Patent application title: IDENTIFICATION OF A XANTHOMONAS EUVESICATORIA RESISTANCE GENE FROM PEPPER (CAPSICUM ANNUUM) AND METHOD FOR GENERATING PLANTS WITH RESISTANCE
Inventors:
György Botund Kiss (Szeged, HU)
Zoltán Szabó (Gárdony, HU)
Carmen E. Illiescu (Gödöllö, HU)
Márta Balogh (Száhalombatta, HU)
IPC8 Class: AC12N1582FI
USPC Class:
800279
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part the polynucleotide confers pathogen or pest resistance
Publication date: 2016-05-19
Patent application number: 20160138041
Abstract:
The present invention relates to the identification of the xcv-1 gene,
which is responsible for a recessive resistance to Xanthomonas
euvesicatoria, by genetic mapping-based cloning from Capsicum annuum. In
addition, the invention relates to methods for generating plants
resistant to an abiotic or biotic factor, in particular to Xanthomonas
euvesicatoria, and the plants themselves, in particular tomato plants.Claims:
1. A nucleic acid molecule that is capable of conferring to a plant
resistance to Xanthomonas euvesicatoria, wherein the nucleic acid
molecule comprises a nucleotide sequence selected from the group
consisting of: (a) a non-naturally occurring nucleotide sequence
comprising at least 80% sequence identity to at least one of the
nucleotide sequences set forth in SEQ ID NO:37, 59, and 66; (b) the
nucleotide sequence set forth in SEQ ID NO:88, 89, or 90; (c) a
nucleotide sequence comprising at least 80% sequence identity to at least
one of the nucleotide sequences set forth in SEQ ID NOS:88, 89, and 90;
(d) a nucleotide sequence encoding an artificial protein comprising at
least 80% sequence identity to the amino acid sequence set forth in SEQ
ID NO:42, wherein the artificial protein comprises a CYSTM region
comprising the deletion of two amino acids relative to the CYSTM region
of the Xcv-1 protein having the amino acid sequence set forth in SEQ ID
NO:42; (e) a nucleotide sequence encoding an artificial protein
comprising at least 80% sequence identity to the amino acid sequence set
forth in SEQ ID NO:38, 60, or 67, wherein the artificial protein
comprises a CYSTM region with a double Leu deletion at the locations
corresponding to positions 87 and 88 of the Xcv-1 protein having the
amino acid sequence set forth in SEQ ID NO:42.
2. An artificial xcv-1 CYSTM protein that is capable of conferring to a plant providing resistance to Xanthomonas euvesicatoria, wherein the artificial xcv-1 CYSTM protein is encoded by the nucleic acid molecule of claim 1.
3-11. (canceled)
12. A nucleic acid molecule for silencing the expression of the SlXcv-1A gene and/or the SlXcv-1B gene, said nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:74; (b) a nucleotide sequence comprising at least 60% identity to the nucleotide sequence set forth in SEQ ID NO:74; (d) the nucleotide sequence set forth in SEQ ID NO: 72; (e) the nucleotide sequence set forth in SEQ ID NO: 73; (f) the nucleotide sequence set forth in SEQ ID NO: 92; and (g) the full-length complement of any one of (a)-(f).
13. The nucleic acid molecule of claim 12, wherein the nucleic acid molecule further comprises an additional nucleotide sequence wherein said additional nucleotide sequence is a miRNA backbone sequences which is operably linked to the nucleic acid molecule nucleotide for silencing the expression of the SlXcv-1A gene and/or the SlXcv-1B gene.
14. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule is capable of silencing at least one of the SlXcv-1A and the SlXcv-1B genes when said nucleic acid molecule expressed in a plant.
15. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule does not silence the expression of, the 6 bp deletion mutant of the SlXcv-1A gene which encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 50 and/or the 6 bp deletion mutant of the SlXcv-1B gene which encodes a protein comprising the amino acid sequence set forth in SEQ ID NO: 52.
16. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule or part thereof is expressed in a plant as an amiRNA.
17. The nucleic acid molecule of claim 12, further comprising an operably linked promoter that is capable of driving the expression of the nucleic acid molecule in a plant.
18. The nucleic acid molecule of claim 12, wherein said nucleic acid molecule is an artificial nucleic acid molecule.
19. A vector comprising a gene according to claim 1.
20. A host cell transformed with a vector according to claim 19.
21. A method for the in vitro preparation of a mutant gene homologous to the xcv-1 gene or its cDNA variant of claim 1, comprising the steps of: a) identifying a gene homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a plant, b) preparing in vitro the genomic and cDNA sequences of the gene identified in step a) in the form of a DNA, and c) creating in vitro the desired deletion of 6 bp in the DNA prepared in step b) in the segment of the gene encoding the CYSTM region.
22. The method of claim 21, wherein in step c), the desired 6-bp deletion is created in those nucleotides of the CYSTM region of the gene that encode the 5th and 6th amino acids from the C-terminus.
23. A mutant plant showing resistance to a biotic or abiotic factor, the genome of which is modified to contain a 6-bp deletion in the segment encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41).
