 of SEQ ID NO:1, wherein the K is substituted with an amino acid other than a K; an S at the position corresponding to position 359 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S; and/or an S at the position corresponding to position 525 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S. In such embodiments, the mutated PPX protein includes a combination of two or more mutations; for example, combinations selected from Tables 4a and 4b. In such embodiments, the mutated PPX protein includes a combination of three or more mutations; for example, combinations selected from Tables 4a and 4b. In some embodiments, the combination of mutations in a mutated PPX gene encode a protein having a mutation at a position corresponding to Y426 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: N85, R144, F145, A180, A220, L226, and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations in a mutated PPX gene encode a protein having a mutation at a position corresponding to L393 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: R144, F145, A220, S244 and S224 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to L403 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: F145, A220 and L226 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to R144 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: N52, N85, A220, S244, L226, M228, K272, S332, L393, L424, Y426 and S525 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to N85 of SEQ ID NO: 1 and a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of: R144, F145, A180, A220, L226, M228, and K272 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to L424 of SEQ ID NO: 1 and a mutation at the amino acid position corresponding to a position selected from the group consisting of: R144, F145, A220, L226 and L393 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to S525 of SEQ ID NO: 1 and a mutation at the amino acid position corresponding to a position N85, F144, F145, A180, L226 and S244 of SEQ ID NO: 1. In some embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to 98 of SEQ ID NO: 9 and a mutation at the amino acid position corresponding to a position selected from the group consisting of 74, 93, 97, 98, 119, 121, 124, 139, 150, 151, 164, 188, 214, 229, 230, 271, 274, 292, 307, 324, 396, 410, 423, 434, 447, 448, 451, 465, 470 and 500 of SEQ ID NO: 9. In certain embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to 98 of SEQ ID NO: 9 and a mutation at the amino acid position corresponding to a position selected from the group consisting of 271, 274, 292, 307, 324, 330, 396, 404, 406, 410, 423, 434, 447, 448, 454, 465, 470 and 500 of SEQ ID NO: 9. In certain embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to 98 of SEQ ID NO: 9 and a mutation at the amino acid position corresponding to a position selected from the group consisting of 307 and 423 of SEQ ID NO: 9. In certain embodiments, the combination of mutations encode a protein having a mutation at a position corresponding to 98 of SEQ ID NO: 9 and a mutation at the amino acid position corresponding to a position selected from the group consisting 124, 188, 214 and 229 of SEQ ID NO: 9.
[0016] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein the PPX protein may be a paralog of Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato mitochondrial PPX protein) and the PPX protein may have one or more corresponding PPX amino acids to SEQ ID NO: 9. In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the one or more mutations in a mutated PPX gene may encode a mutated PPX protein having one or more mutations, two or more mutations, three or more mutations selected from the group consisting of a mutated PPX protein may include one or more mutation at the amino acid position corresponding to one or more positions selected from the group consisting of positions 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 500 of SEQ ID NO: 9.
[0017] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the plant cell may have a mutated PPX gene. In certain embodiments, the mutated PPX gene encodes a mutated PPX protein. In certain embodiments, the plant cell may be part of a herbicide-resistant plant. The method may include introducing into a plant cell a gene repair oligonucleobase (GRON); for example, using a GRON with a targeted mutation in a PPX gene. In certain embodiments, the plant cell produced by the method may include a PPX gene capable of expressing a mutated PPX protein. The method may further include identifying a plant cell or a plant including a plant cell that includes (1) a mutated PPX gene and/or (2) normal growth and/or catalytic activity as compared to a corresponding wild-type plant cell. The herbicide-resistant plant having a plant cell such as described herein may be identified in the presence of a PPX-inhibiting herbicide. In some embodiments, the plant cell is non-transgenic. In some embodiments, the plant cell is transgenic. A plant that includes a plant cell such as described herein may be a non-transgenic or transgenic herbicide-resistant plant; for example, the plant and/or plant cell may have a mutated PPX gene that results in resistance to at least one herbicide. In some embodiments, a plant having a plant cell as described herein may be produced asexually; for example, from one or more plant cells or from plant tissue made up of one or more plant cells; for example, from a tuber. In other embodiments, a plant having a plant cell such as described herein may be produced sexually.
[0018] In another aspect, there is provided a method for producing a herbicide-resistant plant. The method may include introducing into a plant cell a gene repair oligonucleobase (GRON); for example, using a GRON designed with a targeted mutation in a PPX gene. The mutated PPX gene may express a mutated PPX protein. The method may further include identifying a plant that has normal growth and/or catalytic activity as compared to a corresponding wild-type plant cell. The plant may be identified in the presence of a PPX-inhibiting herbicide. In some embodiments, the plant is non-transgenic. The plant may in some embodiments be a non-transgenic herbicide-resistant plant; for example, the plant may include a mutated PPX gene that results in resistance or tolerance to at least one herbicide.
[0019] In another aspect there is provided a seed including a mutated PPX gene. In some embodiments, the seed has a mutated PPX gene. In some embodiments, the mutated PPX encodes a mutated PPX protein. In some embodiments the mutated PPX protein may be resistant to a herbicide; for example, a PPX-inhibiting herbicide. In some embodiments, a plant grown from the seed is resistant to at least one herbicide; for example, a PPX-inhibiting herbicide.
[0020] In another aspect, there is provided a method for increasing the herbicide-resistance of a plant by: (a) crossing a first plant to a second plant, in which the first plant that includes a mutated PPX gene, in which the gene encodes a mutated PPX protein; (b) screening a population resulting from the cross for increased herbicide-resistance; for example, increased resistance to a PPX-inhibiting herbicide (c) selecting a member resulting from the cross having increased herbicide-resistance; and/or (d) producing seeds resulting from the cross. In some embodiments, a hybrid seed is produced by any of the methods such as described herein. In some embodiments, plants are grown from seeds produced by any of the methods such as described herein. In some embodiments, the plants and/or seeds are non-transgenic. In some embodiments, the plants and/or seeds are transgenic.
[0021] In another aspect, there is provided a method of controlling weeds in a field containing plants by applying an effective amount of at least one herbicide to a field containing weeds and plants. In some embodiments of the method, the at least one herbicide is a PPX-inhibiting herbicide. In some embodiments of the method, one or more of the plants in the field includes a mutated PPX gene; for example such as described herein. In some embodiments of the method one or more of the plants in the field includes a non-transgenic or transgenic plant having a mutated PPX gene such as described herein. In some embodiments, the mutated PPX gene encodes a mutated PPX protein. In some embodiments, one more of the plants in the field is herbicide resistant; for example, resistant to a PPX-inhibiting herbicide.
[0022] In another aspect, there is provided an isolated nucleic acid encoding a PPX protein or portion thereof. In some embodiments the isolated nucleic acid includes one or more of the PPX gene mutations such as described herein. In some embodiments, the isolated nucleic acid encodes a mutated PPX protein as disclosed herein. In certain embodiments, the isolated nucleic acid encodes a PPX protein that is herbicide resistant; for example, resistant to a PPX-inhibiting herbicide.
[0023] In another aspect, there is provided an expression vector containing an isolated nucleic acid of a mutated PPX gene. In some embodiments, the expression vector contains an isolated nucleic acid encoding a PPX protein. In some embodiments, the isolated nucleic acid encodes a protein having a mutation selected from the mutations shown in Tables 1, 2, 3a, 3b, 4a, 4b, 8a-f, 9a-d and 10. In certain embodiments, the isolated nucleic acid encodes a protein having two or more mutations. In some embodiments, the two or more mutations are selected from Tables 1, 2, 3a, 3b, 4a, 4b, 8a-f, 9a-d and 10. In certain embodiments, the isolated nucleic acid encodes a PPX protein that is herbicide resistant; for example, resistant to a PPX-inhibiting herbicide.
[0024] As used herein, the term "herbicide" refers to any chemical or substance that can kill a plant or that can halt or reduce growth and/or viability of a plant. In some embodiments, herbicide resistance is the genetically heritable ability of a plant to survive and reproduce following treatment with a concentration of herbicide that would normally kill or severely injure an unmodified wildtype plant. In some embodiments, in conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the herbicide is a PPX-inhibiting herbicide. In some embodiments, a PPX-inhibiting herbicide is a herbicide from a chemical family selected from the group of chemical families listed in Table 5. In some embodiments, a PPX-inhibiting herbicide is a herbicide from a chemical family selected from the group of chemical families consisting of N-phenylphthalimides, triazolinones, and pyrimidindiones. In some embodiments, a PPX-inhibiting herbicide is selected from the group of herbicides listed in Table 5. In some embodiments, PPX-inhibiting herbicide is selected from the group of herbicides consisting of flumoioxazin, sulfentrazone, and saflufenacil. In other embodiments, the PPX-inhibiting herbicide is a flumioxazin herbicide. In other embodiments, the PPX-inhibiting herbicide is a sulfentrazone herbicide. In other embodiments, the PPX-inhibiting herbicide is a saflufenacil herbicide.
[0025] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the plant or plant cell is from a plant crop selected from the group consisting of potato, sunflower, sugar beet, maize, cotton, soybean, wheat, rye, oats, rice, canola, fruits, vegetables, tobacco, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, carrot, lettuce, onion, soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, petunia, tulip, iris, lily, and nut-producing plants insofar as they are not already specifically mentioned. In some embodiments, the plant or plant cell is of a species selected from Table 6. In some embodiments, the plant or plant cell is of a species selected from the group consisting of Arabidopsis thaliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Amaranthus tuberculatus, Zea mays, Brassica napus, and Glycine max. In some embodiments, the plant or plant cell is a Russet Burbank potato cultivar. In some embodiments, a mutated PPX gene encodes a Russet Burbank PPX protein. In some embodiments, a mutated PPX gene encodes an Arabidopsis thaliana PPX protein. In some embodiments, a mutated PPX gene encodes a Solanum tuberosum PPX protein. In some embodiments, a mutated PPX gene encodes a Solanum phureja PPX protein. In some embodiments, a mutated PPX gene encodes a Zea mays PPX protein. In some embodiments, a mutated PPX gene encodes an Oryza sativa PPX protein. In some embodiments, a mutated PPX gene encodes an Amaranthus tuberculatus PPX protein. In some embodiments, a mutated PPX gene encodes a Sorghum bicolor PPX protein. In some embodiments, a mutated PPX gene encodes a Ricinus communis PPX protein. In some embodiments, a mutated PPX gene encodes a Brassica napus PPX protein. In some embodiments, a mutated PPX gene encodes a Glycine max PPX protein. In some embodiments, a mutated PPX gene At4g01690 encodes an Arabidopsis thaliana PPX protein. In some embodiments a mutated PPX gene At5g14220 encodes an Arabidopsis thaliana PPX protein.
[0026] In any of the aspects, embodiments, methods or compositions disclosed herein may include one or more mutated PPX genes. In some embodiments, the methods and compositions involve one or more mutated PPX genes that encode one or more mitochondrial PPX proteins. In other embodiments, the methods and compositions include one or more mutated PPX genes which encode one or more plastidal PPX proteins. In some embodiments, the methods and compositions include one or more mutated PPX genes which encode one or more plastidal PPX proteins and mitochondrial PPX proteins. In some embodiments, the methods and compositions include a mitochondrial mutated PPX gene StmPPX1. In some embodiments, the methods and compositions include a mitochondrial mutated PPX gene StmPPX2. In some embodiments, the plant has the plastidal mutated PPX gene StcPPX1. In some embodiments, the methods and compositions include a mitochondrial mutated PPX gene allele StcPPX2.1. In some embodiments, the methods and compositions include a mitochondrial mutated PPX gene allele StcPPX2.2. In some embodiments, the methods and compositions include a plastidal mutated PPX gene allele StcPPX1. In some embodiments, the methods and compositions include a plastidal mutated PPX gene allele StcPPX1.1.
[0027] As used herein, the term "gene" refers to a DNA sequence that includes control and coding sequences necessary for the production of an RNA, which may have a non-coding function (e.g., a ribosomal or transfer RNA) or which may encode a polypeptide or a polypeptide precursor. The RNA or polypeptide may be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.
[0028] As used herein, the term "coding sequence" refers to a sequence of a nucleic acid or its complement, or a part thereof, that can be transcribed and/or translated to produce the mRNA for and/or the polypeptide or a fragment thereof. Coding sequences include exons in a genomic DNA or immature primary RNA transcripts, which are joined together by the cell's biochemical machinery to provide a mature mRNA. The anti-sense strand is the complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.
[0029] As used herein, the term "non-coding sequence" refers to a sequence of a nucleic acid or its complement, or a part thereof, that is not transcribed into amino acid in vivo, or where tRNA does not interact to place or attempt to place an amino acid. Non-coding sequences include both intron sequences in genomic DNA or immature primary RNA transcripts, and gene-associated sequences such as promoters, enhancers, silencers, etc.
[0030] A nucleobase is a base, which in certain preferred embodiments is a purine, pyrimidine, or a derivative or analog thereof. Nucleosides are nucleobases that contain a pentosefuranosyl moiety, e.g., an optionally substituted riboside or 2'-deoxyriboside. The moiety may be any group that increases DNA binding and/or decreases nuclease degradation as compared to a nucleoside not having the moiety. Nucleosides can be linked by one of several linkage moieties, which may or may not contain phosphorus. Nucleosides that are linked by unsubstituted phosphodiester linkages are termed nucleotides. As used herein, the term "nucleobase" includes peptide nucleobases, the subunits of peptide nucleic acids, and morpholine nucleobases as well as nucleosides and nucleotides.
[0031] An oligonucleobase is a polymer comprising nucleobases; preferably at least a portion of which can hybridize by Watson-Crick base pairing to a DNA having the complementary sequence. An oligonucleobase chain may have a single 5' and 3' terminus, which are the ultimate nucleobases of the polymer. A particular oligonucleobase chain can contain nucleobases of all types. An oligonucleobase compound is a compound comprising one or more oligonucleobase chains that may be complementary and hybridized by Watson-Crick base pairing. Ribo-type nucleobases include pentosefuranosyl containing nucleobases wherein the 2' carbon is a methylene substituted with a hydroxyl, alkyloxy or halogen. Deoxyribo-type nucleobases are nucleobases other than ribo-type nucleobases and include all nucleobases that do not contain a pentosefuranosyl moiety.
[0032] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, an oligonucleobase strand may include both oligonucleobase chains and segments or regions of oligonucleobase chains. An oligonucleobase strand may have a 5' end and a 3' end, and when an oligonucleobase strand is coextensive with a chain, the 5' and 3' ends of the strand are also 5' and 3' termini of the chain.
[0033] As used herein, the term "gene repair oligonucleobase" or "GRON" refers to oligonucleobases, including mixed duplex oligonucleotides, non-nucleotide containing molecules, single stranded oligodeoxynucleotides and other gene repair molecules.
[0034] As used herein, the term "transgenic" refers to an organism or cell that has DNA derived from another organism inserted into its genome. For example, in some embodiments, a transgenic organism or cell includes inserted DNA that includes a foreign promoter and/or coding region.
[0035] As used herein, the term "non-transgenic" refers to an organism or cell that does not have DNA derived from another organism inserted into its genome although a non-transgenic plant or cell may have one or more artificially introduced targeted mutations.
[0036] As used herein, the term "isolated", when referring to a nucleic acid (e.g., an oligonucleotide such as RNA, DNA, or a mixed polymer) refers to a nucleic acid that is apart from a substantial portion of the genome in which it naturally occurs and/or is substantially separated from other cellular components which naturally accompany such nucleic acid. For example, any nucleic acid that has been produced synthetically (e.g., by serial base condensation) is considered to be isolated. Likewise, nucleic acids that are recombinantly expressed, cloned, produced by a primer extension reaction (e.g., PCR), or otherwise excised from a genome are also considered to be isolated.
[0037] As used herein, the term "amino acid sequence" refers to a polypeptide or protein sequence. The convention "AAwt###AAmut" is used to indicate a mutation that results in the wild-type amino acid AAwt at position ### in the polypeptide being replaced with mutant AAmut.
[0038] As used herein, the term "complement" refers to the complementary sequence to a nucleic acid according to standard Watson/Crick pairing rules. A complement sequence can also be a sequence of RNA complementary to the DNA sequence or its complementary sequence, and can also be a cDNA.
[0039] As used herein, the term "substantially complementary" refers to two sequences that hybridize under near stringent hybridization conditions. The skilled artisan will understand that substantially complementary sequences need not hybridize along their entire length.
[0040] As used herein the term "codon" refers to a sequence of three adjacent nucleotides (either RNA or DNA) constituting the genetic code that determines the insertion of a specific amino acid in a polypeptide chain during protein synthesis or the signal to stop protein synthesis. The term "codon" is also used to refer to the corresponding (and complementary) sequences of three nucleotides in the messenger RNA into which the original DNA is transcribed.
[0041] As used herein, the term "homology" refers to sequence similarity among proteins and DNA. The term "homology" or "homologous" refers to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that has less than 100% sequence identity when compared to another sequence.
[0042] As used herein, the term "about" in quantitative terms refers to plus or minus 10%. For example, "about 3%" would encompass 2.7-3.3% and "about 10%" would encompass 9-11%. Moreover, where "about" is used herein in conjunction with a quantitative term it is understood that in addition to the value plus or minus 10%, the exact value of the quantitative term is also contemplated and described. For example, the term "about 3%" expressly contemplates, describes and includes exactly 3%.
BRIEF DESCRIPTION OF THE FIGURES
[0043] FIG. 1 is the amino acid sequence of an Arabidopsis thaliana chloroplast (plastid) PPX protein (SEQ ID NO: 1).
[0044] FIG. 2 is the nucleic acid sequence of an Arabidopsis thaliana chloroplast (plastid) PPX cDNA (SEQ ID NO: 2).
[0045] FIG. 3 is the amino acid sequence of Arabidopsis thaliana mitochondrial PPX protein (SEQ ID NO: 3).
[0046] FIG. 4 is the nucleic acid sequence of an Arabidopsis thaliana mitochondrial PPX cDNA (SEQ ID NO: 4).
[0047] FIG. 5 is the amino acid sequence of Amaranthus tuberculatus mitochondrial PPX protein (SEQ ID NO: 5).
[0048] FIG. 6 is the nucleic acid sequence of Amaranthus tuberculatus mitochondrial PPX cDNA (SEQ ID NO: 6).
[0049] FIG. 7 is the amino acid sequence of Solanum tuberosum plastidal PPX protein StcPPX (SEQ ID NO: 7).
[0050] FIG. 8 is the nucleic acid sequence of Solanum tuberosum plastidal PPX cDNA (SEQ ID NO: 8).
[0051] FIG. 9 is the amino acid sequence of Solanum tuberosum mitochondrial PPX protein (SEQ ID NO: 9).
[0052] FIG. 10 is the nucleic acid sequence of Solanum tuberosum mitochondrial PPX cDNA (SEQ ID NO: 10).
[0053] FIG. 11 is the amino acid sequence of Zea mays plastidal PPX protein (SEQ ID NO: 11).
[0054] FIG. 12 is the nucleic acid sequence of Zea mays plastidal PPX cDNA (SEQ ID NO: 12).
[0055] FIG. 13 is the amino acid sequence of Zea mays mitochondrial PPX protein (SEQ ID NO: 13).
[0056] FIG. 14 is the nucleic acid sequence of Zea mays mitochondrial PPX cDNA (SEQ ID NO: 14).
[0057] FIG. 15 is the amino acid sequence of Oryza sativa plastidal PPX protein (SEQ ID NO: 15).
[0058] FIG. 16 is the nucleic acid sequence of Oryza sativa plastidal PPX cDNA (SEQ ID NO: 16).
[0059] FIG. 17 is the amino acid sequence of Oryza sativa mitochondrial PPX protein cDNA (SEQ ID NO: 17).
[0060] FIG. 18 is the nucleic acid sequence of Oryza sativa mitochondrial PPX cDNA (SEQ ID NO: 18).
[0061] FIG. 19 is the amino acid sequence of Sorghum bicolor plastidal PPX protein (SEQ ID NO: 19).
[0062] FIG. 20 is the nucleic acid sequence of Sorghum bicolor plastidal PPX cDNA (SEQ ID NO: 20).
[0063] FIG. 21 is the amino acid sequence of Sorghum bicolor mitochondrial PPX protein (SEQ ID NO: 21).
[0064] FIG. 22 is the nucleic acid sequence of Sorghum bicolor mitochondrial PPX cDNA (SEQ ID NO: 22).
[0065] FIG. 23 is the amino acid sequence of Ricinus communis plastidal PPX protein (SEQ ID NO: 23).
[0066] FIG. 24 is the nucleic acid sequence of Ricinus communis plastidal PPX cDNA (SEQ ID NO: 24).
[0067] FIG. 25 is the amino acid sequence of Ricinus communis mitochondrial PPX protein (SEQ ID NO: 25).
[0068] FIG. 26 is the nucleic acid sequence of Ricinus communis mitochondrial PPX cDNA (SEQ ID NO: 26).
[0069] FIG. 27 is the amino acid sequence of Solanum tuberosum mitochondrial PPX protein StmPPX1 (SEQ ID NO: 27).
[0070] FIG. 28 is the nucleic acid sequence of Solanum tuberosum mitochondrial PPX cDNA StmPPX1 (SEQ ID NO: 28).
[0071] FIG. 29 is the amino acid sequence of Solanum tuberosum mitochondrial PPX protein StmPPX2.1 (SEQ ID NO: 29).
[0072] FIG. 30 is the nucleic acid sequence of Solanum tuberosum mitochondrial PPX cDNA StmPPX2.1 (SEQ ID NO: 30).
[0073] FIG. 31 is the amino acid sequence of Solanum tuberosum mitochondrial PPX protein StmPPX2.2 (SEQ ID NO: 31).
[0074] FIG. 32 is the nucleic acid sequence of Solanum tuberosum mitochondrial PPX cDNAStmPPX2.2 (SEQ ID NO: 32).
[0075] FIG. 33 is the amino acid sequence of Brassica napus plastidal PPX protein BncPPX1 (SEQ ID NO: 33).
[0076] FIG. 34 is the nucleic acid sequence of Brassica napus PPX cDNA BncPPX1 (SEQ ID NO: 34).
[0077] FIG. 35 is the amino acid sequence of Brassica napus plastidal PPX protein BncPPX2 (SEQ ID NO: 35).
[0078] FIG. 36 is the nucleic acid sequence of Brassica napus PPX cDNA BncPPX2 (SEQ ID NO: 36).
[0079] FIG. 37 is the partial amino acid sequence of Brassica napus plastidal PPX protein BncPPX3 (SEQ ID NO: 37).
[0080] FIG. 38 is the partial nucleic acid sequence of Brassica napus PPX cDNA BncPPX3 (SEQ ID NO: 38).
[0081] FIG. 39 is the amino acid sequence of Glycine max plastidal PPX protein GmcPPX1-1 (SEQ ID NO: 39).
[0082] FIG. 40 is the amino acid sequence of Glycine max plastidal PPX protein GmcPPX1-2 (SEQ ID NO: 40).
[0083] FIG. 41 is the nucleic acid sequence of Glycine max plastidal PPX protein GmcPPX1 (SEQ ID NO: 41).
[0084] FIG. 42 is the amino acid sequence of Glycine max plastidal PPX protein GmcPPX2 (SEQ ID NO: 42).
[0085] FIG. 43 is the nucleic acid sequence of Glycine max plastidal PPX protein GmcPPX2 (SEQ ID NO: 43).
[0086] FIG. 44 is the amino acid sequence of Glycine max mitochondrial PPX protein GmcPPX (SEQ ID NO: 44).
[0087] FIG. 45 is the nucleic acid sequence of Glycine max mitochondrial PPX protein GmcPPX (SEQ ID NO: 45).
[0088] FIG. 46 is an alignment of PPX proteins of various plant species.
[0089] FIG. 47 is a table of homologous amino acid positions in plant PPX amino acid sequences of various species.
[0090] FIG. 48 is a table of homologous amino acid positions in plant PPX amino acid sequences of various species.
DETAILED DESCRIPTION OF THE INVENTION
Rapid Trait Development System (RTDSยฎ)
[0091] In any of the various aspects and embodiments of the compositions and methods disclosed herein, mutations in genes and proteins may be made using, for example, the Rapid Trait Development System (RTDSยฎ) technology developed by Cibus. In combination or alone, plants containing any of the mutations disclosed herein can form the basis of new herbicide-resistant products. Also provided are seeds produced from the mutated plants in which the PPX genes are either homozygous or heterozygous for the mutations. The mutations disclosed herein can be in combination with any other mutation known or with mutations discovered in the future.
[0092] As used herein, the term "heterozygous" refers to having different alleles at one or more genetic loci in homologous chromosome segments. As used herein "heterozygous" may also refer to a sample, a cell, a cell population or an organism in which different alleles at one or more genetic loci may be detected. Heterozygous samples may also be determined via methods known in the art such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows two peaks at a single locus and both peaks are roughly the same size, the sample may be characterized as heterozygous. Or, if one peak is smaller than another, but is at least about 25% the size of the larger peak, the sample may be characterized as heterozygous. In some embodiments, the smaller peak is at least about 15% of the larger peak. In other embodiments, the smaller peak is at least about 10% of the larger peak. In other embodiments, the smaller peak is at least about 5% of the larger peak. In other embodiments, a minimal amount of the smaller peak is detected.
[0093] As used herein, "homozygous" refers to having identical alleles at one or more genetic loci in homologous chromosome segments. "Homozygous" may also refer to a sample, a cell, a cell population or an organism in which the same alleles at one or more genetic loci may be detected. Homozygous samples may be determined via methods known in the art, such as, for example, nucleic acid sequencing. For example, if a sequencing electropherogram shows a single peak at a particular locus, the sample may be termed "homozygous" with respect to that locus.
[0094] The term "hemizygous" refers to a gene or gene segment being present only once in the genotype of a cell or an organism because the second allele is deleted. As used herein "hemizygous" may also refer to a sample, a cell, a cell population or an organism in which an allele at one or more genetic loci may be detected only once in the genotype.
[0095] In some embodiments, RTDS is based on altering a targeted gene by utilizing the cell's own gene repair system to specifically modify the gene sequence in situ and not insert foreign DNA and/or gene expression control sequences. This procedure may effect a precise change in the genetic sequence while the rest of the genome is left unaltered. In contrast to conventional transgenic GMOs, there is no integration of foreign genetic material, nor is any foreign genetic material left in the plant. In many embodiments, the changes in the genetic sequence introduced by RTDS are not randomly inserted. Since affected genes remain in their native location, no random, uncontrolled or adverse pattern of expression occurs.
[0096] The RTDS that effects this change is a chemically synthesized oligonucleotide (e.g., using a gene repair oligonucleobase (GRON)) which may be composed of both DNA and modified RNA bases as well as other chemical moieties, and is designed to hybridize at the targeted gene location to create a mismatched base-pair(s). This mismatched base-pair acts as a signal to attract the cell's own natural gene repair system to that site and correct (replace, insert or delete) the designated nucleotide(s) within the gene. Once the correction process is complete the RTDS molecule is degraded and the now-modified or repaired gene is expressed under that gene's normal endogenous control mechanisms.
Gene Repair Oligonucleobases ("GRON")
[0097] The methods and compositions disclosed herein can be practiced or made with "gene repair oligonucleobases" for example, having the conformations and chemistries as described in detail below. The "gene repair oligonucleobases" as contemplated herein have also been described in published scientific and patent literature using other names including "recombinagenic oligonucleobases;" "RNA/DNA chimeric oligonucleotides;" "chimeric oligonucleotides;" "mixed duplex oligonucleotides" (MDONs); "RNA DNA oligonucleotides (RDOs);" "gene targeting oligonucleotides;" "genoplasts;" "single stranded modified oligonucleotides;" "Single stranded oligodeoxynucleotide mutational vectors" (SSOMVs); "duplex mutational vectors;" and "heteroduplex mutational vectors."
[0098] Oligonucleobases having the conformations and chemistries described in U.S. Pat. No. 5,565,350 by Kmiec (Kmiec I) and U.S. Pat. No. 5,731,181 by Kmiec (Kmiec II), hereby incorporated by reference, are suitable for use as "gene repair oligonucleobases" of the present disclosure. The gene repair oligonucleobases in Kmiec I and/or Kmiec II contain two complementary strands, one of which contains at least one segment of RNA-type nucleotides (an "RNA segment") that are base paired to DNA-type nucleotides of the other strand.
[0099] Kmiec II discloses that purine and pyrimidine base-containing non-nucleotides can be substituted for nucleotides. Additional gene repair molecules that can be used for the present disclosure include, but are not limited to, those described in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Patent No. PCT/US00/23457; and in International Patent Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789, which are each hereby incorporated in their entirety.
[0100] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the gene repair oligonucleobase may be a mixed duplex oligonucleotides (MDON) in which the RNA-type nucleotides of the mixed duplex oligonucleotide are made RNase resistant by replacing the 2'-hydroxyl with a fluoro, chloro or bromo functionality or by placing a substituent on the 2'-O, Suitable substituents include the substituents taught by the Kmiec II. Alternative substituents may include, but are not limited to the substituents taught by U.S. Pat. No. 5,334,711 (Sproat) and the substituents taught by patent publications EP 629 387 and EP 679 657 (collectively, the Martin Applications), which are hereby incorporated by reference. As used herein, a 2'-fluoro, chloro or bromo derivative of a ribonucleotide or a ribonucleotide having a 2'-OH substituted with a substituent described in the Martin Applications or Sproat is termed a "2'-Substituted Ribonucleotide." As used herein the term "RNA-type nucleotide" means a 2'-hydroxyl or 2'-Substituted Nucleotide that is linked to other nucleotides of a mixed duplex oligonucleotide by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II. As used herein the term "deoxyribo-type nucleotide" means a nucleotide having a 2'-H, which can be linked to other nucleotides of a gene repair oligonucleobase by an unsubstituted phosphodiester linkage or any of the non-natural linkages taught by Kmiec I or Kmiec II.
[0101] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the gene repair oligonucleobase may be a mixed duplex oligonucleotides (MDON) that is linked solely by unsubstituted phosphodiester bonds. In alternative embodiments, the linkage is by substituted phosphodiesters, phosphodiester derivatives and non-phosphorus-based linkages as taught by Kmiec II. In yet another embodiment, each RNA-type nucleotide in the mixed duplex oligonucleotide is a 2'-Substituted Nucleotide. Particular preferred embodiments of 2'-Substituted Ribonucleotides include, but are not limited to, 2'-fluoro, 2'-methoxy, 2'-propyloxy, 2'-allyloxy, 2'-hydroxylethyloxy, 2'-methoxyethyloxy, 2'-fluoropropyloxy and 2'-trifluoropropyloxy substituted ribonucleotides. More preferred embodiments of 2'-Substituted Ribonucleotides are 2'-fluoro, 2'-methoxy, 2'-methoxyethyloxy, and 2'-allyloxy substituted nucleotides. In another embodiment the mixed duplex oligonucleotide is linked by unsubstituted phosphodiester bonds.
[0102] Although mixed duplex oligonucleotides (MDONs) having only a single type of 2'-substituted RNA-type nucleotide are more conveniently synthesized, the methods of the invention can also be practiced with mixed duplex oligonucleotides having two or more types of RNA-type nucleotides. The function of an RNA segment may not be affected by an interruption caused by the introduction of a deoxynucleotide between two RNA-type trinucleotides, accordingly, the term RNA segment encompasses terms such as "interrupted RNA segment." An uninterrupted RNA segment is termed a contiguous RNA segment. In an alternative embodiment an RNA segment can contain alternating RNase-resistant and unsubstituted 2'-OH nucleotides. The mixed duplex oligonucleotides preferably have fewer than 100 nucleotides and more preferably fewer than 85 nucleotides, but more than 50 nucleotides. The first and second strands are Watson-Crick base paired. In one embodiment the strands of the mixed duplex oligonucleotide are covalently bonded by a linker, such as a single stranded hexa, penta or tetranucleotide so that the first and second strands are segments of a single oligonucleotide chain having a single 3' and a single 5' end. The 3' and 5' ends can be protected by the addition of a "hairpin cap" whereby the 3' and 5' terminal nucleotides are Watson-Crick paired to adjacent nucleotides. A second hairpin cap can, additionally, be placed at the junction between the first and second strands distant from the 3' and 5' ends, so that the Watson-Crick pairing between the first and second strands is stabilized.
[0103] The first and second strands contain two regions that are homologous with two fragments of the target gene, i.e., have the same sequence as the target gene. A homologous region contains the nucleotides of an RNA segment and may contain one or more DNA-type nucleotides of connecting DNA segment and may also contain DNA-type nucleotides that are not within the intervening DNA segment. The two regions of homology are separated by, and each is adjacent to, a region having a sequence that differs from the sequence of the target gene, termed a "heterologous region." The heterologous region can contain one, two or three mismatched nucleotides. The mismatched nucleotides can be contiguous or alternatively can be separated by one or two nucleotides that are homologous with the target gene. Alternatively, the heterologous region can also contain an insertion or one, two, three or of five or fewer nucleotides. Alternatively, the sequence of the mixed duplex oligonucleotide may differ from the sequence of the target gene only by the deletion of one, two, three, or five or fewer nucleotides from the mixed duplex oligonucleotide. The length and position of the heterologous region is, in this case, deemed to be the length of the deletion, even though no nucleotides of the mixed duplex oligonucleotide are within the heterologous region. The distance between the fragments of the target gene that are complementary to the two homologous regions is identical to the length of the heterologous region where a substitution or substitutions is intended. When the heterologous region contains an insertion, the homologous regions are thereby separated in the mixed duplex oligonucleotide farther than their complementary homologous fragments are in the gene, and the converse is applicable when the heterologous region encodes a deletion.
[0104] The RNA segments of the mixed duplex oligonucleotides are each a part of a homologous region, i.e., a region that is identical in sequence to a fragment of the target gene, which segments together preferably contain at least 13 RNA-type nucleotides and preferably from 16 to 25 RNA-type nucleotides or yet more preferably 18-22 RNA-type nucleotides or most preferably 20 nucleotides. In one embodiment, RNA segments of the homology regions are separated by and adjacent to, i.e., "connected by" an intervening DNA segment. In one embodiment, each nucleotide of the heterologous region is a nucleotide of the intervening DNA segment. An intervening DNA segment that contains the heterologous region of a mixed duplex oligonucleotide is termed a "mutator segment."
[0105] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the gene repair oligonucleobase (GRON) may be a single stranded oligodeoxynucleotide mutational vector (SSOMV), for example, such as disclosed in International Patent Application PCT/US00/23457, U.S. Pat. Nos. 6,271,360, 6,479,292, and 7,060,500 which are incorporated by reference in their entirety. The sequence of the SSOMV is based on the same principles as the mutational vectors described for example in U.S. Pat. Nos. 5,756,325; 5,871,984; 5,760,012; 5,888,983; 5,795,972; 5,780,296; 5,945,339; 6,004,804; and 6,010,907 and in International Publication Nos. WO 98/49350; WO 99/07865; WO 99/58723; WO 99/58702; and WO 99/40789. The sequence of the SSOMV contains two regions that are homologous with the target sequence separated by a region that contains the desired genetic alteration termed the mutator region. The mutator region can have a sequence that is the same length as the sequence that separates the homologous regions in the target sequence, but having a different sequence. Such a mutator region can cause a substitution. Alternatively, the homologous regions in the SSOMV can be contiguous to each other, while the regions in the target gene having the same sequence are separated by one, two or more nucleotides. Such an SSOMV causes a deletion from the target gene of the nucleotides that are absent from the SSOMV. Lastly, the sequence of the target gene that is identical to the homologous regions may be adjacent in the target gene but separated by one, two, or more nucleotides in the sequence of the SSOMV. Such an SSOMV causes an insertion in the sequence of the target gene.
[0106] The nucleotides of the SSOMV are deoxyribonucleotides that are linked by unmodified phosphodiester bonds except that the 3' terminal and/or 5' terminal internucleotide linkage or alternatively the two 3' terminal and/or 5' terminal internucleotide linkages can be a phosphorothioate or phosphoamidate. As used herein an internucleotide linkage is the linkage between nucleotides of the SSOMV and does not include the linkage between the 3' end nucleotide or 5' end nucleotide and a blocking substituent. In a specific embodiment the length of the SSOMV is between 21 and 55 deoxynucleotides and the lengths of the homology regions are, accordingly, a total length of at least 20 deoxynucleotides and at least two homology regions should each have lengths of at least 8 deoxynucleotides.
[0107] The SSOMV can be designed to be complementary to either the coding or the non-coding strand of the target gene. When the desired mutation is a substitution of a single base, it is preferred that both the mutator nucleotide and the targeted nucleotide be a pyrimidine. To the extent that is consistent with achieving the desired functional result, it is preferred that both the mutator nucleotide and the targeted nucleotide in the complementary strand be pyrimidines. Particularly preferred are SSOMVs that encode transversion mutations, i.e., a C or T mutator nucleotide is mismatched, respectively, with a C or T nucleotide in the complementary strand.
[0108] In addition to the oligodeoxynucleotide, the SSOMV can contain a 5' blocking substituent that is attached to the 5' terminal carbons through a linker. The chemistry of the linker is not critical other than its length, which should preferably be at least 6 atoms long and that the linker should be flexible. A variety of non-toxic substituents such as biotin, cholesterol or other steroids or a non-intercalating cationic fluorescent dye can be used. Particularly preferred reagents to make SSOMVs are the reagents sold as Cy3ยฎ and Cy5ยฎ by Glen Research, Sterling Va. (now GE Healthcare), which are blocked phosphoroamidites that upon incorporation into an oligonucleotide yield 3,3,3',3'-tetramethyl N,N'-isopropyl substituted indomonocarbocyanine and indodicarbocyanine dyes, respectively. Cy3 is particularly preferred. When the indocarbocyanine is N-oxyalkyl substituted it can be conveniently linked to the 5' terminal of the oligodeoxynucleotide as a phosphodiester with a 5' terminal phosphate. The chemistry of the dye linker between the dye and the oligodeoxynucleotide is not critical and is chosen for synthetic convenience. When the commercially available Cy3 phosphoramidite is used as directed, the resulting 5' modification consists of a blocking substituent and linker together which are a N-hydroxypropyl, N'-phosphatidylpropyl 3,3,3',3'-tetramethyl indomonocarbocyanine.
[0109] In a preferred embodiment the indocarbocyanine dye is tetra substituted at the 3 and 3' positions of the indole rings. Without limitations as to theory these substitutions prevent the dye from being an intercalating dye. The identity of the substituents at these positions is not critical. The SSOMV can in addition have a 3' blocking substituent. Again the chemistry of the 3' blocking substituent is not critical.
[0110] The mutations herein described might also be obtained by mutagenesis (random, somatic or directed) and any other DNA editing or recombination technologies including, but not limited to, gene targeting using site-specific homologous recombination by zinc finger nucleases.
Delivery of Gene Repair Oligonucleobases into Plant Cells
[0111] Any commonly known method used to transform a plant cell can be used for delivering the gene repair oligonucleobases. Illustrative methods are described below.
Microcarriers and Microfibers
[0112] The use of metallic microcarriers (microspheres) for introducing large fragments of DNA into plant cells having cellulose cell walls by projectile penetration is well known to those skilled in the relevant art (henceforth biolistic delivery). U.S. Pat. Nos. 4,945,050; 5,100,792 and 5,204,253 describe general techniques for selecting microcarriers and devices for projecting them.
[0113] Specific conditions for using microcarriers in the methods disclosed herein are described in International Publication WO 99/07865. In an illustrative technique, ice cold microcarriers (60 mg/mL), mixed duplex oligonucleotide (60 mg/mL) 2.5 M CaCl2 and 0.1 M spermidine are added in that order; the mixture gently agitated, e.g., by vortexing, for 10 minutes and then left at room temperature for 10 minutes, whereupon the microcarriers are diluted in 5 volumes of ethanol, centrifuged and resuspended in 100% ethanol. Good results can be obtained with a concentration in the adhering solution of 8-10 ฮผg/ฮผL microcarriers, 14-17 ฮผg/mL mixed duplex oligonucleotide, 1.1-1.4 M CaCl2 and 18-22 mM spermidine. Optimal results were observed under the conditions of 8 ฮผg/ฮผL microcarriers, 16.5 ฮผg/mL mixed duplex oligonucleotide, 1.3 M CaCl2 and 21 mM spermidine.
[0114] Gene repair oligonucleobases can also be introduced into plant cells for the practice of the present disclosure using microfibers to penetrate the cell wall and cell membrane. U.S. Pat. No. 5,302,523 to Coffee et al. describes the use of 30ร0.5 ฮผm and 10ร0.3 ฮผm silicon carbide fibers to facilitate transformation of suspension maize cultures of Black Mexican Sweet. Any mechanical technique that can be used to introduce DNA for transformation of a plant cell using microfibers can be used to deliver gene repair oligonucleobases for transmutation.
[0115] An illustrative technique for microfiber delivery of a gene repair oligonucleobase is as follows: Sterile microfibers (2 ฮผg) are suspended in 150 ฮผL, of plant culture medium containing about 10 ฮผg of a mixed duplex oligonucleotide. A suspension culture is allowed to settle and equal volumes of packed cells and the sterile fiber/nucleotide suspension are vortexed for 10 minutes and plated. Selective media are applied immediately or with a delay of up to about 120 h as is appropriate for the particular trait.
Protoplast Electroporation
[0116] In an alternative embodiment, the gene repair oligonucleobases can be delivered to the plant cell by electroporation of a protoplast derived from a plant part. The protoplasts are formed by enzymatic treatment of a plant part, particularly a leaf, according to techniques well known to those skilled in the art. See, e.g., Gallois et al., 1996, in Methods in Molecular Biology 55:89-107, Humana Press, Totowa, N.J.; Kipp et al., 1999, in Methods in Molecular Biology 133:213-221, Humana Press, Totowa, N.J. The protoplasts need not be cultured in growth media prior to electroporation. Illustrative conditions for electroporation are 3ร105 protoplasts in a total volume of 0.3 mL with a concentration of gene repair oligonucleobase of between 0.6-4 ฮผg/mL.
Protoplast PEG-Mediated DNA Uptake
[0117] In an alternative embodiment, nucleic acids are taken up by plant protoplasts in the presence of the membrane-modifying agent polyethylene glycol, according to techniques well known to those skilled in the art (see, e.g., Gharti-Chhetri et al., 1992; Datta et al., 1992).
Microinjection
[0118] In an alternative embodiment, the gene repair oligonucleobases can be delivered by injecting it with a microcapillary into plant cells or into protoplasts (see, e.g., Miki et al., 1989; Schnorf et al., 1991).
Transgenics
[0119] In any of the various aspects and embodiments of the compositions and methods disclosed herein, mutations in genes and proteins may be made using, for example, transgenic technology. In some embodiments, the compositions and methods include a plant or plant cell having a transformed nucleic acid construct including a promoter operably linked to a PPX nucleotide disclosed herein. The methods disclosed herein may include introducing a PPX nucleic acid construct disclosed herein into at least one plant cell and regenerating a transformed plant therefrom. The nucleic acid construct comprises at least one nucleotide that encodes a herbicide-resistant PPX protein as disclosed herein, particularly the nucleotide sequences of set forth in FIGS. 2, 4, 6, 8, 10 and 12, and fragments and variants thereof. The methods further involve the use of a promoter that is capable of driving gene expression in a plant cell. In one embodiment, such a promoter is a constitutive promoter or a tissue-preferred promoter. A plant produced by these methods may have increased PPX activity, and/or particularly herbicide-tolerant PPX activity, when compared to an untransformed plant. Thus, the methods find use in enhancing or increasing the resistance of a plant to at least one herbicide that increases the activity of the PPX enzyme, particularly in the presence of a PPX-inhibiting herbicide.
[0120] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the methods for producing a herbicide-resistant plant may include transforming a plant cell with a nucleic acid construct comprising a nucleotide sequence operably linked to a promoter that drives expression in a plant cell and regenerating a transformed plant from said transformed plant cell. The nucleotide sequence is selected from those nucleotide sequences that encode the herbicide-resistant PPX disclosed herein, particularly the nucleotide sequences set forth in FIGS. 2, 4, 6, 8, 10 and 12, and fragments and variants thereof. A herbicide-resistant plant produced by this method comprises enhanced resistance, compared to an untransformed plant, to at least one herbicide, particularly a herbicide that interferes with the activity of the PPX enzyme such as, for example, a PPX-inhibiting herbicide.
[0121] The disclosed nucleic acid molecules can be used in nucleic acid constructs for the transformation of plants, for example, crop plants, such as Solanum tuberosum. In one embodiment, such nucleic acid constructs containing the nucleic acid molecules of the present disclosure can be used to produce transgenic plants to provide for resistance to herbicides, such as herbicides that are known to inhibit PPX activity, such as PPX-inhibiting herbicides. The nucleic acid constructs can be used in expression cassettes, expression vectors, transformation vectors, plasmids and the like. The transgenic plants obtained following transformation with such constructs demonstrate increased resistance to PPX-inhibiting herbicides such as, for example, flumioxazin and sulfentrazone herbicides.
Constructs
[0122] The nucleic acid molecules disclosed herein (e.g., mutated PPX genes) can be used in the production of recombinant nucleic acid constructs. In one embodiment, the nucleic acid molecules of the present disclosure can be used in the preparation of nucleic acid constructs, for example, expression cassettes for expression in the plant of interest.
[0123] Expression cassettes may include regulatory sequences operably linked to the PPX nucleic acid sequences disclosed herein. The cassette may additionally contain at least one additional gene to be co-transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes.
[0124] The nucleic acid constructs may be provided with a plurality of restriction sites for insertion of the PPX nucleic acid sequence to be under the transcriptional regulation of the regulatory regions. The nucleic acid constructs may additionally contain nucleic acid molecules encoding for selectable marker genes.
[0125] Any promoter can be used in the production of the nucleic acid constructs. The promoter may be native or analogous, or foreign or heterologous, to the plant host and/or to the PPX nucleic acid sequences disclosed herein. Additionally, the promoter may be the natural sequence or alternatively a synthetic sequence. Where the promoter is "foreign" or "heterologous" to the plant host, it is intended that the promoter is not found in the native plant into which the promoter is introduced. Where the promoter is "foreign" or "heterologous" to the PPX nucleic acid sequences disclosed herein, it is intended that the promoter is not the native or naturally occurring promoter for the operably linked PPX nucleic acid sequences disclosed herein. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence.
[0126] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the PPX nucleic acid sequences disclosed herein may be expressed using heterologous promoters, the native promoter sequences may be used in the preparation of the constructs. Such constructs would change expression levels of the PPX protein in the plant or plant cell. Thus, the phenotype of the plant or plant cell is altered.
[0127] Any promoter can be used in the preparation of constructs to control the expression of the PPX coding sequence, such as promoters providing for constitutive, tissue-preferred, inducible, or other promoters for expression in plants. Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43 838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689); pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten et al. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.
[0128] Tissue-preferred promoters can be utilized to direct PPX expression within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2):157-168; Rinehart et al. (1996) Plant Physiol. 1 12(3):1331-1341; Van Camp et al. (1996) Plant Physiol. 1 12(2):525-535; Canevascini et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol. Biol. 23(6):1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505.
[0129] The nucleic acid constructs may also include transcription termination regions. Where transcription terminations regions are used, any termination region may be used in the preparation of the nucleic acid constructs. For example, the termination region may be native to the transcriptional initiation region, may be native to the operably linked PPX sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the PPX nucleic acid molecule of interest, the plant host, or any combination thereof). Examples of termination regions that are available for use in the constructs of the present disclosure include those from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
[0130] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the nucleic acids may be optimized for increased expression in the transformed plant. That is, the nucleic acids encoding the mutant PPX proteins can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498.
[0131] In addition, other sequence modifications can be made to the nucleic acid sequences disclosed herein. For example, additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon/intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may also be adjusted to levels average for a target cellular host, as calculated by reference to known genes expressed in the host cell. In addition, the sequence can be modified to avoid predicted hairpin secondary mRNA structures.
[0132] Other nucleic acid sequences may also be used in the preparation of the constructs of the present disclosure, for example to enhance the expression of the PPX coding sequence. Such nucleic acid sequences include the introns of the maize AdhI, intron1 gene (Callis et al. (1987) Genes and Development 1:1183-1200), and leader sequences, (W-sequence) from the Tobacco Mosaic virus (TMV), Maize Chlorotic Mottle Virus and Alfalfa Mosaic Virus (Gallie et al. (1987) Nucleic Acid Res. 15:8693-8711, and Skuzeski et al. (1990) Plant Mol. Biol. 15:65-79, 1990). The first intron from the shrunken-1 locus of maize has been shown to increase expression of genes in chimeric gene constructs. U.S. Pat. Nos. 5,424,412 and 5,593,874 disclose the use of specific introns in gene expression constructs, and Gallie et al. ((1994) Plant Physiol. 106:929-939) also have shown that introns are useful for regulating gene expression on a tissue specific basis. To further enhance or to optimize PPX gene expression, the plant expression vectors disclosed herein may also contain DNA sequences containing matrix attachment regions (MARs). Plant cells transformed with such modified expression systems, then, may exhibit overexpression or constitutive expression of a nucleotide sequence of the disclosure.
[0133] The expression constructs disclosed herein can also include nucleic acid sequences capable of directing the expression of the PPX sequence to the chloroplast. Such nucleic acid sequences include chloroplast targeting sequences that encodes a chloroplast transit peptide to direct the gene product of interest to plant cell chloroplasts. Such transit peptides are known in the art. With respect to chloroplast-targeting sequences, "operably linked" means that the nucleic acid sequence encoding a transit peptide (i.e., the chloroplast-targeting sequence) is linked to the PPX nucleic acid molecules disclosed herein such that the two sequences are contiguous and in the same reading frame. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481. While the PPX proteins disclosed herein may include a native chloroplast transit peptide, any chloroplast transit peptide known in the art can be fused to the amino acid sequence of a mature PPX protein by operably linking a chloroplast-targeting sequence to the 5'-end of a nucleotide sequence encoding a mature PPX protein.
[0134] Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5):3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb. 22(6):789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem. 270(1 1):6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem. 272(33):20357-20363); chorismate synthase (Schmidt et al. (1993) J. Biol. Chem. 268(36):27447-27457); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem. 263:14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem. 264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; and Shah et al. (1986) Science 233:478-481.
[0135] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the nucleic acid constructs may be prepared to direct the expression of the mutant PPX coding sequence from the plant cell chloroplast. Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab and Maliga (1993) EMBO J. 12:601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.
[0136] The nucleic acids of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the nucleic acids of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.
[0137] The nucleic acid constructs can be used to transform plant cells and regenerate transgenic plants comprising the mutant PPX coding sequences. Numerous plant transformation vectors and methods for transforming plants are available. See, for example, U.S. Pat. No. 6,753,458, An, G. et al. (1986) Plant Physiol., 81:301-305; Fry, J. et al. (1987) Plant Cell Rep. 6:321-325; Block, M. (1988) Theor. Appl Genet. 76:767-774; Hinchee et al. (1990) Stadler. Genet. Symp. 203212.203-212; Cousins et al. (1991) Aust. J. Plant Physiol. 18:481-494; Chee, P. P. and Slightom, J. L. (1992) Gene. 118:255-260; Christou et al. (1992) Trends. Biotechnol. 10:239-246; D'Halluin et al. (1992) Bio/Technol. 10:309-3 14; Dhir et al. (1992) Plant Physiol. 99:81-88; Casas et al. (1993) Proc. Nat. Acad. Sci. USA 90:11212-11216; Christou, P. (1993) In Vitro Cell. Dev. Biol.-Plant; 29P:1 19-124; Davies, et al. (1993) Plant Cell Rep. 12:180-183; Dong, J. A. and Mc Hughen, A. (1993) Plant Sci. 91:139-148; Franklin, C. I. and Trieu, T. N. (1993) Plant. Physiol. 102:167; Golovkin et al. (1993) Plant Sci. 90:41-52; Guo Chin Sci. Bull. 38:2072-2078; Asano, et al. (1994) Plant Cell Rep. 13; Ayeres N. M. and Park, W. D. (1994) Crit. Rev. Plant. Sci. 13:219-239; Barcelo et al. (1994) Plant. J. 5:583-592; Becker, et al. (1994) Plant. J. 5:299-307; Borkowska et al. (1994) Acta. Physiol Plant. 16:225-230; Christou, P. (1994) Agro. Food. Ind. Hi Tech. 5: 17-27; Eapen et al. (1994) Plant Cell Rep. 13:582-586; Hartman et al. (1994) Bio-Technology 12: 919923; Ritala et al. (1994) Plant. Mol. Biol. 24:317-325; and Wan, Y. C. and Lemaux, P. G. (1994) Plant Physiol. 104:3748. The constructs may also be transformed into plant cells using homologous recombination.
[0138] The disclosed constructs comprising the PPX nucleic acid sequences disclosed herein can be used in various methods to produce transgenic host cells, such as bacteria, yeast, and to transform plant cells and in some cases regenerate transgenic plants. For example, methods of producing a transgenic crop plant containing the PPX mutant proteins disclosed herein, where expression of the nucleic acid(s) in the plant results in herbicide tolerance as compared to wild-type plants or to known PPX mutant type plants comprising: (a) introducing into a plant cell an expression vector comprising nucleic acid encoding a mutant PPX protein, and (b) generating from the plant cell a transgenic plant which is herbicide tolerant.
PPX Mutations
[0139] The compositions and methods may relate at least in part to mutations in a PPX gene, for example mutations that render a plant resistant or tolerant to a herbicide of the PPX-inhibiting family of herbicides. The compositions and methods also in certain embodiments relate to the use of a gene repair oligonucleobase to make a desired mutation in the chromosomal or episomal sequences of a plant in the gene encoding for a PPX protein. The mutated protein, which may in some embodiments substantially maintain the catalytic activity of the wild-type protein, allowing for increased resistance or tolerance of the plant to a herbicide of the PPX-inhibiting family, and thus in some embodiments allowing for substantially normal growth or development of the plant, its organs, tissues, or cells as compared to the wild-type plant irrespective of the presence or absence of the herbicide. The compositions and methods also relate to a non-transgenic or transgenic plant cell in which a PPX gene has been mutated, a non-transgenic plant or transgenic regenerated therefrom, as well as a plant resulting from a cross using a regenerated non-transgenic or transgenic plant to a plant having a mutation in a different PPX gene, for example. These mutations may also be applied to target tolerance to these inhibitors in plants including crop plants, algae, bacteria, fungi and mammalian systems.
In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, at least one mutation of a mutated PPX protein may be at the amino acid position corresponding to a position selected from the group consisting of 52, 85, 105, 111, 130, 139, 143, 144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219, 220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 305, 311, 316, 318, 332, 343, 354, 357, 359, 360, 366, 393, 403, 424, 426, 430, 438, 440, 444, 455, 457, 470, 478, 483, 484, 485, 487, 490, 503, 508, and 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at one or more amino acid positions corresponding to a position selected from the group consisting of 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 50 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 52 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 85 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 111 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 130 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 139 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 143 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 147 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 165 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 180 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 192 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 193 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 199 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 206 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 219 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 226 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 228 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 229 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 230 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 256 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 270 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 271 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 305 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 311 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 316 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 318 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 332 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 357 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 359 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 360 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 366 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 438 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 440 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 444 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 455 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 457 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 470 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 478 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position a phenylalanine to glycine at a position corresponding to position 483 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 484 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 485 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 487 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 490 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 503 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 508 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 58 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 64 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 74 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 84 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 93 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 97 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 98 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 101 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 119 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 121 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 124 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 139 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 150 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 151 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 157 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 164 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 170 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 177 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 187 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 188 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 195 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 214 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 215 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 229 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 230 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 271 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 274 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 278 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 283 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 292 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 296 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 324 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 330 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 396 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 404 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 406 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 410 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 421 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 434 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 447 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 448 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 449 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 451 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 454 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 465 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 470 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 500 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes two or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of 52, 85, 105, 111, 130, 139, 143, 144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219, 220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 305, 311, 316, 318, 332, 343, 354, 357, 359, 360, 366, 393, 403, 424, 426, 430, 438, 440, 444, 455, 457, 470, 478, 483, 484, 485, 487, 490, 503, 508 and 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes two or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and 500 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes three or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of 52, 85, 105, 111, 130, 139, 143, 144, 145, 147, 165, 167, 170, 180, 185, 192, 193, 199, 206, 212, 219, 220, 221, 226, 228, 229, 230, 237, 244, 256, 257, 270, 271, 272, 305, 311, 316, 318, 332, 343, 354, 357, 359, 360, 366, 393, 403, 424, 426, 430, 438, 440, 444, 455, 457, 470, 478, 483, 484, 485, 487, 490, 503, 508 and 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes three or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of 58, 64, 74, 84, 93, 97, 98, 101, 119, 121, 124, 139, 150, 151, 157, 164, 170, 177, 187, 188, 195, 214, 215, 229, 230, 271, 274, 278, 283, 292, 296, 307, 324, 330, 396, 404, 406, 410, 421, 423, 434, 447, 448, 449, 451, 454, 465, 470 and
500 of SEQ ID NO: 9.
In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, at least one mutation of a mutated PPX protein may be at the amino acid position corresponding to a position selected from the group consisting of G52, N85, N105, E111, G130, D139, P143, R144, F145, L147, F165, L167, I170, A180, P185, E192, S193, R199, V206, E212, Y219, A220, G221, L226, M228, K229, A230, K237, S244, 8256, R257, K270, P271, Q272, 5305, E311, T316, T318, S332, S343, A354, L357, K359, L360, A366, L393, L403, L424, Y426, S430, K438, E440, V444, L455, K457, V470, F478, F483, D484, I485, D487, K490, L503, V508 and I525 of SEQ ID NO: 1. In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, at least one mutation of a mutated PPX protein may be at the amino acid position corresponding to a position selected from the group consisting of D58, E64, G74, G84, L93, K97, K98, A101, S119, F121, T124, N139, E150, S151, Q157, V164, D170, C177, H187, L188, N195, P214, I215, K229, K230, C271, D274, F283, A292, S296, C307, N324, D330, S396, A404, R406, K410, L421, A423, C434, D447, S448, V449, D451, D454, Y465, K470 and T500 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position G52 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position N85 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position E111 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position G130 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D139 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position P143 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position R144 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position F145 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L147 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position F165 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L167 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position I170 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A180 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position P185 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position E192 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S193 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position R199 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position V206 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position E212 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position Y219 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A220 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position G221 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L226 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position M228 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K229 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A230 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K237 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S244 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position R256 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position R257 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K270 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position P271 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position Q272 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S305 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position E311 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position T316 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position T318 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S332 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 5343 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A354 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L357 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K359 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L360 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A366 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position Y426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S430 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K438 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position E440 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position V444 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L455 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K457 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position V470 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position F478 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position F483 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D484 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position I485 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D487 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K490 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L503 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position V508 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position I525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D58 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position E64 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position G74 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position G84 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L93 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K97 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K98 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A101 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S119 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position F121 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position T124 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position N139 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position E150 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 5151 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position Q157 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position V164 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D170 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position C177 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position H187 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L188 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position N195 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position P214 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position I215 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K229 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K230 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position C271 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D274 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position F283 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A292 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S296 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position C307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position N324 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D330 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S396 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A404 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position R406 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K410 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L421 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position C434 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D447 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S448 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position V449 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D451 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position D454 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position Y465 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K470 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position T500 of SEQ ID NO: 9. In some embodiments, a PPX protein is a paralog of
[0141] Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato plastidal PPX protein) and the PPX protein may have an N at the position corresponding to position 52 of SEQ ID NO:1, wherein the N is substituted with an amino acid other than an N; a K at the position corresponding to position 272 of SEQ ID NO:1, wherein the K is substituted with an amino acid other than a K; an S at the position corresponding to position 359 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S; and/or an S at the position corresponding to position 525 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S. In such embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position N52 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position N85 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position R144 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position F145 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A180 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position P185 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position A220 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L226 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position M228 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S244 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position K272 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to S305 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S332 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L357 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position 5359 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position L424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position Y426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position F478 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a mutation at the amino acid position corresponding to position S525 of SEQ ID NO: 1.
[0142] In some embodiments, a mutated PPX protein includes two or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of G52, N85, R144, F145, A180, P185, A220, L226, M228, S244, Q272, 5305, S332, L357, K359, L393, L403, L424, Y426, F478 and I525 of SEQ ID NO: 1. In some embodiments, a PPX protein is a paralog of Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato PPX protein) and the PPX protein has two or more mutations and has one or more of: (1) an N at the position corresponding to position 52 of SEQ ID NO:1, wherein the N is substituted with an amino acid other than an N; (2) a K at the position corresponding to position 272 of SEQ ID NO:1, wherein the K is substituted with an amino acid other than a K; (3) an S at the position corresponding to position 359 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S; and/or (4) an S at the position corresponding to position 525 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S. In such embodiments, a mutated PPX protein includes two or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of N52, N85, R144, F145, A180, P185, A220, L226, M228, S244, K272, S305, S332, L357, S359, L393, L403, L424, Y426, F478 and S525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes three or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of G52, N85, R144, F145, A180, P185, A220, L226, M228, S244, Q272, S305, S332, L357, K359, L393, L403, L424, Y426, F478 and I525 of SEQ ID NO: 1. In some embodiments, a PPX protein is a paralog of Arabidopsis thaliana PPX protein (for example the PPX protein may be a potato PPX protein) and the PPX protein has three or more mutations and has one or more of: (1) an N at the position corresponding to position 52 of SEQ ID NO:1, wherein the N is substituted with an amino acid other than an N; (2) a K at the position corresponding to position 272 of SEQ ID NO:1, wherein the K is substituted with an amino acid other than a K; (3) an S at the position corresponding to position 359 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S; and/or (4) an S at the position corresponding to position 525 of SEQ ID NO:1, wherein the S is substituted with an amino acid other than an S. In such embodiments, a mutated PPX protein includes three or more mutations, at least one mutation of which is at the amino acid position corresponding to a position selected from the group consisting of N52, N85, R144, F145, A180, P185, A220, L226, M228, S244, K272, S305, S332, L357, S359, L393, L403, L424, Y426, F478 and S525 of SEQ ID NO: 1.
[0143] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the mutated PPX protein may include one or more mutations selected from the mutations shown in Table 1.
TABLE-US-00001 TABLE 1 Amino acid mutations in the Arabidopsis thaliana PPX protein G52K F145Y A220I M228L S332C L393S Y426C Y426R N85D A180T A220L S244G L357I L393V Y426F Y426T R144C P185H A220T S244T K359R L403R Y426H Y426V R144H P185R A220V Q272F K359T L403S Y426I F478S F145L A220C L226M S305L L393M L424S Y426L I525T E111V L147V S193T A230F P271R L360K L455V I485E G130N F165N R199L R256H E311R A366E K457V K490N D139H P185Y V206F R256S T318G K438S V470S L503F P143R E192D Y219S K270E S332L E440K V470Y V508T R144L E192K K229Q K270Q L360D V444I D484A
[0144] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the one or more mutations in a mutated PPX gene may encode a mutated PPX protein having one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of a glycine to lysine at a position corresponding to position 52 of SEQ ID NO: 1; an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1; a glutamic acid to valine at a position corresponding to position 111 of SEQ ID NO: 1; a glycine to asparagine at a position corresponding to position 130 of SEQ ID NO: 1; an aspartic acid to histidine at a position corresponding to position 139 of SEQ ID NO: 1; a proline to arginine at a position corresponding to position 143 of SEQ ID NO: 1; an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1; an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1; an arginine to leucine at a position corresponding to position 144 of SEQ ID NO: 1; a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1, a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1; a leucine to valine at a position corresponding to position 147 of SEQ ID NO: 1; a phenylalanine to asparagine at a position corresponding to position 165 of SEQ ID NO: 1; an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1; a proline to histidine at a position corresponding to position 185 of SEQ ID NO: 1; a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 1; a proline to tyrosine at a position corresponding to position 185 of SEQ ID NO: 1; a glutamic acid to aspartic acid at a position corresponding to position 192 of SEQ ID NO: 1; a glutamic acid to lysine at a position corresponding to position 192 of SEQ ID NO: 1; a serine to threonine at a position corresponding to position 193 of SEQ ID NO: 1; an arginine to leucine at a position corresponding to position 199 of SEQ ID NO: 1; a valine to phenylalanine at a position corresponding to position 206 of SEQ ID NO: 1; a tyrosine to serine at a position corresponding to position 219 of SEQ ID NO: 1; an alanine to cysteine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to leucine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to valine at a position corresponding to position 220 of SEQ ID NO: 1; a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1; a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1; a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 1; an alanine to phenylalanine at a position corresponding to position 230 of SEQ ID NO: 1; a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1; a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1; an arginine to histidine at a position corresponding to position 256 of SEQ ID NO: 1; an arginine to serine at a position corresponding to position 256 of SEQ ID NO: 1; a lysine to glutamic acid at a position corresponding to position 270; a lysine to glutamine at a position corresponding to position 270; a proline to arginine at a position corresponding to position 271 of SEQ ID NO: 1; a glutamine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1; a serine to leucine at a position corresponding to position 305 of SEQ ID NO: 1; a glutamic acid to arginine at a position corresponding to position 311 of SEQ ID NO: 1; a threonine to glycine at a position corresponding to position 316 of SEQ ID NO: 1; a threonine to glycine at a position corresponding to position 318 of SEQ ID NO: 1; a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1; a serine to leucine at a position corresponding to position 332 of SEQ ID NO: 1; a leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 1; a lysine to arginine at a position corresponding to position 359 of SEQ ID NO: 1; a lysine to threonine at a position corresponding to position 359 of SEQ ID NO: 1; a leucine to lysine at a position corresponding to position 360 of SEQ ID NO 1; a leucine to aspartic acid at a position corresponding to position 360 of SEQ ID NO: 1; an alanine to glutamic acid at a position corresponding to position 366 of SEQ ID NO: 1; a leucine to methionine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 403 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1; a tyrosine to cysteine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to valine at a position corresponding to position 426 of SEQ ID NO: 1; a lysine to serine at a position corresponding to position 438 of SEQ ID NO: 1; a glutamic acid to lysine at a position corresponding to position 440 of SEQ ID NO: 1; a valine to isoleucine at a position corresponding to position 444 of SEQ ID NO: 1; a leucine to valine at a position corresponding to position 455 of SEQ ID NO: 1; a lysine to valine at a position corresponding to position 457 of SEQ ID NO: 1; a valine to serine at a position corresponding to position 470 of SEQ ID NO: 1; a valine to tyrosine at a position corresponding to position 470 of SEQ ID NO: 1; a phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO: 1; a phenylalanine to glycine at a position corresponding to position 483 of SEQ ID NO: 1; an aspartic acid to alanine at a position corresponding to position 484 of SEQ ID NO: 1; an isoleucine to glutamic acid at a position corresponding to position 485 of SEQ ID NO: 1; a lysine to asparagine at a position corresponding to position 490 of SEQ ID NO: 1; a leucine to phenylalanine at a position corresponding to position 503 of SEQ ID NO: 1; a valine to threonine at a position corresponding to position 508 of SEQ ID NO: 1; and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1.
[0145] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX gene may encode a mutated PPX protein that includes an glycine to lysine at a position corresponding to position 52 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a glutamic acid to valine at a position corresponding to position 111 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a glycine to asparagine at a position corresponding to position 130 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an aspartic acid to histidine at a position corresponding to position 139 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to arginine at a position corresponding to position 143 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an arginine to leucine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to valine at a position corresponding to position 147 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to asparagine at a position corresponding to position 165 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to histidine at a position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to tyrosine at a position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a glutamic acid to aspartic acid at a position corresponding to position 192 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a glutamic acid to lysine at a position corresponding to position 192 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 193 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an arginine to leucine at a position corresponding to position 199 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a valine to phenylalanine at a position corresponding to position 206 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to serine at a position corresponding to position 219 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to cysteine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to leucine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to valine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to phenylalanine at a position corresponding to position 230 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an arginine to histidine at a position corresponding to position 256 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an arginine to serine at a position corresponding to position 256 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to glutamic acid at a position corresponding to position 270. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to glutamine at a position corresponding to position 270. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to arginine at a position corresponding to position 271 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a glutamine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to leucine at a position corresponding to position 305 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a glutamic acid to arginine at a position corresponding to position 311 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a threonine to glycine at a position corresponding to position 316 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a threonine to glycine at a position corresponding to position 318 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to leucine at a position corresponding to position 332 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to arginine at a position corresponding to position 359 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to threonine at a position corresponding to position 359 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to lysine at a position corresponding to position 360 of SEQ ID NO 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to aspartic acid at a position corresponding to position 360 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to glutamic acid at a position corresponding to position 366 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to methionine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes leucine to serine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to serine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to cysteine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to valine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to serine at a position corresponding to position 438 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a glutamic acid to lysine at a position corresponding to position 440 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a valine to isoleucine at a position corresponding to position 444 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to valine at a position corresponding to position 455 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to valine at a position corresponding to position 457 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a valine to serine at a position corresponding to position 470 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a valine to tyrosine at a position corresponding to position 470 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to glycine at a position corresponding to position 483 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an aspartic acid to alanine at a position corresponding to position 484 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an isoleucine to glutamic acid at a position corresponding to position 485 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to asparagine at a position corresponding to position 490 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to phenylalanine at a position corresponding to position 503 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a valine to threonine at a position corresponding to position 508 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1.
TABLE-US-00002 TABLE 2 Summary of nucleotide/codon mutations in the Arabidopsis plastidal PPX gene that lead to amino acid changes that confer tolerance to PPX inhibitors. Position numbers based on numbering of Arabidopsis plastidal PPX gene number At4g01690 (SEQ ID NO: 1). AA mtn NA mtn G52K GGG โ AAA N85D AAT โ GAT R144C AGG โ TGC AGG โ TGT R144H AGG โ CAC AGG โ CAT F145L TTT โ CTT F145Y TTT โ TAT A180T GCA โ ACA P185H CCG โ CAC CCG โ CAT P185R CCG โ CGG A220C GCT โ TGT A220I GCT โ ATT A220L GCT โ CTT A220T GCT โ ACT A220V GCT โ GTT L226M GTG โ ATG M228L ATG โ CTG S244G AGC โ GGC S244T AGC โ ACC Q272F CAG โ TTC CAG โ TTT S305L TCA โ TTA S332C TCT โ TGT L3571 CTC โ ATC K359R AAA โ AGA K359T AAA โ ACT L393M TTG โ ATG L393S TTG โ TCG L393V TTG โ GTG L403R TTA โ CGA L403S TTA โ TCA L424S TTG โ TCG Y426C TAC โ TGC Y426F TAC โ TTC Y426H TAC โ CAC Y426I TAC โ ATC Y426L TAC โ TTA TAC โ CTC Y426R TAC โ CGC Y426T TAC โ ACC Y426V TAC โ GTC F478S TTT โ TCT S525T ATT โ ACT * "AA mtn" refers to amino acid mutation; "NA mtn" refers to nucleic acid mutation
[0146] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX gene may include a GGGโAAA which encodes a mutated PPX protein that includes an glycine to lysine at a position corresponding to position 52 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a AATโGAT nucleic acid mutation that encodes a mutated PPX protein that includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a AGGโTGC or TGT nucleic acid mutation that encodes a mutated PPX protein that includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a AGGโCAC or CAT nucleic acid mutation that encodes a mutated PPX protein that includes an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTTโCTT nucleic acid mutation that encodes a mutated PPX protein that includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTTโTAT nucleic acid mutation that encodes a mutated PPX protein that includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a GCAโACA nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a CCGโCAC or CAT nucleic acid mutation that encodes a mutated PPX protein that includes a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a CCGโCGT nucleic acid mutation that encodes a mutated PPX protein that includes a proline to histidine at a position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a CCGโCGG nucleic acid mutation that encodes a mutated PPX protein that includes a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a GCTโTGT nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to cysteine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a GCTโATT nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a GCTโCTT nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to leucine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a GCTโACT nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a GCTโGTT nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to valine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a GTGโATG nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a ATGโCTG nucleic acid mutation that encodes a mutated PPX protein that includes a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a AGCโGGC nucleic acid mutation that encodes a mutated PPX protein that includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a AGCโACC nucleic acid mutation that encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a CAGโTTC or TTT nucleic acid mutation that encodes a mutated PPX protein that includes a glutamine to asparagine at a position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TCAโTTA nucleic acid mutation that encodes a mutated PPX protein that includes a serine to leucine at a position corresponding to position 305 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TCTโTGT nucleic acid mutation that encodes a mutated PPX protein that includes a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a CTCโATC nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a AAAโAGA nucleic acid mutation that encodes a mutated PPX protein that includes a lysine to arginine at a position corresponding to position 359 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a AAAโACT nucleic acid mutation that encodes a mutated PPX protein that includes a lysine to threonine at a position corresponding to position 359 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTGโATG nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to methionine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTGโTCG nucleic acid mutation that encodes a mutated PPX protein that includes leucine to serine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTGโGTG nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTAโCGA nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTAโTCA nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to serine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTGโTCG nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TACโTGC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to cysteine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TACโTTC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TACโCAC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TACโATC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TACโTTA or CTC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TACโCGC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TACโACC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TACโGTC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to valine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a TTTโTCT nucleic acid mutation that encodes a mutated PPX protein that includes a phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a ATTโACT nucleic acid mutation that encodes a mutated PPX protein that includes an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1.
TABLE-US-00003 TABLE 3A Summary of nucleotide/codon mutations in a potato plastidal PPX gene that lead to amino acid changes that confer tolerance to PPX inhibitors. Position numbers are based on numbering of the Arabidopsis plastidal PPX gene number At4g01690 (SEQ ID NO: 1). AA mtn NA mtn N52K AAT โ AAA N85D AAT โ GAT R144C CGC โ TGC R144H CGC โ CAC F145L TTT โ CTT F145Y TTT โ TAT A180T GCC โ ACC P185H CCT โ CAT P185R CCT โ CGT A220C GCC โ TGC A220I GCC โ ATC A220L GCC โ CTC A220T GCC โ ACC A220V GCC โ GTC L226M TTG โ ATG M228L ATG โ CTG S244G AGC โ GGC S244T AGC โ ACC K272F AAA โ TTT AAA โ TTC S305L TCT โ CTT S332C AGT โ TGT L357I CTT โ ATT S359R AGT โ AGA S359T AGT โ ACT L393M TTG โ ATG L393S TTG โ TCG L393V TTG โ GTG L403R CTA โ CGA L403S CTA โ TCA L424S TTG โ TCG Y426C TAC โ TGC Y426F TAC โ TTC Y426H TAC โ CAC Y426I TAC โ ATC Y426L TAC โ TTA TAC โ CTC Y426R TAC โ CGC Y426T TAC โ ACC Y426V TAC โ GTC F478S TTT โ TCT S525T TCT โ ACT * "AA mtn" refers to amino acid mutation; "NA mtn" refers to nucleic acid mutation
[0147] In some embodiments, in conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, the one or more mutations in a mutated PPX gene may encode a mutated PPX protein having one or more mutations, two or more mutations, or three or more mutations selected from the group consisting of a asparagine to lysine at a position corresponding to position 52 of SEQ ID NO: 1; an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1; an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1; an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1; a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1, a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1; an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1; a proline to histidine at a position corresponding to position 185 of SEQ ID NO: 1; a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 1; an alanine to cysteine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to leucine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1; an alanine to valine at a position corresponding to position 220 of SEQ ID NO: 1; a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1; a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1; a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1; a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1; a lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1; a serine to leucine at a position corresponding to position 305 of SEQ ID NO: 1; a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1; a leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 1; a serine to arginine at a position corresponding to position 359 of SEQ ID NO: 1; a serine to threonine at a position corresponding to position 359 of SEQ ID NO: 1; a leucine to methionine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1; a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 403 of SEQ ID NO: 1; a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1; a tyrosine to cysteine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO: 1; a tyrosine to valine at a position corresponding to position 426 of SEQ ID NO: 1; a phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO: 1; and a isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1.
[0148] In some embodiments, in conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX gene may encode a mutated PPX protein that includes an asparagine to lysine at a position corresponding to position 52 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to histidine at a position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to cysteine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to leucine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an alanine to valine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to leucine at a position corresponding to position 305 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to arginine at a position corresponding to position 359 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 359 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to methionine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes leucine to serine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to serine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to cysteine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a tyrosine to valine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1.