24. A method for generating a transgenic plant resistant to a biotic or abiotic factor according to claim 23 by transformation, comprising the steps of: a) transforming the cells of a sensitive plant by a gene comprising the nucleotide sequence set forth in SEQ ID NO: 37 or 90 or a gene encoding a protein homologous to xcv-1 CYSTM and which comprises a deletion of two amino acids in the CYSTM region in comparison with CYSTM region of the wild-type homologous protein, and provides resistance in a manner ensuring the functional expression thereof, b) inactivating one or more resident genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41), or the mRNA or protein thereof, in the sensitive transformant plant cells obtained in step a), and c) regenerating the plant from the transformants and selecting the resistant individuals.
25. A method according to claim 24, wherein in step b), the mRNA products of the resident gene(s) are functionally silenced by amiRNA technique or an engineered nuclease deletion method.
26. (canceled)
27. A method for generating a mutant plant showing an abiotic or biotic resistance according to claim 23 using a genome editing method, comprising the steps of: a) identifying one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a sensitive plant, b) preparing DNA constructs encoding TALEN-L and TALEN-R or ZFN-L and ZFN-R engineered nuclease proteins specific to the gene sequence encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene and identified in step a), more specifically those specific to the 6 nucleotides to be deleted and the surrounding nucleotides as target sequence in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas nuclease construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence, c) cloning the construct obtained in step b) into an appropriate vector, d) transforming sensitive plant cells with a vector comprising the construct obtained in step b) in a manner ensuring the functional expression of the transgenes and the generation of the 6-bp deletion in the CYSTM region by the nuclease, e) identifying the transformants carrying mutations showing the 6-bp deletion in the CYSTM region of the genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41), f) regenerating the plant cells having the mutation identified in step e) and selecting the resistant plants, and g) removing the transgenes comprising the TALEN or ZFN constructs or CRISPR/Cas constructs containing the gene of the sgRNS protein by genetic segregation.
28. (canceled)
29. A method for generating a tomato plant resistant to Xanthomonas euvesicatoria, comprising the steps of: a) identifying the resident genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and 51, respectively), which are homologous to the wild-type Xcv-1 gene in the tomato plant, b) preparing in vitro the genomic and cDNA sequences of the identified genes in the form of a DNA, c) creating in vitro the 6-bp deletion in the positions corresponding to the deletions in the xcv-1 gene thereby generating constructs carrying the mutant genes Slxcv-1A and Slxcv-1B (SEQ ID NO:59 and 66, respectively) or their cDNA sequences (SEQ ID NO:88 and 89, respectively), d) cloning the constructs obtained in step c) into an appropriate vector and transforming the cells of the tomato plant with the resulting vector in a manner ensuring the functional expression of the transgenes, e) preparing an amiRNA gene construct specific for silencing the SlXcv-1A and/or the SlXcv-1B gene f) cloning the construct obtained in step e) into an appropriate vector, g) transforming the plant cells generated in step d) with the vector obtained in step f) in a manner ensuring the expression of the amiRNA and inactivation of the mRNA products of the wild-type CYSTM genes SlXcv-1A and SlXcv-1B, and h) regenerating the transformants obtained in step g) and selecting the resistant plant.
30. The method of claim 29, wherein step (e) comprises preparing an amiRNA gene construct specific to the complementary ribonucleotide sequence corresponding to the CYSTM region of the wild-type genes SlXcv-1A and/or SlXcv-1B (SEQ ID NO:49 and 51, respectively), more specifically an amiRNA gene construct specific to the 6 nucleotides to be deleted and the surrounding nucleotides (SlXe1-amiRNA, SEQ ID NO: 74).
31. The method of claim 29, wherein the amiRNA gene construct comprises the nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:74; (b) a nucleotide sequence comprising at least 60% identity to the nucleotide sequence set forth in SEQ ID NO:74; (d) the nucleotide sequence set forth in SEQ ID NO: 72; (e) the nucleotide sequence set forth in SEQ ID NO: 73; (f) the nucleotide sequence set forth in SEQ ID NO: 92; and (g) the full-length complement of any one of (a)-(f).
32. A method for generating a tomato plant according to claim 23 that is resistant to Xanthomonas euvesicatoria, comprising the steps of: a) identifying the resident genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and 51, respectively), which are homologous to the wild-type Xcv-1 gene in the tomato plant, b) preparing in vitro the genomic and cDNA sequences of the identified genes in the form of a DNA, c) creating in vitro the 6-bp deletion in the positions corresponding to the deletions in the xcv-1 gene thereby generating constructs carrying the mutant genes Slxcv-1A and Slxcv-1B (SEQ ID NO:59 and 66, respectively) or their cDNA sequences (SEQ ID NO:88 and 89, respectively), d) cloning the constructs obtained in step c) into an appropriate vector and transforming the cells of the tomato plant with the resulting vector in a manner ensuring the functional expression of the transgenes, e) preparing engineered nuclease proteins TALEN-L (SEQ ID NO:78) and TALEN-R (SEQ ID NO:79, and 80) specific to the target sequences (SEQ ID NO:75, 76, 77) specific to the CYSTM region of the wild-type SlXcv-1A and SlXcv-1B gene, or DNA constructs encoding engineered nuclease proteins ZFN-L and ZFN-R specific to SEQ ID NO: 86 and 87 in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas nuclease construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence (SEQ ID NO: 81), f) cloning the construct obtained in step e) into an appropriate vector, g) transforming the plant cells generated in step d) with the vector obtained in step f) in a manner ensuring the functional expression of the above TALEN or ZFN or CRISPR/Cas nuclease transgenes and the functional inactivation of genes SlXcv-1A and SlXcv-1B by them, h) identifying deletion or insertion knock-out mutations in the transformant cells, i) regenerating the plant cells having the mutation identified in step h) and selecting the resistant plants, and j) removing the nuclease transgenes TALEN-L plus TALEN-R or ZFN-L plus ZFN-R or CRISPR/Cas by genetic segregation.