[0149] In some embodiments, a mutated PPX gene includes a AATโAAA nucleic acid mutation that encodes a mutated PPX protein that includes an asparagine to lysine at a position corresponding to position 52 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a AATโGAT nucleic acid mutation that encodes a mutated PPX protein that includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a CGCโTGC nucleic acid mutation that encodes a mutated PPX protein that includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a CGCโCAC nucleic acid mutation that encodes a mutated PPX protein that includes an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TTTโCTT nucleic acid mutation that encodes a mutated PPX protein that includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TTTโTAT nucleic acid mutation that encodes a mutated PPX protein that includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a GCCโACC nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a CCTโCAT nucleic acid mutation that encodes a mutated PPX protein that includes a proline to arginine at a position corresponding to position 185 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a CCTโCGT nucleic acid mutation that encodes a mutated PPX protein that includes a proline to histidine at a position corresponding to position 185 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a GCCโTGC nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to cysteine at a position corresponding to position 220 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a GCCโATC nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to isoleucine at a position corresponding to position 220 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a GCCโCTC nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to leucine at a position corresponding to position 220 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a GCCโACC nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a GCCโGTC nucleic acid mutation that encodes a mutated PPX protein that includes an alanine to valine at a position corresponding to position 220 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TTGโATG nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a ATGโCTG nucleic acid mutation that encodes a mutated PPX protein that includes a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a AGCโGGC nucleic acid mutation that encodes a mutated PPX protein that includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a AGCโACC nucleic acid mutation that encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a AAAโAAT nucleic acid mutation that encodes a mutated PPX protein that includes a lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TCTโCTT nucleic acid mutation that encodes a mutated PPX protein that includes a serine to leucine at a position corresponding to position 305 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a AGTโTGT nucleic acid mutation that encodes a mutated PPX protein that includes a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a CTTโATT nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to isoleucine at a position corresponding to position 357 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a AGTโAGA nucleic acid mutation that encodes a mutated PPX protein that includes a serine to arginine at a position corresponding to position 359 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a AGTโACT nucleic acid mutation that encodes a mutated PPX protein that includes a serine to threonine at a position corresponding to position 359 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TTGโATG nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to methionine at a position corresponding to position 393 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TTGโTCG nucleic acid mutation that encodes a mutated PPX protein that includes leucine to serine at a position corresponding to position 393 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TTGโGTG nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a CTAโCGA nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a CTAโTCA nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to serine at a position corresponding to position 403 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TTGโTCG nucleic acid mutation that encodes a mutated PPX protein that includes a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TACโTGC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to cysteine at a position corresponding to position 426 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TACโAAC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TACโCAC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TACโATC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to isoleucine at a position corresponding to position 426 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TACโTTC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to leucine at a position corresponding to position 426 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TACโCGC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to arginine at a position corresponding to position 426 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TACโACC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to threonine at a position corresponding to position 426 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TACโGTC nucleic acid mutation that encodes a mutated PPX protein that includes a tyrosine to valine at a position corresponding to position 426 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TTTโTCT nucleic acid mutation that encodes a mutated PPX protein that includes a phenylalanine to serine at a position corresponding to position 478 of SEQ ID NO: 7. In some embodiments, a mutated PPX gene includes a TCTโACT nucleic acid mutation that encodes a mutated PPX protein that includes an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 7.
TABLE-US-00004 TABLE 3B Summary of nucleotide/codon mutations in a potato mitochondrial PPX gene that lead to amino acid changes that confer tolerance to PPX inhibitors. Position numbers are based on numbering of the Solanum tuberosum mitochondrial PPX gene number AJ225108 (SEQ ID NO: 9). AA mtn NA mtn D58N GAT -> AAT E64V GAA -> GTA G74C GGT -> TGT G84N GGA -> GAT R98C CGC -> CAC R98H CGC -> TGC R98L CGC -> CTC N139Y CCT -> TAT E150D GAA -> GAT E150K GAA -> AAA T500S ACC _> AGC S151T AGT -> ACT Q157L CAG -> CTG V164F GTT -> TTT D170E GAT -> GAA H187Q AAG -> CAG L188F CTT -> TTT N195K AAT -> AAA P214H CCT -> CAT P214S CCT -> TCT K229E AAG -> GAG K229Q AAG -> CAG K230R AAG -> AGG F283G GAC -> GGC A292G GCA -> GGA S296L TCA -> TTA C307S TGT -> AGT N324D AAT -> GAT N324K AAT -> AAA D330E GAT -> GAA A404S GCC -> TCC R406K AGG -> AAG K410I AAA -> ATA A423V GCT -> GTT C434S TGC -> AGC C434Y TGC -> TAC S448A TCA -> GCA D451G GAT -> GGT D454N GAC -> AAC Y465F TAT -> TTT K470T AAG -> ACG * "AA mtn" refers to amino acid mutation; "NA mtn"refers to nucleic acid mutation
[0150] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX gene may encode a mutated PPX protein that includes an aspartic acid to asparagine at a position corresponding to position 58 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes glutamic acid to valine at a position corresponding to position 64 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes glycine to cysteine at a position corresponding to position 74 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes glycine to asparagine at a position corresponding to position 84 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes leucine to histidine at a position corresponding to position 93 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes lysine to arginine at a position corresponding to position 97 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes alanine to valine at a position corresponding to position 101 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes serine to asparagine at a position corresponding to position 119 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes phenylalanine to leucine at a position corresponding to position 121 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes asparagine to tyrosine at a position corresponding to position 139 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes glutamic acid to aspartic acid at a position corresponding to position 150 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes glutamic acid to lysine at a position corresponding to position 150 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes serine to threonine at a position corresponding to position 151 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes glutamine to leucine at a position corresponding to position 157 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes valine to phenylalanine at a position corresponding to position 164 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes valine to alanine at a position corresponding to position 164 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes aspartic acid to glutamic acid at a position corresponding to position 170 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes cysteine to serine at a position corresponding to position 177 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes histidine to glutamine at a position corresponding to position 187 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes asparagine to lysine at a position corresponding to position 195 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a proline to serine at a position corresponding to position 214 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an isoleucine to histidine at a position corresponding to position 215 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes an isoleucine to serine at a position corresponding to position 215 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes lysine to glutamic acid at a position corresponding to position 229 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes lysine to arginine at a position corresponding to position 230 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes cysteine to arginine at a position corresponding to position 271 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes aspartic acid to glycine at a position corresponding to position 274 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes aspartic acid to glycine at a position corresponding to position 278 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes a phenylalanine to glycine at a position corresponding to position 283 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes alanine to glycine at a position corresponding to position 292 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes serine to leucine at a position corresponding to position 296 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes asparagine to aspartic acid at a position corresponding to position 324 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes asparagine to lysine at a position corresponding to position 324 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes aspartic acid to glutamic acid at a position corresponding to position 330 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes serine to leucine at a position corresponding to position 396 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes alanine to serine at a position corresponding to position 404 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes arginine to lysine at a position corresponding to position 406 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes lysine to isoleucine at a position corresponding to position 410 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes leucine to valine at a position corresponding to position 421 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes cysteine to serine at a position corresponding to position 434 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes cysteine to tyrosine at a position corresponding to position 434 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes aspartic acid to glycine at a position corresponding to position 447 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes serine to alanine at a position corresponding to position 448 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes valine to glutamic acid at a position corresponding to position 449 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes aspartic acid to glycine at a position corresponding to position 451 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes aspartic acid to asparagine at a position corresponding to position 454 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes tyrosine to phenylalanine at a position corresponding to position 465 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes lysine to threonine at a position corresponding to position 470 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene encodes a mutated PPX protein that includes threonine to serine at a position corresponding to position 500 of SEQ ID NO: 9.
[0151] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX gene includes a GATโAAT nucleic acid mutation that encodes a mutated PPX protein that includes an aspartic acid to asparagine at a position corresponding to position 58 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GAAโGTA nucleic acid mutation that encodes a mutated PPX protein that includes glutamic acid to valine at a position corresponding to position 64 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GGTโTGT nucleic acid mutation that encodes a mutated PPX protein that includes glycine to cysteine at a position corresponding to position 74 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GGAโGAT nucleic acid mutation that encodes a mutated PPX protein that includes glycine to asparagine at a position corresponding to position 84 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a CGCโTGC nucleic acid mutation that encodes a mutated PPX protein that includes arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a CGCโCAC nucleic acid mutation that encodes a mutated PPX protein that includes arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a CGCโCTC nucleic acid mutation that encodes a mutated PPX protein that includes arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AATโTAT nucleic acid mutation that encodes a mutated PPX protein that includes asparagine to tyrosine at a position corresponding to position 139 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GAAโGAT nucleic acid mutation that encodes a mutated PPX protein that includes glutamic acid to aspartic acid at a position corresponding to position 150 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GAAโAAA nucleic acid mutation that encodes a mutated PPX protein that includes glutamic acid to lysine at a position corresponding to position 150 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AGTโACT nucleic acid mutation that encodes a mutated PPX protein that includes serine to threonine at a position corresponding to position 151 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a CAGโCTG nucleic acid mutation that encodes a mutated PPX protein that includes glutamine to leucine at a position corresponding to position 157 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GTTโTTT nucleic acid mutation that encodes a mutated PPX protein that includes valine to phenylalanine at a position corresponding to position 164 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GATโGAA nucleic acid mutation that encodes a mutated PPX protein that includes aspartic acid to glutamic acid at a position corresponding to position 170 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a CACโCAG nucleic acid mutation that encodes a mutated PPX protein that includes histidine to glutamine at a position corresponding to position 187 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a CTTโTTT nucleic acid mutation that encodes a mutated PPX protein that includes leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AATโAAA nucleic acid mutation that encodes a mutated PPX protein that includes asparagine to lysine at a position corresponding to position 195 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a CCTโCAT nucleic acid mutation that encodes a mutated PPX protein that includes proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a CCTโTCT nucleic acid mutation that encodes a mutated PPX protein that includes proline to serine at a position corresponding to position 214 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AAGโGAG nucleic acid mutation that encodes a mutated PPX protein that includes lysine to glutamic acid at a position corresponding to position 229 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AAGโCAG nucleic acid mutation that encodes a mutated PPX protein that includes lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AAGโAGG nucleic acid mutation that encodes a mutated PPX protein that includes lysine to arginine at a position corresponding to position 230 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GACโGGC nucleic acid mutation that encodes a mutated PPX protein that includes aspartic acid to glycine at a position corresponding to position 283 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a TCAโTTA nucleic acid mutation that encodes a mutated PPX protein that includes serine to leucine at a position corresponding to position 296 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a TGTโAGT nucleic acid mutation that encodes a mutated PPX protein that includes cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AATโGAT nucleic acid mutation that encodes a mutated PPX protein that includes asparagine to aspartic acid at a position corresponding to position 324 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AATโAAA nucleic acid mutation that encodes a mutated PPX protein that includes asparagine to lysine at a position corresponding to position 324 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GATโGAA nucleic acid mutation that encodes a mutated PPX protein that includes aspartic acid to glutamic acid at a position corresponding to position 330 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GCCโTCC nucleic acid mutation that encodes a mutated PPX protein that includes alanine to serine at a position corresponding to position 404 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AGGโAAG nucleic acid mutation that encodes a mutated PPX protein that includes arginine to lysine at a position corresponding to position 406 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AAAโATA nucleic acid mutation that encodes a mutated PPX protein that includes lysine to isoleucine at a position corresponding to position 410 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a XXX GCTโGTT nucleic acid mutation that encodes a mutated PPX protein that includes alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a TGCโAGC nucleic acid mutation that encodes a mutated PPX protein that includes cysteine to serine at a position corresponding to position 434 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a TGCโTAC nucleic acid mutation that encodes a mutated PPX protein that includes cysteine to tyrosine at a position corresponding to position 434 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a TCAโGCA nucleic acid mutation that encodes a mutated PPX protein that includes serine to alanine at a position corresponding to position 448 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GATโGGT nucleic acid mutation that encodes a mutated PPX protein that includes aspartic acid to glycine at a position corresponding to position 451 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a GACโAAC nucleic acid mutation that encodes a mutated PPX protein that includes aspartic acid to asparagine at a position corresponding to position 454 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a TATโTTT nucleic acid mutation that encodes a mutated PPX protein that includes tyrosine to phenylalanine at a position corresponding to position 465 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a AAGโACG nucleic acid mutation that encodes a mutated PPX protein that includes lysine to threonine at a position corresponding to position 470 of SEQ ID NO: 9. In some embodiments, a mutated PPX gene includes a ACCโAGC nucleic acid mutation that encodes a mutated PPX protein that includes threonine to serine at a position corresponding to position 500 of SEQ ID NO: 9.
[0152] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX gene may include a GGGโAAA which encodes a mutated PPX protein that includes an glycine to lysine at a position corresponding to position 52 of SEQ ID NO: 1. In some embodiments, a mutated PPX gene includes a AATโGAT nucleic acid mutation that encodes a mutated PPX protein that includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1.
[0153] In some embodiments, in conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX gene may include a combination of mutations, for example, two or more, three or more, four or more, five or more or six or more mutations in a PPX gene. In certain embodiments, the combination of mutations is selected from the combinations of mutations shown in Tables 4a and 4b.
TABLE-US-00005 TABLE 4A Combinations of Amino Acid Mutations (each row of each of the three grouped columns represents a combination of mutations). Position numbers are based on numbering of the Arabidopsis plastidal PPX gene number At4g01690 (SEQ ID NO: 1) R144C A220T L226M L424S F145L L424S R144H S332C L226M Y426F A220T Y426H R144C Q272F A220T Y426F F145Y L393V R144C K272F A220T Y426H S244T Y426F G52K R144H S244T R144C Y426F F145Y L424S N52K R144H S244T N85D Y426H A220T L403R N85D A220T R144C Y426H L226M Y426F R144H S244T S244T Y426H N85D Y426H R144C L226M S244G Y426H L226M L424S N85D L226M A180T Y426H F145Y L403R N85D F145Y L226M Y426H S244G L393V R144C M228L F145L Y426H A180T Y426H N85D A180T A220T Y426H R144C Y426H N85D R144C N85D Y426H N85D S525T N85D Q272F F145L L393V L226M S525T N85D K272F L226M L424S F145Y S525T N85D M228L L226M Y426F F145L S525T A180T Y426F A220T L393V S244G S525T F145L Y426H A220T Y426F A180T S525T S244G Y426F R144C Y426F R144C S525T F145L L403R N85D I525T F145Y L424S L226M I525T R144C L424S F145Y I525T L226M Y426H F145L I525T A220T L424S R144C I525T F145Y Y426F R144C Y426H R144C L393V A180T Y426H S244G I525T A220T Y426H A180T I525T L226M Y426H S244G L393V S244T L393V L226M L403R F145Y Y426H
In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1 and a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1. While in other embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a glutamine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an glycine to lysine at a position corresponding to position 52 of SEQ ID NO:1, an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1 and a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1 and a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a glutamine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1.
In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1 and a serine to cysteine at a position corresponding to position 332 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 7 and a lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 7. In some embodiments, a mutated PPX protein includes an asparagine to lysine at a position corresponding to position 52 of SEQ ID NO: 7, an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 7 and a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 7. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 144 of SEQ ID NO: 1 and a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and an lysine to phenylalanine at a position corresponding to position 272 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a methionine to leucine at a position corresponding to position 228 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 7 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 7. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to threonine at a position corresponding to position 244 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 220 of SEQ ID NO: 1 and a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a tyrosine to phenylalanine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 1 and a leucine to serine at a position corresponding to position 424 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 1 and a leucine to arginine at a position corresponding to position 403 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and a leucine to valine at a position corresponding to position 393 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 144 of SEQ ID NO: 1 and a tyrosine to histidine at a position corresponding to position 426 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 1 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 1. In some embodiments, a mutated PPX protein includes a serine to glycine at a position corresponding to position 244 of SEQ ID NO: 7 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 7. In some embodiments, a mutated PPX protein includes an alanine to threonine at a position corresponding to position 180 of SEQ ID NO: 7 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 7. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 145 of SEQ ID NO: 7 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 7. In some embodiments, a mutated PPX protein includes a phenylalanine to tyrosine at a position corresponding to position 145 of SEQ ID NO: 7 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 7. In some embodiments, a mutated PPX protein includes an asparagine to aspartic acid at a position corresponding to position 85 of SEQ ID NO: 7 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 7. In some embodiments, a mutated PPX protein includes a leucine to methionine at a position corresponding to position 226 of SEQ ID NO: 7 and an isoleucine to threonine at a position corresponding to position 525 of SEQ ID NO: 7.
TABLE-US-00006 [0155] TABLE 4B Combinations of Amino Acid Mutations (each row of each of the two grouped columns represents a combination of mutations). Position numbers are based on numbering of the Solanum tuberosum mitochondrial PPX gene number AJ225108 (SEQ ID NO: 9). G74C R98C R98C P214H L93H V164A R98C T124I L188F K229Q R98L P214H R98C T124I P214H K229Q R98L T124I L188F K229Q R98C T124I K229Q R98L T124I P214H K229Q R98C P214H A423V R98L T124I K229Q R98C T124I L188F K229Q A423V S119N N139Y R98C T124I P214H K229Q A423V F121L E150D R98C T124I K229Q A423V S151T K229E K230R R98C P214H C307S Q157L H187Q R98C T124I L188F K229Q C307S C271R D274G R98C T124I P214H K229Q C307S C307S A423V R98C T124I K229Q C307S S396L K4101 R98H P214H C434S T500S R98H T124I L188F K229Q D447G A292G R98H T124I P214H K229Q S448A N324D R98H T124I K229Q Y465F K470T R98H P214H A423V R98L P214H A243V R98H T124I L188F K229Q A423V R98L T124I L188F K229Q A243V R98H T124I P214H K229Q A423V R98L T124I P214H K229Q A243V R98H T124I K229Q A423V R98L T124I K229Q A423V R98H P214H C307S R98L P214H C307S R98H T124I L188F K229Q C307S R98L T124I L188F K229Q C307S R98H T124I P214H K229Q C307S R98L T124I P214H K229Q C307S R98H T124I K229Q C307S R98L T124I K229Q C307S
In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, a mutated PPX protein includes a glycine to cysteine at a position corresponding to position 74 of SEQ ID NO: 9 and an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an leucine to histidine at a position corresponding to position 93 of SEQ ID NO: 9 and a valine to alanine at a position corresponding to position 164 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9 and a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9, and a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, a proline to histidine at a position corresponding to position 214, and a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, and a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a serine to asparagine at a position corresponding to position 119 of SEQ ID NO: 9 and an asparagine to tyrosine at a position corresponding to position 139 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a phenylalanine to leucine at a position corresponding to position 121 of SEQ ID NO: 9 and a glutamic acid to aspartic acid at a position corresponding to position 150 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a serine to threonine at a position corresponding to position 151 of SEQ ID NO: 9, a lysine to glutamic acid at a position corresponding to position 229 of SEQ ID NO: 9, and a lysine to arginine at a position corresponding to position 230 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a glutamine to leucine at a position corresponding to position 157 of SEQ ID NO: 9 and a histidine to glutamine at a position corresponding to position 187 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a cysteine to arginine at a position corresponding to position 271 of SEQ ID NO: 9 and a aspartic acid to glycine at a position corresponding to position 274 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9 and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a serine to leucine at a position corresponding to position 396 of SEQ ID NO: 9 and a lysine to isoleucine at a position corresponding to position 410 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a cysteine to serine at a position corresponding to position 434 of SEQ ID NO: 9 and a threonine to serine at a position corresponding to position 500 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an aspartic acid to glycine at a position corresponding to position 447 of SEQ ID NO: 9 and an alanine to glycine at a position corresponding to position 292 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a serine to alanine at a position corresponding to position 448 of SEQ ID NO: 9 and an asparagine to aspartic acid at a position corresponding to position 324 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes a tyrosine to phenylalanine at a position corresponding to position 465 of SEQ ID NO: 9 and a lysine to threonine at a position corresponding to position 470 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9, and an alanine to valine at a position corresponding to position 243 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9, a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9, and an alanine to valine at a position corresponding to position 243 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9, a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9, and an alanine to valine at a position corresponding to position 243 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9, and an alanine to valine at a position corresponding to position 243 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9, and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9, a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9, and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9, a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9, and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to leucine at a position corresponding to position 98 of SEQ ID NO: 9, a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9, a lysine to glutamine at a position corresponding to position 229 of SEQ ID NO: 9, and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9 and a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9 and a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a proline to hisitidine at a position corresponding to position 214 of SEQ ID NO: 9 and a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; and a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to cysteine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9 and a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9 and a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a proline to hisitidine at a position corresponding to position 214 of SEQ ID NO: 9 and a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; and a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and an alanine to valine at a position corresponding to position 423 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a leucine to phenylalanine at a position corresponding to position 188 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a proline to histidine at a position corresponding to position 214 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9. In some embodiments, a mutated PPX protein includes an arginine to histidine at a position corresponding to position 98 of SEQ ID NO: 9; a threonine to isoleucine at a position corresponding to position 124 of SEQ ID NO: 9; a lysine to glutamine at a position corresponding to position 229 SEQ ID NO: 9; and a cysteine to serine at a position corresponding to position 307 of SEQ ID NO: 9.
[0156] Paralogs
[0157] The subject mutations in the PPX gene are generally described herein using the Solanum tuberosum plastidal PPX genes and proteins (see e.g., FIGS. 8 and 7 respectively) with amino acid positions referenced to positions in Arabidopsis thaliana (SEQ ID NO: 1). The compositions and methods also encompass mutant PPX genes and proteins of other species (paralogs). However, due to variations in the PPX genes of different species, the number of the amino acid residue to be changed in one species may be different in another species. Nevertheless, the analogous position is readily identified by one of skill in the art by sequence homology. For example, Table 6 summarizes the homologous amino acid positions in various plant PPX coding sequence paralogs and FIG. 33 shows an amino acid sequence alignment of PPX paralogs from various plants. Thus, analogous positions in these and other paralogs can be identified and mutated.
Herbicides
[0158] The compositions and methods provided herein include PPX genes and PPX proteins that confer resistance to PPX-inhibiting herbicides. In some embodiments, PPX-inhibiting herbicides include the chemical families of diphenylethers, phenylpyrazoles N-phenylphthalimides, thiadiazoles, oxadiazoles, triazolinones, oxazolidinediones, pyrimidindiones. Exemplary PPX-inhibiting herbicide active ingredients and their respective chemical family are summarized in Table 5.
TABLE-US-00007 TABLE 5 Exemplary PPX-inhibiting Herbicides. Chemical Family Herbicide Active Ingredient Diphenylethers acifluorfen-Na Bifenox Chlomethoxyfen fluoroglycofen-ethyl Fomesafen Halosafen Lactofen Oxyfluorfen Phenylpyrazoles Fluazolate pyraflufen-ethyl N-phenylphthalimides cinidon-ethyl Flumioxazin flumiclorac-pentyl Thiadiazoles fluthiacet-methyl Thidiazimin Oxadiazoles Oxadiazon Oxadiargyl Triazolinones Azafenidin carfentrazone-ethyl Sulfentrazone Oxazolidinediones Pentoxazone Pyrimidindiones Benzfendizone Butafenacil Saflufenacil Others Pyrazogyl Profluazol
[0159] In some embodiments, PPX-inhibiting herbicide is acifluorfen-Na. In some embodiments, PPX-inhibiting herbicide is bifenox. In some embodiments, PPX-inhibiting herbicide is chlomethoxyfen. In some embodiments, PPX-inhibiting herbicide is fluoroglycofen-ethyl. In some embodiments, PPX-inhibiting herbicide is fomesafen. In some embodiments, PPX-inhibiting herbicide is halosafen. In some embodiments, PPX-inhibiting herbicide is lactofen. In some embodiments, PPX-inhibiting herbicide is oxyfluorfen. In some embodiments, PPX-inhibiting herbicide is fluazolate. In some embodiments, PPX-inhibiting herbicide is pyraflufen-ethyl. In some embodiments, PPX-inhibiting herbicide is cinidon-ethyl. In some embodiments, PPX-inhibiting herbicide is flumioxazin. In some embodiments, PPX-inhibiting herbicide is flumiclorac-pentyl. In some embodiments, PPX-inhibiting herbicide is fluthiacet-methyl. In some embodiments, PPX-inhibiting herbicide is thidiazimin. In some embodiments, PPX-inhibiting herbicide is oxadiazon. In some embodiments, PPX-inhibiting herbicide is oxadiargyl. In some embodiments, PPX-inhibiting herbicide is azafenidin. In some embodiments, PPX-inhibiting herbicide is carfentrazone-ethyl. In some embodiments, PPX-inhibiting herbicide is sulfentrazone. In some embodiments, PPX-inhibiting herbicide is pentoxazone. In some embodiments, PPX-inhibiting herbicide is benzfendizone. In some embodiments, PPX-inhibiting herbicide is butafenacil. In some embodiments, PPX-inhibiting herbicide is saflufenacil. In some embodiments, PPX-inhibiting herbicide is pyrazogyl. In some embodiments, PPX-inhibiting herbicide is profluazol.
[0160] Also provided is a transgenic or non-transgenic plant or plant cell having one or more mutations in the PPX gene, for example, such as disclosed herein. In certain embodiments, the plant or plant cell having one or more mutations in a PPX gene has increased resistance or tolerance to a member of PPX-inhibiting herbicides. In certain embodiments, the plant or plant cell having one or more mutations in a PPX gene may exhibit substantially normal growth or development of the plant, its organs, tissues or cells, as compared to the corresponding wild-type plant or cell. In particular aspects and embodiments provided are transgenic or non-transgenic plants having a mutation in a PPX gene, for example, such as disclosed herein, which in certain embodiments has increased resistance or tolerance to one or more members of the PPX-inhibiting herbicide chemical families and may exhibit substantially normal growth or development of the plant, its organs, tissues or cells, as compared to the corresponding wild-type plant or cell, i.e., in the presence of one or more herbicide such as for example, flumioxazin, sulfentrazone or saflufenacil, the mutated PPX protein has substantially the same catalytic activity as compared to the wild-type PPX protein.
[0161] Further provided are methods for producing a plant having a mutated PPX gene, for example, having one or more mutations as described herein; preferably the plant substantially maintains the catalytic activity of the wild-type protein irrespective of the presence or absence of a relevant herbicide. In certain embodiments, the methods include introducing into a plant cell a gene repair oligonucleobase with one or more targeted mutations in the PPX gene (for example, such as disclosed herein) and identifying a cell, seed, or plant having a mutated PPX gene.
Plant Species
[0162] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, plants as disclosed herein can be of any species of dicotyledonous, monocotyledonous or gymnospermous plant, including any woody plant species that grows as a tree or shrub, any herbaceous species, or any species that produces edible fruits, seeds or vegetables, or any species that produces colorful or aromatic flowers. For example, the plant or plant cell may be selected from a species of plant from the group consisting of potato, sunflower, sugar beet, maize, cotton, soybean, wheat, rye, oats, rice, canola, fruits, vegetables, tobacco, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, carrot, lettuce, onion, soya spp, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, turf and forage grasses, flax, oilseed rape, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, petunia, tulip, iris, lily, and nut-producing plants insofar as they are not already specifically mentioned. The plant or plant cell may also be of a species selected from Table 6. The plant or plant cell may also be of a species selected from the group consisting of Arabidopsis thaliana, Solanum tuberosum, Solanum phureja, Oryza sativa, Amaranthus tuberculatus, Sorghum bicolor, Ricinus communis and Zea mays.
TABLE-US-00008 TABLE 6 Summary of homologous amino acid positions in plant PPX amino acid sequences of various species. See also Figures 47 and 48 for additional homologous amino acid position summaries. Genbank G N R F A P A L M S Q S S L K L L L Y F I Species Accession # Loc 52 85 144 145 180 185 220 226 228 244 272 305 332 357 359 393 403 424 426 478 525 Arabidopsis AX084732 P 52 85 144 145 180 185 220 226 228 244 272 305 332 357 359 393 403 424 426 478 525 thaliana- At4g01690 Arabidopsis NM_121426 M NA 41 101 Y P K 182 188 190 206 G L H F L 358 371 T F Y D thaliana- 102 137 142 235 269 298 323 325 392 394 444 489 At5g14220 Arnaranthus DQ386117 B NA NA 128 Y P K G 216 218 234 R L 324 F L 384 397 T F Y E tuberculatus 129 164 169 210 261 295 349 351 418 420 470 515 Solanum AJ225107 P N 105 164 165 200 205 240 246 248 264 K 325 352 377 S 413 423 444 446 498 S tuberosum 76 292 379 545 Solarium NA see Fig 27. M NA NA 98 Y P N G 184 186 202 R L 296 F L 356 369 T F Y D tuberosum 99 134 139 178 231 265 321 323 390 392 442 487 Zea mays AF218052 P NA NA 142 143 178 P18 218 224 226 242 K T 330 355 R 391 401 422 424 476 523 3 270 303 357 Zea mays AF273767 M NA 70 130 Y P K 215 221 I 239 N 302 T V L 396 410 T F Y D 131 166 171 223 268 336 361 363 431 433 483 528 Orysa sativa NM 001049312 P G NA 143 144 P 184 219 225 227 243 K T T L I 392 402 423 425 477 S Os01g0286600 51 179 271 304 331 356 358 524 Orysa sativa NA see Fig 17. M D Q 139 Y P K G 230 I 248 N L 345 F L 405 419 T F Y D Os04g0490000 50 79 140 175 180 224 232 277 311 370 372 440 442 492 537 Sorghum XM_002455439 P NA NA 143 144 179 P 219 225 227 243 K T 331 L R 392 402 423 425 477 A bicolor- 184 271 304 356 358 524 Sb03g011670 Sorghum XM_002446665 M NA 70 130 Y P K 215 221 I 239 N L T F L 396 410 T F Y D bicolor- 131 166 171 223 268 302 336 361 363 431 433 483 528 Sb06g020950 Ricinus XM 002515127 P N 84 143 144 179 184 219 225 227 243 K 304 331 356 358 392 402 423 425 477 A cottontails- 51 271 524 Rc1343150 Ricinus XM_0025095 02 M NA NA 99 Y P K 181 187 V 205 234 F 299 F L 359 372 T F Y D cottontails- 100 135 140 189 268 324 326 393 395 445 490 Rc1678480 โ G210 deleted in DQ386118 leading to tolerance to PPX inhibitor P is plastidal; M is mitochondrial; B is both
[0163] The gene repair oligonucleobase can be introduced into a plant cell using any method commonly used in the art, including but not limited to, microcarriers (biolistic delivery), microfibers, polyethylene glycol (PEG)-mediated uptake, electroporation, and microinjection.
[0164] Also provided are methods and compositions related to the culture of cells mutated according to methods as disclosed herein in order to obtain a plant that produces seeds, henceforth a "fertile plant", and the production of seeds and additional plants from such a fertile plant.
[0165] Also provided are methods of selectively controlling weeds in a field, the field comprising plants with the disclosed PPX gene alterations and weeds, the method comprising application to the field of a herbicide to which the plants have been rendered resistant.
[0166] Also provided are mutations in the PPX gene that confer resistance or tolerance to a member of the relevant herbicide to a plant or wherein the mutated PPX gene has substantially the same enzymatic activity as compared to wild-type PPX.
Selection of Herbicide Resistant Plants and Application of Herbicide
[0167] Plants and plant cells can be tested for resistance or tolerance to a herbicide using commonly known methods in the art, e.g., by growing the plant or plant cell in the presence of a herbicide and measuring the rate of growth as compared to the growth rate in the absence of the herbicide.
[0168] As used herein, substantially normal growth of a plant, plant organ, plant tissue or plant cell is defined as a growth rate or rate of cell division of the plant, plant organ, plant tissue, or plant cell that is at least 35%, at least 50%, at least 60%, or at least 75% of the growth rate or rate of cell division in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild-type PPX protein.
[0169] As used herein, substantially normal development of a plant, plant organ, plant tissue or plant cell is defined as the occurrence of one or more development events in the plant, plant organ, plant tissue or plant cell that are substantially the same as those occurring in a corresponding plant, plant organ, plant tissue or plant cell expressing the wild-type PPX protein.
[0170] In conjunction with any of the aspects, embodiments, methods and/or compositions disclosed herein, plant organs provided herein may include, but are not limited to, leaves, stems, roots, vegetative buds, floral buds, meristems, embryos, cotyledons, endosperm, sepals, petals, pistils, carpels, stamens, anthers, microspores, pollen, pollen tubes, ovules, ovaries and fruits, or sections, slices or discs taken therefrom. Plant tissues include, but are not limited to, callus tissues, ground tissues, vascular tissues, storage tissues, meristematic tissues, leaf tissues, shoot tissues, root tissues, gall tissues, plant tumor tissues, and reproductive tissues. Plant cells include, but are not limited to, isolated cells with cell walls, variously sized aggregates thereof, and protoplasts.
[0171] Plants are substantially "tolerant" to a relevant herbicide when they are subjected to it and provide a dose/response curve which is shifted to the right when compared with that provided by similarly subjected non-tolerant like plant. Such dose/response curves have "dose" plotted on the X-axis and "percentage kill", "herbicidal effect", etc., plotted on the y-axis. Tolerant plants will require more herbicide than non-tolerant like plants in order to produce a given herbicidal effect. Plants that are substantially "resistant" to the herbicide exhibit few, if any, necrotic, lytic, chlorotic or other lesions, when subjected to herbicide at concentrations and rates which are typically employed by the agrochemical community to kill weeds in the field. Plants which are resistant to a herbicide are also tolerant of the herbicide.
[0172] In some embodiments an "increased resistance to a herbicide" or "increased tolerance to a herbicide" refers to a level of resistance or tolerance that a plant, seed, or plant part having a mutated PPX gene or protein as disclosed herein has to plant herbicides above a defined reference level. The defined reference level of resistance to a herbicide is the level of resistance displayed by a plant of the same species without the corresponding mutation(s). In some embodiments, resistance is substantially increased above the defined reference level, e.g., greater than or equal to 20% above, 50% above, 75% above; or 100% above the defined reference level.
EXAMPLES
[0173] The following are examples, which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
Example 1
Plastidal and Mitochondrial PPX Gene Cloning and Characterization
[0174] Plastidal and mitochondrial PPX genes were amplified from both cDNA and genomic DNA from a Russet Burbank cultivar. The plastidal PPX clones fall into two classes, given the names StcPPX1 and StcPPX1.1, likely representing alleles of a single PPX gene in potato. Within the amino acid coding sequence, these clones differ by 10 polymorphisms, 3 of which lead to amino acid differences with only two being found in the mature protein. One amino acid difference is in the chloroplast transit peptide. In one of the StcPPX1.1 clones, intron 3 was unspliced.
[0175] A full length error-free genomic clone of the plastidal PPX was obtained. The analysis of about 5 Kb of genomic sequence from 5 independent clones and StcPPX cDNA sequencing results indicates that the Russet Burbank variety subject to characterization is heterozygous, with very few polymorphisms existing between the two alleles.
[0176] First, five full-length StmPPX genomic DNA clones were cloned and sequenced. These five represented both alleles, having the same SNPs as found in the cDNA. Genomic DNA fragments of a shorter amplicon were cloned and sequenced to test for additional alleles. Cloning this internal amplicon of the mitochondrial PPX indicated that there were three alleles; 6 out of 22 clones had a deletion within one of the introns and the other 16 clones had an even distribution of the two alleles observed in the cDNA clones. Next, another 12 full length StmPPX genomic DNA clones were sequenced.
[0177] The completed sequencing of the mitochondrial PPX genes in Russet Burbank potato indicated that there are two genes, which we have termed StmPPX1 and StmPPX2. There are two StmPPX2 alleles with 8 SNPs identified between them. Between StmPPX1 and StmPPX2.1 there is 1 insertion, 4 deletions and 30 SNPs, whereas between StmPPX1 and StmPPX2.2 there is 1 insertion, 4 deletions and 29 SNPs. Additional detail is presented in Table 7.
[0178] Gene sequences of the plastidal and mitochondrial potato PPX genes from Russet Burbank (Solanum tuberosum) were compared, using the Basic Local Alignment Search Tool (BLAST), with our locally installed database based on scaffolds for the recent release of the first full potato (Solanum phureja) genome. Only a single plastidal and a single mitochondrial PPX gene were found.
TABLE-US-00009 TABLE 7 Allelic differences between the mitochondrial forms of PPX in Russet Burbank potato. StmPPX nt aa nt Gene aa Gene nt Gene aa Gene StmPPX AtcPPX nt Position Gene 1 Gene 1 2, Allele 1 2, Allele 1 2, Allele 2 2, Allele 2 aa position aa position 296 A (TAC) Y A (TAC) Y T (TTC) F 99 145 360 C (AAC) N T (AAT) N T (AAT) N 120 166 402 T (CCT) P A (CCA) P A (CCA) P 134 180 528 G (ACG) T A (ACA) T A (ACA) T 176 218 680 T (GTA) V T (GTA) V A (GAA) E 227 269|270 692 A (CAC) H G (CGC) R G (CGC) R 231 272
Example 2
PPX Complementation
[0179] StcPPX1, less its chloroplast transit peptide was cloned from cDNA into Cibus' proprietary functional screening vector. This vector may be used both for functional screening, and for GRON QC. The potato PPX genes were used to complement the HemG mutant strain of E. coli, which lacks a functional HemG gene, a bacterial homolog of PPX. Without a complementing gene, the media must be supplemented with hematin for E. coli growth. Clones for the plastidal PPX gene (pACYStcPPX Col6) and the mitochondrial PPX gene (pACYStmPPX Col 6, 12 and 21) were transformed and all genes/alleles were shown to complement the HemG mutant E. coli strain, allowing it to grow in the absence of hematin.
[0180] In order to assess mutations that confer tolerance to PPX inhibitors such as Chateau (flumioxazin-Valent/Sumitomo), Naja (diphenylether-MAI) or Kixor (saflufenacil-BASF). PPX inhibiting herbicides are shown in Table 5. Pure active ingredients for the PPX inhibiting herbicides Chateau (flumioxazin-Valent/Sumitomo), Spartan (sulfentrazone-FMC) and Kixor/Sharpen (saflufenacil-BASF) were obtained. The wild-type potato plastidal PPX clone (pACYStcPPX Col6) was transformed to complement the hemG mutant E. coli strain and selected with a series of concentrations of the active ingredient for the PPX inhibiting herbicide Spartan (sulfentrazone-FMC) to determine the concentration at which the complemented HemG minus strain does not grow. The wildtype construct did not grow on 2.5 mM sulfentrazone, therefore, selection for tolerant mutants was performed at this concentration. This was further refined, and 0.75 mM sulfentrazone was also used to select for tolerance. The wildtype construct had limited growth at 10 mM flumioxazin in liquid selection and no growth at 0.3 mM saflufenacil in plate based selection, concentrations used to test for tolerant mutants. All potato mitochondrial PPX genes and alleles were tested for natural tolerance to sulfentrazone and flumioxazin, but none were tolerant.
Example 3
PPX PCR-Mutagenesis and Selection of Mutagenized Clones
[0181] Mutagenesis experiments were initially performed on two overlapping fragments (5' and 3') of the potato plastidal PPX gene, to identify mutations in the potato plastidal PPX coding sequence that confer herbicide tolerance.
Liquid Selection Standardization
[0182] Liquid culture selection conditions were developed for both sulfentrazone and flumioxazin. Cultures of 1 mL volume were tested with sulfentrazone and flumioxazin concentrations ranging from 0 to 10 mM. The 0 mM samples had 25 ฮผL DMSO (2.5%) to mimic the concentration of DMSO in the samples containing 10 mM herbicide. Each tube was inoculated with 10 ฮผL of an overnight culture of HemG cells complemented with the wildtype PPX plasmid to ensure uniformity. Spectrophotometric readings (OD600) were taken for each sample (1:4 dilution) and a sample of each plated on LB-Chlor-IPTG plates to determine whether the OD600 correlated with the number of viable colonies. A 10% dilution of the overnight culture was plated for the sulfentrazone-treated cells and a 1% dilution for the cells treated with flumioxazin was plated (see results in Tables 8a-f and 9a-d).
[0183] Both sulfentrazone and flumioxazin precipitate out in the liquid media, causing it to appear opaque even before inoculating bacteria. As the ODs for sulfentrazone show, this herbicide eventually goes into solution while flumioxazin does not. Flumioxazin's deficient solubility skews the OD readings, however, flumioxazin demonstrated steadily decreasing colony numbers against the WT gene, indicating the cells ability to absorb the flumioxazin from the media.
5' End Mutagenesis
[0184] The 5' end of the PPX gene was mutagenized using Stratagene GeneMorph II Random Mutagenesis Kit and cloned into XL-1 Blue to check the mutation rate. Results showed 14 out of 16 colonies sequenced were mutants. The 90% XL-1 Blue plates (approximately 4000 colonies) were scraped, plasmid prepped, and transformed into HemG and plated on 2.5 mM sulfentrazone. The sulfentrazone plates grew approximately 200 colonies and the 10% LB-Chlor-IPTG plates grew a lawn of colonies. Tables 9a-d describe the nucleotide and amino acid substitutions found in the tolerant clones.
Selection of Mutagenized Clones
[0185] Randomly mutagenized plasmids (5' and 3' ends) were transformed into XL1-Blue E. coli cells. The resulting colonies were pooled, plasmid DNA isolated and transformed into HemG (PPX mutant E. coli) cells. For selection with flumioxazin, cells were recovered for 1 h in liquid minimal media followed by the addition of herbicide and overnight recovery of the cells. The next day, the cells were plated at an appropriate dilution on LB plates containing antibiotic to select for the complementing plasmid. Colonies from each plate were sequenced. After liquid selection in 10 mM flumioxazin, approximately 30 colonies appeared on plates with the wild type (WT) PPX compared to approximately 200-1200 colonies with mutagenized plasmids. For sulfentrazone selection, cultures were grown in minimal media overnight, diluted and plated plates with 0 and 0.75 mM concentration of sulfentrazone. Colony counts were compared between the two and mutation tolerance determined based on the percentage of colonies on the 0.75 mM sulfentrazone plates as compared with those on the 0 mM plates. The number of colonies appearing on the plates served as a method to rank the mutations.
3' End Mutagenesis
[0186] Mutagenesis was performed on the 3' end of the PPX gene. Clones were transformed into HemG and grown overnight in 2.5, 5, and 10 mM flumioxazin for selection. Selection at 5 mM flumioxazin had many more colonies than on the 10 mM selection plates. Selection for mutagenized clones using 10 mM flumioxazin yielded four clones. Selection for mutagenized clones using 5 mM flumioxazin yielded 200 colonies. The top third of the 200 colonies obtained for 3' end mutagenesis were screened in 10 mM flumioxazin. All tolerant colonies (approximately 130) were sequenced and the best flumioxazin tolerant mutants, assessed by colony count on flumioxazin, the 3' end assessed for tolerance to sulfentrazone.
Example 4
Analysis of Amino Acid Substitutions Conferring Tolerance
[0187] Tolerance of all possible amino acid substitutions at each position displaying tolerance to sulfentrazone or flumioxazin were tested. Next, single amino acid substitutions were combined in all permutations and combinations to assess complementation and herbicide tolerance. Results of single and multiple mutant combinations to flumioxazin are shown in Tables 8a, 8b, and 8c, where the last column shows the number of colonies reported for each mutation on 10 mM flumioxazin. Results of single and multiple mutant combinations to sulfentrazone are shown in Tables 9a and 9b, where the last column shows the number of colonies reported for each mutation on 0.75 mM sulfentrazone. Results of single and multiple mutant combinations to saflufenacil are shown in Table 10, where the fourth column shows the number of colonies reported for each mutation on 0.3 mM saflufenacil.
TABLE-US-00010 TABLE 8a Tolerance of single and multiple mutant combinations in the potato plastidal PPX coding sequence to flumioxazin. Avg # of Flumioxazin Mutation Plasmid Resistant Clones -- L393V F1125 0 -- L403R F1155 2 A180T Y426F SD5083 2 -- L424S F1154 2 F145L Y426H SD5055 3 S244G Y426F SD5087 10 F145L L403R SD5115 10 -- Y426F F1165 11 F145Y L424S SD5106 13 R144C L424S SD5102 41 L226M Y426H SD5059 55 A220T L403R SD5114 69 -- Y426H F1180 71 A220T L424S SD5104 72 F145Y Y426F SD5086 75 R144C L393V SD5092 81 A220T L393V SD5094 107 S244G L393V SD5097 114 L226M L403R SD5119 123 A180T Y426H SD5053 129 L226M L424S SD5109 137 L226M Y426F SD5089 143 A220T Y426F SD5084 147 A220T Y426H SD5054 151 R144C Y426F SD5082 155 -- -- wt 0
TABLE-US-00011 TABLE 8b Tolerance of single and multiple mutant combinations in the potato plastidal PPX coding sequence to flumioxazin. Avg # of Flumioxazin Mutation Plasmid Resistant Clones R144C A220T SD5011 Col 1 701 A220T F113 667 R144H S332C F76 517 R144C K272F SD5014 Col 1 300 N52K R144H S244T F72 202 N85D A220T SD5007 Col 1 196 R144H S244T F96 174 R144C S7 139 R144C L226M SD5012 Col 1 117 M228L S37 107 L226M F114 102 N85D L226M SD5008 Col 2 50 F145Y S32 38 N85D F145Y SD5004 Col 3 37 S244G S120 31 R144C M228L SD5013 Col 2 31 P185R SD5016 Col 1 27 N52K SD5001 Col 3 23 N85D A180T SD5005 Col 1 23 A180T F17 22 N85D R144C SD5002 Col 1 21 S332C SD5019 Col 1 20 F145L S118 19 N85D F80 16 K272F F7 15 S244T SD5018 Col 3 10 N85D K272F SD5010 Col 4 7 N85D M228L SD5009 Col 3 6 WT 43
TABLE-US-00012 TABLE 8c Tolerance of single and multiple mutant combinations in the potato plastidal PPX coding sequence to flumioxazin. Avg # of Flumioxazin Mutation Plasmid Resistant Clones N85D Y426H SD5051 30 R144C Y426H SD5052 48 F145Y Y426H SD5056 52 S244T Y426H SD5058 73 S244G Y426H SD5057 80 A180T Y426H SD5053 128 L226M Y426H SD5059 228 F145L Y426H SD5055 305 A220T Y426H SD5054 391 -- Y426H F1180 210 -- -- wt 32
TABLE-US-00013 TABLE 8d Tolerance of single and multiple mutant combinations in the potato mitochondrial PPX coding sequence to 5 mM flumioxazin. A404S C271R D274G C307S A423V C434S T500S C434Y D330E D447G A292G D454N N324K R406K S396L K410I S448A N324D Y465F K470T
TABLE-US-00014 TABLE 8e Tolerance of single and multiple mutant combinations in the potato mitochondrial PPX coding sequence to 5 mM flumioxazin. A101V C177S D170E D58N E150K E64V F121L E150D G74C R98C G84N K97R L93H V164A N195K P214S Q157L H187Q R98C R98H R98L R98L P214H R98L T124I L188F K229Q S119N N139Y S151T K229E K230R V164F
TABLE-US-00015 TABLE 8f Tolerance of single and multiple mutant combinations in the potato mitochondrial PPX coding sequence to 10 mM flumioxazin. # Colonies with 10 Mutant mM Flumioxazin wt 42 R98L 83 P214H 51 R98L/P214H 88 R98L/P214H/T124I 110 R98L/P214H/T124I/K229Q 109
TABLE-US-00016 TABLE 9a Tolerance of single and multiple mutant combinations in the potato plastidal PPX coding sequence to sulfentrazone. Avg 0.75 Avg 0 0.75 mM/ Mutation Plasmid mM Sulf mM 0 mM -- Y426F F1165 8 91 0.8% -- L393V F1125 6 57 1.0% L226M Y426H SD5059 14 79 1.8% S244T L393V SD5098 11 53 2.1% F145Y Y426H SD5056 16 72 2.2% -- L403R F1155 16 72 2.2% R144C S525T SD5072 19 76 2.6% A220T L393V SD5094 20 55 3.6% -- L424S F1154 21 56 3.7% F145L L424S SD5105 18 48 3.8% -- Y426H F1180 29 68 4.2% A220T Y426H SD5054 19 39 5.0% F145Y L393V SD5096 55 86 6.3% S244T Y426F SD5088 58 88 6.5% F145Y L424S SD5106 49 69 7.1% A220T L403R SD5114 50 63 7.9% L226M Y426F SD5089 117 67 17.4% N85D Y426H SD5051 200 92 21.8% L226M L424S SD5109 91 40 22.6% F145Y L403R SD5116 315 110 28.5% F145L L393V SD5095 381 121 31.6% L226M L403R SD5119 252 75 33.8% R144C Y426F SD5082 400 117 34.3% S244G L393V SD5097 433 125 34.7% -- S525T F1061 278 79 35.3% A180T Y426H SD5053 331 80 41.4% R144C Y426H SD5052 709 125 56.6% -- -- wt 0 85 0.0%
TABLE-US-00017 TABLE 9b Tolerance of single and multiple mutant combinations in the potato plastidal PPX coding sequence to sulfentrazone. Avg # of Sulfentrazone Mutation(s) Plasmid Resistant Clones R144C M228L SD5013 Col 2 267 R144C A220T SD5011 Col 1 260 F145Y S32 233 R144C L226M SD5012 Col 1 186 A220T F113 149 P185R SD5016 Col 1 133 R144C K272F SD5014 Col 1 118 N52K SD5001 Col 3 90 M228L S37 78 N85D A180T SD5005 Col 1 75 S244G S120 68 R144H S332C F76 64 N85D F145Y SD5004 Col 3 62 K272F F7 61 L226M F114 57 R144C S7 55 S244T SD5018 Col 3 32 R144H S244T F96 31 F145L S118 29 S332C SD5019 Col 1 28 N85D R144C SD5002 Col 1 25 N85D A220T SD5007 Col 1 19 N85D L226M SD5008 Col 2 18 A180T F17 18 N52K R144H S244T F72 18 N85D M228L SD5009 Col 3 12 N85D F80 6 N85D K272F SD5010 Col 4 6 WT 23
TABLE-US-00018 TABLE 9c Tolerance of single and multiple mutant combinations in the potato mitochondrial PPX coding sequence to sulfentrazone. Sulf Conc Mutant(s) Colonies % age 0 mM R98L/P214H 1341 0.5 mM R98L/P214H 349 26.0% 0.9 mM R98L/P214H 127 9.5% 1.0 mM R98L/P214H 77 5.7% 1.1 mM R98L/P214H 67 5.0% 1.2 mM R98L/P214H 48 3.6% 0 mM R98L 1541 0.5 mM R98L 339 22.0% 0.9 mM R98L 145 9.4% 1.0 mM R98L 110 7.1% 1.1 mM R98L 76 4.9% 1.2 mM R98L 54 3.5% 0 mM R98L/T124I/K229Q 1220 0.5 mM R98L/T124I/K229Q 312 25.6% 0.9 mM R98L/T124I/K229Q 88 7.2% 1.0 mM R98L/T124I/K229Q 66 5.4% 1.1 mM R98L/T124I/K229Q 40 3.3%
TABLE-US-00019 TABLE 9d Tolerance of single and multiple mutant combinations in the potato mitochondrial PPX coding sequence to sulfentrazone. Sulf Conc Mutant(s) Colonies % age 0 mM R98L/P214H 1088 0.4 mM R98L/P214H 251 23.1% 0.5 mM R98L/P214H 150 13.8% 0.6 mM R98L/P214H 105 9.7% 0.7 mM R98L/P214H 108 9.9% 0.8 mM R98L/P214H 51 4.7% 0 mM R98L 1171 0.4 mM R98L 174 14.9% 0.5 mM R98L 104 8.9% 0.6 mM R98L 98 8.4% 0.7 mM R98L 92 7.9% 0.8 mM R98L 51 4.4% 0 mM R98L/T124I/P214H/K229Q 1134 0.4 mM R98L/T124I/P214H/K229Q 724 63.8% 0.5 mM R98L/T124I/P214H/K229Q 654 57.7% 0.6 mM R98L/T124I/P214H/K229Q 402 35.4% 0.7 mM R98L/T124I/P214H/K229Q 302 26.6% 0.8 mM R98L/T124I/P214H/K229Q 280 24.7% 0 mM P214H 1184 0.4 mM P214H 0 0.0% 0.5 mM P214H 0 0.0% 0.6 mM P214H 0 0.0% 0.7 mM P214H 0 0.0% 0.8 mM P214H 0 0.0%
TABLE-US-00020 TABLE 10 Tolerance of single and multiple mutant combinations in the potato plastidal PPX coding sequence to saflufenacil measured by number of colonies reported. Avg # of saflufenacil Avg 0 0.3 mM/ Mutation Plasmid resistant clones mM 0 mM -- S525T F1061 0 69 0.0% N85D Y426H SD5051 0 64 0.0% F145L L393V SD5095 0 114 0.0% S244G L393V SD5097 0 111 0.0% F145Y L403R SD5116 0 104 0.0% L226M L424S SD5109 6 50 1.3% L226M Y426F SD5089 15 96 1.5% A220T L393V SD5094 116 99 11.7% A220T Y426F SD5084 190 87 22.0% L226M L403R SD5119 225 69 32.5% R144C Y426F SD5082 319 61 52.6% R144C Y426H SD5052 415 75 55.4% A180T Y426H SD5053 394 60 65.7% A220T Y426H SD5054 356 46 77.5% -- -- wt 0 96 0.0%
Example 5
Plant Cell Culture-Herbicide Kill Curves
Flumioxazin Kill Curves
[0188] Herbicide selection experiments were performed to determine the concentration of herbicide necessary to kill protoplast derived microcalli in a defined treatment period. In light of an initial kill curve result where a concentration of 125 ฮผM was sufficient to kill all cells within a week, a new kill curve was designed using lower concentrations of flumioxazin aimed at determining the concentration at which 99% of the cells are killed (see Table 11). The herbicide was suspended in DMSO, with the final concentration of DMSO in the herbicide treatments being 1%. Development of cells was evaluated under the microscope once a week. Excepting the control treatments, division in all treatments with flumioxazin was prevented after one week and after one month no microcalli developed at any concentration tested. A flumioxazin concentration of 0.032 mM is sufficient to prevent microcallus development from potato protoplasts.
TABLE-US-00021 TABLE 11 Summary of results of flumioxazin kill curve experiments with cell suspension and shoot tip-derived protoplasts. Protoplasts were exposed to flumioxazin for a period of one month. Non treated Stop-GFP + protoplast + PEG # Of calli Correcting GRON # of calli Flumioxazin in 3 beads Flumioxazin in 3 beads Conc. ฮผM # % Conc. ฮผM # % Control #1* 185 Control 230 Control #2** 150 Control 190 Av. Cont 1 + Cont 2 157.5 100 Av. Cont 1 + Cont 2 210 100 0.0156 40 25 0.0156 83 39 0.0312 8 5 0.0312 11 4 0.0468 0.0 0.0 0.0468 0.0 0.0 0.0624 0 0 0.0624 0 0 *Culture medium, no herbicide **Culture medium with 1% DMSO, no herbicide
Sulfentrazone Kill Curves
[0189] Kill curves with sulfentrazone on shoot tip-derived protoplasts and cell suspension showed concentrations of 7.8 ฮผM sulfentrazone are sufficient to kill all protoplast-derived cells (see Table 12). Therefore, new kill curves were initiated with lower concentrations of 0, 0.5, 0.6, 0.7 and 0.8 ฮผM of sulfentrazone, shown in Table 13. The results suggest that GRON treated protoplasts may be selected at concentrations between 0.6 ฮผM and 0.7 ฮผM of the herbicide.
TABLE-US-00022 TABLE 12 Summary of results of sulfentrazone kill curve experiments with cell suspension and shoot tip-derived protoplasts. Protoplasts were exposed to sulfentrazone for a period of one month. Sulfentrazone treatment [ฮผM] Microcallus formation Control: 1* Yes; abundant Control: 2** Yes; abundant 62.5 No 31.25 No 15.6 No 7.8 No *Culture medium, no herbicide **Culture medium with 1% DMSO, no herbicide
TABLE-US-00023 TABLE 13 Summary of results of sulfentrazone kill curve experiments with cell suspension and shoot tip-derived protoplasts. Protoplasts were exposed to the herbicide for a period of one month. Non treated Stop-GFP + protoplast + PEG # Of calli Correcting GRON # of Sulfentrazon in 3 beads Sulfentrazonel calli in beads Conc. ฮผM # % Conc. ฮผM # % 0.0 115 100 0.0 125 100% 0.5 45 30 0.5 38 30.2 0.6 8 6.9 0.6 12 9.6 0.7 2 1.7 0.7 7 5.6 0.8 0.0 0.0 0.8 0.0 0.0 *Culture medium, no herbicide **Culture medium with 1% DMSO, no herbicide
Example 6
Leaf Disk Kill Curves
[0190] Aimed at establish the concentration of sulfentrazone that will inhibit callus formation in leaf disc explants, leaf discs were punched with a sterile hole punch from 5 weeks old in vitro grown potato plants. The leaf discs were cultured in petri dishes containing solid Haberlach culture medium containing various concentrations of Sulfentrazone in a final concentration of 1% DMSO. Six leaf discs cultured at each herbicide concentration. Plates were sealed with micropore tape and incubated at room temperature (approximately 23ยฐ C.). In an initial experiment, 7.8 ฮผM sulfentrazone, the lowest concentration tested in that experiment, was sufficient to stop callus formations and bleach all leaf discs within 20 days. Results of kill curve with lower concentrations of sulfentrazone showed that a concentration of 3.0 ฮผM of Sulfentrazone was sufficient to inhibit callus formation in almost all leaf discs after 20 days, whereas callus initiated after 13 days on the leaf veins of some leaf discs grown on 2.0 ฮผM of Sulfentrazone. Similar leaf disc kill curve experiments were performed using saflufenacil where 0.5 ฮผM of this herbicide was sufficient to inhibit callus formation in almost all leaf discs after 20 days.
Example 7
Materials and Methods for Cell Culture and GRON Introduction
TABLE-US-00024 [0191] TABLE 14 GRON Sequences GRON Sequence StcPPX1144/C/ VGTTGGGAGATCCTGATGCGCCTTGCTTTGTCTTG 47/5'Cy3/3'idC TGGAAGGATAAACH (SEQ ID NO: 33) StcPPX1144/NC/ VGTTTATCCTTCCACAAGACAAAGCAAGGCGCATC 47/5'Cy3/3'idC AGGATCTCCCAACH (SEQ ID NO: 34) StcPPX1220/C/ VCATCATTTTACAGGTGTTTACACCGGTGACCCCT 47/5'Cy3/3'idC CAAAATTGH (SEQ ID NO: 35) StcPPX1220/NC/ VCAATTTTGAGGGGTCACCGGTGTAAACACCTGTA 47/5'Cy3/3'idC AAATGATGH (SEQ ID NO: 36) The converting base is shown in bold. V = CY3; H = 3'DMT dC CPG
[0192] Cell Culture Work Description. Shoots, for example, derived from seeds, tubers, axillary buds, leaves, steams, roots, callus, or from microspore-derived embryos, are propagated under sterile conditions in vitro. Explants are subcultured, for example, every 3-4 weeks and cultured in Magenta GA7 culture vessels (Phytotechnology Laboratories, Shawnee Mission, Kans., USA) with vented lids in a volume of about 100 mL culture medium, for example MS medium, according to Murashige and Skoog (A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant 15 (1962) 473-49), or modifications thereof. The vessels may be sealed with Micropore tape (3M Company). Young leaves, shoot tips, roots, microtubers or long stem segments possessing a leaf and axillary bud, as well as callus derived from these tissues, may be used for protoplast isolation. Protoplasts may also be isolated from suspension culture cells derived from young leaves, shoot tips, roots, microtubers or long stem segments possessing a leaf and axillary bud, as well as callus derived from these tissues.
Protoplast Isolation from Shoot Tips
[0193] About 200 shoot tips of 2-8 week-old in vitro shoots that have been cultured under a regular day/night regime, or, preferably were kept for two days before protoplast isolation in the dark, shoots may be cut into small pieces with a scalpel in a petri dish with sterile water. After all tips have been cut, the water is replaced with protoplast culture medium, preferably BN (B5 Salts and Vitamins (Phytotechnology Laboratories), glucose 20 g/L, mannitol 70 g/L, alpha naphthalene acetic acid 5 mg/L, additional CaCl2ร2H2O 600 mg/L, casein hydrolysate 250 mg/L, cysteine-HCL 10 mg/L, polyvinylpyrrolidone (MW 10,000) 5 g/L. After approximately 1-2 h, the protoplast culture medium is replaced with enzyme solution, for example consisting of medium BN, in which 0.5% (w/v) Cellulase YC and 0.75% (w/v) Macerozyme R10 (both from Karlan Research Products, Cottonwood, Ariz.), 1 g/L bovine serum albumin, and 1 g/L 2-morpholinoethanesulfonic acid are dissolved. The ratio of the number of shoot tips over the volume of enzyme solution can be between 10 and 16, preferably 13. The dish with shoot tip pieces in enzyme solution is incubated for at 25ยฐ C.-30ยฐ C., preferably 28ยฐ C., in darkness on a shaker set to about 50 rpm. After overnight incubation the protoplast suspension is purified using an iodixanol density gradient (adapted from Optiprep Application Sheet C18; Purification of Intact Plant Protoplasts; Axis-Shield USA, 10 Commerce Way, Norton, Mass. 02776). After the density gradient centrifugation, the band with purified protoplasts is removed together with about 5 mL W5 medium (Frigerio et al., 1998). The protoplast density and yield are determined with a hemocytometer. The protoplasts density is adjusted to 1ร106/mL in BN medium containing 2 mg/L 2,6-dichlorobenzonitrile (cellulose synthase inhibitor), and the protoplasts are cultured in darkness at 30ยฐ C. for about 16 h.
Protoplast Isolation from Cell Suspensions
[0194] The isolation of protoplasts from cell suspensions follows the same protocol as described for the isolation of protoplasts from shoot tips, with the following exceptions:
1. Fast growing cell suspensions are used, preferably three days after their last subculture. 1.5 mL settled cell volume is transferred to about 15 ml BN medium, which after 2 h is replaced with enzyme solution. 2. The protoplast purification is followed immediately by the GRON/PEG treatment.
Gene Repair Oligonucleotide (GRON) Introduction
[0195] The protoplast suspension is mixed with an equal volume of W5 medium, transferred to a 50 mL centrifuge tube, and centrifuged for 10 min at the lowest setting of a clinical centrifuge (about 50รg). The supernatant is removed and replaced with TM medium (Klaus, S. Markerfreie transplastome Tabakpflanzen (Marker-free transplastomic tobacco plants). PhD Dissertation, 2002, Ludwig-Maximilians-Universitat Munchen, 109 pp), adjusting the protoplast density to 5ร106/mL. Aliquots of 100 ฮผL containing 5ร105 protoplasts each are distributed into 12 mL round bottom centrifuge tubes. GRONs (such as those shown in Table 14) targeted at one or more mutations in one or both of the mitochondrial and plastidal PPX genes are then introduced into the protoplasts using a PEG treatment. To introduce the GRONs into the protoplasts, 12.5 ฮผg GRON dissolved in 25 ฮผL purified water and 125 ฮผL of a polyethylene glycol solution (5 g PEG MW 1500, 638 mg mannitol, 207 mg CaNO3ร4H2O and 8.75 mL purified water; pH adjusted to about 9.0) is added. After a 10-30 min incubation on ice, the protoplast-PEG suspension is washed with W5 medium and resuspended in medium BN. The suspension is kept overnight in darkness at room temperature.
[0196] GRONs may be introduced into protoplasts by electroporation, cationic lipids, nanoparticles, polycations such as hexadimethrine bromide (polybrene) or spermidine, or by using GRONs complexed to a variety of cell penetrating peptides (CPPs) including but not limited to TAT, pVEC, transportan, nona-arginine, BAX inhibiting peptide (VPMLK), or such as those listed in Patel et al. Cell Penetrating Peptides: Intracellular Pathways and Pharmaceutical Perspectives. Pharmaceutical Research, 24 (2007) 1977-1992, or Veldhoen et al. Recent developments in peptide-based nucleic acid delivery. International Journal of Molecular Science (2008) 1276-1320. In another embodiment, GRONs are introduced into protoplasts through negatively charged polymers including, but not limited to dendrimers such as Polyamidoamine (PAMAM).
[0197] GRONs may also be delivered into whole tissues or cells using methods that may include microinjection, biolistics with the GRONs coated on carriers such as gold or directly in the form of droplets of a GRON suspension, GRON coated whiskers or using GRONs complexed to a variety of cell penetrating peptides (CPPs) negatively charged polymers as mentioned in the preceding paragraph. Other embodiments envision the use of ultrasound, imbibition in GRON containing solutions, or permeabilization of cell walls, for example through agents such as toluene or saponin.
Embedding of Protoplasts in Calcium Alginate
[0198] One day after the GRON introduction, protoplasts are embedded in calcium alginate. The embedding of protoplasts in gel substrates (e.g., agarose, alginate) has been shown to enhance protoplast survival and to increase division frequencies of protoplast-derived cells. The method applied is based on that described in Dovzhenko et al. (Thin-alginate-layer technique for protoplast culture of tobacco leaf protoplasts: shoot formation in less than two weeks. Protoplasma 204 (1998) 114-118).
Protoplast Culture and Selection of Herbicide-Resistant Calli
[0199] The selection of herbicide-resistant calli is carried out using sequential subcultures of the alginates in liquid media according to Pelletier et al. (1983). Selection may be started one week after the PEG/GRON treatment at an appropriate concentration of PPX-inhibiting herbicide; for example, 32 ฮผM flumioxazin, or 0.25 ฮผM, 0.5 ฮผM, 1 ฮผM, 2 ฮผM, 3 ฮผM, 4 ฮผM, 5 ฮผM, 6 ฮผM, 7 ฮผM, 7.8 ฮผM, 15.6 ฮผM, 31.2 ฮผM or 62.5 ฮผM sulfentrazone.
[0200] Before the end of the selection phase in liquid medium, cells and colonies are released from the alginate by treating them for 30-45 min with culture medium containing 50 mM sodium citrate. At the moment of transferring released colonies from liquid to solid medium CuI (Haberlach et al. Isolation, culture and regeneration of protoplasts from potato and several related Solanum species. Plant Science, 39 (1985) 67-74), the majority of colonies may be either dead, or consist of a greenish center, covered with outer layers of dead cells. On the solidified selection medium (Cul+herbicide) the majority of microcalli that still contain living cells may stop growing and turn brownish. Limited growth of individual calli continues occasionally, but all non-resistant calli eventually turn brown and die. Two to three weeks after the transfer to solidified selection medium (occasionally earlier), actively growing calli may appear among a background of brownish cells and microcalli.
[0201] Regeneration of plants from protoplast-derived, herbicide-tolerant calli with a confirmed mutation in a PPX gene is performed. PPX-inhibiting herbicide-tolerant calli that develop on solidified selection medium and whose DNA upon analysis shows the presence of a mutation are transferred to herbicide-free medium CuI to accelerate development. Individual callus lines vary in their growth rates and morphologies. In general, the development towards shoot regeneration follows these steps:
Undifferentiated, green callusโcallus with dark green areasโdevelopment of shoot initialsโdevelopment of a plant.
[0202] The development of individual callus lines is variable, but through continuous subculture and multiplication on Cul medium or by changing the media formulation to differentiation medium including but not limited to Haberlach differentiation medium, for an acceptable period of time (1-6 months) followed by transfer of the callus lines to regeneration media including but not limited to Bokelmann regeneration medium (Bokelmann G. S, and Roest S., Z. Pflanzenphysiol. vol. 109, p. 259-265 (1983)) eventually many produce shoots.
[0203] Once shoots with three to four leaves are formed on regeneration medium, they are transferred to propagation medium including but not limited to MS medium. On this medium, over time, shoots and leaves develop that are morphologically `normal`. After in vitro plantlets produce roots, standard protocols are used for the adaptation to greenhouse conditions.
Molecular Screening
[0204] Using standard molecular techniques and more sensitive PCR based technologies can be used to monitor the frequency of PPX mutations following an RTDS treatment. These molecular techniques include and are not limited to, allele specific PCR, DNA sequencing and other SNP identification technologies using non-PCR techniques. These techniques allow for monitoring the frequency of PPX targeted mutations early in the procedure. In certain embodiments, the mutations can be measured in populations of single cells. These techniques can then be applicable throughout the culture process to confirm and monitor mutations are present in selected calli and regenerated plants.
Example 8
Herbicide Spray
[0205] Solanum tuberosum or Russet Burbank potato cultivar plants when they are 2-6'' tall (generally the 5-6 leaf stage) are sprayed with various PPX-inhibiting herbicides. Herbicides are sprayed in the presence of 0.25% AU391 surfactant. The herbicides are sprayed, for example, at the following rates:
Flumioxazin 2 oz active ingredient/Acre (ai/A) Sulfentrazone 4.5 oz ai/A Saflufenacil 1-13 oz ai/A
[0206] Herbicides are applied by foliar spray with control plants being left unsprayed. PPX-inhibiting herbicide trials are evaluated 14 days post spraying using a damage scale of 1-10 with 1 being dead, and 10 being the undamaged unsprayed controls. Individual plant lines are scored at each spray rate compared to the performance of the controls at that particular rate. PPX inhibiting herbicides have a potentially wide window of application and can be used as a "pre" or "post" application for crops including potato. Herbicide evaluations include both greenhouse and field applications to monitor plant (crop) damage and/or weed control. Products from RTDS work can allow farmers to plant crops like potato and apply PPX inhibiting herbicides to eliminate or control weeds in the fields while not damaging crops.
[0207] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0208] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. It is recognized that various modifications are possible within the scope of the invention claimed.
[0209] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement, and variation of the inventions disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.
[0210] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
[0211] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.
[0212] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.
[0213] Other embodiments are set forth within the following claims.