33. (canceled)
34. The mutant plant of claim 23, in which the resistance is recessive resistance to Xanthomonas sp.
35. The mutant plant of claim 23, which is a monocotyledonous plant or a dicotyledonous plant.
36. (canceled)
37. The mutant plant of claim 23, which is a plant selected from the group consisting of: rice, maize, wheat, rye, barley, millet, banana orange, mandarin, lemon, grapefruit, pomelo, potato, tomato, pepper, eggplant, cabbage, radish, cauliflower, rape, alfalfa, bean, pea, pumpkin, cucumber, melon, apple, quince, cherry, plum, apricot, peach and cotton.
38-43. (canceled)
44. A food product produced from the mutant plant of claim 23 or part thereof.
45. (canceled)
46. Seeds of the mutant plant of claim 23.
47-52. (canceled)
Description:
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention relates to the identification of the xcv-1 gene, which is responsible for a recessive resistance to Xanthomonas euvesicatoria, by genetic mapping-based cloning from Capsicum annuum. In addition, the invention relates to methods for generating resistant plants, in particular plants resistant to Xanthomonas euvesicatoria.
BACKGROUND OF THE INVENTION AND PRIOR ART
[0002] It is well-known that in arable plant production, sensitive crops are infested by various pathogens (viruses, bacteria, fungi etc.) the serious consequences of which include yield reduction or yield loss. Therefore, one fundamental requirement for advanced and competitive plant varieties is to show resistance to pests and pathogens causing major yield losses. Resistant plants allow cheaper and more environmentally friendly production because no spray liquids containing hazardous substances and toxins are released into nature. The environmentally friendly production technology of resistant plants also increases yield safety; produces of higher quality and quality can be harvested at lower costs.
[0003] Identification, isolation and characterisation of the genes providing resistance is of vital importance for both theory and practice: the process can be understood by exploring the genes involved in the infection processes, their products and the functions thereof, and this forms the indispensable basis for developing control strategies.
[0004] As regards the molecular processes of plant protection, two basic mechanisms can be distinguished. The first one is dominant resistance and the second one is recessive resistance. In case of a dominant protective mechanism, a signal molecule (typically, a protein) from the pathogen initiates a self destruction process (apoptosis) resulting in the death of the infected cells and their neighbours thereby halting the spreading of the pathogen (Pontier D. et al., C R Acad Sci III. 321:721-34, 1998.). The result of the cell destruction, i.e., programmed cell death, is a dried and discoloured necrotic patch, which is the manifestation of the so-called hypersensitive reaction (HR) (Klement Z. et al., Phytopathology 54: 474-477, 1964). Dominant resistance is race-specific and can be easily abolished as new races may break the resistance.
[0005] In case of recessive resistance, a mutation causes a loss of a function that is indispensable for the development of virulence. As yet, the steps of the development of recessive resistance, as well as the functions thereof and the plant genes of such functions are mostly unknown, however, it was found that more than one gene is involved in the development of resistance on the part of both the bacterium and plant host. Recessive resistance genes can be identified when a difference exists between the genes involved in the process induced by the pathogen and causing the disease (virulence) and the wild-type genes. Genes providing recessive resistance have been identified from a few plants, including among others the RRS1-R gene from Arabidopsis thaliana (Deslandes L. et al., PNAS 99: 2404-2409, 2002) and a gene against Xanthomonas oryzae from rice (Iyer-Pacuzzi A. S., Pathosystem. Mol. Plant Microb. Interaction 20:731-739, 2007). A number of genes providing dominant resistance have been identified from pepper on the basis of mutant phenotypes, but no genes providing recessive resistance. In general, recessive resistance is not race-specific and is more difficult; therefore, it is more stable.
[0006] In the field growing of edible and spice pepper, the bacterium Xanthomonas campestris pv. vesicatoria (Xcv), recently renamed as Xanthomonas euvesicatoria (Xe) causes the most significant damage (Jones J. B. et al., System. Appl. Microbiol. 27: 755-762, 2004). The Xe bacterium is mediated mostly by water and enters the plant through wounds and leaf gaps. Under warm and moist climatic conditions, the bacterium spreads rapidly. The symptoms of the disease mostly appear on the leaves. Scar-like patches develop on the back of the leaves and later become necrotic areas on the face of the leaves. The infected leaves of sensitive plants die and fall off within one to two weeks, and the yield is burnt by the sun.
[0007] Similar to a group of plant and animal bacteria, Xanthomonas euvesicatoria is also capable of growing so-called pili through the Type Three Secretion System (TTSS), and the ends of these pili extend until the eukaryotic cell membrane. The pilus is permeable for the effector molecules of the bacterium. One or more of the effector molecules create a so-called translocon in the cell membrane through which the effector molecules enter the eukaryotic cytoplasm; in several cases, they also enter the nucleus from the cytoplasm if they comprise a Nuclear Localization Signal (NLS). For their growth, the bacteria use the nutrient molecules present in the plant cells, which are released upon the loss of integrity of the plant cells. Disintegration of the cells is induced by the effector molecules introduced through the Type Three Secretion System of Xe via an infection mechanism the details of which are yet unclear. The proliferation of the bacteria damages plant tissue to such an extent as to cause the majority of the leaves to fall off and the plant to dry out sooner or later.