Sequence CWU
1
501537PRTArabidopsis thaliana 1Met Glu Leu Ser Leu Leu Arg Pro Thr Thr Gln
Ser Leu Leu Pro Ser1 5 10
15Phe Ser Lys Pro Asn Leu Arg Leu Asn Val Tyr Lys Pro Leu Arg Leu
20 25 30Arg Cys Ser Val Ala Gly Gly
Pro Thr Val Gly Ser Ser Lys Ile Glu 35 40
45Gly Gly Gly Gly Thr Thr Ile Thr Thr Asp Cys Val Ile Val Gly
Gly 50 55 60Gly Ile Ser Gly Leu Cys
Ile Ala Gln Ala Leu Ala Thr Lys His Pro65 70
75 80Asp Ala Ala Pro Asn Leu Ile Val Thr Glu Ala
Lys Asp Arg Val Gly 85 90
95Gly Asn Ile Ile Thr Arg Glu Glu Asn Gly Phe Leu Trp Glu Glu Gly
100 105 110Pro Asn Ser Phe Gln Pro
Ser Asp Pro Met Leu Thr Met Val Val Asp 115 120
125Ser Gly Leu Lys Asp Asp Leu Val Leu Gly Asp Pro Thr Ala
Pro Arg 130 135 140Phe Val Leu Trp Asn
Gly Lys Leu Arg Pro Val Pro Ser Lys Leu Thr145 150
155 160Asp Leu Pro Phe Phe Asp Leu Met Ser Ile
Gly Gly Lys Ile Arg Ala 165 170
175Gly Phe Gly Ala Leu Gly Ile Arg Pro Ser Pro Pro Gly Arg Glu Glu
180 185 190Ser Val Glu Glu Phe
Val Arg Arg Asn Leu Gly Asp Glu Val Phe Glu 195
200 205Arg Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala
Gly Asp Pro Ser 210 215 220Lys Leu Ser
Met Lys Ala Ala Phe Gly Lys Val Trp Lys Leu Glu Gln225
230 235 240Asn Gly Gly Ser Ile Ile Gly
Gly Thr Phe Lys Ala Ile Gln Glu Arg 245
250 255Lys Asn Ala Pro Lys Ala Glu Arg Asp Pro Arg Leu
Pro Lys Pro Gln 260 265 270Gly
Gln Thr Val Gly Ser Phe Arg Lys Gly Leu Arg Met Leu Pro Glu 275
280 285Ala Ile Ser Ala Arg Leu Gly Ser Lys
Val Lys Leu Ser Trp Lys Leu 290 295
300Ser Gly Ile Thr Lys Leu Glu Ser Gly Gly Tyr Asn Leu Thr Tyr Glu305
310 315 320Thr Pro Asp Gly
Leu Val Ser Val Gln Ser Lys Ser Val Val Met Thr 325
330 335Val Pro Ser His Val Ala Ser Gly Leu Leu
Arg Pro Leu Ser Glu Ser 340 345
350Ala Ala Asn Ala Leu Ser Lys Leu Tyr Tyr Pro Pro Val Ala Ala Val
355 360 365Ser Ile Ser Tyr Pro Lys Glu
Ala Ile Arg Thr Glu Cys Leu Ile Asp 370 375
380Gly Glu Leu Lys Gly Phe Gly Gln Leu His Pro Arg Thr Gln Gly
Val385 390 395 400Glu Thr
Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala
405 410 415Pro Pro Gly Arg Ile Leu Leu
Leu Asn Tyr Ile Gly Gly Ser Thr Asn 420 425
430Thr Gly Ile Leu Ser Lys Ser Glu Gly Glu Leu Val Glu Ala
Val Asp 435 440 445Arg Asp Leu Arg
Lys Met Leu Ile Lys Pro Asn Ser Thr Asp Pro Leu 450
455 460Lys Leu Gly Val Arg Val Trp Pro Gln Ala Ile Pro
Gln Phe Leu Val465 470 475
480Gly His Phe Asp Ile Leu Asp Thr Ala Lys Ser Ser Leu Thr Ser Ser
485 490 495Gly Tyr Glu Gly Leu
Phe Leu Gly Gly Asn Tyr Val Ala Gly Val Ala 500
505 510Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu Thr Ala
Ile Glu Val Asn 515 520 525Asn Phe
Met Ser Arg Tyr Ala Tyr Lys 530 53521719DNAArabidopsis
thaliana 2tgacaaaatt ccgaattctc tgcgatttcc atggagttat ctcttctccg
tccgacgact 60caatcgcttc ttccgtcgtt ttcgaagccc aatctccgat taaatgttta
taagcctctt 120agactccgtt gttcagtggc cggtggacca accgtcggat cttcaaaaat
cgaaggcgga 180ggaggcacca ccatcacgac ggattgtgtg attgtcggcg gaggtattag
tggtctttgc 240atcgctcagg cgcttgctac taagcatcct gatgctgctc cgaatttaat
tgtgaccgag 300gctaaggatc gtgttggagg caacattatc actcgtgaag agaatggttt
tctctgggaa 360gaaggtccca atagttttca accgtctgat cctatgctca ctatggtggt
agatagtggt 420ttgaaggatg atttggtgtt gggagatcct actgcgccaa ggtttgtgtt
gtggaatggg 480aaattgaggc cggttccatc gaagctaaca gacttaccgt tctttgattt
gatgagtatt 540ggtgggaaga ttagagctgg ttttggtgca cttggcattc gaccgtcacc
tccaggtcgt 600gaagaatctg tggaggagtt tgtacggcgt aacctcggtg atgaggtttt
tgagcgcctg 660attgaaccgt tttgttcagg tgtttatgct ggtgatcctt caaaactgag
catgaaagca 720gcgtttggga aggtttggaa actagagcaa aatggtggaa gcataatagg
tggtactttt 780aaggcaattc aggagaggaa aaacgctccc aaggcagaac gagacccgcg
cctgccaaaa 840ccacagggcc aaacagttgg ttctttcagg aagggacttc gaatgttgcc
agaagcaata 900tctgcaagat taggtagcaa agttaagttg tcttggaagc tctcaggtat
cactaagctg 960gagagcggag gatacaactt aacatatgag actccagatg gtttagtttc
cgtgcagagc 1020aaaagtgttg taatgacggt gccatctcat gttgcaagtg gtctcttgcg
ccctctttct 1080gaatctgctg caaatgcact ctcaaaacta tattacccac cagttgcagc
agtatctatc 1140tcgtacccga aagaagcaat ccgaacagaa tgtttgatag atggtgaact
aaagggtttt 1200gggcaattgc atccacgcac gcaaggagtt gaaacattag gaactatcta
cagctcctca 1260ctctttccaa atcgcgcacc gcccggaaga attttgctgt tgaactacat
tggcgggtct 1320acaaacaccg gaattctgtc caagtctgaa ggtgagttag tggaagcagt
tgacagagat 1380ttgaggaaaa tgctaattaa gcctaattcg accgatccac ttaaattagg
agttagggta 1440tggcctcaag ccattcctca gtttctagtt ggtcactttg atatccttga
cacggctaaa 1500tcatctctaa cgtcttcggg ctacgaaggg ctatttttgg gtggcaatta
cgtcgctggt 1560gtagccttag gccggtgtgt agaaggcgca tatgaaaccg cgattgaggt
caacaacttc 1620atgtcacggt acgcttacaa gtaaatgtaa aacattaaat ctcccagctt
gcgtgagttt 1680tattaaatat tttgagatat ccaaaaaaaa aaaaaaaaa
17193508PRTArabidopsis thaliana 3Met Ala Ser Gly Ala Val Ala
Asp His Gln Ile Glu Ala Val Ser Gly1 5 10
15Lys Arg Val Ala Val Val Gly Ala Gly Val Ser Gly Leu
Ala Ala Ala 20 25 30Tyr Lys
Leu Lys Ser Arg Gly Leu Asn Val Thr Val Phe Glu Ala Asp 35
40 45Gly Arg Val Gly Gly Lys Leu Arg Ser Val
Met Gln Asn Gly Leu Ile 50 55 60Trp
Asp Glu Gly Ala Asn Thr Met Thr Glu Ala Glu Pro Glu Val Gly65
70 75 80Ser Leu Leu Asp Asp Leu
Gly Leu Arg Glu Lys Gln Gln Phe Pro Ile 85
90 95Ser Gln Lys Lys Arg Tyr Ile Val Arg Asn Gly Val
Pro Val Met Leu 100 105 110Pro
Thr Asn Pro Ile Glu Leu Val Thr Ser Ser Val Leu Ser Thr Gln 115
120 125Ser Lys Phe Gln Ile Leu Leu Glu Pro
Phe Leu Trp Lys Lys Lys Ser 130 135
140Ser Lys Val Ser Asp Ala Ser Ala Glu Glu Ser Val Ser Glu Phe Phe145
150 155 160Gln Arg His Phe
Gly Gln Glu Val Val Asp Tyr Leu Ile Asp Pro Phe 165
170 175Val Gly Gly Thr Ser Ala Ala Asp Pro Asp
Ser Leu Ser Met Lys His 180 185
190Ser Phe Pro Asp Leu Trp Asn Val Glu Lys Ser Phe Gly Ser Ile Ile
195 200 205Val Gly Ala Ile Arg Thr Lys
Phe Ala Ala Lys Gly Gly Lys Ser Arg 210 215
220Asp Thr Lys Ser Ser Pro Gly Thr Lys Lys Gly Ser Arg Gly Ser
Phe225 230 235 240Ser Phe
Lys Gly Gly Met Gln Ile Leu Pro Asp Thr Leu Cys Lys Ser
245 250 255Leu Ser His Asp Glu Ile Asn
Leu Asp Ser Lys Val Leu Ser Leu Ser 260 265
270Tyr Asn Ser Gly Ser Arg Gln Glu Asn Trp Ser Leu Ser Cys
Val Ser 275 280 285His Asn Glu Thr
Gln Arg Gln Asn Pro His Tyr Asp Ala Val Ile Met 290
295 300Thr Ala Pro Leu Cys Asn Val Lys Glu Met Lys Val
Met Lys Gly Gly305 310 315
320Gln Pro Phe Gln Leu Asn Phe Leu Pro Glu Ile Asn Tyr Met Pro Leu
325 330 335Ser Val Leu Ile Thr
Thr Phe Thr Lys Glu Lys Val Lys Arg Pro Leu 340
345 350Glu Gly Phe Gly Val Leu Ile Pro Ser Lys Glu Gln
Lys His Gly Phe 355 360 365Lys Thr
Leu Gly Thr Leu Phe Ser Ser Met Met Phe Pro Asp Arg Ser 370
375 380Pro Ser Asp Val His Leu Tyr Thr Thr Phe Ile
Gly Gly Ser Arg Asn385 390 395
400Gln Glu Leu Ala Lys Ala Ser Thr Asp Glu Leu Lys Gln Val Val Thr
405 410 415Ser Asp Leu Gln
Arg Leu Leu Gly Val Glu Gly Glu Pro Val Ser Val 420
425 430Asn His Tyr Tyr Trp Arg Lys Ala Phe Pro Leu
Tyr Asp Ser Ser Tyr 435 440 445Asp
Ser Val Met Glu Ala Ile Asp Lys Met Glu Asn Asp Leu Pro Gly 450
455 460Phe Phe Tyr Ala Gly Asn His Arg Gly Gly
Leu Ser Val Gly Lys Ser465 470 475
480Ile Ala Ser Gly Cys Lys Ala Ala Asp Leu Val Ile Ser Tyr Leu
Glu 485 490 495Ser Cys Ser
Asn Asp Lys Lys Pro Asn Asp Ser Leu 500
50541822DNAArabidopsis thaliana 4tttccgtcac tgctttcgac tggtcagaga
ttttgactct gaattgttgc agatagcaat 60ggcgtctgga gcagtagcag atcatcaaat
tgaagcggtt tcaggaaaaa gagtcgcagt 120cgtaggtgca ggtgtaagtg gacttgcggc
ggcttacaag ttgaaatcga ggggtttgaa 180tgtgactgtg tttgaagctg atggaagagt
aggtgggaag ttgagaagtg ttatgcaaaa 240tggtttgatt tgggatgaag gagcaaacac
catgactgag gctgagccag aagttgggag 300tttacttgat gatcttgggc ttcgtgagaa
acaacaattt ccaatttcac agaaaaagcg 360gtatattgtg cggaatggtg tacctgtgat
gctacctacc aatcccatag agctggtcac 420aagtagtgtg ctctctaccc aatctaagtt
tcaaatcttg ttggaaccat ttttatggaa 480gaaaaagtcc tcaaaagtct cagatgcatc
tgctgaagaa agtgtaagcg agttctttca 540acgccatttt ggacaagagg ttgttgacta
tctcatcgac ccttttgttg gtggaacaag 600tgctgcggac cctgattccc tttcaatgaa
gcattctttc ccagatctct ggaatgtaga 660gaaaagtttt ggctctatta tagtcggtgc
aatcagaaca aagtttgctg ctaaaggtgg 720taaaagtaga gacacaaaga gttctcctgg
cacaaaaaag ggttcgcgtg ggtcattctc 780ttttaagggg ggaatgcaga ttcttcctga
tacgttgtgc aaaagtctct cacatgatga 840gatcaattta gactccaagg tactctcttt
gtcttacaat tctggatcaa gacaggagaa 900ctggtcatta tcttgtgttt cgcataatga
aacgcagaga caaaaccccc attatgatgc 960tgtaattatg acggctcctc tgtgcaatgt
gaaggagatg aaggttatga aaggaggaca 1020accctttcag ctaaactttc tccccgagat
taattacatg cccctctcgg ttttaatcac 1080cacattcaca aaggagaaag taaagagacc
tcttgaaggc tttggggtac tcattccatc 1140taaggagcaa aagcatggtt tcaaaactct
aggtacactt ttttcatcaa tgatgtttcc 1200agatcgttcc cctagtgacg ttcatctata
tacaactttt attggtggga gtaggaacca 1260ggaactagcc aaagcttcca ctgacgaatt
aaaacaagtt gtgacttctg accttcagcg 1320actgttgggg gttgaaggtg aacccgtgtc
tgtcaaccat tactattgga ggaaagcatt 1380cccgttgtat gacagcagct atgactcagt
catggaagca attgacaaga tggagaatga 1440tctacctggg ttcttctatg caggtaatca
tcgagggggg ctctctgttg ggaaatcaat 1500agcatcaggt tgcaaagcag ctgaccttgt
gatctcatac ctggagtctt gctcaaatga 1560caagaaacca aatgacagct tataacattg
tcaaggttcg tcccttttta tcacttactt 1620tgtaaacttg taaaatgcaa caagccgccg
tgcgattagc caacaactca gcaaaaccca 1680gattctcata aggctcacta attccagaat
aaactattta tgtattgttt ggtctgtttt 1740cttgttgcat cactggtatg gtctgtctag
gtagaagaat atgatagggt gagggatttt 1800aggattgaag aatctttaaa ac
18225534PRTAmaranthus tuberculatus 5Met
Val Ile Gln Ser Ile Thr His Leu Ser Pro Asn Leu Ala Leu Pro1
5 10 15Ser Pro Leu Ser Val Ser Thr
Lys Asn Tyr Pro Val Ala Val Met Gly 20 25
30Asn Ile Ser Glu Arg Glu Glu Pro Thr Ser Ala Lys Arg Val
Ala Val 35 40 45Val Gly Ala Gly
Val Ser Gly Leu Ala Ala Ala Tyr Lys Leu Lys Ser 50 55
60His Gly Leu Ser Val Thr Leu Phe Glu Ala Asp Ser Arg
Ala Gly Gly65 70 75
80Lys Leu Lys Thr Val Lys Lys Asp Gly Phe Ile Trp Asp Glu Gly Ala
85 90 95Asn Thr Met Thr Glu Ser
Glu Ala Glu Val Ser Ser Leu Ile Asp Asp 100
105 110Leu Gly Leu Arg Glu Lys Gln Gln Leu Pro Ile Ser
Gln Asn Lys Arg 115 120 125Tyr Ile
Ala Arg Ala Gly Leu Pro Val Leu Leu Pro Ser Asn Pro Ala 130
135 140Ala Leu Leu Thr Ser Asn Ile Leu Ser Ala Lys
Ser Lys Leu Gln Ile145 150 155
160Met Leu Glu Pro Phe Leu Trp Arg Lys His Asn Ala Thr Glu Leu Ser
165 170 175Asp Glu His Val
Gln Glu Ser Val Gly Glu Phe Phe Glu Arg His Phe 180
185 190Gly Lys Glu Phe Val Asp Tyr Val Ile Asp Pro
Phe Val Ala Gly Thr 195 200 205Cys
Gly Gly Asp Pro Gln Ser Leu Ser Met His His Thr Phe Pro Glu 210
215 220Val Trp Asn Ile Glu Lys Arg Phe Gly Ser
Val Phe Ala Gly Leu Ile225 230 235
240Gln Ser Thr Leu Leu Ser Lys Lys Glu Lys Gly Gly Glu Asn Ala
Ser 245 250 255Ile Lys Lys
Pro Arg Val Arg Gly Ser Phe Ser Phe Gln Gly Gly Met 260
265 270Gln Thr Leu Val Asp Thr Met Cys Lys Gln
Leu Gly Glu Asp Glu Leu 275 280
285Lys Leu Gln Cys Glu Val Leu Ser Leu Ser Tyr Asn Gln Lys Gly Ile 290
295 300Pro Ser Leu Gly Asn Trp Ser Val
Ser Ser Met Ser Asn Asn Thr Ser305 310
315 320Glu Asp Gln Ser Tyr Asp Ala Val Val Val Thr Ala
Pro Ile Arg Asn 325 330
335Val Lys Glu Met Lys Ile Met Lys Phe Gly Asn Pro Phe Ser Leu Asp
340 345 350Phe Ile Pro Glu Val Thr
Tyr Val Pro Leu Ser Val Met Ile Thr Ala 355 360
365Phe Lys Lys Asp Lys Val Lys Arg Pro Leu Glu Gly Phe Gly
Val Leu 370 375 380Ile Pro Ser Lys Glu
Gln His Asn Gly Leu Lys Thr Leu Gly Thr Leu385 390
395 400Phe Ser Ser Met Met Phe Pro Asp Arg Ala
Pro Ser Asp Met Cys Leu 405 410
415Phe Thr Thr Phe Val Gly Gly Ser Arg Asn Arg Lys Leu Ala Asn Ala
420 425 430Ser Thr Asp Glu Leu
Lys Gln Ile Val Ser Ser Asp Leu Gln Gln Leu 435
440 445Leu Gly Thr Glu Asp Glu Pro Ser Phe Val Asn His
Leu Phe Trp Ser 450 455 460Asn Ala Phe
Pro Leu Tyr Gly His Asn Tyr Asp Ser Val Leu Arg Ala465
470 475 480Ile Asp Lys Met Glu Lys Asp
Leu Pro Gly Phe Phe Tyr Ala Gly Asn 485
490 495His Lys Gly Gly Leu Ser Val Gly Lys Ala Met Ala
Ser Gly Cys Lys 500 505 510Ala
Ala Glu Leu Val Ile Ser Tyr Leu Asp Ser His Ile Tyr Val Lys 515
520 525Met Asp Glu Lys Thr Ala
53061605DNAAmaranthus tuberculatus 6atggtaattc aatccattac ccacctttca
ccaaaccttg cattgccatc gccattgtca 60gtttcaacca agaactaccc agtagctgta
atgggcaaca tttctgagcg ggaagaaccc 120acttctgcta aaagggttgc tgttgttggt
gctggagtta gtggacttgc tgctgcatat 180aagctaaaat cccatggttt gagtgtgaca
ttgtttgaag ctgattctag agctggaggc 240aaacttaaaa ctgttaaaaa agatggtttt
atttgggatg agggggcaaa tactatgaca 300gaaagtgagg cagaggtctc gagtttgatc
gatgatcttg ggcttcgtga gaagcaacag 360ttgccaattt cacaaaataa aagatacata
gctagagccg gtcttcctgt gctactacct 420tcaaatcccg ctgcactact cacgagcaat
atcctttcag caaaatcaaa gctgcaaatt 480atgttggaac catttctctg gagaaaacac
aatgctactg aactttctga tgagcatgtt 540caggaaagcg ttggtgaatt ttttgagcga
cattttggga aagagtttgt tgattatgtt 600attgaccctt ttgttgcggg tacatgtggt
ggagatcctc aatcgctttc catgcaccat 660acatttccag aagtatggaa tattgaaaaa
aggtttggct ctgtgtttgc cggactaatt 720caatcaacat tgttatctaa gaaggaaaag
ggtggagaaa atgcttctat taagaagcct 780cgtgtacgtg gttcattttc atttcaaggt
ggaatgcaga cacttgttga cacaatgtgc 840aaacagcttg gtgaagatga actcaaactc
cagtgtgagg tgctgtcctt gtcatataac 900cagaagggga tcccctcact agggaattgg
tcagtctctt ctatgtcaaa taataccagt 960gaagatcaat cttatgatgc tgtggttgtc
actgctccaa ttcgcaatgt caaagaaatg 1020aagattatga aatttggaaa tccattttca
cttgacttta ttccagaggt gacgtacgta 1080cccctttccg ttatgattac tgcattcaaa
aaggataaag tgaagagacc tcttgagggc 1140ttcggagttc ttatcccctc taaagagcaa
cataatggac tgaagactct tggtacttta 1200ttttcctcca tgatgtttcc tgatcgtgct
ccatctgaca tgtgtctctt tactacattt 1260gtcggaggaa gcagaaatag aaaacttgca
aacgcttcaa cggatgaatt gaagcaaata 1320gtttcttctg accttcagca gctgttgggc
actgaggacg aaccttcatt tgtcaatcat 1380ctcttttgga gcaacgcatt cccattgtat
ggacacaatt acgattctgt tttgagagcc 1440atagacaaga tggaaaagga tcttcctgga
tttttttatg caggtaacca taagggtgga 1500ctttcagtgg gaaaagcgat ggcctccgga
tgcaaggctg cggaacttgt aatatcctat 1560ctggactctc atatatacgt gaagatggat
gagaagaccg cgtaa 16057557PRTSolanum tuberosum 7Met Thr
Thr Thr Ala Val Ala Asn His Pro Ser Ile Phe Thr His Arg1 5
10 15Ser Pro Leu Pro Ser Pro Ser Ser
Ser Ser Ser Ser Pro Ser Phe Leu 20 25
30Phe Leu Asn Arg Thr Asn Phe Ile Pro Tyr Phe Ser Thr Ser Lys
Arg 35 40 45Asn Ser Val Asn Cys
Asn Gly Trp Arg Thr Arg Cys Ser Val Ala Lys 50 55
60Asp Tyr Thr Val Pro Pro Ser Glu Val Asp Gly Asn Gln Phe
Pro Glu65 70 75 80Leu
Asp Cys Val Val Val Gly Ala Gly Ile Ser Gly Leu Cys Ile Ala
85 90 95Lys Val Ile Ser Ala Asn Tyr
Pro Asn Leu Met Val Thr Glu Ala Arg 100 105
110Asp Arg Ala Gly Gly Asn Ile Thr Thr Val Glu Arg Asp Gly
Tyr Leu 115 120 125Trp Glu Glu Gly
Pro Asn Ser Phe Gln Pro Ser Asp Pro Met Leu Thr 130
135 140Met Ala Val Asp Cys Gly Leu Lys Asp Asp Leu Val
Leu Gly Asp Pro145 150 155
160Asp Ala Pro Arg Phe Val Leu Trp Lys Asp Lys Leu Arg Pro Val Pro
165 170 175Gly Lys Leu Thr Asp
Leu Pro Phe Phe Asp Leu Met Ser Ile Pro Gly 180
185 190Lys Leu Arg Ala Gly Phe Gly Ala Ile Gly Leu Arg
Pro Ser Pro Pro 195 200 205Gly Tyr
Glu Glu Ser Val Glu Gln Phe Val Arg Arg Asn Leu Gly Ala 210
215 220Glu Val Phe Glu Arg Leu Ile Glu Pro Phe Cys
Ser Gly Val Tyr Ala225 230 235
240Gly Asp Pro Ser Lys Leu Ile Met Lys Ala Ala Phe Gly Lys Val Trp
245 250 255Lys Leu Glu Gln
Thr Gly Gly Ser Ile Ile Gly Gly Thr Phe Lys Ala 260
265 270Ile Lys Glu Arg Ser Ser Asn Pro Lys Pro Pro
Arg Asp Pro Arg Leu 275 280 285Pro
Thr Pro Lys Gly Gln Thr Val Gly Ser Phe Arg Lys Gly Leu Arg 290
295 300Met Leu Pro Asp Ala Ile Cys Glu Arg Leu
Gly Ser Lys Val Lys Leu305 310 315
320Ser Trp Lys Leu Ser Ser Ile Thr Lys Ser Glu Lys Gly Gly Tyr
Leu 325 330 335Leu Thr Tyr
Glu Thr Pro Glu Gly Val Val Ser Leu Arg Ser Arg Ser 340
345 350Ile Val Met Thr Val Pro Ser Tyr Val Ala
Ser Asn Ile Leu Arg Pro 355 360
365Leu Ser Val Ala Ala Ala Asp Ala Leu Ser Ser Phe Tyr Tyr Pro Pro 370
375 380Val Ala Ala Val Thr Ile Ser Tyr
Pro Gln Glu Ala Ile Arg Asp Glu385 390
395 400Arg Leu Val Asp Gly Glu Leu Lys Gly Phe Gly Gln
Leu His Pro Arg 405 410
415Ser Gln Gly Val Glu Thr Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe
420 425 430Pro Asn Arg Ala Pro Asn
Gly Arg Val Leu Leu Leu Asn Tyr Ile Gly 435 440
445Gly Ala Thr Asn Thr Glu Ile Val Ser Lys Thr Glu Ser Gln
Leu Val 450 455 460Glu Ala Val Asp Arg
Asp Leu Arg Lys Met Leu Ile Lys Pro Lys Ala465 470
475 480Gln Asp Pro Phe Val Thr Gly Val Arg Val
Trp Pro Gln Ala Ile Pro 485 490
495Gln Phe Leu Val Gly His Leu Asp Thr Leu Gly Thr Ala Lys Thr Ala
500 505 510Leu Ser Asp Asn Gly
Leu Asp Gly Leu Phe Leu Gly Gly Asn Tyr Val 515
520 525Ser Gly Val Ala Leu Gly Arg Cys Val Glu Gly Ala
Tyr Glu Ile Ala 530 535 540Ser Glu Val
Thr Gly Phe Leu Ser Gln Tyr Ala Tyr Lys545 550
55581674DNASolanum tuberosum 8atgacaacaa cggccgtcgc caaccatcct
agcattttca ctcaccggtc gccgctgccg 60tcgccgtcgt cctcctcctc atcgccgtca
tttttatttt taaaccgtac gaatttcatt 120ccttactttt ccacctccaa gcgcaatagt
gtcaattgca atggctggag aacacgatgt 180tccgttgcca aggattatac agttcctccc
tcggaagtcg acggtaatca gttcccggag 240ctggattgtg tggtagttgg agcaggaatt
agtggactct gcattgctaa ggtgatttcg 300gctaattatc ccaatttgat ggtgacggag
gcgagggatc gtgccggtgg aaacataacg 360acggtggaaa gagatggata cttatgggaa
gaaggtccta acagtttcca gccttcggat 420cctatgttga caatggctgt agattgtgga
ttgaaggatg atttggtgtt gggagatcct 480gatgcgcctc gctttgtctt gtggaaggat
aaactaaggc ctgttcccgg caagctcact 540gatcttccct tctttgattt gatgagtatc
cctggcaagc tcagagctgg ttttggtgcc 600attggccttc gcccttcacc tccaggttat
gaggaatcag ttgagcagtt cgtgcgtcgt 660aatcttggtg cagaagtctt tgaacgtttg
attgaaccat tttgttctgg tgtttacgcc 720ggtgacccct caaaattgat tatgaaagca
gcatttggga aagtgtggaa gctagaacaa 780actggtggta gcattattgg gggaaccttt
aaagcaatta aggagagatc cagtaaccct 840aaaccgcctc gtgatccgcg tttaccaaca
ccaaaaggac aaactgttgg atcatttagg 900aagggtctga gaatgctgcc ggatgcaatt
tgtgaaagac tgggaagcaa agtaaaacta 960tcatggaagc tttctagcat tacaaagtca
gaaaaaggag gatatctctt gacatacgag 1020acaccagaag gagtagtttc tctgcgaagt
cgaagcattg tcatgactgt tccatcctat 1080gtagcaagca acatattacg ccctctttcg
gtcgctgcag cagatgcact ttcaagtttc 1140tactatcccc cagtagcagc agtgacaatt
tcatatcctc aagaggctat tcgtgatgag 1200cgtctggttg atggtgaact aaagggattt
gggcagttgc atccacgttc acagggagtg 1260gaaacactag gaacaatata tagttcatca
ctctttccta accgtgctcc aaatggccgg 1320gtgctactct tgaactacat tggaggagca
acaaatactg aaattgtgtc taagacggag 1380agccaacttg tggaagcagt tgaccgtgac
ctcagaaaaa tgcttataaa acccaaagca 1440caagatccct ttgttacggg tgtgcgagta
tggccacaag ctatcccaca gtttttggtc 1500ggacatctgg atacactagg tactgcaaaa
actgctctaa gtgataatgg gcttgacggg 1560ctattccttg ggggtaatta tgtgtctggt
gtagcattgg gaaggtgtgt tgaaggtgct 1620tatgaaatag catctgaggt aactggattt
ctgtctcagt atgcatacaa atga 16749504PRTSolanum tuberosum 9Met Ala
Pro Ser Ala Gly Glu Asp Lys Gln Asn Cys Pro Lys Arg Val1 5
10 15Ala Val Ile Gly Ala Gly Val Ser
Gly Leu Ala Ala Ala Tyr Lys Leu 20 25
30Lys Ile His Gly Leu Asp Val Thr Val Phe Glu Ala Glu Gly Arg
Ala 35 40 45Gly Gly Lys Leu Arg
Ser Leu Ser Gln Asp Gly Leu Ile Trp Asp Glu 50 55
60Gly Ala Asn Thr Met Thr Glu Ser Glu Gly Asp Val Thr Phe
Leu Leu65 70 75 80Asp
Ser Leu Gly Leu Arg Glu Lys Gln Gln Phe Pro Leu Ser Gln Asn
85 90 95Lys Arg Tyr Ile Ala Arg Asn
Gly Thr Pro Thr Leu Ile Pro Ser Asn 100 105
110Pro Ile Asp Leu Ile Lys Ser Asn Phe Leu Ser Thr Gly Ser
Lys Leu 115 120 125Gln Met Leu Phe
Glu Pro Leu Leu Trp Lys Asn Lys Lys Leu Thr Lys 130
135 140Val Ser Asp Glu His Glu Ser Val Ser Gly Phe Phe
Gln Arg His Phe145 150 155
160Gly Lys Glu Val Val Asp Tyr Leu Ile Asp Pro Phe Val Ala Gly Thr
165 170 175Cys Gly Gly Asp Pro
Asp Ser Leu Ser Met His Leu Ser Phe Pro Glu 180
185 190Leu Trp Asn Leu Glu Lys Arg Phe Gly Ser Val Ile
Val Gly Ala Ile 195 200 205Arg Ser
Lys Leu Ser Pro Ile Lys Glu Lys Lys Gln Gly Pro Pro Lys 210
215 220Thr Ser Val Asn Lys Lys His Gln Arg Gly Ser
Phe Ser Phe Leu Gly225 230 235
240Gly Met Gln Thr Leu Thr Asp Ala Ile Cys Asn Asp Leu Lys Glu Asp
245 250 255Glu Leu Arg Leu
Asn Ser Arg Val Leu Glu Leu Ser Cys Ser Cys Ser 260
265 270Gly Asp Ser Ala Thr Asp Ser Trp Ser Ile Phe
Ser Ala Ser Pro His 275 280 285Lys
Arg Gln Ala Glu Glu Asp Ser Phe Asp Ala Val Ile Met Thr Ala 290
295 300Pro Leu Cys Asp Val Lys Gly Met Lys Ile
Ala Lys Arg Gly Asn Pro305 310 315
320Phe Leu Leu Asn Phe Ile Pro Glu Val Asp Tyr Val Pro Leu Ser
Val 325 330 335Val Ile Thr
Thr Phe Lys Lys Glu Ser Val Lys His Pro Leu Glu Gly 340
345 350Phe Gly Val Leu Val Pro Ser Glu Glu Gln
Lys His Gly Leu Lys Thr 355 360
365Leu Gly Thr Leu Phe Ser Ser Met Met Phe Pro Asp Arg Ala Pro Asn 370
375 380Asn Val Tyr Leu Tyr Thr Thr Phe
Val Gly Gly Ser Arg Asn Arg Glu385 390
395 400Leu Ala Lys Ala Ser Arg Thr Glu Leu Lys Glu Ile
Val Thr Ser Asp 405 410
415Leu Lys Gln Leu Leu Gly Ala Glu Gly Glu Pro Thr Tyr Val Asn His
420 425 430Val Cys Trp Ser Lys Ala
Phe Pro Leu Tyr Gly His Asn Tyr Asp Ser 435 440
445Val Leu Asp Ala Ile Asp Lys Met Glu Lys Asn Leu Pro Gly
Leu Phe 450 455 460Tyr Ala Gly Asn His
Lys Gly Gly Leu Ser Val Gly Lys Ala Leu Ser465 470
475 480Ser Gly Cys Asn Ala Ala Asp Leu Val Ile
Ser Tyr Leu Glu Ala Val 485 490
495Ser Thr Asp Thr Lys Asn His Arg 500101608DNASolanum
tuberosum 10atggctccat ctgccggaga agataaacaa aattgtccca agagagttgc
agtcattggt 60gctggcgtca gtggacttgc tgcagcatac aagttgaaaa ttcatggctt
ggatgtcaca 120gtattcgaag cagaagggag agctggaggg aagttacgaa gcctgagtca
agatggccta 180atatgggatg aaggcgcaaa tactatgact gaaagtgaag gtgatgtcac
atttttgctt 240gattcgcttg gactccgaga aaaacaacaa tttccacttt cacagaacaa
gcgctacatt 300gccagaaatg gtactcctac tctgatacct tcaaatccaa ttgacctgat
caaaagcaac 360tttctttcca ctggatcaaa gcttcagatg cttttcgagc ctcttttgtg
gaagaataaa 420aagcttacaa aggtgtctga cgaacacgaa agtgtcagtg gattcttcca
gcgtcatttt 480ggaaaggagg ttgttgacta tctaattgat ccttttgttg ctggaacgtg
tggtggtgat 540cctgactcgc tttcaatgca cctttcgttt ccagagttgt ggaatttaga
gaaaaggttt 600ggctcagtca tagttggggc aattcgatcc aagttatcac ctataaagga
aaagaaacaa 660ggaccaccca aaacttcagt aaataagaag caccagcggg ggtccttttc
atttttgggc 720ggaatgcaaa cacttactga cgcaatatgc aatgatctca aagaagatga
acttaggcta 780aactctagag ttctggaatt atcttgtagc tgtagtgggg actctgcgac
agatagctgg 840tcaatttttt ctgcctcacc acacaagcgg caagcagaag aagattcatt
tgatgctgta 900attatgacgg cccctctctg tgacgttaag ggtatgaaga ttgctaagag
aggaaatcca 960tttctgctca actttattcc tgaggttgat tatgtaccac tatctgttgt
tataaccaca 1020tttaagaagg agagtgtaaa gcatcctctt gagggttttg gagtgcttgt
accttccgag 1080gagcaaaaac atggtctgaa gacattaggc accctcttct cttctatgat
gtttccagat 1140cgtgcaccca acaatgtcta tctctatact acatttgttg gtggaagccg
aaatagagaa 1200ctcgcgaaag cctcgaggac tgagctgaaa gagatagtaa cttctgacct
taagcagttg 1260ttgggtgctg agggagagcc aacatatgtg aatcatgtat gctggagtaa
agcatttccg 1320ttgtacgggc ataactatga ttcagtcctc gatgcaattg acaaaatgga
gaaaaatctt 1380cctggattat tctatgcagg taaccacaag ggaggattgt cagttggcaa
agcactatct 1440tctggatgta atgcagcaga tcttgttata tcatatcttg aagccgtttc
aacggacacc 1500aaaaaccata ggtgaaatct attctctcat gcagcttgcc gttctttgtt
ccacaaaatc 1560gtttaacttc atgacgagga gcaactttaa cgtgcagcca gtgacgca
160811535PRTZea mays 11Met Val Ala Ala Thr Ala Thr Ala Met Ala
Thr Ala Ala Ser Pro Leu1 5 10
15Leu Asn Gly Thr Arg Ile Pro Ala Arg Leu Arg His Arg Gly Leu Ser
20 25 30Val Arg Cys Ala Ala Val
Ala Gly Gly Ala Ala Glu Ala Pro Ala Ser 35 40
45Thr Gly Ala Arg Leu Ser Ala Asp Cys Val Val Val Gly Gly
Gly Ile 50 55 60Ser Gly Leu Cys Thr
Ala Gln Ala Leu Ala Thr Arg His Gly Val Gly65 70
75 80Asp Val Leu Val Thr Glu Ala Arg Ala Arg
Pro Gly Gly Asn Ile Thr 85 90
95Thr Val Glu Arg Pro Glu Glu Gly Tyr Leu Trp Glu Glu Gly Pro Asn
100 105 110Ser Phe Gln Pro Ser
Asp Pro Val Leu Thr Met Ala Val Asp Ser Gly 115
120 125Leu Lys Asp Asp Leu Val Phe Gly Asp Pro Asn Ala
Pro Arg Phe Val 130 135 140Leu Trp Glu
Gly Lys Leu Arg Pro Val Pro Ser Lys Pro Ala Asp Leu145
150 155 160Pro Phe Phe Asp Leu Met Ser
Ile Pro Gly Lys Leu Arg Ala Gly Leu 165
170 175Gly Ala Leu Gly Ile Arg Pro Pro Pro Pro Gly Arg
Glu Glu Ser Val 180 185 190Glu
Glu Phe Val Arg Arg Asn Leu Gly Ala Glu Val Phe Glu Arg Leu 195
200 205Ile Glu Pro Phe Cys Ser Gly Val Tyr
Ala Gly Asp Pro Ser Lys Leu 210 215
220Ser Met Lys Ala Ala Phe Gly Lys Val Trp Arg Leu Glu Glu Thr Gly225
230 235 240Gly Ser Ile Ile
Gly Gly Thr Ile Lys Thr Ile Gln Glu Arg Ser Lys 245
250 255Asn Pro Lys Pro Pro Arg Asp Ala Arg Leu
Pro Lys Pro Lys Gly Gln 260 265
270Thr Val Ala Ser Phe Arg Lys Gly Leu Ala Met Leu Pro Asn Ala Ile
275 280 285Thr Ser Ser Leu Gly Ser Lys
Val Lys Leu Ser Trp Lys Leu Thr Ser 290 295
300Ile Thr Lys Ser Asp Asp Lys Gly Tyr Val Leu Glu Tyr Glu Thr
Pro305 310 315 320Glu Gly
Val Val Ser Val Gln Ala Lys Ser Val Ile Met Thr Ile Pro
325 330 335Ser Tyr Val Ala Ser Asn Ile
Leu Arg Pro Leu Ser Ser Asp Ala Ala 340 345
350Asp Ala Leu Ser Arg Phe Tyr Tyr Pro Pro Val Ala Ala Val
Thr Val 355 360 365Ser Tyr Pro Lys
Glu Ala Ile Arg Lys Glu Cys Leu Ile Asp Gly Glu 370
375 380Leu Gln Gly Phe Gly Gln Leu His Pro Arg Ser Gln
Gly Val Glu Thr385 390 395
400Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala Pro Asp
405 410 415Gly Arg Val Leu Leu
Leu Asn Tyr Ile Gly Gly Ala Thr Asn Thr Gly 420
425 430Ile Val Ser Lys Thr Glu Ser Glu Leu Val Glu Ala
Val Asp Arg Asp 435 440 445Leu Arg
Lys Met Leu Ile Asn Ser Thr Ala Val Asp Pro Leu Val Leu 450
455 460Gly Val Arg Val Trp Pro Gln Ala Ile Pro Gln
Phe Leu Val Gly His465 470 475
480Leu Asp Leu Leu Glu Ala Ala Lys Ala Ala Leu Asp Arg Gly Gly Tyr
485 490 495Asp Gly Leu Phe
Leu Gly Gly Asn Tyr Val Ala Gly Val Ala Leu Gly 500
505 510Arg Cys Val Glu Gly Ala Tyr Glu Ser Ala Ser
Gln Ile Ser Asp Phe 515 520 525Leu
Thr Lys Tyr Ala Tyr Lys 530 535121608DNAZea mays
12atggtcgccg ccacagccac cgccatggcc accgctgcat cgccgctact caacgggacc
60cgaatacctg cgcggctccg ccatcgagga ctcagcgtgc gctgcgctgc tgtggcgggc
120ggcgcggccg aggcaccggc atccaccggc gcgcggctgt ccgcggactg cgtcgtggtg
180ggcggaggca tcagtggcct ctgcaccgcg caggcgctgg ccacgcggca cggcgtcggg
240gacgtgcttg tcacggaggc ccgcgcccgc cccggcggca acattaccac cgtcgagcgc
300cccgaggaag ggtacctctg ggaggagggt cccaacagct tccagccctc cgaccccgtt
360ctcaccatgg ccgtggacag cggactgaag gatgacttgg tttttgggga cccaaacgcg
420ccgcgtttcg tgctgtggga ggggaagctg aggcccgtgc catccaagcc cgccgacctc
480ccgttcttcg atctcatgag catcccaggg aagctcaggg ccggtctagg cgcgcttggc
540atccgcccgc ctcctccagg ccgcgaagag tcagtggagg agttcgtgcg ccgcaacctc
600ggtgctgagg tctttgagcg cctcattgag cctttctgct caggtgtcta tgctggtgat
660ccttctaagc tcagcatgaa ggctgcattt gggaaggttt ggcggttgga agaaactgga
720ggtagtatta ttggtggaac catcaagaca attcaggaga ggagcaagaa tccaaaacca
780ccgagggatg cccgccttcc gaagccaaaa gggcagacag ttgcatcttt caggaagggt
840cttgccatgc ttccaaatgc cattacatcc agcttgggta gtaaagtcaa actatcatgg
900aaactcacga gcattacaaa atcagatgac aagggatatg ttttggagta tgaaacgcca
960gaaggggttg tttcggtgca ggctaaaagt gttatcatga ctattccatc atatgttgct
1020agcaacattt tgcgtccact ttcaagcgat gctgcagatg ctctatcaag attctattat
1080ccaccggttg ctgctgtaac tgtttcgtat ccaaaggaag caattagaaa agaatgctta
1140attgatgggg aactccaggg ctttggccag ttgcatccac gtagtcaagg agttgagaca
1200ttaggaacaa tatacagttc ctcactcttt ccaaatcgtg ctcctgacgg tagggtgtta
1260cttctaaact acataggagg tgctacaaac acaggaattg tttccaagac tgaaagtgag
1320ctggtcgaag cagttgaccg tgacctccga aaaatgctta taaattctac agcagtggac
1380cctttagtcc ttggtgttcg agtttggcca caagccatac ctcagttcct ggtaggacat
1440cttgatcttc tggaagccgc aaaagctgcc ctggaccgag gtggctacga tgggctgttc
1500ctaggaggga actatgttgc aggagttgcc ctgggcagat gcgttgaggg cgcgtatgaa
1560agtgcctcgc aaatatctga cttcttgacc aagtatgcct acaagtga
160813544PRTZea mays 13Met Leu Ala Leu Thr Ala Ser Ala Ser Ser Ala Ser
Ser His Pro Tyr1 5 10
15Arg His Ala Ser Ala His Thr Arg Arg Pro Arg Leu Arg Ala Val Leu
20 25 30Ala Met Ala Gly Ser Asp Asp
Pro Arg Ala Ala Pro Ala Arg Ser Val 35 40
45Ala Val Val Gly Ala Gly Val Ser Gly Leu Ala Ala Ala Tyr Arg
Leu 50 55 60Arg Gln Ser Gly Val Asn
Val Thr Val Phe Glu Ala Ala Asp Arg Ala65 70
75 80Gly Gly Lys Ile Arg Thr Asn Ser Glu Gly Gly
Phe Val Trp Asp Glu 85 90
95Gly Ala Asn Thr Met Thr Glu Gly Glu Trp Glu Ala Ser Arg Leu Ile
100 105 110Asp Asp Leu Gly Leu Gln
Asp Lys Gln Gln Tyr Pro Asn Ser Gln His 115 120
125Lys Arg Tyr Ile Val Lys Asp Gly Ala Pro Ala Leu Ile Pro
Ser Asp 130 135 140Pro Ile Ser Leu Met
Lys Ser Ser Val Leu Ser Thr Lys Ser Lys Ile145 150
155 160Ala Leu Phe Phe Glu Pro Phe Leu Tyr Lys
Lys Ala Asn Thr Arg Asn 165 170
175Ser Gly Lys Val Ser Glu Glu His Leu Ser Glu Ser Val Gly Ser Phe
180 185 190Cys Glu Arg His Phe
Gly Arg Glu Val Val Asp Tyr Phe Val Asp Pro 195
200 205Phe Val Ala Gly Thr Ser Ala Gly Asp Pro Glu Ser
Leu Ser Ile Arg 210 215 220His Ala Phe
Pro Ala Leu Trp Asn Leu Glu Arg Lys Tyr Gly Ser Val225
230 235 240Ile Val Gly Ala Ile Leu Ser
Lys Leu Ala Ala Lys Gly Asp Pro Val 245
250 255Lys Thr Arg His Asp Ser Ser Gly Lys Arg Arg Asn
Arg Arg Val Ser 260 265 270Phe
Ser Phe His Gly Gly Met Gln Ser Leu Ile Asn Ala Leu His Asn 275
280 285Glu Val Gly Asp Asp Asn Val Lys Leu
Gly Thr Glu Val Leu Ser Leu 290 295
300Ala Cys Thr Phe Asp Gly Val Pro Ala Leu Gly Arg Trp Ser Ile Ser305
310 315 320Val Asp Ser Lys
Asp Ser Gly Asp Lys Asp Leu Ala Ser Asn Gln Thr 325
330 335Phe Asp Ala Val Ile Met Thr Ala Pro Leu
Ser Asn Val Arg Arg Met 340 345
350Lys Phe Thr Lys Gly Gly Ala Pro Val Val Leu Asp Phe Leu Pro Lys
355 360 365Met Asp Tyr Leu Pro Leu Ser
Leu Met Val Thr Ala Phe Lys Lys Asp 370 375
380Asp Val Lys Lys Pro Leu Glu Gly Phe Gly Val Leu Ile Pro Tyr
Lys385 390 395 400Glu Gln
Gln Lys His Gly Leu Lys Thr Leu Gly Thr Leu Phe Ser Ser
405 410 415Met Met Phe Pro Asp Arg Ala
Pro Asp Asp Gln Tyr Leu Tyr Thr Thr 420 425
430Phe Val Gly Gly Ser His Asn Arg Asp Leu Ala Gly Ala Pro
Thr Ser 435 440 445Ile Leu Lys Gln
Leu Val Thr Ser Asp Leu Lys Lys Leu Leu Gly Val 450
455 460Glu Gly Gln Pro Thr Phe Val Lys His Val Tyr Trp
Gly Asn Ala Phe465 470 475
480Pro Leu Tyr Gly His Asp Tyr Ser Ser Val Leu Glu Ala Ile Glu Lys
485 490 495Met Glu Lys Asn Leu
Pro Gly Phe Phe Tyr Ala Gly Asn Ser Lys Asp 500
505 510Gly Leu Ala Val Gly Ser Val Ile Ala Ser Gly Ser
Lys Ala Ala Asp 515 520 525Leu Ala
Ile Ser Tyr Leu Glu Ser His Thr Lys His Asn Asn Ser His 530
535 540142042DNAZea mays 14ctctcctacc tccacctcca
cgacaacaag caaatcccca tccagttcca aaccctaact 60caaatgctcg ctttgactgc
ctcagcctca tccgcttcgt cccatcctta tcgccacgcc 120tccgcgcaca ctcgtcgccc
ccgcctacgt gcggtcctcg cgatggcggg ctccgacgac 180ccccgtgcag cgcccgccag
atcggtcgcc gtcgtcggcg ccggggtcag cgggctcgcg 240gcggcgtaca ggctcagaca
gagcggcgtg aacgtaacgg tgttcgaagc ggccgacagg 300gcgggaggaa agatacggac
caattccgag ggcgggtttg tctgggatga aggagctaac 360accatgacag aaggtgaatg
ggaggccagt agactgattg atgatcttgg tctacaagac 420aaacagcagt atcctaactc
ccaacacaag cgttacattg tcaaagatgg agcaccagca 480ctgattcctt cggatcccat
ttcgctaatg aaaagcagtg ttctttcgac aaaatcaaag 540attgcgttat tttttgaacc
atttctctac aagaaagcta acacaagaaa ctctggaaaa 600gtgtctgagg agcacttgag
tgagagtgtt gggagcttct gtgaacgcca ctttggaaga 660gaagttgttg actattttgt
tgatccattt gtagctggaa caagtgcagg agatccagag 720tcactatcta ttcgtcatgc
attcccagca ttgtggaatt tggaaagaaa gtatggttca 780gttattgttg gtgccatctt
gtctaagcta gcagctaaag gtgatccagt aaagacaaga 840catgattcat cagggaaaag
aaggaataga cgagtgtcgt tttcatttca tggtggaatg 900cagtcactaa taaatgcact
tcacaatgaa gttggagatg ataatgtgaa gcttggtaca 960gaagtgttgt cattggcatg
tacatttgat ggagttcctg cactaggcag gtggtcaatt 1020tctgttgatt cgaaggatag
cggtgacaag gaccttgcta gtaaccaaac ctttgatgct 1080gttataatga cagctccatt
gtcaaatgtc cggaggatga agttcaccaa aggtggagct 1140ccggttgttc ttgactttct
tcctaagatg gattatctac cactatctct catggtgact 1200gcttttaaga aggatgatgt
caagaaacct ctggaaggat ttggggtctt aataccttac 1260aaggaacagc aaaaacatgg
tctgaaaacc cttgggactc tcttttcctc aatgatgttc 1320ccagatcgag ctcctgatga
ccaatattta tatacaacat ttgttggggg tagccacaat 1380agagatcttg ctggagctcc
aacgtctatt ctgaaacaac ttgtgacctc tgaccttaaa 1440aaactcttgg gcgtagaggg
gcaaccaact tttgtcaagc atgtatactg gggaaatgct 1500tttcctttgt atggccatga
ttatagttct gtattggaag ctatagaaaa gatggagaaa 1560aaccttccag ggttcttcta
cgcaggaaat agcaaggatg ggcttgctgt tggaagtgtt 1620atagcttcag gaagcaaggc
tgctgacctt gcaatctcat atcttgaatc tcacaccaag 1680cataataatt cacattgaaa
gtgtctgacc tatcctctag cagttgtcga caaatttctc 1740cagttcatgt acagtagaaa
ccgatgcgtt gcagtttcag aacatcttca cttcttcaga 1800tattaaccct tcgttgaaca
tccaccagaa aggtagtcac atgtgtaagt gggaaaatga 1860ggttaaaaac tattatggcg
gccgaaatgt tcctttttgt tttcctcaca agtggcctac 1920gacacttgat gttggaaata
catttaaatt tgttgaattg tttgagaaca catgcgtgac 1980gtgtaatatt tgcctattgt
gattttagca gtagtcttgg ccagattatg ctttacgcct 2040tt
204215536PRTOryza sativa
15Met Ala Ala Ala Ala Ala Ala Met Ala Thr Ala Thr Ser Ala Thr Ala1
5 10 15Ala Pro Pro Leu Arg Ile
Arg Asp Ala Ala Arg Arg Thr Arg Arg Arg 20 25
30Gly His Val Arg Cys Ala Val Ala Ser Gly Ala Ala Glu
Ala Pro Ala 35 40 45Ala Pro Gly
Ala Arg Val Ser Ala Asp Cys Val Val Val Gly Gly Gly 50
55 60Ile Ser Gly Leu Cys Thr Ala Gln Ala Leu Ala Thr
Lys His Gly Val65 70 75
80Gly Asp Val Leu Val Thr Glu Ala Arg Ala Arg Pro Gly Gly Asn Ile
85 90 95Thr Thr Ala Glu Arg Ala
Gly Glu Gly Tyr Leu Trp Glu Glu Gly Pro 100
105 110Asn Ser Phe Gln Pro Ser Asp Pro Val Leu Thr Met
Ala Val Asp Ser 115 120 125Gly Leu
Lys Asp Asp Leu Val Phe Gly Asp Pro Asn Ala Pro Arg Phe 130
135 140Val Leu Trp Glu Gly Lys Leu Arg Pro Val Pro
Ser Lys Pro Gly Asp145 150 155
160Leu Pro Phe Phe Asp Leu Met Ser Ile Pro Gly Lys Leu Arg Ala Gly
165 170 175Leu Gly Ala Leu
Gly Val Arg Ala Pro Pro Pro Gly Arg Glu Glu Ser 180
185 190Val Glu Asp Phe Val Arg Arg Asn Leu Gly Ala
Glu Val Phe Glu Arg 195 200 205Leu
Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala Gly Asp Pro Ser Lys 210
215 220Leu Ser Met Lys Ala Ala Phe Gly Lys Val
Trp Arg Leu Glu Asp Thr225 230 235
240Gly Gly Ser Ile Ile Gly Gly Thr Ile Lys Thr Ile Gln Glu Arg
Gly 245 250 255Lys Asn Pro
Lys Pro Pro Arg Asp Pro Arg Leu Pro Thr Pro Lys Gly 260
265 270Gln Thr Val Ala Ser Phe Arg Lys Gly Leu
Thr Met Leu Pro Asp Ala 275 280
285Ile Thr Ser Arg Leu Gly Ser Lys Val Lys Leu Ser Trp Lys Leu Thr 290
295 300Ser Ile Thr Lys Ser Asp Asn Lys
Gly Tyr Ala Leu Val Tyr Glu Thr305 310
315 320Pro Glu Gly Val Val Ser Val Gln Ala Lys Thr Val
Val Met Thr Ile 325 330
335Pro Ser Tyr Val Ala Ser Asp Ile Leu Arg Pro Leu Ser Ser Asp Ala
340 345 350Ala Asp Ala Leu Ser Ile
Phe Tyr Tyr Pro Pro Val Ala Ala Val Thr 355 360
365Val Ser Tyr Pro Lys Glu Ala Ile Arg Lys Glu Cys Leu Ile
Asp Gly 370 375 380Glu Leu Gln Gly Phe
Gly Gln Leu His Pro Arg Ser Gln Gly Val Glu385 390
395 400Thr Leu Gly Thr Ile Tyr Ser Ser Ser Leu
Phe Pro Asn Arg Ala Pro 405 410
415Ala Gly Arg Val Leu Leu Leu Asn Tyr Ile Gly Gly Ser Thr Asn Thr
420 425 430Gly Ile Val Ser Lys
Thr Glu Ser Glu Leu Val Glu Ala Val Asp Arg 435
440 445Asp Leu Arg Lys Met Leu Ile Asn Pro Lys Ala Val
Asp Pro Leu Val 450 455 460Leu Gly Val
Arg Val Trp Pro Gln Ala Ile Pro Gln Phe Leu Ile Gly465
470 475 480His Leu Asp His Leu Glu Ala
Ala Lys Ser Ala Leu Gly Lys Gly Gly 485
490 495Tyr Asp Gly Leu Phe Leu Gly Gly Asn Tyr Val Ala
Gly Val Ala Leu 500 505 510Gly
Arg Cys Val Glu Gly Ala Tyr Glu Ser Ala Ser Gln Ile Ser Asp 515
520 525Tyr Leu Thr Lys Tyr Ala Tyr Lys
530 535161907DNAOryza sativa 16atccactcct ctccagtctc
cccgccgctc cgcatcccgc agccgctcgt cagcgacgga 60catggccgcc gccgccgcag
ccatggccac cgccacctcc gccacggcag cgccgccgct 120ccgcattcgc gacgccgcga
ggaggacccg ccgacgcggc cacgttcgct gcgccgtcgc 180cagcggcgcg gccgaggcgc
ccgcggcgcc cggggcgcgg gtgtcggcgg actgcgtcgt 240ggtgggcggc ggcatcagcg
ggctctgcac cgcgcaggcg ctggccacaa agcacggcgt 300cggcgacgtg ctcgtcacgg
aggcccgcgc ccgccccggc ggcaacatca ccaccgccga 360gcgcgccggc gagggctacc
tctgggagga ggggcccaac agcttccagc cttccgaccc 420cgtcctcacc atggccgtgg
acagcgggct caaggacgat ctcgtgttcg gggaccccaa 480cgcgccgcgg ttcgtgctgt
gggaggggaa gctaaggccg gtgccgtcca agcccggcga 540cctgccgttc ttcgacctca
tgagcatccc cggcaagctc agggccggcc ttggcgcgct 600cggcgttcga gcgccacctc
cagggcgtga ggagtcggtg gaggacttcg tgcggcgcaa 660cctcggcgcg gaggtctttg
agcgcctcat tgagcctttc tgctcaggtg tgtatgctgg 720tgatccttca aagctcagta
tgaaggctgc atttgggaag gtgtggaggc tggaggatac 780tggaggtagc attattggtg
gaaccatcaa aacaatccag gagaggggga aaaaccccaa 840accgccgagg gatccccgcc
ttccaacgcc aaaggggcag acagttgcat ctttcaggaa 900gggtctgact atgctcccgg
atgctattac atctaggttg ggtagcaaag tcaaactttc 960atggaagttg acaagcatta
caaagtcaga caacaaagga tatgcattag tgtatgaaac 1020accagaaggg gtggtctcgg
tgcaagctaa aactgttgtc atgaccatcc catcatatgt 1080tgctagtgat atcttgcggc
cactttcaag tgatgcagca gatgctctgt caatattcta 1140ttatccacca gttgctgctg
taactgtttc atatccaaaa gaagcaatta gaaaagaatg 1200cttaattgac ggagagctcc
agggtttcgg ccagctgcat ccgcgtagtc agggagttga 1260gactttagga acaatatata
gctcatcact ctttccaaat cgtgctccag ctggaagggt 1320gttacttctg aactacatag
gaggttctac aaatacaggg attgtttcca agactgaaag 1380tgagctggta gaagcagttg
accgtgacct caggaagatg ctgataaatc ctaaagcagt 1440ggaccctttg gtccttggcg
tccgggtatg gccacaagcc ataccacagt tcctcattgg 1500ccatcttgat catcttgagg
ctgcaaaatc tgccctgggc aaaggtggtt atgatggatt 1560gttcctcgga gggaactatg
ttgcaggagt tgccctgggc cgatgcgttg aaggtgcata 1620tgagagtgcc tcacaaatat
ctgactactt gaccaagtac gcctacaagt gatcaaagtt 1680ggcctgctcc ttttggcaca
tagatgtgag gcttctagca gcaaaaattt catgggcatc 1740tttttatcct gattctaatt
agttagaatt tagaattgta gaggaatgtt ccatttgcag 1800ttcataatag ttgttcagat
ttcagccatt caatttgtgc agccatttac tatatgtagt 1860atgatcttgt aagtactact
aagaacaaat caattatatt ttcctgc 190717551PRTOryza sativa
17Met Leu Ser Pro Ala Thr Thr Phe Ser Ser Ser Ser Ser Ser Ser Ser1
5 10 15Pro Ser Arg Ala His Ala
Arg Ala Pro Thr Arg Phe Ala Val Ala Ala 20 25
30Ser Ala Arg Ala Ala Arg Phe Arg Pro Ala Arg Ala Met
Ala Ala Ser 35 40 45Asp Asp Pro
Arg Gly Gly Arg Ser Val Ala Val Val Gly Ala Gly Val 50
55 60Ser Gly Leu Ala Ala Ala Tyr Arg Leu Arg Lys Arg
Gly Val Gln Val65 70 75
80Thr Val Phe Glu Ala Ala Asp Arg Ala Gly Gly Lys Ile Arg Thr Asn
85 90 95Ser Glu Gly Gly Phe Ile
Trp Asp Glu Gly Ala Asn Thr Met Thr Glu 100
105 110Ser Glu Leu Glu Ala Ser Arg Leu Ile Asp Asp Leu
Gly Leu Gln Gly 115 120 125Lys Gln
Gln Tyr Pro Asn Ser Gln His Lys Arg Tyr Ile Val Lys Asp 130
135 140Gly Ala Pro Thr Leu Ile Pro Ser Asp Pro Ile
Ala Leu Met Lys Ser145 150 155
160Thr Val Leu Ser Thr Lys Ser Lys Leu Lys Leu Phe Leu Glu Pro Phe
165 170 175Leu Tyr Glu Lys
Ser Ser Arg Arg Thr Ser Gly Lys Val Ser Asp Glu 180
185 190His Leu Ser Glu Ser Val Ala Ser Phe Phe Glu
Arg His Phe Gly Lys 195 200 205Glu
Val Val Asp Tyr Leu Ile Asp Pro Phe Val Ala Gly Thr Ser Gly 210
215 220Gly Asp Pro Glu Ser Leu Ser Ile Arg His
Ala Phe Pro Ala Leu Trp225 230 235
240Asn Leu Glu Asn Lys Tyr Gly Ser Val Ile Ala Gly Ala Ile Leu
Ser 245 250 255Lys Leu Ser
Thr Lys Gly Asp Ser Val Lys Thr Gly Gly Ala Ser Pro 260
265 270Gly Lys Gly Arg Asn Lys Arg Val Ser Phe
Ser Phe His Gly Gly Met 275 280
285Gln Ser Leu Ile Asp Ala Leu His Asn Glu Val Gly Asp Gly Asn Val 290
295 300Lys Leu Gly Thr Glu Val Leu Ser
Leu Ala Cys Cys Cys Asp Gly Val305 310
315 320Ser Ser Ser Gly Gly Trp Ser Ile Ser Val Asp Ser
Lys Asp Ala Lys 325 330
335Gly Lys Asp Leu Arg Lys Asn Gln Ser Phe Asp Ala Val Ile Met Thr
340 345 350Ala Pro Leu Ser Asn Val
Gln Arg Met Lys Phe Thr Lys Gly Gly Val 355 360
365Pro Phe Val Leu Asp Phe Leu Pro Lys Val Asp Tyr Leu Pro
Leu Ser 370 375 380Leu Met Val Thr Ala
Phe Lys Lys Glu Asp Val Lys Lys Pro Leu Glu385 390
395 400Gly Phe Gly Ala Leu Ile Pro Tyr Lys Glu
Gln Gln Lys His Gly Leu 405 410
415Lys Thr Leu Gly Thr Leu Phe Ser Ser Met Met Phe Pro Asp Arg Ala
420 425 430Pro Asn Asp Gln Tyr
Leu Tyr Thr Ser Phe Ile Gly Gly Ser His Asn 435
440 445Arg Asp Leu Ala Gly Ala Pro Thr Ala Ile Leu Lys
Gln Leu Val Thr 450 455 460Ser Asp Leu
Arg Lys Leu Leu Gly Val Glu Gly Gln Pro Thr Phe Val465
470 475 480Lys His Val His Trp Arg Asn
Ala Phe Pro Leu Tyr Gly Gln Asn Tyr 485
490 495Asp Leu Val Leu Glu Ala Ile Ala Lys Met Glu Asn
Asn Leu Pro Gly 500 505 510Phe
Phe Tyr Ala Gly Asn Asn Lys Asp Gly Leu Ala Val Gly Asn Val 515
520 525Ile Ala Ser Gly Ser Lys Ala Ala Asp
Leu Val Ile Ser Tyr Leu Glu 530 535
540Ser Cys Thr Asp Gln Asp Asn545 550181972DNAOryza
sativa 18cgatccgaag gacgaacccc gcacaagaca acaagtaaat ccccatccat
agctatccaa 60gagccccaaa tcagatgctc tctcctgcca ccaccttctc ctcctcctcc
tcctcctcgt 120cgccgtcgcg cgcccacgct cgcgctccca cccgcttcgc ggtcgcagca
tccgcgcgcg 180ccgcacggtt ccgccccgcg cgcgccatgg ccgcctccga cgacccccgc
ggcgggaggt 240ccgtcgccgt cgtcggcgcc ggcgtcagtg ggctcgcggc ggcgtacagg
ctgaggaagc 300gcggcgtgca ggtgacggtg ttcgaggcgg ccgacagggc gggtgggaag
atacggacca 360actccgaggg cgggttcatc tgggacgaag gggccaacac catgacagag
agtgaattgg 420aggcaagcag gcttattgac gatcttggcc tacaaggcaa acagcagtat
cctaactcac 480aacacaagcg ttacattgtc aaagatggag caccaacact gattccctca
gatcccattg 540cgctcatgaa aagcactgtt ctttctacaa aatcaaagct caagctattt
ctggaaccat 600ttctctatga gaaatctagc agaaggacct cgggaaaagt gtctgatgaa
catttaagtg 660agagtgttgc aagtttcttt gaacgccact ttggaaaaga ggttgttgat
tatcttattg 720atccatttgt ggctggaaca agcggaggag atcctgagtc attatcaatt
cgtcatgcat 780ttccagcatt atggaatttg gagaataagt atggctctgt cattgctggt
gccatcttgt 840ccaaactatc cactaagggt gattcagtga agacaggagg tgcttcgcca
gggaaaggaa 900ggaataaacg tgtgtcattt tcatttcatg gtggaatgca gtcactaata
gatgcacttc 960acaatgaagt tggagatggt aacgtgaagc ttggtacaga agtgttgtca
ttggcatgtt 1020gctgtgatgg agtctcttct tctggtggtt ggtcaatttc tgttgattca
aaagatgcta 1080aagggaaaga tctcagaaag aaccaatctt tcgatgctgt tataatgact
gctccattgt 1140ctaatgtcca gaggatgaag tttacaaaag gtggagttcc ctttgtgcta
gactttcttc 1200ctaaggtcga ttatctacca ctatctctca tggtaacagc ttttaagaag
gaagatgtca 1260aaaaaccatt ggaaggattt ggtgccttga taccctataa ggaacagcaa
aagcatggtc 1320tcaaaaccct tgggaccctc ttctcctcga tgatgtttcc agatcgagct
cctaatgatc 1380aatatctata tacatctttc attgggggga gccataatag agacctcgct
ggggctccaa 1440cggctattct gaaacaactt gtgacctctg acctaagaaa gctcttgggt
gttgagggac 1500aacctacttt tgtgaagcat gtacattgga gaaatgcttt tcctttatat
ggccagaatt 1560atgatctggt actggaagct atagcaaaaa tggagaacaa tcttccaggg
ttcttttacg 1620caggaaataa caaggatggg ttggctgttg gaaatgttat agcttcagga
agcaaggctg 1680ctgaccttgt gatctcttat cttgaatctt gcacagatca ggacaattag
catttaaggt 1740atctgacctt aagcaatttc agacaaattt gctcacttta tgtaaattga
aaaggttcac 1800atgatttcca gtttcatatt tttctcttgc tatagtatat ccactcatgt
aaagatggga 1860acatagtcct aaaagacatt atggtcgctt gagatgctca tgtttttttg
aacagtgatt 1920cttgacttgt actatttttt gacaaccaaa taaatttctc aatgtttccg
ag 197219551PRTSorghum bicolor 19Met Leu Ser Pro Ala Thr Thr
Phe Ser Ser Ser Ser Ser Ser Ser Ser1 5 10
15Pro Ser Arg Ala His Ala Arg Ala Pro Thr Arg Phe Ala
Val Ala Ala 20 25 30Ser Ala
Arg Ala Ala Arg Phe Arg Pro Ala Arg Ala Met Ala Ala Ser 35
40 45Asp Asp Pro Arg Gly Gly Arg Ser Val Ala
Val Val Gly Ala Gly Val 50 55 60Ser
Gly Leu Ala Ala Ala Tyr Arg Leu Arg Lys Arg Gly Val Gln Val65
70 75 80Thr Val Phe Glu Ala Ala
Asp Arg Ala Gly Gly Lys Ile Arg Thr Asn 85
90 95Ser Glu Gly Gly Phe Ile Trp Asp Glu Gly Ala Asn
Thr Met Thr Glu 100 105 110Ser
Glu Leu Glu Ala Ser Arg Leu Ile Asp Asp Leu Gly Leu Gln Gly 115
120 125Lys Gln Gln Tyr Pro Asn Ser Gln His
Lys Arg Tyr Ile Val Lys Asp 130 135
140Gly Ala Pro Thr Leu Ile Pro Ser Asp Pro Ile Ala Leu Met Lys Ser145
150 155 160Thr Val Leu Ser
Thr Lys Ser Lys Leu Lys Leu Phe Leu Glu Pro Phe 165
170 175Leu Tyr Glu Lys Ser Ser Arg Arg Thr Ser
Gly Lys Val Ser Asp Glu 180 185
190His Leu Ser Glu Ser Val Ala Ser Phe Phe Glu Arg His Phe Gly Lys
195 200 205Glu Val Val Asp Tyr Leu Ile
Asp Pro Phe Val Ala Gly Thr Ser Gly 210 215
220Gly Asp Pro Glu Ser Leu Ser Ile Arg His Ala Phe Pro Ala Leu
Trp225 230 235 240Asn Leu
Glu Asn Lys Tyr Gly Ser Val Ile Ala Gly Ala Ile Leu Ser
245 250 255Lys Leu Ser Thr Lys Gly Asp
Ser Val Lys Thr Gly Gly Ala Ser Pro 260 265
270Gly Lys Gly Arg Asn Lys Arg Val Ser Phe Ser Phe His Gly
Gly Met 275 280 285Gln Ser Leu Ile
Asp Ala Leu His Asn Glu Val Gly Asp Gly Asn Val 290
295 300Lys Leu Gly Thr Glu Val Leu Ser Leu Ala Cys Cys
Cys Asp Gly Val305 310 315
320Ser Ser Ser Gly Gly Trp Ser Ile Ser Val Asp Ser Lys Asp Ala Lys
325 330 335Gly Lys Asp Leu Arg
Lys Asn Gln Ser Phe Asp Ala Val Ile Met Thr 340
345 350Ala Pro Leu Ser Asn Val Gln Arg Met Lys Phe Thr
Lys Gly Gly Val 355 360 365Pro Phe
Val Leu Asp Phe Leu Pro Lys Val Asp Tyr Leu Pro Leu Ser 370
375 380Leu Met Val Thr Ala Phe Lys Lys Glu Asp Val
Lys Lys Pro Leu Glu385 390 395
400Gly Phe Gly Ala Leu Ile Pro Tyr Lys Glu Gln Gln Lys His Gly Leu
405 410 415Lys Thr Leu Gly
Thr Leu Phe Ser Ser Met Met Phe Pro Asp Arg Ala 420
425 430Pro Asn Asp Gln Tyr Leu Tyr Thr Ser Phe Ile
Gly Gly Ser His Asn 435 440 445Arg
Asp Leu Ala Gly Ala Pro Thr Ala Ile Leu Lys Gln Leu Val Thr 450
455 460Ser Asp Leu Arg Lys Leu Leu Gly Val Glu
Gly Gln Pro Thr Phe Val465 470 475
480Lys His Val His Trp Arg Asn Ala Phe Pro Leu Tyr Gly Gln Asn
Tyr 485 490 495Asp Leu Val
Leu Glu Ala Ile Ala Lys Met Glu Asn Asn Leu Pro Gly 500
505 510Phe Phe Tyr Ala Gly Asn Asn Lys Asp Gly
Leu Ala Val Gly Asn Val 515 520
525Ile Ala Ser Gly Ser Lys Ala Ala Asp Leu Val Ile Ser Tyr Leu Glu 530
535 540Ser Cys Thr Asp Gln Asp Asn545
550201922DNASorghum bicolor 20atggtcgccg ccgccgccat
ggccaccgct gcatcggcgg ccgcgccgct actcaacggg 60acccgaaggc ctgcgaggct
ccgccgtcgc ggactccgcg tgcgctgcgc cgctgtggcg 120ggcggcgcgg ccgaggcacc
ggcctccacc ggcgcgcggc tgtccgcgga ctgcgtcgtg 180gtgggcggcg ggatcagtgg
cctctgcacc gcgcaggcgc tggccacgcg gcacggcgtc 240ggggaggtgc ttgtcacgga
ggcccgcgcc cgacccggcg gcaacatcac caccgtcgag 300cgccccgagg aagggtacct
ctgggaggag ggtcccaaca gcttccagcc atccgacccc 360gttctctcca tggccgtgga
cagcgggctg aaggatgacc tggtttttgg ggatcccaac 420gcgccgcggt tcgtgctgtg
ggaggggaag ctgaggcccg tgccatccaa gcccgccgac 480ctcccgttct tcgatctcat
gagcatccct ggcaagctca gggccggtct cggcgcgctt 540ggcatccgcc cgcctcctcc
aggccgcgag gagtcagtgg aggagtttgt gcgccgcaac 600ctcggtgctg aggtctttga
gcgcctaatt gagcctttct gctcaggtgt ctatgctggt 660gatccttcca agctcagtat
gaaggctgca tttgggaagg tgtggcggtt agaagaagct 720ggaggtagta ttattggtgg
aaccatcaag acgattcagg agagaggcaa gaatccaaaa 780ccaccgaggg atccccgcct
tccgaagcca aaagggcaga cagttgcatc tttcaggaag 840ggtcttgcca tgcttccaaa
tgccatcaca tccagcttgg gtagtaaagt caaactatca 900tggaaactca cgagcattac
aaaatcagat ggcaaggggt atgttttgga gtatgaaaca 960ccagaagggg ttgttttggt
gcaggctaaa agtgttatca tgaccattcc atcatatgtt 1020gctagcgaca ttttgcgtcc
actttcaggt gatgctgcag atgctctatc aagattctat 1080tatccaccag ttgctgctgt
aacggtttcg tatccaaagg aagcaattag aaaagaatgc 1140ttaattgatg gggaactcca
gggttttggc cagttgcatc cacgtagtca aggagttgag 1200acattaggaa caatatacag
ctcatcactc tttccaaatc gtgctcctgc tggtagggtg 1260ttacttctaa actacatagg
aggtgctaca aacacaggaa ttgtttccaa gactgaaagt 1320gagctggtag aagcagttga
ccgtgacctc cgaaaaatgc ttataaattc tacagcagtg 1380gaccctttag tccttggtgt
ccgagtttgg ccacaagcca tacctcagtt cctggtagga 1440catcttgatc ttctggaggt
cgcaaaatct gccctggacc aaggtggcta tgatgggctg 1500ttcctaggag ggaactatgt
tgcaggagtt gccctgggca gatgcattga gggcgcatat 1560gagagtgccg cacaaatata
tgacttcttg accaagtatg cctacaagtg atggaagaag 1620tggagcgctg cttgttaatt
gttatgttgc atagatgagg tgagaccagg agtagtaaaa 1680ggcattacga gtatttttca
ttcttatttt gtaaattgca cttctgtttt tttttcctgt 1740cagtaattag ttagatttta
gttctgtagg agattgttgt gttcactgcc ctgcaaaaga 1800atttttattt tgcattcgtt
tatgagagct gtgcagactt atgtaacgtt ttactgtaag 1860tatcaacaaa atcagatact
attctgcaag agctaacaga atgtgcaact gagattgcct 1920tg
192221544PRTSorghum bicolor
21Met Leu Ala Arg Thr Ala Thr Val Ser Ser Thr Ser Ser His Ser His1
5 10 15Pro Tyr Arg Pro Thr Ser
Ala Arg Ser Leu Arg Leu Arg Pro Val Leu 20 25
30Ala Met Ala Gly Ser Asp Asp Ser Arg Ala Ala Pro Ala
Arg Ser Val 35 40 45Ala Val Val
Gly Ala Gly Val Ser Gly Leu Val Ala Ala Tyr Arg Leu 50
55 60Arg Lys Ser Gly Val Asn Val Thr Val Phe Glu Ala
Ala Asp Arg Ala65 70 75
80Gly Gly Lys Ile Arg Thr Asn Ser Glu Gly Gly Phe Leu Trp Asp Glu
85 90 95Gly Ala Asn Thr Met Thr
Glu Gly Glu Leu Glu Ala Ser Arg Leu Ile 100
105 110Asp Asp Leu Gly Leu Gln Asp Lys Gln Gln Tyr Pro
Asn Ser Gln His 115 120 125Lys Arg
Tyr Ile Val Lys Asp Gly Ala Pro Ala Leu Ile Pro Ser Asp 130
135 140Pro Ile Ser Leu Met Lys Ser Ser Val Leu Ser
Thr Lys Ser Lys Ile145 150 155
160Ala Leu Phe Phe Glu Pro Phe Leu Tyr Lys Lys Ala Asn Thr Arg Asn
165 170 175Pro Gly Lys Val
Ser Asp Glu His Leu Ser Glu Ser Val Gly Ser Phe 180
185 190Phe Glu Arg His Phe Gly Arg Glu Val Val Asp
Tyr Leu Ile Asp Pro 195 200 205Phe
Val Ala Gly Thr Ser Ala Gly Asp Pro Glu Ser Leu Ser Ile Cys 210
215 220His Ala Phe Pro Ala Leu Trp Asn Leu Glu
Arg Lys Tyr Gly Ser Val225 230 235
240Val Val Gly Ala Ile Leu Ser Lys Leu Thr Ala Lys Gly Asp Pro
Val 245 250 255Lys Thr Arg
Arg Asp Ser Ser Ala Lys Arg Arg Asn Arg Arg Val Ser 260
265 270Phe Ser Phe His Gly Gly Met Gln Ser Leu
Ile Asn Ala Leu His Asn 275 280
285Glu Val Gly Asp Asp Asn Val Lys Leu Gly Thr Glu Val Leu Ser Leu 290
295 300Ala Cys Thr Leu Asp Gly Ala Pro
Ala Pro Gly Gly Trp Ser Ile Ser305 310
315 320Asp Asp Ser Lys Asp Ala Ser Gly Lys Asp Leu Ala
Lys Asn Gln Thr 325 330
335Phe Asp Ala Val Ile Met Thr Ala Pro Leu Ser Asn Val Gln Arg Met
340 345 350Lys Phe Thr Lys Gly Gly
Ala Pro Phe Val Leu Asp Phe Leu Pro Lys 355 360
365Val Asp Tyr Leu Pro Leu Ser Leu Met Val Thr Ala Phe Lys
Lys Glu 370 375 380Asp Val Lys Lys Pro
Leu Glu Gly Phe Gly Val Leu Ile Pro Tyr Lys385 390
395 400Glu Gln Gln Lys His Gly Leu Lys Thr Leu
Gly Thr Leu Phe Ser Ser 405 410
415Met Met Phe Pro Asp Arg Ala Pro Asp Asp Gln Tyr Leu Tyr Thr Thr
420 425 430Phe Val Gly Gly Ser
His Asn Arg Asp Leu Ala Gly Ala Pro Thr Ser 435
440 445Ile Leu Lys Gln Leu Val Thr Ser Asp Leu Lys Lys
Leu Leu Gly Val 450 455 460Gln Gly Gln
Pro Thr Phe Val Lys His Ile Tyr Trp Gly Asn Ala Phe465
470 475 480Pro Leu Tyr Gly His Asp Tyr
Asn Ser Val Leu Glu Ala Ile Glu Lys 485
490 495Met Glu Lys Asn Leu Pro Gly Phe Phe Tyr Ala Gly
Asn Asn Lys Asp 500 505 510Gly
Leu Ala Val Gly Ser Val Ile Ala Ser Gly Ser Lys Ala Ala Asp 515
520 525Leu Ala Ile Ser Tyr Leu Glu Ser His
Thr Lys His Asn Asn Leu His 530 535
540221635DNASorghum bicolor 22atgctcgctc ggactgccac ggtctcctcc acttcgtccc
actcccatcc ttatcgcccc 60acctccgctc gcagtctccg cctacgtccg gtcctcgcga
tggcgggctc cgacgactcc 120cgcgcagctc ccgccaggtc ggtcgccgtc gtcggcgccg
gggtcagcgg gctcgtggcg 180gcgtacaggc tcaggaagag cggcgtgaat gtgacggtgt
tcgaggcggc cgacagggcg 240ggaggaaaga tacggaccaa ttccgagggc gggtttctct
gggatgaagg agcgaacacc 300atgacagaag gtgaattgga ggccagtaga ctgatagatg
atctcggtct acaagacaaa 360cagcagtatc ctaactccca acacaagcgt tacattgtca
aagatggagc accagcactg 420attccttcgg atcccatttc gctgatgaaa agcagtgttc
tttctacaaa atcaaagatt 480gcgttatttt ttgaaccatt tctctacaag aaagctaaca
caagaaaccc tggaaaagta 540tctgatgagc atttgagtga gagtgttggg agcttctttg
aacgccactt cggaagagaa 600gttgttgact atcttattga tccatttgta gctggaacaa
gtgcaggaga tccagagtca 660ctatctattt gtcatgcatt cccagcactg tggaatttgg
aaagaaaata tggttcagtt 720gttgttggtg ccatcttgtc taagctaaca gctaaaggtg
atccagtaaa gacaagacgt 780gattcatcag cgaaaagaag gaatagacgc gtgtcgtttt