[0008] The complete genome of Xanthomonas euvesicatoria (X. campestris pv. vesicatoria strain 85-10) has been determined (Thieme F. et al., J. Bacteriol. 187:7254-7266, 2005), and the genes of the effector proteins involved in the induction of a dominant hypersensitive reaction from pepper have been identified. Genes for recessive resistance to Xanthomonas euvesicatoria have not been identified from pepper yet.
[0009] The literature describes a few pepper varieties resistant to Xe. These plants carry resistance genes including but not limited to Bs1 (Cook A. A. and Stall R. E., Plant Dis. 53:1060-1062, 1963), Bs2 (Cook A. A. and Guevara Y. G., Plant Dis. 68:329-330, 1984), Bs3 (Kim B. S. and Hartmann R. W., Plant Dis. 69:233-235, 1985), Bs4 (Hibberd et al., Phytopathology 77:1304-1307, 1987), bs5 (Jones J. B. et al., System. Appl. Microbiol. 27:755-762, 2004) and bs6 (Vallejos C. E. et al., Theor. Appl. Genet. 121:37-46, 2010). The latter two--bs5 and bs6--are recessive types of resistance genes.
[0010] Despite the existing resistant pepper varieties, there is still an extreme need for pepper varieties resistant to Xanthomonas species and for other plant varieties that are resistant to biotic or abiotic factors.
[0011] The objective of the present study is to identify and isolate a gene from pepper (Capsicum annuum) providing recessive resistance to Xanthomonas euvesicatoria, which can be used to develop single or double (pyramided) resistance varieties--mostly to Xanthomonas sp., but presumably to other biotic or abiotic factors as well--in sensitive pepper species and other plant species such as tomato, potato, rice, citruses, banana, etc.
SUMMARY OF THE INVENTION
[0012] The above objective could be achieved by the present invention. From a Capsicum annuum carrying a recessive resistance, a gene designated as xcv-1 was isolated using genetic map-based cloning. The sequence of the isolated gene was determined (SEQ ID NO:37) and it was found that the xcv-1 protein (SEQ ID NO:38) encoded by the gene comprises a double Leu deletion at the locations corresponding to positions 87 and 88 of the wild-type Xcv-1 protein (SEQ ID NO:42). The mutant xcv-1 protein is a tail-anchored (TA) transmembrane (TM) protein, more specifically a CYSTM protein, which carries the double leucine deletion in its cysteine-rich transmembrane region (hereinafter referred to as `CYSTM region`). This CYSTM region shows structural relatedness to the CYSTM region of other known transmembrane proteins (Venancio T. M. and Aravind L., Bioinformatics 26:149-152, 2010) in that it is a common feature that they are rich in cysteine (comprising at least 3 cysteines), that they are boardered by an amino acid with negative charge (aspartic acid or glutamic acid) or a polar amino acid (asparagine) in position 4 from the C-terminus of the protein, and that the Asp, Glu or Asn is preceded by two hydrophobic amino acids (here: leucine), and less frequently, these positions contain isoleucine, methionine, tryptophan, glycine, alanine, threonine, phenylalanine, valine and cysteine.
[0013] It is interesting to note that certain fungi (e.g., Schizosaccharomyces pombe, Saccharomyces cerevisiae) which are resistant to certain abiotic factors such as UV radiation, and certain drugs (e.g., canavanine), lose such resistance and their sporulation capacity if a CYSTM-type protein loses function as a result of a gene mutation (Lee J. K. et al., Biochem. Biophys, Res. Comm. 202:1113-1119, 1994; Lee, J. K. et al., Mol. Gen. Genet. 246:663-670, 1995; Venancio T. M. et al., Mol Biosyst. 6:175-181, 2010; Venancio, T. M. and Aravind, L. Bioinformatics 26:149-152, 2010). Similar to fungi, Arabidopsis thaliana plants also suffer severe disturbances in megasporogenesis in case of a loss of function in its genes homologous to the above CYSTM proteins (WIH1, WIH2 double mutation) (Lieber, D. et al., Current Biology 21:1009-1017, 2011).
[0014] It was found that by removing two amino acids from the C-terminal CYSTM region--preferably those in positions 5 and 6 from the C-terminus--of a protein homologous to the wild-type Xcv-1 protein but derived from a plant organism other than pepper (for example, tomato), advantageous properties, primarily recessive resistance can be induced in the plant organism. The deletion of the two codons, i.e., 6 base pairs, encoding these two amino acids in the CYSTM region--which include but is not limited to leucine, isoleucine, methionine, tryptophan and cysteine--is referred to as `the desired 6-bp deletion`.