catttcatgg tggaatgcag 840tcactaataa atgcacttca caatgaagtt ggagatgata
atgtgaagct tggtacagaa 900gtgttgtcat tggcgtgtac attagatgga gcccctgcac
caggcgggtg gtcaatttct 960gatgattcga aggatgctag tggcaaggac cttgctaaaa
accaaacctt tgatgctgtt 1020ataatgacag ctccattgtc aaatgtccag aggatgaagt
tcacaaaagg tggagctcct 1080tttgttctag actttcttcc taaggtggat tatctaccac
tatctctcat ggtgactgct 1140tttaagaagg aagatgtcaa gaaacctctg gaaggatttg
gcgtcttaat accctacaag 1200gaacagcaaa aacatggtct aaaaaccctt gggactctct
tctcctcaat gatgttccca 1260gatcgagctc ctgacgacca atatttatat acaacatttg
ttgggggtag ccacaataga 1320gatcttgctg gagctccaac gtctattctg aaacaacttg
tgacctctga ccttaaaaaa 1380ctcttaggcg tacaggggca accaactttt gtcaagcata
tatactgggg aaatgctttt 1440cctttgtatg gtcatgatta caattctgta ttggaagcta
tagaaaagat ggagaaaaat 1500cttccagggt tcttctacgc aggaaataac aaggatgggc
ttgctgttgg gagtgttata 1560gcttcaggaa gcaaggctgc tgaccttgca atctcgtatc
ttgaatctca caccaagcat 1620aataatttac attga
163523536PRTRicinus communis 23Met Ala Asn Leu Ala
Asp Phe Ser Leu Phe Leu Arg Ser Thr Pro Ser1 5
10 15Leu Val Pro Ser Tyr Pro Lys Thr Thr Ile Asn
Arg Thr Leu Lys Leu 20 25
30Gln Leu Arg Cys Ser Ile Thr Glu Gln Ser Thr Thr Thr Ile Ser Pro
35 40 45Gly Gly Asn Ser Gln Ser Pro Ala
Asp Cys Val Ile Val Gly Gly Gly 50 55
60Ile Ser Gly Leu Cys Ile Ala Gln Ala Leu Ser Thr Lys His Arg Asp65
70 75 80Ile Ala Thr Asn Val
Ile Val Thr Glu Ala Arg Asp Arg Val Gly Gly 85
90 95Asn Ile Thr Thr Ile Glu Arg Asp Gly Tyr Leu
Trp Glu Glu Gly Pro 100 105
110Asn Ser Phe Gln Pro Ser Asp Pro Met Leu Thr Met Val Val Asp Ser
115 120 125Gly Leu Lys Asp Asp Leu Val
Leu Gly Asp Pro Asn Ala Pro Arg Phe 130 135
140Val Leu Trp Asn Gly Lys Leu Arg Pro Val Pro Ser Lys Pro Thr
Asp145 150 155 160Leu Pro
Phe Phe Asp Leu Met Ser Phe Gly Gly Lys Ile Arg Ala Gly
165 170 175Phe Gly Ala Leu Gly Leu Arg
Pro Pro Pro Pro Gly His Glu Glu Ser 180 185
190Val Glu Glu Phe Val Arg Arg Asn Leu Gly Asp Glu Val Phe
Glu Arg 195 200 205Leu Ile Glu Pro
Phe Cys Ser Gly Val Tyr Ala Gly Asp Pro Ser Lys 210
215 220Leu Ser Met Lys Ala Ala Phe Gly Lys Val Trp Lys
Leu Glu Gln Ile225 230 235
240Gly Gly Ser Val Ile Gly Gly Thr Phe Lys Thr Ile Gln Glu Arg Asn
245 250 255Lys Ile Pro Lys Pro
Pro Arg Asp Pro Arg Leu Pro Thr Pro Lys Gly 260
265 270Gln Thr Val Gly Ser Phe Arg Lys Gly Leu Ile Met
Leu Pro Asp Ala 275 280 285Ile Ala
Lys Arg Leu Gly Ser Asn Val Lys Leu Ser Trp Lys Leu Ser 290
295 300Ser Ile Thr Lys Leu Glu Asn Gly Gly Tyr Ser
Leu Thr Phe Glu Thr305 310 315
320Pro Asp Gly Ser Val Ser Leu Gln Thr Lys Ser Val Val Met Thr Val
325 330 335Pro Ser His Ile
Ala Ser Ser Phe Leu His Pro Leu Ser Ala Ala Ala 340
345 350Ala Asp Ala Leu Ser Lys Phe Tyr Tyr Pro Pro
Val Ala Ala Val Ser 355 360 365Val
Ser Tyr Pro Lys Asp Ala Ile Arg Ala Glu Cys Leu Ile Asp Gly 370
375 380Glu Leu Lys Gly Phe Gly Gln Leu His Pro
Arg Ser Gln Gly Val Glu385 390 395
400Thr Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala
Pro 405 410 415Ala Gly Arg
Ile Leu Leu Leu Asn Tyr Ile Gly Gly Ala Thr Asn Pro 420
425 430Gly Ile Leu Ser Lys Thr Glu Thr Glu Leu
Val Glu Ala Val Asp Arg 435 440
445Asp Leu Arg Lys Met Leu Ile Lys Pro Asn Ala Lys Asp Pro Phe Val 450
455 460Leu Gly Val Arg Val Trp Pro Gln
Ala Ile Pro Gln Phe Leu Val Gly465 470
475 480His Leu Asp Ile Leu Asp Ser Ala Lys Gly Ala Leu
Gly Asp Ala Gly 485 490
495Leu Glu Gly Leu Phe Leu Gly Gly Asn Tyr Val Ser Gly Val Ala Leu
500 505 510Gly Arg Cys Val Glu Gly
Ala Tyr Glu Val Ala Ala Glu Val Thr Asn 515 520
525Phe Leu Ser Gln Asn Ala Tyr Lys 530
535241671DNARicinus communis 24caccacctga gttacagaag agtcatccgg
tgtgattgcc tctcgaattc gaattctgcc 60atggccaatc tcgcagactt ctctcttttt
ctccggtcaa caccctccct tgtcccctcc 120tatccgaaaa ccacaatcaa cagaacgtta
aaactccaac tccggtgctc aatcacagag 180caatcgacta ctacaatttc ccctggcgga
aattcccaat caccagcgga ttgcgtgatt 240gtaggaggcg gaattagcgg cctatgcatc
gcccaagctc tctctaccaa gcaccgtgat 300atagctacca atgtgattgt cactgaggcc
agagaccgcg ttggtggcaa catcacaacc 360atcgaaagag acggttatct ttgggaagag
ggccccaata gtttccagcc ctccgatcct 420atgctaacca tggtggtgga tagtgggtta
aaagatgatt tagttttggg agatcctaat 480gcgcctcgtt ttgtgctctg gaatgggaaa
ttgagaccag ttccgtcaaa gcctactgac 540ttgccctttt ttgacttgat gagctttggt
gggaaaatta gagctggatt tggtgctctt 600ggacttcgac ctccaccacc aggacatgag
gagtcagttg aagagtttgt ccggcgtaat 660cttggtgatg aagtttttga gcgtctaatc
gagccctttt gttcaggtgt ttatgcaggt 720gatccttcaa aactaagcat gaaagcagca
tttggaaaag tttggaagct ggagcaaatt 780ggtggcagtg tcattggcgg cactttcaaa
acaattcagg agagaaataa gatacccaag 840cctcctcgag acccgcgctt accaacaccg
aagggtcaaa cagtaggatc ttttagaaag 900ggacttatca tgttgcctga tgcgattgcc
aaaaggttgg gtagcaatgt taaattgtct 960tggaagcttt caagtattac taaattggaa
aatggagggt atagtctaac atttgaaaca 1020cctgatgggt cagtttcgct gcaaacgaaa
agtgttgtaa tgacagttcc atcccacatt 1080gcaagcagct tcttacatcc tctttctgct
gctgctgctg acgccctatc aaaattttat 1140tacccgccag ttgcagcagt gtcagtttca
tacccaaaag atgcaattcg ggcagaatgc 1200ttaatagatg gtgagcttaa ggggttcggc
cagttgcatc cacggagcca aggggtagaa 1260acattaggaa ctatatacag ctcctcactt
tttcccaatc gtgcaccagc aggaaggatt 1320ttgctcttga actacattgg aggggcgacc
aatcctggga ttttgtccaa gacggaaact 1380gaacttgtag aggcagttga ccgtgatttg
aggaagatgc tcataaaacc caatgcgaag 1440gatccatttg ttctaggtgt gcgagtgtgg
ccccaagcca ttccacaatt cttggttggt 1500catttagata tcctagatag tgcaaaaggt
gctctgggag atgcaggctt ggaagggctg 1560tttcttgggg gcaactatgt atcaggcgtt
gctttgggcc gatgtgtgga aggagcatat 1620gaagttgcag cagaggtgac caatttcctc
tcgcagaatg cttataaatg a 167125511PRTRicinus communis 25Met Ser
Ser Val Ile Lys Glu Asp Arg Asn Pro Ser His Val Lys Arg1 5
10 15Val Ala Val Val Gly Ala Gly Val
Ser Gly Leu Ala Ala Ala Tyr Lys 20 25
30Leu Lys Ser His Gly Leu Lys Val Thr Val Phe Glu Ala Glu Glu
Arg 35 40 45Ala Gly Gly Lys Leu
Arg Ser Val Asn His Asp Gly Leu Ile Trp Asp 50 55
60Glu Gly Ala Asn Thr Met Thr Glu Ser Glu Met Glu Val Lys
Ser Leu65 70 75 80Ile
Gly Asn Leu Gly Ile Arg Glu Lys Gln Gln Phe Pro Ile Ser Gln
85 90 95Asn Lys Arg Tyr Ile Val Arg
Asn Gly Lys Pro Ile Leu Ile Pro Thr 100 105
110Asn Pro Ile Ala Leu Ile Thr Ser Asn Ile Leu Ser Ala Gln
Ser Lys 115 120 125Phe Gln Ile Ile
Leu Glu Pro Phe Leu Trp Lys Lys Arg Glu Ser Ser 130
135 140Glu Thr His Asn Ala Tyr Thr Glu Glu Ser Val Gly
Glu Phe Phe Gln145 150 155
160Arg His Phe Gly Lys Glu Val Val Asp Tyr Leu Ile Asp Pro Phe Val
165 170 175Ala Gly Thr Ser Ala
Gly Asp Pro Glu Ser Leu Ser Val Cys His Ser 180
185 190Phe Pro Glu Leu Trp Asn Leu Glu Lys Arg Phe Gly
Ser Ile Ile Ala 195 200 205Gly Val
Val Gln Ala Lys Leu Ser Thr Lys Arg Gly Lys Ser Gln Glu 210
215 220Thr Lys Gly Ser Ser Val Lys Lys Lys Gln Gln
Arg Gly Ser Phe Ser225 230 235
240Phe Phe Gly Gly Met Gln Thr Leu Thr Asp Thr Leu Cys Lys Ala Leu
245 250 255Ala Lys Asp Glu
Leu Arg Leu Glu Ser Lys Val Phe Ser Leu Ser Tyr 260
265 270Asn Pro Asp Ser Lys Ser Ala Val Glu Asn Trp
Ser Leu Ser Tyr Ala 275 280 285Phe
Lys Gly Ala Lys His Leu Gln Asn Ser Ser Tyr Asp Ala Ile Val 290
295 300Met Thr Ala Pro Leu Cys Asn Val Lys Glu
Met Lys Ile Thr Lys Asn305 310 315
320Arg Asn Ile Phe Ser Leu Asn Phe Leu Pro Glu Val Ser Tyr Met
Pro 325 330 335Leu Ser Val
Val Ile Thr Thr Phe Lys Lys Asp Asn Val Lys Ser Pro 340
345 350Leu Glu Gly Phe Gly Val Leu Val Pro Ser
Lys Glu Gln Gln Asn Gly 355 360
365Leu Lys Thr Leu Gly Thr Leu Phe Ser Ser Met Met Phe Pro Asp Arg 370
375 380Ala Pro Asn Asp Leu Tyr Leu Tyr
Thr Thr Phe Val Gly Gly Ser Arg385 390
395 400Asn Lys Glu Leu Ala Lys Ala Ser Thr Asp Asp Leu
Lys Gln Ile Val 405 410
415Thr Ser Asp Leu Arg Gln Leu Leu Gly Ala Glu Gly Glu Pro Thr Phe
420 425 430Val Asn His Phe Tyr Trp
Ser Lys Ala Phe Pro Leu Tyr Gly Arg Asn 435 440
445Tyr Asp Ala Val Leu Glu Ala Ile Asp Thr Met Glu Lys Asp
Leu Pro 450 455 460Gly Phe Phe Tyr Ala
Gly Asn His Lys Gly Gly Leu Ser Val Gly Lys465 470
475 480Ala Ile Ala Ser Gly Cys Lys Ala Ala Asp
Leu Val Ile Ser Tyr Leu 485 490
495Glu Ser Ser Ser Asp Asp Lys Met Leu Lys Glu Gly Pro Ser Asn
500 505 510261536DNARicinus communis
26atgtcttcag ttatcaaaga agacagaaac ccaagtcatg ttaaaagagt agctgttgta
60ggtgctgggg ttagtgggct tgctgcagct tacaaactga aatcacatgg cttgaaagtt
120acagtatttg aagctgaaga aagagctgga gggaagctga gaagcgttaa ccatgatggt
180ttaatttggg atgaaggtgc aaataccatg actgagagtg aaatggaggt caaaagttta
240attggcaatc ttgggattcg tgaaaagcaa caatttccga tttcacagaa caaaaggtat
300attgtaagaa atgggaagcc aatattaata cccacaaatc ccatcgcact gatcaccagc
360aacattctct ctgcacagtc aaagtttcaa atcattctgg agccattttt gtggaagaaa
420cgtgaatctt cagaaacgca caatgcttat actgaggaaa gtgttggtga gtttttccaa
480cgtcattttg gtaaagaggt tgttgattat cttattgacc cttttgttgc gggcactagt
540gctggagatc ctgaatctct ttctgtatgc cattcttttc cagagctatg gaatctggag
600aaacgatttg gatctattat agctggggta gttcaggcaa aattatctac caaaagaggg
660aagagccaag aaaccaaagg atcatcggta aagaagaagc agcagcgtgg ttcattctct
720ttttttggtg gaatgcagac gctaactgat acattgtgca aagcacttgc gaaggatgag
780cttagattag aatcaaaggt cttctctttg tcttacaatc ctgattctaa gtcagcagta
840gagaattggt cactttctta tgcttttaag ggcgccaagc atttgcaaaa ctcatcttat
900gatgctattg tcatgacggc accattgtgc aatgttaaag aaatgaagat cacaaaaaac
960agaaatatct tttcactgaa ttttcttcct gaggtgagtt atatgccgct atcagttgtt
1020attaccactt ttaagaagga taatgtcaag agcccccttg aaggctttgg agttcttgtt
1080ccttctaagg agcaacagaa tggtctaaaa acccttggta cactcttttc ctctatgatg
1140tttccagatc gtgcacccaa tgatctgtat ctctatacaa cctttgttgg agggagtcga
1200aacaaggaac tggcaaaagc ttcaacggat gatctgaagc agattgttac ctccgacctt
1260aggcaattgc taggagcaga aggcgagccc acatttgtta atcatttcta ctggagtaaa
1320gcatttccat tatatgggag gaactatgat gcagtacttg aagccattga tacgatggaa
1380aaagatcttc ctggattctt ctatgcaggt aaccacaaag gtggactatc ggttggcaaa
1440gcaatagcct ctggatgcaa agcagctgat cttgtaatat cctatttgga atcttcttca
1500gatgacaaga tgctgaagga agggccatca aattag
153627504PRTSolanum tuberosumMOD_RES(38)..(38)Any amino acid 27Met Ala
Pro Ser Ala Gly Glu Asp Lys Gln Asn Cys Pro Lys Arg Val1 5
10 15Ala Val Ile Gly Ala Gly Val Ser
Gly Leu Ala Ala Ala Tyr Lys Leu 20 25
30Lys Ile His Gly Leu Xaa Val Thr Val Phe Glu Ala Glu Gly Arg
Ala 35 40 45Gly Gly Lys Leu Arg
Ser Leu Ser Gln Asp Gly Xaa Ile Trp Asp Glu 50 55
60Gly Ala Asn Thr Met Thr Glu Ser Glu Gly Asp Val Thr Phe
Leu Leu65 70 75 80Asp
Ser Leu Gly Leu Arg Glu Lys Gln Gln Phe Pro Leu Ser Gln Asn
85 90 95Lys Arg Tyr Ile Ala Arg Asn
Gly Thr Pro Thr Leu Ile Pro Ser Asn 100 105
110Pro Ile Asp Leu Ile Lys Ser Asn Phe Leu Ser Thr Gly Ser
Lys Leu 115 120 125Gln Met Leu Phe
Glu Pro Leu Leu Trp Lys Asn Xaa Lys Leu Thr Lys 130
135 140Val Ser Asp Glu His Glu Ser Val Ser Gly Phe Phe
Gln Arg His Phe145 150 155
160Gly Lys Glu Val Val Asp Tyr Leu Ile Asp Pro Phe Val Ala Gly Thr
165 170 175Cys Gly Gly Asp Pro
Asp Ser Leu Ser Met His Leu Ser Phe Pro Glu 180
185 190Leu Trp Asn Leu Glu Lys Arg Phe Gly Ser Val Ile
Val Gly Ala Ile 195 200 205Arg Ser
Lys Leu Ser Pro Ile Lys Glu Lys Lys Gln Gly Pro Pro Lys 210
215 220Thr Ser Val Asn Lys Lys Arg Gln Arg Gly Ser
Phe Ser Phe Leu Gly225 230 235
240Gly Met Gln Thr Leu Thr Asp Ala Ile Cys Lys Asp Leu Lys Glu Asp
245 250 255Glu Leu Arg Leu
Asn Ser Arg Val Leu Glu Leu Ser Cys Ser Cys Ser 260
265 270Gly Asp Ser Ala Ile Asp Ser Trp Ser Ile Phe
Ser Ala Ser Pro His 275 280 285Lys
Arg Gln Ala Glu Glu Glu Ser Phe Asp Ala Val Ile Met Thr Ala 290
295 300Pro Leu Cys Asp Val Lys Ser Met Lys Ile
Ala Lys Arg Gly Asn Pro305 310 315
320Phe Leu Leu Asn Phe Ile Pro Glu Val Asp Tyr Val Pro Leu Ser
Val 325 330 335Val Ile Thr
Thr Phe Lys Lys Glu Ser Val Lys His Pro Leu Glu Gly 340
345 350Phe Gly Val Leu Val Pro Ser Xaa Glu Gln
Lys His Gly Leu Lys Thr 355 360
365Leu Gly Thr Leu Phe Ser Ser Met Met Phe Pro Asp Arg Ala Pro Asn 370
375 380Asn Val Tyr Leu Tyr Thr Thr Phe
Val Gly Gly Ser Arg Asn Arg Glu385 390
395 400Leu Ala Lys Ala Ser Arg Thr Glu Leu Lys Glu Ile
Val Thr Ser Asp 405 410
415Leu Lys Gln Leu Leu Gly Ala Glu Gly Glu Pro Thr Tyr Val Asn His
420 425 430Val Cys Trp Ser Lys Ala
Phe Pro Leu Tyr Gly His Asn Tyr Asp Ser 435 440
445Val Leu Asp Ala Ile Asp Lys Met Glu Lys Asn Leu Pro Gly
Leu Phe 450 455 460Tyr Ala Gly Asn His
Lys Gly Gly Leu Ser Val Gly Lys Ala Leu Ser465 470
475 480Ser Gly Cys Asn Ala Ala Asp Leu Val Ile
Ser Tyr Leu Glu Ala Val 485 490
495Ser Thr Asp Xaa Lys Asn His Ser 500281515DNASolanum
tuberosum 28atggctccat ctgccggaga agataaacaa aattgtccma agagagttgc
agtcattggt 60gctggcgtca gtggacttgc tgcagcatac aagttgaaaa tycatggstt
gratgtcaca 120gtattygaag cagaagggag agctggaggg aagttacgaa gcctgagtca
agatggsmta 180atatgggatg aaggcgcaaa tactatgact gaaagtgaag gtgatgtcac
atttttgctt 240gattcgcttg gactccgaga aaarcaacaa tttccacttt cacagaacaa
rcgctacatt 300gccagaaatg gyactcctac tctgatacct tcaaatccaa ttgacctgat
caaaagcaat 360tttctttcca ctggatcaaa gcttcagatg cttttcgagc cacttttgtg
gaagaataaw 420aagcttacaa aggtgtctga cgaacacgaa agtgtcagtg gattcttcca
gcgtcatttt 480ggraaggagg ttgttgacta tctaattgay ccttttgttg ctggaacatg
tggtggtgat 540cctgactcgc tttcaatgca cctttcgttt ccagagttgt ggaatttaga
gaaaaggttt 600ggctcagtca tagttggggc aattcgatcc aagttatcac ctataaagga
aaagaaacaa 660gggccaccca aaacttcagt aaataagaag cgccagcggg ggtccttttc
atttttgggc 720ggaatgcaaa cacttactga cgcaatatgc aaagatctca aagaagatga
acttaggcta 780aactctagag ttctggaatt atcttgtagc tgtagtgggg actctgcgat
agatagctgg 840tcaatttttt ctgcctcacc acacaagcgg caagcagaag aagaatcatt
tgatgctgta 900attatgacgg cccctctctg tgacgttaag agtatgaaga ttgctaagag
aggaaatcca 960tttctgctca actttattcc tgaggtygat tatgtaccac tatctgttgt
tataaccaca 1020tttaagaagg agagtgtaaa gcatccyctt gagggttttg gagtgcttgt
accytccsag 1080gagcaaaaac atggtctgaa gacaytaggc accctcttct cttctatgat
gtttccagat 1140cgtgcaccca acaatgtcta tctctatact acatttgttg gtggaagccg
aaatagagaa 1200ctygcgaaag cctcgaggac tgagctgaaa gagatagtaa cttctgacct
taagcagttg 1260ttgggtgctg agggagagcc aacatatgtg aatcatgtat gctggagtaa
agcatttccg 1320ttgtacgggc ataactatga ttcagtmctc gatgcaattg acaaaatgga
gaaaaatctt 1380cctggattat tctatgcagg taaccacaag ggaggattgt cagttggcaa
agcactatct 1440tctggatgta atgcagcaga tcttgttata tcatatcttg aagccgtttc
aacggacwcc 1500aaaaaccata gctga
151529504PRTSolanum tuberosum 29Met Ala Pro Ser Ala Gly Glu
Asp Lys Gln Asn Cys Pro Lys Arg Val1 5 10
15Ala Val Ile Gly Ala Gly Val Ser Gly Leu Ala Ala Ala
Tyr Lys Leu 20 25 30Lys Ile
His Gly Leu Asp Val Thr Val Phe Glu Ala Glu Gly Arg Ala 35
40 45Gly Gly Lys Leu Arg Ser Leu Ser Gln Asp
Gly Leu Ile Trp Asp Glu 50 55 60Gly
Ala Asn Thr Met Thr Glu Ser Glu Gly Asp Val Thr Phe Leu Leu65
70 75 80Asp Ser Leu Gly Leu Arg
Glu Lys Gln Gln Phe Pro Leu Ser Gln Asn 85
90 95Lys Arg Tyr Ile Ala Arg Asn Gly Thr Pro Thr Leu
Ile Pro Ser Asn 100 105 110Pro
Ile Asp Leu Ile Lys Ser Asn Phe Leu Ser Thr Gly Ser Lys Leu 115
120 125Gln Met Leu Phe Glu Pro Leu Leu Trp
Lys Asn Lys Lys Leu Thr Lys 130 135
140Val Ser Asp Glu His Glu Ser Val Ser Gly Phe Phe Gln Arg His Phe145
150 155 160Gly Lys Glu Val
Val Asp Tyr Leu Ile Asp Pro Phe Val Ala Gly Thr 165
170 175Cys Gly Gly Asp Pro Asp Ser Leu Ser Met
His Leu Ser Phe Pro Glu 180 185
190Leu Trp Asn Leu Glu Lys Arg Phe Gly Ser Val Ile Val Gly Ala Ile
195 200 205Arg Ser Lys Leu Ser Pro Ile
Lys Glu Lys Lys Gln Gly Pro Pro Lys 210 215
220Thr Ser Val Asn Lys Lys Arg Gln Arg Gly Ser Phe Ser Phe Leu
Gly225 230 235 240Gly Met
Gln Thr Leu Thr Asp Ala Ile Cys Asn Asp Leu Lys Glu Asp
245 250 255Glu Leu Arg Leu Asn Ser Arg
Val Leu Glu Leu Ser Cys Ser Cys Ser 260 265
270Gly Asp Ser Ala Thr Asp Ser Trp Ser Ile Phe Ser Ala Ser
Pro His 275 280 285Lys Arg Gln Ala
Glu Glu Asp Ser Phe Asp Ala Val Ile Met Thr Ala 290
295 300Pro Leu Cys Asp Val Lys Gly Met Lys Ile Ala Lys
Arg Gly Asn Pro305 310 315
320Phe Leu Leu Asn Phe Ile Pro Glu Val Asp Tyr Val Pro Leu Ser Val
325 330 335Val Ile Thr Thr Phe
Lys Lys Glu Ser Val Lys His Pro Leu Glu Gly 340
345 350Phe Gly Val Leu Val Pro Ser Glu Glu Gln Lys His
Gly Leu Lys Thr 355 360 365Leu Gly
Thr Leu Phe Ser Ser Met Met Phe Pro Asp Arg Ala Pro Asn 370
375 380Asn Val Tyr Leu Tyr Thr Thr Phe Val Gly Gly
Ser Arg Asn Arg Glu385 390 395
400Leu Ala Lys Ala Ser Arg Thr Glu Leu Lys Glu Ile Val Thr Ser Asp
405 410 415Leu Lys Gln Leu
Leu Gly Ala Glu Gly Glu Pro Thr Tyr Val Asn His 420
425 430Val Cys Trp Ser Lys Ala Phe Pro Leu Tyr Gly
His Asn Tyr Asp Ser 435 440 445Val
Leu Asp Ala Ile Asp Lys Met Glu Lys Asn Leu Pro Gly Leu Phe 450
455 460Tyr Ala Gly Asn His Lys Gly Gly Leu Ser
Val Gly Lys Ala Leu Ser465 470 475
480Ser Gly Cys Asn Ala Ala Asp Leu Val Ile Ser Tyr Leu Glu Ala
Val 485 490 495Ser Thr Asp
Thr Lys Asn His Arg 500301608DNASolanum tuberosum 30atggctccat
ctgccggaga agataaacaa aattgtccca agagagttgc agtcattggt 60gctggcgtca
gtggacttgc tgcagcatac aagttgaaaa ttcatggctt ggatgtcaca 120gtattcgaag
cagaagggag agctggaggg aagttacgaa gcctgagtca agatggccta 180atatgggatg
aaggcgcaaa tactatgact gaaagtgaag gtgatgtcac atttttgctt 240gattcgcttg
gactccgaga aaaacaacaa tttccacttt cacagaacaa gcgctacatt 300gccagaaatg
gtactcctac tctgatacct tcaaatccaa ttgacctgat caaaagcaat 360tttctttcca
ctggatcaaa gcttcagatg cttttcgagc cacttttgtg gaagaataaa 420aagcttacaa
aggtgtctga cgaacacgaa agtgtcagtg gattcttcca gcgtcatttt 480ggaaaggagg
ttgttgacta tctaattgat ccttttgttg ctggaacatg tggtggtgat 540cctgactcgc
tttcaatgca cctttcgttt ccagagttgt ggaatttaga gaaaaggttt 600ggctcagtca
tagttggggc aattcgatcc aagttatcac ctataaagga aaagaaacaa 660ggaccaccca
aaacttcagt aaataagaag cgccagcggg ggtccttttc atttttgggc 720ggaatgcaaa
cacttactga cgcaatatgc aatgatctca aagaagatga acttaggcta 780aactctagag
ttctggaatt atcttgtagc tgtagtgggg actctgcgac agatagctgg 840tcaatttttt
ctgcctcacc acacaagcgg caagcagaag aagattcatt tgatgctgta 900attatgacgg
cccctctctg tgacgttaag ggtatgaaga ttgctaagag aggaaatcca 960tttctgctca
actttattcc tgaggttgat tatgtaccac tatctgttgt tataaccaca 1020tttaagaagg
agagtgtaaa gcatcctctt gagggttttg gagtgcttgt accttccgag 1080gagcaaaaac
atggtctgaa gacattaggc accctcttct cttctatgat gtttccagat 1140cgtgcaccca
acaatgtcta tctctatact acatttgttg gtggaagccg aaatagagaa 1200ctcgcgaaag
cctcgaggac tgagctgaaa gagatagtaa cttctgacct taagcagttg 1260ttgggtgctg
agggagagcc aacatatgtg aatcatgtat gctggagtaa agcatttccg 1320ttgtacgggc
ataactatga ttcagtcctc gatgcaattg acaaaatgga gaaaaatctt 1380cctggattat
tctatgcagg taaccacaag ggaggattgt cagttggcaa agcactatct 1440tctggatgta
atgcagcaga tcttgttata tcatatcttg aagccgtttc aacggacacc 1500aaaaaccata
ggtgaaatct attctctcat gcagcttgcc gttctttgtt ccacaaaatc 1560gtttaacttc
atgacgagga gcaactttaa cgtgcagcca gtgacgca
160831504PRTSolanum tuberosum 31Met Ala Pro Ser Ala Gly Glu Asp Lys Gln
Asn Cys Pro Lys Arg Val1 5 10
15Ala Val Ile Gly Ala Gly Val Ser Gly Leu Ala Ala Ala Tyr Lys Leu
20 25 30Lys Ile His Gly Leu Asp
Val Thr Val Phe Glu Ala Glu Gly Arg Ala 35 40
45Gly Gly Lys Leu Arg Ser Leu Ser Gln Asp Gly Leu Ile Trp
Asp Glu 50 55 60Gly Ala Asn Thr Met
Thr Glu Ser Glu Gly Asp Val Thr Phe Leu Leu65 70
75 80Asp Ser Leu Gly Leu Arg Glu Lys Gln Gln
Phe Pro Leu Ser Gln Asn 85 90
95Lys Arg Phe Ile Ala Arg Asn Gly Thr Pro Thr Leu Ile Pro Ser Asn
100 105 110Pro Ile Asp Leu Ile
Lys Ser Asn Phe Leu Ser Thr Gly Ser Lys Leu 115
120 125Gln Met Leu Phe Glu Pro Leu Leu Trp Lys Asn Lys
Lys Leu Thr Lys 130 135 140Val Ser Asp
Glu His Glu Ser Val Ser Gly Phe Phe Gln Arg His Phe145
150 155 160Gly Lys Glu Val Val Asp Tyr
Leu Ile Asp Pro Phe Val Ala Gly Thr 165
170 175Cys Gly Gly Asp Pro Asp Ser Leu Ser Met His Leu
Ser Phe Pro Glu 180 185 190Leu
Trp Asn Leu Glu Lys Arg Phe Gly Ser Val Ile Val Gly Ala Ile 195
200 205Arg Ser Lys Leu Ser Pro Ile Lys Glu
Lys Lys Gln Gly Pro Pro Lys 210 215
220Thr Ser Glu Asn Lys Lys Arg Gln Arg Gly Ser Phe Ser Phe Leu Gly225
230 235 240Gly Met Gln Thr
Leu Thr Asp Ala Ile Cys Asn Asp Leu Lys Glu Asp 245
250 255Glu Leu Arg Leu Asn Ser Arg Val Leu Glu
Leu Ser Cys Ser Cys Ser 260 265
270Gly Asp Ser Ala Thr Asp Ser Trp Ser Ile Phe Ser Ala Ser Pro His
275 280 285Lys Arg Gln Ala Glu Glu Asp
Ser Phe Asp Ala Val Ile Met Thr Ala 290 295
300Pro Leu Cys Asp Val Lys Gly Met Lys Ile Ala Lys Arg Gly Asn
Pro305 310 315 320Phe Leu
Leu Asn Phe Ile Pro Glu Val Asp Tyr Val Pro Leu Ser Val
325 330 335Val Ile Thr Thr Phe Lys Lys
Glu Ser Val Lys His Pro Leu Glu Gly 340 345
350Phe Gly Val Leu Val Pro Ser Glu Glu Gln Lys His Gly Leu
Lys Thr 355 360 365Leu Gly Thr Leu
Phe Ser Ser Met Met Phe Pro Asp Arg Ala Pro Asn 370
375 380Asn Val Tyr Leu Tyr Thr Thr Phe Val Gly Gly Ser
Arg Asn Arg Glu385 390 395
400Leu Ala Lys Ala Ser Arg Thr Glu Leu Lys Glu Ile Val Thr Ser Asp
405 410 415Leu Lys Gln Leu Leu
Gly Ala Glu Gly Glu Pro Thr Tyr Val Asn His 420
425 430Val Cys Trp Ser Lys Ala Phe Pro Leu Tyr Gly His
Asn Tyr Asp Ser 435 440 445Val Leu
Asp Ala Ile Asp Lys Met Glu Lys Asn Leu Pro Gly Leu Phe 450
455 460Tyr Ala Gly Asn His Lys Gly Gly Leu Ser Val
Gly Lys Ala Leu Ser465 470 475
480Ser Gly Cys Asn Ala Ala Asp Leu Val Ile Ser Tyr Leu Glu Ala Val
485 490 495Ser Thr Asp Thr
Lys Asn His Arg 500321608DNASolanum tuberosum 32atggctccat
ctgccggaga agataaacaa aattgtccca agagagttgc agtcattggt 60gctggcgtca
gtggacttgc tgcagcatac aagttgaaaa ttcatggctt ggatgtcaca 120gtattcgaag
cagaagggag agctggaggg aagttacgaa gcctgagtca agatggccta 180atatgggatg
aaggcgcaaa tactatgact gaaagtgaag gtgatgtcac atttttgctt 240gattcgcttg
gactccgaga aaaacaacaa tttccacttt cacagaacaa gcgcttcatt 300gccagaaatg
gtactcctac tctgatacct tcaaatccaa ttgacctgat caaaagcaat 360tttctttcca
ctggatcaaa gcttcagatg cttttcgagc cacttttgtg gaagaataaa 420aagcttacaa
aggtgtctga cgaacacgaa agtgtcagtg gattcttcca gcgtcatttt 480ggaaaggagg
ttgttgacta tctaattgat ccttttgttg ctggaacatg tggtggtgat 540cctgactcgc
tttcaatgca cctttcgttt ccagagttgt ggaatttaga gaaaaggttt 600ggctcagtca
tagttggggc aattcgatcc aagttatcac ctataaagga aaagaaacaa 660ggaccaccca
aaacttcaga aaataagaag cgccagcggg ggtccttttc atttttgggc 720ggaatgcaaa
cacttactga cgcaatatgc aatgatctca aagaagatga acttaggcta 780aactctagag
ttctggaatt atcttgtagc tgtagtgggg actctgcgac agatagctgg 840tcaatttttt
ctgcctcacc acacaagcgg caagcagaag aagattcatt tgatgctgta 900attatgacgg
cccctctctg tgacgttaag ggtatgaaga ttgctaagag aggaaatcca 960tttctgctca
actttattcc tgaggttgat tatgtaccac tatctgttgt tataaccaca 1020tttaagaagg
agagtgtaaa gcatcctctt gagggttttg gagtgcttgt accttccgag 1080gagcaaaaac
atggtctgaa gacattaggc accctcttct cttctatgat gtttccagat 1140cgtgcaccca
acaatgtcta tctctatact acatttgttg gtggaagccg aaatagagaa 1200ctcgcgaaag
cctcgaggac tgagctgaaa gagatagtaa cttctgacct taagcagttg 1260ttgggtgctg
agggagagcc aacatatgtg aatcatgtat gctggagtaa agcatttccg 1320ttgtacgggc
ataactatga ttcagtcctc gatgcaattg acaaaatgga gaaaaatctt 1380cctggattat
tctatgcagg taaccacaag ggaggattgt cagttggcaa agcactatct 1440tctggatgta
atgcagcaga tcttgttata tcatatcttg aagccgtttc aacggacacc 1500aaaaaccata
ggtgaaatct attctctcat gcagcttgcc gttctttgtt ccacaaaatc 1560gtttaacttc
atgacgagga gcaactttaa cgtgcagcca gtgacgca
160833535PRTBrassica napus 33Met Asp Leu Ser Leu Leu Arg Pro Gln Pro Phe
Leu Ser Pro Phe Ser1 5 10
15Asn Pro Phe Pro Arg Ser Arg Pro Tyr Lys Pro Leu Asn Leu Arg Cys
20 25 30Ser Val Ser Gly Gly Ser Val
Val Ser Ser Thr Ile Glu Gly Gly Gly 35 40
45Gly Gly Lys Thr Val Thr Ala Asp Cys Val Ile Val Gly Gly Gly
Ile 50 55 60Ser Gly Leu Cys Ile Ala
Gln Ala Leu Val Thr Lys His Pro Asp Ala65 70
75 80Ala Lys Asn Val Met Val Thr Glu Ala Lys Asp
Arg Val Gly Gly Asn 85 90
95Ile Ile Thr Arg Glu Glu Gln Gly Phe Leu Trp Glu Glu Gly Pro Asn
100 105 110Ser Phe Gln Pro Ser Asp
Pro Met Leu Thr Met Val Val Asp Ser Gly 115 120
125Leu Lys Asp Asp Leu Val Leu Gly Asp Pro Thr Ala Pro Arg
Phe Val 130 135 140Leu Trp Asn Gly Lys
Leu Arg Pro Val Pro Ser Lys Leu Thr Asp Leu145 150
155 160Pro Phe Phe Asp Leu Met Ser Ile Gly Gly
Lys Ile Arg Ala Gly Phe 165 170
175Gly Ala Ile Gly Ile Arg Pro Ser Pro Pro Gly Arg Glu Glu Ser Val
180 185 190Glu Glu Phe Val Arg
Arg Asn Leu Gly Asp Glu Val Phe Glu Arg Leu 195
200 205Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala Gly Asp
Pro Ala Lys Leu 210 215 220Ser Met Lys
Ala Ala Phe Gly Lys Val Trp Lys Leu Glu Glu Asn Gly225
230 235 240Gly Ser Ile Ile Gly Gly Ala
Phe Lys Ala Ile Gln Ala Lys Asn Lys 245
250 255Ala Pro Lys Thr Thr Arg Asp Pro Arg Leu Pro Lys
Pro Lys Gly Gln 260 265 270Thr
Val Gly Ser Phe Arg Lys Gly Leu Thr Met Leu Pro Asp Ala Ile 275
280 285Ser Ala Arg Leu Gly Asp Lys Val Lys
Val Ser Trp Lys Leu Ser Ser 290 295
300Ile Ser Lys Leu Pro Ser Gly Gly Tyr Ser Leu Thr Tyr Glu Thr Pro305
310 315 320Glu Gly Ile Val
Thr Val Gln Ser Lys Ser Val Val Met Thr Val Pro 325
330 335Ser His Val Ala Ser Ser Leu Leu Arg Pro
Leu Ser Asp Ser Ala Ala 340 345
350Glu Ala Leu Ser Lys Leu Tyr Tyr Pro Pro Val Ala Ala Val Ser Ile
355 360 365Ser Tyr Pro Lys Glu Ala Ile
Arg Ser Glu Cys Leu Ile Asp Gly Glu 370 375
380Leu Lys Gly Phe Gly Gln Leu His Pro Arg Thr Gln Lys Val Glu
Thr385 390 395 400Leu Gly
Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala Pro Pro
405 410 415Gly Arg Val Leu Leu Leu Asn
Tyr Ile Gly Gly Ala Thr Asn Thr Gly 420 425
430Ile Leu Ser Lys Ser Glu Gly Glu Leu Val Glu Ala Val Asp
Arg Asp 435 440 445Leu Arg Lys Met
Leu Ile Lys Pro Ser Ser Thr Asp Pro Leu Val Leu 450
455 460Gly Val Lys Val Trp Pro Gln Ala Ile Pro Gln Phe
Leu Ile Gly His465 470 475
480Ile Asp Leu Val Asp Ala Ala Lys Ala Ser Leu Ser Ser Ser Gly His
485 490 495Glu Gly Leu Phe Leu
Gly Gly Asn Tyr Val Ala Gly Val Ala Leu Gly 500
505 510Arg Cys Val Glu Gly Ala Tyr Glu Thr Ala Thr Gln
Val Asn Asp Phe 515 520 525Met Ser
Arg Tyr Ala Tyr Lys 530 535341830DNABrassica napus