[0015] Without being limited to any theory regarding the development of resistance, it is likely that a double Leu deletion in the CYSTM region (the last 13 amino acids in the C-terminus of the protein) of the mutant xcv-1 protein encoded by the xcv-1 gene prevents or reduces the entry of the effector molecules of bacteria with type three secretion system into plant cells. This hypothesis is preliminaryly substantiated by the result of double resistant papper lines carrying Bs2 and xcv-1 in homozygous configuration. These plants upon infection with AvrBs2 containing Xe do not show the HR phenotype characteristic of AvrBs2 effector of the infecting Xe most probably because AvrBs2 is not entering the plant cells, on the other hand the phenotype of this infection very similar to that caused by the xcv-1/xcv-1 containing plants. The above result indicate that xcv-1 is epistatic over Bs2.
[0016] The identification and characterization of the xcv1 bacterial spot disease resistance has revealed that this gene contains a six nucleotide in-frame deletion that removes two leucine amino acids from the carboxy terminal portion of the protein. Computational analyses suggest that this protein is a membrane protein with an unknown function. Since this mutation confers resistance to several strains of Xanthomonas that cause disease on pepper and tomato, it would be informative to test whether this mutation affects the type three secretion delivery of type three effector proteins into plant cells. One could use a reporter gene assay to examine the biochemical activity of translational fusion proteins between the N-terminal domains of various type three effector proteins and the reporter gene adenylate cyclase (Direct biochemical evidence for type Ill secretion-dependent translocation of the AvrBs2 effector protein into plant cells. Casper-Lindley C. et al., PNAS 99:8336-8341, 2002). Using this assay, xcv-1 and other plants, including wild-type peppers, tomato, citrus, walnut, lettuce, brassica, soybean, bean, rice, etc., can be tested for their ability to receive type three secreted effector proteins from strains of Xanthomonas euvesicatoria, X. perforans, X. gardneri and other type-three secretion system dependent bacteria. The adenylate cyclase assay will allow a means of monitoring the mechanism of resistance in xcv-1 plants or with combinations of resistance genes.
[0017] Accordingly, it was assumed that a transgenic plant having recessive resistance can be generated by the removal of the gene encoding the original CYSTM region through knocking out homologous resident genes of a plant, and by the simultaneous replacement with a mutant CYSTM region comprising a desired 6-bp deletion through transformation. Similarly, it is assumed that if a derivative or derivatives carrying the desired 6-bp deletion is/are generated in a plant by spontaneous or induced mutation of the gene segment encoding the CYSTM region of solitary (one-copy) or multiple-copy (double-, triple-copy etc.) CYSTM proteins, or by nuclease-based "genome editing" methods such as, for example, Zinc Finger Nuclease (ZFN), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease technique--or CRISPR/Cas nuclease technique in short--, or in any other ways, then plants with recessive resistance may be generated. Presumably, the mutant homologues will show an advantageous property, i.e., will render the plant resistant.
[0018] This hypothesis was proven by transgenic technique using Xe-sensitive tomato (Solanum lycopersicum) as follows: recessive resistance to Xe was generated in tomato by inactivating two resident genes (SlXcv-1A, SEQ ID NO:49 and SlXcv-1B, SEQ ID NO:51) homologous to the wild-type Xcv-1 gene and by creating in vitro the 6-bp deletions in the homologous genes at the positions corresponding to the xcv-1 gene (Slxcv-1A, SEQ ID NO:59 and Slxcv-1B, SEQ ID NO:66), followed by the introduction of the mutant genes into the plant cells.
[0019] Additionally, without limiting the scope of the invention, three methods using genome editing techniques (TALEN, CRISPR/Cas nuclease and ZFN) are described for creating the desired 6-bp deletion and thereby generating recessive resistance to Xanthomonas species in tomato.
[0020] Slxcv-1A and Slxcv-1B, the mutant genes responsible for the resistance, could be isolated from the transgenic tomato plants, their sequences were determined (SEQ ID NO:59 and SEQ ID NO:66, respectively), and they encode proteins Slxcv-1A and Slxcv-1B (SEQ ID NO:60 and SEQ ID NO:67, respectively). The sequences of the latter were compared to the wild-type protein sequences (SEQ ID NO:50 and SEQ ID NO:52), and the deletions were confirmed at the locations corresponding to positions 86 to 87 and 88 to 89.
[0021] Accordingly, one object of the present invention is the xcv-1 gene isolated from Capsicum annuum, which is responsible for a recessive resistance to Xanthomonas euvesicatoria and has the nucleotide sequence of SEQ ID NO:37. The present invention also relates to the cDNA of the xcv-1 gene, which has the nucleotide sequence of SEQ ID NO:90.
[0022] Another object of the present invention is the xcv-1 CYSTM protein providing the resistance, which is encoded by the xcv-1 gene and its cDNA sequence, and has the amino acid sequence of SEQ ID NO:38, wherein the CYSTM region of the protein carries a double Leu deletion at the locations corresponding to positions 87 and 88 of the wild-type protein of SEQ ID NO:42.
[0023] Another object of the present invention is a protein homologous to the xcv-1 CYSTM protein, which comprises a deletion of two amino acids in the CYSTM region in comparison with the CYSTM region of the wild-type homologous protein, and provides resistance. Preferably, the mutant homologous protein carries the deletion of two amino acids in the CYSTM region, at positions 5 and 6 from the C-terminus of the wild-type protein. Preferred examples of such mutant proteins include proteins Slxcv-1A or Slxcv-1B, which have the amino acid sequences of SEQ ID NO:60 or SEQ ID NO:67, respectively.
[0024] In addition, the invention relates to the homologues of the xcv-1 gene encoding the above homologous mutant CYSTM proteins. Preferred examples include the Slxcv-1A gene having the nucleotide sequence of SEQ ID NO:59 or its cDNA variant having the nucleotide sequence of SEQ ID NO:88, which encode the Slxcv-1A mutant homologous protein (SEQ ID NO:60), or the Slxcv-1B gene having the nucleotide sequence of SEQ ID NO:66 or its cDNA variant having the nucleotide sequence of SEQ ID NO:89, which encode the Slxcv-1B mutant protein (SEQ ID NO:67).
[0025] Furthermore, the present invention relates to engineered nuclease proteins specific to the DNA sequence of a gene homologous to the wild-type Xcv-1 gene (SEQ ID NO:41), which selectively recognise the DNA segment encoding the CYSTM region of the gene homologous to the wild-type Xcv-1 gene, or certain partial sequences thereof. Preferred engineered nuclease proteins include those specific to the DNA sequences of genes SlXcv-1A and SlXcv-1B. Additional preferred engineered nuclease proteins include a ZFN nuclease pair selectively recognising the gene segments represented by SEQ ID NO:86 and 87, or a TALEN nuclease pair selectively recognising the gene segments represented by SEQ ID NO:78 and 79 or 78 and 80, or a sgRNS-CRISPR/Cas nuclease selectively recognising the gene segments represented by SEQ ID NO:81.
[0026] The present invention also relates to the genes encoding said engineered nuclease proteins.
[0027] The present invention also relates to artificial nucleic acid molecules (amiRNA) for the silencing the SlXcv-1A and SlXcv-1B which are complementer to the CYSTM region of the mRNA of the plant cells. These nucleic acid molecules comprises additional nucleotide sequences for expression. Important to note, that these nucleic acid molecules do not silence those genes carrying the desired 6 bp deletion.
[0028] Another object of the present invention is a vector comprising the xcv-1 gene (SEQ ID NO:37), one or more homologues thereof, preferably genes Slxcv-1A (SEQ ID NO:59) and/or Slxcv-1B (SEQ ID NO:66), or the genes of the TALEN, CRISPR/Cas and ZFN nucleases specific to the Xcv-1 genes suitable for genome editing and other nucleic acid molecules described in this invention.
[0029] Another object of the invention is a host cell transformed with said vector.
[0030] Another object of the present invention is a method for the in vitro preparation of a mutant gene homologous to the xcv-1 gene or its cDNA variant, comprising the steps of: a) identifying a gene homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a plant; b) preparing in vitro the genomic and cDNA sequences of the gene identified in step a) in the form of a DNA; and c) creating in vitro a deletion of the desired 6 bp in the DNA prepared in step b) in the portion of the gene encoding the CYSTM region. Using the method of the invention, the 6-bp deletion is preferably created in those nucleotides of the CYSTM region of the gene that encode the 5th and 6th amino acids from the C-terminus.
[0031] The present invention also relates to mutant plants showing resistance to a biotic or abiotic factor, the genomes of which are modified to contain a 6-bp deletion in the segment encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) or its cDNA sequence (SEQ ID NO:91).
[0032] Another object of the present invention is a method for generating a transgenic plant resistant to biotic or abiotic factors by transformation, comprising the steps of: a) transforming the cells of a sensitive plant by one or more mutant genes homologous to the xcv-1 gene of the invention in a manner ensuring the functional expression thereof, b) inactivating one or more resident genes homologous to the wild-type Xcv-1 gene, or the mRNA or protein product thereof, in the sensitive transformant plant cells obtained in step a); and c) regenerating the plant from the transformants and selecting the resistant individuals.
[0033] Another object of the invention is a method based on genome editing, which comprises a step of creating the desired 6-bp deletion in the wild-type homologous gene using ZFN, TALEN or CRISPR/Cas nucleases specific to the sequence of the wild-type Xcv-1 gene.
[0034] Yet another object of the invention is a method based on genome editing, in which the ZFN and TALEN nuclease proteins recognising the DNA sequences homologous and specific to the Xcv-1 gene are introduced into plant-derived host cells using bacteria having type three secretion system but not causing diseases (non-pathogenic bacteria) (see e.g., WO/2005/085417).
[0035] Yet another object of the present invention is a plant and its progeny resistant to biotic or abiotic factors, which can be generated by the methods of the invention and carries a deletion of two amino acids in the CYSTM region of one or more of its transmembrane proteins in comparison with the wild-type protein. Preferably, the plant is a tomato plant (Solanum lycopersium), in which recessive resistance to Xanthomonas euvesicatoria has been created.
[0036] Furthermore, the invention relates to a method to generate resistant plant by combining (pyramiding) at least two resistance genes against the same pathogene (e.g. Xanthomonas sp.). Accordingly, the present invention relates to a tomato plant containing more than one resistance genes conferring resistance against Xanthomonas euvesicatoria which is generated by one of the procedures described in the invention of which one resistance gene is based on the creation of the desired 6 bp deletion in a gene homologues to xcv-1 and the other resistance gene or genes is/are including but not limited to e.g. Bs2, Bs2, Bs3, Bs4, bs5, bs6 or their combination.
[0037] The present invention also relates to a rice plant containing more than one resistance genes conferring resistance against Xanthomonas oryzae pv. oryzae which is generated by one of the procedures described in the invention where said rice plant carries in combination another resistance gene or genes against Xanthomonas oryzae pv. oryzae which is/are including but not limited to e.g. Xa-4+xa-5+Xa-7+xa-13+Xa-21 genes.