34ttgaacaaag aggctggacc ggtccggaat tcccgggata tcgtcgaccc acgcgtccgg
60tcgacgctga tcggagataa gagtcgacaa aattgaggat tctccttctc gcgggcgatc
120gccatggatt tatctcttct ccgtccgcag ccattcctat cgccattctc aaatccattt
180cctcggtcgc gtccctacaa gcctctcaac ctccgttgct ccgtatccgg tggatccgtc
240gtctcttcta caatcgaagg cggaggagga ggtaaaaccg tcacggcgga ctgcgtgatc
300gtcggcggag gaatcagcgg cctgtgcatt gcgcaagcgc tcgtgacgaa gcacccagac
360gctgcaaaga atgtgatggt gacggaggcg aaggaccgtg tgggagggaa tatcatcacg
420cgagaggagc aagggtttct atgggaagaa ggtcccaata gctttcagcc gtctgatcct
480atgctcacta tggtggtaga tagtggtttg aaagatgatc tagtcttggg agatcctact
540gctccgaggt ttgtgttgtg gaatgggaag ctgaggccgg ttccgtcgaa gctaactgac
600ttgcctttct ttgacttgat gagtattgga gggaagatta gagctgggtt tggtgccatt
660ggtattcgac cttcacctcc gggtcgtgag gaatcagtgg aagagtttgt aaggcgtaat
720cttggtgatg aggtttttga gcgcttgatt gaaccctttt gctcaggtgt ttatgcggga
780gatcctgcga aactgagtat gaaagcagct tttgggaagg tttggaagct agaggagaat
840ggtgggagca tcattggtgg tgcttttaag gcaattcaag cgaaaaataa agctcccaag
900acaacccgag acccgcgtct gccaaagcca aagggccaaa cagttggttc tttcaggaaa
960ggactcacaa tgctgccaga cgcaatctct gcaaggttgg gtgacaaggt gaaagtttct
1020tggaagctct caagtatcag taagctgccc agcggaggat atagcttaac ttacgaaact
1080ccggagggga tagtcactgt acagagcaaa agtgttgtga tgactgtgcc atctcatgtt
1140gctagtagtc tcttgcgccc tctctctgac tctgcagctg aagcgctctc aaaactctac
1200tatccaccag ttgcagcagt atctatctca tacccgaaag aagcaatccg aagcgaatgt
1260ttaatagatg gtgaactaaa agggttcggc cagttgcatc cacgcacgca gaaagtggaa
1320actcttggaa caatatacag ttcatcgctc tttcctaacc gagcaccacc tggaagagtg
1380ttgctactga actacatcgg tggagctacc aacactggga tcttatcaaa gtcagaaggt
1440gagttagtgg aagcagtgga tagagacttg aggaagatgc tgataaagcc aagctcgacc
1500gatccacttg tacttggagt aaaagtttgg cctcaagcca ttcctcagtt tctgataggt
1560cacattgatt tggtagacgc agcgaaagca tctctctcgt catctggcca tgagggctta
1620ttcttgggtg gaaattacgt tgccggtgta gcattgggtc ggtgtgtgga aggtgcttat
1680gaaactgcaa cccaagtgaa cgatttcatg tcgaggtacg cttacaagta atgtaacgca
1740gcaacggttt gatactaagt tgtagattgc agttttgact ctgtttgtga aaaattcaag
1800tctatgattg agtaaattta tatgtattaa
183035537PRTBrassica napus 35Met Asp Leu Ser Leu Leu Arg Pro Gln Pro Phe
Leu Ser Pro Phe Ser1 5 10
15Asn Pro Phe Pro Arg Ser Arg Pro Tyr Lys Pro Leu Asn Leu Arg Cys
20 25 30Ser Val Ser Gly Gly Ser Val
Val Val Gly Ser Ser Thr Ile Glu Gly 35 40
45Gly Gly Gly Gly Lys Thr Val Ala Ala Asp Cys Val Ile Val Gly
Gly 50 55 60Gly Ile Ser Gly Leu Cys
Ile Ala Gln Ala Leu Val Thr Lys His Pro65 70
75 80Asp Ala Ala Lys Ser Val Met Val Thr Glu Ala
Lys Asp Arg Val Gly 85 90
95Gly Asn Ile Ile Thr Arg Glu Glu Gln Gly Phe Leu Trp Glu Glu Gly
100 105 110Pro Asn Ser Phe Gln Pro
Ser Asp Pro Met Leu Thr Met Val Val Asp 115 120
125Ser Gly Leu Lys Asp Asp Leu Val Leu Gly Asp Pro Thr Ala
Pro Arg 130 135 140Phe Val Leu Trp Asn
Gly Lys Leu Arg Pro Val Pro Ser Lys Leu Thr145 150
155 160Asp Leu Pro Phe Phe Asp Leu Met Ser Ile
Gly Gly Lys Ile Arg Ala 165 170
175Gly Phe Gly Ala Ile Gly Ile Arg Pro Ser Pro Pro Gly Arg Glu Glu
180 185 190Ser Val Glu Glu Phe
Val Arg Arg Asn Leu Gly Asp Glu Val Phe Glu 195
200 205Arg Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala
Gly Asp Pro Ala 210 215 220Lys Leu Ser
Met Lys Ala Ala Leu Gly Lys Val Trp Lys Leu Lys Glu225
230 235 240Asn Gly Gly Ser Ile Ile Gly
Gly Ala Phe Lys Ala Ile Gln Ala Lys 245
250 255Asn Lys Ala Pro Lys Thr Thr Arg Asp Pro Arg Leu
Pro Lys Pro Lys 260 265 270Gly
Gln Thr Val Gly Ser Phe Arg Lys Gly Leu Thr Met Leu Pro Asp 275
280 285Ala Ile Ser Ala Arg Leu Gly Asp Lys
Val Lys Val Ser Trp Lys Leu 290 295
300Ser Ser Ile Ser Lys Leu Pro Ser Gly Gly Tyr Ser Leu Thr Tyr Glu305
310 315 320Thr Pro Glu Gly
Ile Val Thr Val Gln Ser Lys Ser Val Val Met Thr 325
330 335Val Pro Ser His Val Ala Ser Ser Leu Leu
Arg Pro Leu Ser Asp Ser 340 345
350Ala Ala Glu Ala Leu Ser Lys Leu Tyr Tyr Pro Pro Val Ala Ala Val
355 360 365Ser Ile Ser Tyr Pro Lys Glu
Ala Ile Arg Ser Glu Cys Leu Ile Asp 370 375
380Gly Glu Leu Lys Gly Phe Gly Gln Leu His Pro Arg Thr Gln Lys
Val385 390 395 400Glu Thr
Leu Gly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala
405 410 415Pro Pro Gly Arg Val Leu Leu
Leu Asn Tyr Ile Gly Gly Ala Thr Asn 420 425
430Thr Gly Ile Leu Ser Lys Ser Glu Gly Glu Leu Val Glu Ala
Val Asp 435 440 445Arg Asp Leu Arg
Lys Met Leu Ile Lys Pro Ser Ser Thr Asp Pro Leu 450
455 460Val Leu Gly Val Lys Val Trp Pro Gln Ala Ile Pro
Gln Phe Leu Ile465 470 475
480Gly His Ile Asp Leu Val Asp Ala Ala Lys Ala Ser Leu Ser Ser Ser
485 490 495Gly His Glu Gly Leu
Phe Leu Gly Gly Asn Tyr Val Ala Gly Val Ala 500
505 510Leu Gly Arg Cys Val Glu Gly Ala Tyr Glu Thr Ala
Thr Gln Val Asn 515 520 525Asp Phe
Met Ser Arg Tyr Ala Tyr Lys 530 535361668DNABrassica
napus 36gatcggagat aaggttgacg aaattgagaa tcctcctcct cgcgggccat cgccatggat
60ttatctcttc tccgtccgca gccattccta tcgccattct caaatccatt tcctcggtcg
120cgtccctaca agcctctcaa cctccgttgc tccgtatccg gtggatccgt cgtcgtcggc
180tcgtccacaa tcgaaggcgg aggaggaggt aaaaccgtcg cggcggattg cgtgatcgtc
240ggcggaggaa tcagcggcct gtgcattgcg caagcgctcg tgacgaagca cccggacgct
300gcgaagagtg tgatggtgac ggaggcgaag gaccgcgtgg gagggaatat catcacgcga
360gaggagcaag ggtttctatg ggaagaaggt cccaacagct ttcagccgtc tgatcctatg
420ctcactatgg tggtagatag tggtttgaag gatgatctag tcttgggaga tcctactgcg
480ccgaggttcg tgttgtggaa tgggaagctg aggccggttc cgtcgaagct aactgacttg
540cctttctttg acttgatgag cattggaggg aagattagag ctgggtttgg tgccattggc
600attcgaccgt cacctccagg tcgtgaggaa tctgtggaag agtttgtaag gcgtaacctt
660ggtgatgagg tttttgagcg tttgattgaa cccttttgtt caggtgttta tgcgggagat
720cctgcgaaac tgagtatgaa agcagctttg gggaaggttt ggaaactaaa ggagaatggt
780ggaagcatca taggtggtgc ttttaaggca attcaagcga aaaataaagc tcccaagaca
840acccgagacc cgcgtctgcc aaagccaaag ggccaaacag ttggttcttt caggaaagga
900ctcacaatgc tgccagacgc aatctctgca aggttgggtg acaaggtgaa agtttcttgg
960aagctctcaa gtatcagtaa gctgcccagc ggaggatata gcttaactta cgaaactccg
1020gaggggatag tcactgtaca gagcaaaagt gttgtgatga ctgtgccatc tcatgttgct
1080agtagtctct tgcgccctct ctctgactct gcagctgaag cgctctcaaa actctactat
1140ccaccagttg cagcagtatc tatctcatac ccgaaagaag caatccgaag cgaatgttta
1200atagatggtg aactaaaagg gttcggccag ttgcatccac gcacgcagaa agtggaaact
1260cttggaacaa tatacagttc atcgctcttt cctaaccgag caccacctgg aagagtgttg
1320ctactgaact acatcggtgg agctaccaac actgggatct tatcaaagtc agaaggtgag
1380ttagtggaag cagtggatag agacttgagg aagatgctga taaagccaag ctcgaccgat
1440ccacttgtac ttggagtaaa agtttggcct caagccattc ctcagtttct gataggtcac
1500attgatttgg tagacgcagc gaaagcatct ctctcgtcat ctggccatga gggcttattc
1560ttgggtggaa attacgttgc cggtgtagca ttgggtcggt gtgtggaagg tgcttatgaa
1620actgcaaccc aagtgaacga tttcatgtcg aggtacgctt acaagtaa
166837212PRTBrassica napus 37Val Thr Val Gln Ser Lys Ser Val Val Met Thr
Val Pro Ser His Val1 5 10
15Ala Ser Ser Leu Leu Arg Pro Leu Ser Asp Ser Ala Ala Glu Ala Leu
20 25 30Ser Lys Leu Tyr Tyr Pro Pro
Val Ala Ala Val Ser Ile Ser Tyr Ala 35 40
45Lys Glu Ala Ile Arg Ser Glu Cys Leu Ile Asp Gly Glu Leu Lys
Gly 50 55 60Phe Gly Gln Leu His Pro
Arg Thr Gln Lys Val Glu Thr Leu Gly Thr65 70
75 80Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala
Pro Pro Gly Arg Val 85 90
95Leu Leu Leu Asn Tyr Ile Gly Gly Ala Thr Asn Thr Gly Ile Leu Ser
100 105 110Lys Ser Glu Gly Glu Leu
Val Glu Ala Val Asp Arg Asp Leu Arg Lys 115 120
125Met Leu Ile Lys Pro Ser Ser Thr Asp Pro Leu Val Leu Gly
Val Lys 130 135 140Leu Trp Pro Gln Ala
Ile Pro Gln Phe Leu Ile Gly His Ile Asp Leu145 150
155 160Val Asp Ala Ala Lys Ala Ser Leu Ser Ser
Ser Gly His Glu Gly Leu 165 170
175Phe Leu Gly Gly Asn Tyr Val Ala Gly Val Ala Leu Gly Arg Cys Val
180 185 190Glu Gly Ala Tyr Glu
Thr Ala Thr Gln Val Asn Asp Phe Met Ser Arg 195
200 205Tyr Ala Tyr Lys 21038757DNABrassica napus
38tagtcactgt acagagcaaa agtgtagtga tgactgtgcc atctcatgta gctagtagtc
60tcttgcgccc tctctctgat tctgcagctg aagcgctctc aaaactctac tatccgccag
120ttgcagccgt atccatctca tacgcgaaag aagcaatccg aagcgaatgc ttaatagatg
180gtgaactaaa agggttcggc cagttgcatc cacgcacgca aaaagtggaa actcttggaa
240caatatacag ttcatcgctc tttcccaacc gagcaccgcc tggaagagta ttgctattga
300actacatcgg tggagctacc aacactggga tcttatcaaa gtcggaaggt gagttagtgg
360aagcagtaga tagagacttg aggaagatgc tgataaagcc aagctcgacc gatccacttg
420tacttggagt aaaattatgg cctcaagcca ttcctcagtt tctgataggt cacattgatt
480tggtagacgc agcgaaagca tcgctctcgt catctggtca tgagggctta ttcttgggtg
540gaaattacgt tgccggtgta gcattgggtc ggtgtgtgga aggtgcttat gaaactgcaa
600cccaagtgaa tgatttcatg tcaaggtatg cttacaagta atgtaacgca gcaacgattt
660gatactaagt agtagatttc gcagttctga ctttaagaac actctgtttg tgaaaaattc
720aagtctgtga ttgagtaaat ttatgtatta ttactaa
75739536PRTGlycine max 39Met Val Ala Ala Ala Ala Met Ala Thr Ala Ala Ser
Ala Ala Ala Pro1 5 10
15Leu Leu Asn Gly Thr Arg Arg Pro Ala Arg Leu Arg Arg Arg Gly Leu
20 25 30Arg Val Arg Cys Ala Ala Val
Ala Gly Gly Ala Ala Glu Ala Pro Ala 35 40
45Ser Thr Gly Ala Arg Leu Ser Ala Asp Cys Val Val Val Gly Gly
Gly 50 55 60Ile Ser Gly Leu Cys Thr
Ala Gln Ala Leu Ala Thr Arg His Gly Val65 70
75 80Gly Glu Val Leu Val Thr Glu Ala Arg Ala Arg
Pro Gly Gly Asn Ile 85 90
95Thr Thr Val Glu Arg Pro Glu Glu Gly Tyr Leu Trp Glu Glu Gly Pro
100 105 110Asn Ser Phe Gln Pro Ser
Asp Pro Val Leu Ser Met Ala Val Asp Ser 115 120
125Gly Leu Lys Asp Asp Leu Val Phe Gly Asp Pro Asn Ala Pro
Arg Phe 130 135 140Val Leu Trp Glu Gly
Lys Leu Arg Pro Val Pro Ser Lys Pro Ala Asp145 150
155 160Leu Pro Phe Phe Asp Leu Met Ser Ile Pro
Gly Lys Leu Arg Ala Gly 165 170
175Leu Gly Ala Leu Gly Ile Arg Pro Pro Pro Pro Gly Arg Glu Glu Ser
180 185 190Val Glu Glu Phe Val
Arg Arg Asn Leu Gly Ala Glu Val Phe Glu Arg 195
200 205Leu Ile Glu Pro Phe Cys Ser Gly Val Tyr Ala Gly
Asp Pro Ser Lys 210 215 220Leu Ser Met
Lys Ala Ala Phe Gly Lys Val Trp Arg Leu Glu Glu Ala225
230 235 240Gly Gly Ser Ile Ile Gly Gly
Thr Ile Lys Thr Ile Gln Glu Arg Gly 245
250 255Lys Asn Pro Lys Pro Pro Arg Asp Pro Arg Leu Pro
Lys Pro Lys Gly 260 265 270Gln
Thr Val Ala Ser Phe Arg Lys Gly Leu Ala Met Leu Pro Asn Ala 275
280 285Ile Thr Ser Ser Leu Gly Ser Lys Val
Lys Leu Ser Trp Lys Leu Thr 290 295
300Ser Ile Thr Lys Ser Asp Gly Lys Gly Tyr Val Leu Glu Tyr Glu Thr305
310 315 320Pro Glu Gly Val
Val Leu Val Gln Ala Lys Ser Val Ile Met Thr Ile 325
330 335Pro Ser Tyr Val Ala Ser Asp Ile Leu Arg
Pro Leu Ser Gly Asp Ala 340 345
350Ala Asp Ala Leu Ser Arg Phe Tyr Tyr Pro Pro Val Ala Ala Val Thr
355 360 365Val Ser Tyr Pro Lys Glu Ala
Ile Arg Lys Glu Cys Leu Ile Asp Gly 370 375
380Glu Leu Gln Gly Phe Gly Gln Leu His Pro Arg Ser Gln Gly Val
Glu385 390 395 400Thr Leu
Gly Thr Ile Tyr Ser Ser Ser Leu Phe Pro Asn Arg Ala Pro
405 410 415Ala Gly Arg Val Leu Leu Leu
Asn Tyr Ile Gly Gly Ala Thr Asn Thr 420 425
430Gly Ile Val Ser Lys Thr Glu Ser Glu Leu Val Glu Ala Val
Asp Arg 435 440 445Asp Leu Arg Lys
Met Leu Ile Asn Ser Thr Ala Val Asp Pro Leu Val 450
455 460Leu Gly Val Arg Val Trp Pro Gln Ala Ile Pro Gln
Phe Leu Val Gly465 470 475
480His Leu Asp Leu Leu Glu Val Ala Lys Ser Ala Leu Asp Gln Gly Gly
485 490 495Tyr Asp Gly Leu Phe
Leu Gly Gly Asn Tyr Val Ala Gly Val Ala Leu 500
505 510Gly Arg Cys Ile Glu Gly Ala Tyr Glu Ser Ala Ala
Gln Ile Tyr Asp 515 520 525Phe Leu
Thr Lys Tyr Ala Tyr Lys 530 53540551PRTGlycine max
40Met Val Ser Val Phe Asn Asp Ile Leu Phe Pro Pro Asn Gln Thr Leu1
5 10 15Ser Pro Thr Ser Phe Phe
Thr Ser Pro Thr Arg Lys Phe Pro Arg Ser 20 25
30Arg Pro Asn Pro Ile Leu Arg Cys Ser Ile Ala Glu Glu
Ser Thr Glu 35 40 45Ser Arg Pro
Lys Thr Gly Asp Ser Pro Pro Pro Pro Leu Met Glu Ala 50
55 60Leu Ala Val Trp His Arg Pro Gly Pro Arg His Gln
Ala Arg Gln Cys65 70 75
80Gln His Cys Trp Gly Asp Ser Arg Ala Arg Asp Arg Val Gly Gly Gly
85 90 95Asn Ile Thr Thr Met Glu
Ser Gly Gly Tyr Leu Trp Glu Glu Gly Pro 100
105 110Asn Ser Phe Gln Pro Ser Asp Pro Met Leu Thr Met
Val Val Asp Ser 115 120 125Gly Leu
Lys Asp Gln Leu Val Leu Gly Asp Pro Asp Ala Pro Arg Phe 130
135 140Val Leu Trp Asn Gly Lys Leu Arg Pro Val Pro
Gly Lys Pro Thr Asp145 150 155
160Leu Pro Phe Phe Asp Leu Met Ser Ile Gly Gly Lys Ile Arg Ala Gly
165 170 175Phe Gly Val Leu
Gly Ile Arg Pro Pro Pro Pro Val Glu Glu Phe Val 180
185 190Arg Arg Asn Leu Gly Asp Asp Val Phe Glu Arg
Leu Ile Glu Pro Phe 195 200 205Cys
Ser Gly Gly Asn Thr Cys Ile Phe Lys Phe Val Gly Ala Leu Leu 210
215 220Ile Leu Trp Gly Leu Cys Arg Arg Ser Phe
Lys Ile Lys Tyr Glu Ser225 230 235
240Ser Ile Trp Glu Ser Leu Glu Ala Gly Lys Asn Gly Gly Ser Ile
Ile 245 250 255Gly Gly Thr
Phe Lys Ala Ile Gln Glu Arg Asn Gly Ala Ser Lys Pro 260
265 270Pro Arg Asp Pro Arg Leu Pro Lys Pro Lys
Gly Gln Thr Val Gly Ser 275 280
285Phe Arg Lys Gly Leu Ile Met Leu Pro Asp Ala Ile Ser Ala Arg Leu 290
295 300Gly Asn Lys Val Lys Leu Ser Trp
Lys Leu Ser Ser Ile Ser Lys Leu305 310
315 320Asp Ser Gly Glu Tyr Ser Leu Thr Tyr Glu Thr Pro
Glu Gly Val Val 325 330
335Ser Leu Gln Cys Lys Thr Val Val Leu Thr Ile Pro Ser Tyr Val Ala
340 345 350Ser Thr Leu Leu Arg Pro
Leu Ser Ala Ala Ala Ala Asp Thr Leu Ser 355 360
365Lys Phe Tyr Tyr Pro Pro Val Val Ala Val Ser Ile Ser Tyr
Pro Lys 370 375 380Glu Ala Ile Arg Ser
Glu Cys Leu Ile Asp Gly Glu Leu Lys Gly Phe385 390
395 400Gly Ala Ile Tyr Ser Ser Ser Leu Phe Ser
Asn Arg Ala Pro Pro Gly 405 410
415Arg Val Leu Leu Leu Asn Tyr Ile Gly Gly Ala Thr Asn Thr Gly Ile
420 425 430Tyr Gln Ser Phe Ser
Gly Lys Leu Gln Gly Trp Phe Lys Glu Leu Ile 435
440 445Ile Phe Thr Ser Gly Leu Phe Gly Cys Phe Lys Gln
Leu Arg Pro Asn 450 455 460Gly Leu Val
Ser Asn Thr Asp Ser Glu Leu Val Ala Thr Val Asp Arg465
470 475 480Asp Leu Arg Lys Ile Leu Ile
Asn Pro Asn Ala Gln Asp Pro Phe Val 485
490 495Val Gly Val Arg Leu Trp Pro Gln Ala Ile Pro Gln
Phe Leu Ile Gly 500 505 510His
Leu Asp Leu Leu Asp Val Ala Lys Ala Ser Leu Arg Asn Thr Gly 515
520 525Phe Glu Gly Leu Phe Leu Gly Gly Asn
Tyr Val Ser Gly Val Ala Leu 530 535
540Gly Arg Trp Val Glu Gly Ala545 550411656DNAGlycine max
41atggtttccg tcttcaacga catcctattc ccgcctaacc aaaccctttc cccaacgtcc
60ttcttcacct ctcccactcg aaaattccct cgctctcgcc ctaaccctat tctccgctgc
120tccatcgccg aggagtccac cgagtctcgg cccaaaaccg gagactcccc ccccccgccg
180ttgatggagg cgttagcggt ctggcatcgc ccaggccctc gccaccaagc acgccaatgc
240caacactgtt ggggagattc gagggcccga gaccgtgtcg gcggcggcaa catcaccacg
300atggagagtg gcggatacct ctgggaagaa ggccccaaca gctttcagcc ctctgatcca
360atgctcacca tggtggtgga cagtggctta aaggatcagc ttgttttggg ggatcctgat
420gcacctcggt ttgtgttgtg gaatgggaag ttgaggccag tgcctgggaa gccgactgat
480ttgcctttct ttgacttgat gagcatcggt ggcaaaatca gggctggctt tggtgtgctt
540ggtattcggc ctcctcctcc agttgaagag tttgttcgtc ggaaccttgg tgatgatgtt
600tttgaacgat tgatagagcc tttttgttca gggggcaata cttgtatatt taaatttgtg
660ggagcattac tcatattgtg gggtctatgc aggcgatcct tcaaaattaa gtatgaaagc
720agcatttggg aaagtttgga ggctggaaaa aatggtggta gcataattgg tggaactttc
780aaagcaatac aagagagaaa tggagcttca aaaccacctc gagatccacg tctgccaaaa
840ccaaagggtc agactgttgg atcttttcgg aagggactta tcatgttgcc tgatgcaatt
900tctgcaagat taggcaacaa agtaaagtta tcttggaagc tttcaagtat tagtaaactg
960gatagtggag agtacagttt gacatatgaa acacccgaag gagtggtttc tttgcagtgc
1020aaaaccgttg tcctgaccat tccttcctat gttgctagta cattgctgcg tcctctgtct
1080gctgctgctg cagatacgct ttcaaagttt tattaccctc cagttgttgc agtttccata
1140tcctatccaa aagaagctat tagatcagaa tgcttgatag atggtgagtt gaaggggttt
1200ggagctatat acagctcatc actattctcc aatcgagcac cacctggaag ggttctactc
1260ttgaattaca ttggaggagc tactaatact ggaatttatc aaagtttttc tgggaaactt
1320caaggatggt ttaaagaact aatcattttc accagcgggt tatttgggtg ttttaaacaa
1380ctcaggccta atggtcttgt ttcgaatacg gacagtgaac ttgtcgcaac agttgatcga
1440gatttgagaa aaatccttat aaacccaaat gcccaggatc catttgtagt gggggtgaga
1500ctgtggcctc aagctattcc acagttctta attggccatc ttgatcttct agatgttgct
1560aaagcttctc tcagaaatac tgggtttgaa gggctgttcc ttgggggtaa ctatgtgtct
1620ggtgttgcct tgggacgatg ggttgaggga gcctga
165642543PRTGlycine max 42Met Val Ser Val Phe Asn Glu Ile Leu Phe Pro Pro
Asn Gln Thr Leu1 5 10
15Leu Arg Pro Ser Leu His Ser Pro Thr Ser Phe Phe Thr Ser Pro Thr
20 25 30Arg Lys Phe Pro Arg Ser Arg
Pro Asn Pro Ile Leu Arg Cys Ser Ile 35 40
45Ala Glu Glu Ser Thr Ala Ser Pro Pro Lys Thr Arg Asp Ser Ala
Pro 50 55 60Val Asp Cys Val Val Val
Gly Gly Gly Val Ser Gly Leu Cys Ile Ala65 70
75 80Gln Ala Leu Ala Thr Lys His Ala Asn Ala Asn
Val Val Val Thr Glu 85 90
95Ala Arg Asp Arg Val Gly Gly Asn Ile Thr Thr Met Glu Arg Asp Gly
100 105 110Tyr Leu Trp Glu Glu Gly
Pro Asn Ser Phe Gln Pro Ser Asp Pro Met 115 120
125Leu Thr Met Val Val Asp Ser Gly Leu Lys Asp Glu Leu Val
Leu Gly 130 135 140Asp Pro Asp Ala Pro
Arg Phe Val Leu Trp Asn Arg Lys Leu Arg Pro145 150
155 160Val Pro Gly Lys Leu Thr Asp Leu Pro Phe
Phe Asp Leu Met Ser Ile 165 170
175Gly Gly Lys Ile Arg Ala Gly Phe Gly Ala Leu Gly Ile Arg Pro Pro
180 185 190Pro Pro Gly His Glu
Glu Ser Val Glu Glu Phe Val Arg Arg Asn Leu 195
200 205Gly Asp Glu Val Phe Glu Arg Leu Ile Glu Pro Phe
Cys Ser Gly Val 210 215 220Tyr Ala Gly
Asp Pro Ser Lys Leu Ser Met Lys Ala Ala Phe Gly Lys225
230 235 240Val Trp Lys Leu Glu Lys Asn
Gly Gly Ser Ile Ile Gly Gly Thr Phe 245
250 255Lys Ala Ile Gln Glu Arg Asn Gly Ala Ser Lys Pro
Pro Arg Asp Pro 260 265 270Arg
Leu Pro Lys Pro Lys Gly Gln Thr Val Gly Ser Phe Arg Lys Gly 275
280 285Leu Thr Met Leu Pro Asp Ala Ile Ser
Ala Arg Leu Gly Asn Lys Val 290 295
300Lys Leu Ser Trp Lys Leu Ser Ser Ile Ser Lys Leu Asp Ser Gly Glu305
310 315 320Tyr Ser Leu Thr
Tyr Glu Thr Pro Glu Gly Val Val Ser Leu Gln Cys 325
330 335Lys Thr Val Val Leu Thr Ile Pro Ser Tyr
Val Ala Ser Thr Leu Leu 340 345
350Arg Pro Leu Ser Ala Ala Ala Ala Asp Ala Leu Ser Lys Phe Tyr Tyr
355 360 365Pro Pro Val Ala Ala Val Ser
Ile Ser Tyr Pro Lys Glu Ala Ile Arg 370 375
380Ser Glu Cys Leu Ile Asp Gly Glu Leu Lys Gly Phe Gly Gln Leu
His385 390 395 400Pro Arg
Ser Gln Gly Val Glu Thr Leu Gly Thr Ile Tyr Ser Ser Ser
405 410 415Leu Phe Pro Asn Arg Ala Pro
Pro Gly Arg Val Leu Leu Leu Asn Tyr 420 425
430Ile Gly Gly Ala Thr Asn Thr Gly Ile Leu Ser Lys Thr Asp
Ser Glu 435 440 445Leu Val Glu Thr
Val Asp Arg Asp Leu Arg Lys Ile Leu Ile Asn Pro 450
455 460Asn Ala Gln Asp Pro Phe Val Val Gly Val Arg Leu
Trp Pro Gln Ala465 470 475
480Ile Pro Gln Phe Leu Val Gly His Leu Asp Leu Leu Asp Val Ala Lys
485 490 495Ala Ser Ile Arg Asn
Thr Gly Phe Glu Gly Leu Phe Leu Gly Gly Asn 500
505 510Tyr Val Ser Gly Val Ala Leu Gly Arg Cys Val Glu
Gly Ala Tyr Glu 515 520 525Val Ala
Ala Glu Val Asn Asp Phe Leu Thr Asn Arg Val Tyr Lys 530
535 540431632DNAGlycine max 43atggtttccg tcttcaacga
gatcctattc ccgccgaacc aaacccttct tcgcccctcc 60ctccattccc caacctcttt
cttcacctct cccactcgaa aattccctcg ctctcgccct 120aaccctattc tacgctgctc
cattgcggag gaatccaccg cgtctccgcc caaaaccaga 180gactccgccc ccgtggactg
cgtcgtcgtc ggcggaggcg tcagcggcct ctgcatcgcc 240caggccctcg ccaccaaaca
cgccaatgcc aacgtcgtcg tcacggaggc ccgagaccgc 300gtcggcggca acatcaccac
gatggagagg gacggatacc tctgggaaga aggccccaac 360agcttccagc cttctgatcc
aatgctcacc atggtggtgg acagtggttt aaaggatgag 420cttgttttgg gggatcctga
tgcacctcgg tttgtgttgt ggaacaggaa gttgaggccg 480gtgcccggga agctgactga
tttgcctttc tttgacttga tgagcattgg tggcaaaatc 540agggctggct ttggtgcgct
tggaattcgg cctcctcctc caggtcatga ggaatcggtt 600gaagagtttg ttcgtcggaa
ccttggtgat gaggtttttg aacggttgat agagcctttt 660tgttcagggg tctatgcagg
cgatccttca aaattaagta tgaaagcagc attcgggaaa 720gtttggaagc tggaaaaaaa
tggtggtagc attattggtg gaactttcaa agcaatacaa 780gagagaaatg gagcttcaaa
accacctcga gatccgcgtc tgccaaaacc aaaaggtcag 840actgttggat ctttccggaa
gggacttacc atgttgcctg atgcaatttc tgccagacta 900ggcaacaaag taaagttatc
ttggaagctt tcaagtatta gtaaactgga tagtggagag 960tacagtttga catatgaaac
accagaagga gtggtttctt tgcagtgcaa aactgttgtc 1020ctgaccattc cttcctatgt
tgctagtaca ttgctgcgtc ctctgtctgc tgctgctgca 1080gatgcacttt caaagtttta
ttaccctcca gttgctgcag tttccatatc ctatccaaaa 1140gaagctatta gatcagaatg
cttgatagat ggtgagttga aggggtttgg tcaattgcat 1200ccacgtagcc aaggagtgga
aacattagga actatataca gctcatcact attccccaac 1260cgagcaccac ctggaagggt
tctactcttg aattacattg gaggagcaac taatactgga 1320attttatcga agacggacag
tgaacttgtg gaaacagttg atcgagattt gaggaaaatc 1380cttataaacc caaatgccca
ggatccattt gtagtggggg tgagactgtg gcctcaagct 1440attccacagt tcttagttgg
ccatcttgat cttctagatg ttgctaaagc ttctatcaga 1500aatactgggt ttgaagggct
cttccttggg ggtaattatg tgtctggtgt tgccttggga 1560cgatgcgttg agggagccta
tgaggtagca gctgaagtaa acgattttct cacaaataga 1620gtgtacaaat ag
163244515PRTGlycine max 44Met
Ala Ser Ser Ala Thr Asp Asp Asn Pro Arg Ser Val Lys Arg Val1
5 10 15Ala Val Val Gly Ala Gly Val
Ser Gly Leu Ala Ala Ala Tyr Lys Leu 20 25
30Lys Ser His Gly Leu Asp Val Thr Val Phe Glu Ala Glu Gly
Arg Ala 35 40 45Gly Gly Arg Leu
Arg Ser Val Ser Gln Asp Gly Leu Ile Trp Asp Glu 50 55
60Gly Ala Asn Thr Met Thr Glu Ser Glu Ile Glu Val Lys
Gly Leu Ile65 70 75
80Asp Ala Leu Gly Leu Gln Glu Lys Gln Gln Phe Pro Ile Ser Gln His
85 90 95Lys Arg Tyr Ile Val Lys
Asn Gly Ala Pro Leu Leu Val Pro Thr Asn 100
105 110Pro Ala Ala Leu Leu Lys Ser Lys Leu Leu Ser Ala
Gln Ser Lys Ile 115 120 125His Leu
Ile Phe Glu Pro Phe Met Trp Lys Arg Ser Asp Pro Ser Asn 130
135 140Val Cys Asp Glu Asn Ser Val Glu Ser Val Gly
Arg Phe Phe Glu Arg145 150 155
160His Phe Gly Lys Glu Val Val Asp Tyr Leu Ile Asp Pro Phe Val Gly
165 170 175Gly Thr Ser Ala
Ala Asp Pro Glu Ser Leu Ser Met Arg His Ser Phe 180
185 190Pro Glu Leu Trp Asn Leu Glu Lys Arg Phe Gly
Ser Ile Ile Ala Gly 195 200 205Ala
Leu Gln Ser Lys Leu Phe Ala Lys Arg Glu Lys Thr Gly Glu Asn 210
215 220Arg Thr Ala Leu Arg Lys Asn Lys His Lys
Arg Gly Ser Phe Ser Phe225 230 235
240Gln Gly Gly Met Gln Thr Leu Thr Asp Thr Leu Cys Lys Glu Leu
Gly 245 250 255Lys Asp Asp
Leu Lys Leu Asn Glu Lys Val Leu Thr Leu Ala Tyr Gly 260
265 270His Asp Gly Ser Ser Ser Ser Gln Asn Trp
Ser Ile Thr Ser Ala Ser 275 280
285Asn Gln Ser Thr Gln Asp Val Asp Ala Val Ile Met Thr Asn Leu His 290
295 300Tyr Leu Lys His Ser Leu His Asn
Gly Gln Ala Pro Leu Tyr Asn Val305 310
315 320Lys Asp Ile Lys Ile Thr Lys Arg Gly Thr Pro Phe
Pro Leu Asn Phe 325 330
335Leu Pro Glu Val Ser Tyr Val Pro Ile Ser Val Met Ile Thr Thr Phe
340 345 350Lys Lys Glu Asn Val Lys
Arg Pro Leu Glu Gly Phe Gly Val Leu Val 355 360
365Pro Ser Lys Glu Gln Lys Asn Gly Leu Lys Thr Leu Gly Thr
Leu Phe 370 375 380Ser Ser Met Met Phe
Pro Asp Arg Ala Pro Ser Asp Leu Tyr Leu Tyr385 390
395 400Thr Thr Phe Ile Gly Gly Thr Gln Asn Arg
Glu Leu Ala Gln Ala Ser 405 410
415Thr Asp Glu Leu Arg Lys Ile Val Thr Ser Asp Leu Arg Lys Leu Leu
420 425 430Gly Ala Glu Gly Glu
Pro Thr Phe Val Asn His Phe Tyr Trp Ser Lys 435
440 445Gly Phe Pro Leu Tyr Gly Arg Asn Tyr Gly Ser Val
Leu Gln Ala Ile 450 455 460Asp Lys Ile
Glu Lys Asp Leu Pro Gly Phe Phe Phe Ala Gly Asn Tyr465
470 475 480Lys Gly Gly Leu Ser Val Gly
Lys Ala Ile Ala Ser Gly Cys Lys Ala 485
490 495Ala Asp Leu Val Ile Ser Tyr Leu Asn Ser Ala Ser
Asp Asn Thr Val 500 505 510Pro
Asp Lys 515451548DNAGlycine max 45atggcttcct ctgcaacaga cgataaccca
agatctgtaa aaagagtagc tgttgttggt 60gctggggtaa gtgggcttgc tgcggcttac
aaattgaaat cacatggtct ggatgtcact 120gtatttgaag ctgagggaag agctggaggg
aggttgagaa gtgtttctca ggatggtcta 180atttgggatg agggagctaa tacaatgact
gaaagtgaaa ttgaggttaa aggtttgatt 240gatgctcttg gacttcaaga aaagcagcag
tttccaatat cacagcataa gcgctatatt 300gtgaaaaatg gggcaccact tctggtaccc
acaaatcctg ctgcactact gaagagtaaa 360ctgctttctg cacaatcaaa gatccatctc
atttttgaac catttatgtg gaaaagaagt 420gacccctcta atgtgtgtga tgaaaattct
gtggaaagtg taggcaggtt ctttgaacgt 480cattttggaa aagaggttgt ggactatctg
attgatcctt ttgttggggg cactagtgca 540gcagatcctg aatctctctc tatgcgccat
tctttcccag agctatggaa tttggagaaa 600aggtttggct ccattatagc cggggcattg
caatctaagt tattcgccaa aagggaaaaa 660actggagaaa ataggactgc actaagaaaa
aacaaacaca agcgtggttc gttttctttc 720cagggtggga tgcagacact gacagataca
ttgtgcaaag agcttggcaa agacgacctt 780aaattaaatg aaaaggtttt gacattagct
tatggtcatg atggaagttc ctcttcacaa 840aactggtcta ttactagtgc ttctaaccaa
agtacacaag atgttgatgc agtaatcatg 900acgaatctgc attatttaaa gcattcgttg
cataatggtc aagctcctct atataatgtc 960aaggacatca agatcacaaa aaggggaact
ccctttccac ttaattttct tcccgaggta 1020agctacgtgc caatctcagt catgattact
accttcaaaa aggagaatgt aaagagacct 1080ttggagggat ttggagttct tgttccttct
aaagagcaaa aaaatggttt aaaaaccctt 1140ggtacacttt tttcctctat gatgttccca
gatcgtgcac ctagtgattt atatctctat 1200accaccttca ttggcggaac tcaaaacagg
gaacttgctc aagcttcaac tgacgagctt 1260aggaaaattg ttacttctga cctgagaaag
ttgttgggag cagaggggga accaacattt 1320gttaaccatt tctattggag taaaggcttt
cctttgtatg gacgtaacta tgggtcagtt 1380cttcaagcaa ttgataagat agaaaaagat
cttcccggat ttttctttgc aggtaactac 1440aaaggtggac tctcagttgg caaagcaata
gcctcaggct gcaaagcagc tgatcttgtg 1500atatcctacc tcaactctgc ttcagacaac
acagtgcctg ataaatga 15484648DNAArtificial
SequenceDescription of Artificial Sequence Synthetic oligonucleotide
46gttgggagat cctgatgcgc cttgctttgt cttgtggaag gataaacc
484748DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 47gtttatcctt ccacaagaca aagcaaggcg catcaggatc
tcccaacc 484843DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 48catcatttta
caggtgttta caccggtgac ccctcaaaat tgc
434943DNAArtificial SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 49caattttgag gggtcaccgg tgtaaacacc tgtaaaatga tgc
43505PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 50Val Pro Met Leu Lys1 5
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