[0038] The present invention also relates to a citrus plant containing more than one resistance genes conferring resistance against Xanthomonas citri pv. citri, Xanthomonas axonopodis pv. citri which is generated by one of the procedures described in the invention where said citrus plant carries in combination another resistance gene or genes against Xanthomonas citri, Xanthomonas axonopodis strains.
[0039] Furthermore, the invention relates to antibodies against the proteins of the invention, which are specific to the mutant CYSTM region of the proteins and bind to the resistant mutant protein but not to the wild-type protein, and are useful as probes in in vitro methods for determining whether a plant carries such resistant mutant proteins or not.
[0040] The present invention also relates to genetic probes, which are specific to the mutant region of the xcv-1 gene and of its homologues, and are useful for the identification of resistance genes in an in vitro method.
DESCRIPTION OF THE FIGURES
[0041] FIG. 1: The xcv contig physically covering the xcv-1 gene with overlapping BAC clones. Scheme of the identified and overlapping BAC clones (horizontal lines). The numbering of the BAC clones is indicated above the lines. The two ends of the BAC clones are indicated by "-40" and "op", respectively. The initial marker is indicated by an arrow.
[0042] FIG. 2: Hydrophobicity curve of the Xcv-1 protein. The part above the line marked by "0" is that part of the protein which is presumably localised in the membrane. TM=transmembrane, DAS: "Dense Alignment Surface" algorithm.
[0043] FIG. 3: The point of attack (middle line) of the target mRNA (SlXcv1A1/SlXcv1B genes), the amino acid sequence deducible from that (upper line), and the designed 21-bp SlXe1-amiRNA sequence (lower line).
[0044] FIG. 4: "Northern" autoradiogram of the RNA hybridisation of the SlXe1-amiRNA. The Northern blot of the maturing SlXe1-amiRNAs of various lengths (21, 22 and 24 bp) was hybridised to an alpha-32ATP-labelled probe encoding the SlXe1-amiRNA. Samples: 1.=RNA sample prepared from a control (untransformed) plant, 2-3. RNA sample prepared from a plant containing the SlXe1-amiRNA expression construct, M.=smallRNA molecular weight marker (20 bp, 21 bp, 30 bp).
[0045] FIG. 5. TALEN, ZFN and CRISPR target sequences and RVDs specific to the genes SlXcv-1A and SlXcv-1B. 5A. Portion of the CYSTM region of genes SlXcv-1A and SlXcv-1B. The vertical lines and serial numbers above the sequences indicate the nucleotide positions according to SEQ ID NO:49 and SEQ ID NO:51. Partial amino acid sequences of proteins SlXcv-1A and SlXcv-1B are shown below the double-stranded DNA sequences. The two leucines which are missing from the mutant proteins (SlXcv-1A and SlXcv-1B) are underlined. The amino acids are indicated by the internationally accepted one-letter codes. The * indicates the stop codon. The target sequences to be recognised by the TALEN-L and TALEN-R nuclease pairs are indicated by arrow heads pointing to the right and left above the target sequences and by lines above the target sequences, and the target sequences to be recognised by the ZFN-L and ZFN-R nuclease pairs are indicated by arrow heads pointing to the right and left below the target sequences and by dotted lines below the target sequences. The arrows are in the 5'>3' direction. The sequences to be recognised by the CRISPR/Cas complex are indicated by grey background and bold letters. The six nucleotides present in the upper strand of the DNA in the mutant genes (SlXcv-1A, SlXcv-1B, Slxcv-1A and Slxcv-1A) are underlined. 5B. Amino acid doublets (RVDs) of the proteins SlXcv-1AB TALEN-L, SlXcv-1A TALEN-R1 and SlXcv-1B TALEN-R2. The numbers above the amino acid doublets of the SlXcv-1AB TALEN-L protein are the serial numbers of the RVDs.
[0046] FIG. 6. Functional map of the vectors containing the TALEN pairs specific to genes SlXcv-1A and SlXcv-1B. Abbreviations: 35S pr=35S promoter, TAL-N'=sequence of the N-terminus of the TAL effector, TAL-C'=sequence of the C-terminus of the TAL effector, NLS=Nuclear Localization Signal, SlXcv-1A_TAL-R, SlXcv-1AB_TAL-L=repeat sequences containing 17 RVDs specific to the SlXcv-1A gene, SlXcv-1B_TAL-R, SlXcv-1AB_TAL-L=repeat sequences containing 17 RVDs specific to the SlXcv-1B gene, N=Nopaline synthase polyA, pA=35S polyA, RB=right border sequence of the t-DNA, LB=left border sequence of the t-DNA, HYG R=Hygromycin resistance gene, KAN R=Kanamycin resistance gene, B=BamHI, S=SacI, the arrows (---->) indicate the direction of transcription.
[0047] FIG. 7. Possible variants of genes SlXcv-1A and SlXcv-1B in TALEN-treated plants
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0048] As used herein, the term "resistance to biotic or abiotic factors" means that the plant is resistant to various biotic factors such as plant-pathogenic bacteria, fungi and viruses, or abiotic factors such as salt stress, drought stress etc.
[0049] As used herein, the term "recessive resistance to Xanthomonas sp." means that the plant is resistant to at least one Xanthomonas species including, but not limited to, Xanthomonas euvesicatoria, Xanthomonas gardneri, Xanthomonas perforans, Xanthomonas oryzae pv. oryzae, Xanthomonas citri pv. citri, Xanthomonas axonopodis pv. citri, Xanthomonas campestris pv. musacearum. In a preferred embodiment of the invention, the plant is resistant to at least Xanthomonas euvesicatoria. In some embodiments of the invention, the plant is resistant to two, three, four, or more Xanthomonas species and preferably, one of the species is Xanthomonas euvesicatoria.
[0050] As used herein, the term "plant" means an organism capable of photosynthesising, the parts of which, e.g., root, stem, leaf, flower, fruit etc., the progeny of which after sexual reproduction, e.g., F1, F2, F3 etc. generation after crossing or self-pollination, and progeny after vegetative reproduction, e.g., cloning from root cuttings or stem cuttings, grafting, budding, micropropagation, etc.
[0051] As used herein, the term "resident gene" means genes naturally occurring in living organisms not engineered by humans.
[0052] As used herein a "tail-anchored protein" (TA protein) refers to a protein the NH2-terminal portion (domain) of which is anchored to the double phospholipid membrane through a single hydrophobic portion located near to its COOH-terminus, as described by Borgese N. et al. (J. Cell Biol. 161: 1013-1019, 2003).
[0053] As used herein, the term "transmembrane" (TM in short) means a hydrophobic protein portion spanning the double phospholipid membrane.
[0054] As used herein, the term "transmembrane protein" (TM protein) means a protein comprising a transmembrane protein domain.
[0055] As used herein, the term "CYSTM protein" means a TA protein having a cysteine-rich TM region close to the COOH-terminus (CYSTM region) in the sense described by Venancio T. M. and Aravind L. (Bioinformatics 26:149-152, 2010).
[0056] As used herein, the term "CYSTM region" in relation to proteins means a cysteine-rich TM protein segment in the sense described by Venancio T. M. and Aravind L. (Bioinformatics 26:149-152, 2010).
[0057] As used herein, the term "homologous" refers to the situation where nucleic acid or protein sequences are similar because they have a common evolutionary origin.
[0058] As used herein, the term "proteins homologous to the wild-type Xcv-1 protein" refers to CYSTM proteins in the sense described by Venancio T. M. and Aravind L. (Bioinformatics 26:149-152, 2010).
[0059] As used herein, the term "proteins homologous to the mutant xcv-1 protein" means CYSTM protein variants in which the CYSTM region contains a deletion of 2 amino acids compared to its wild type protein and which provide resistance.
[0060] As used herein, the term "genes homologous to the Xcv-1 gene" means gene variants or its cDNA sequence without intron(s) encoding the above "proteins homologous to the Xcv-1 protein".
[0061] As used herein, the term "genes homologous to the xcv-1 gene" means gene variants or its cDNA sequence without intron(s) encoding the above "proteins homologous to the xcv-1 protein".
[0062] Fragments and variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the polynucleotide or a portion of the amino acid sequence and hence protein encoded thereby. Fragments of polynucleotides comprising coding sequences may encode protein fragments that retain biological activity of the full-length or native protein and hence retain the ability to initiate in a plant a hypersensitive response in the presence of a effector protein from a plant pathogen. Alternatively, fragments of a polynucleotide that are useful as hybridization probes generally do not encode proteins that retain biological activity or do not retain promoter activity. Thus, fragments of a nucleotide sequence may range from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length polynucleotide of the invention.
[0063] Polynucleotides that are fragments of a native polynucleotide of the present invention comprise at least 16, 20, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, or 3500 contiguous nucleotides, or up to the number of nucleotides present in a full-length polynucleotide disclosed herein (for example, 3859 nucleotides for SEQ ID NOS: 79, 80 and 81, respectively).
[0064] "Variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polynucleotides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode a polynucleotide of the invention or can be used in decreasing the level of Xcv-1 or a protein homologous to Xcv-1 in a plant by the methods disclosed herein. Variant polynucleotides further include homologous polynucleotides isolated from other species. Generally, variants of a particular polynucleotide of the invention (for example, SEQ ID NO:37 or 39 or 41 or 49 or 51 or 59 or 66 or 69 or 72 or 73 or 74 or 75 or 76 or 77 or 78 or 79 or 80 or 81 or 86 or 87 or 88 or 89 or 90 or 92), will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
[0065] Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, a polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 38 or 40 or 42 or 50 or 52 or 60 or 67 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
[0066] "Variant" protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Such variants also include homologous proteins in other species. Biologically active variants of a protein of the present invention will have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. Such biologically active variants include, for example, wild-type Xcv-1 and homologous proteins as well as mutant versions thereof (e.g. xcv-1) that confer to a plant resistance to at least one plant pathogenic Xanthomonas species. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
[0067] The polynucleotides of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire (i.e full-length) sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. "Orthologs" is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species.
[0068] The following terms are used to describe the sequence relationships between two or more polynucleotides or polypeptides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", and, (d) "percentage of sequence identity."
[0069] (a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
[0070] (b) As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two polynucleotides. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
[0071] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers, E. W. and Miller, W. CABIOS 4:11-17, 1988; the local alignment algorithm of Smith, T. F. et al., Adv. Appl. Math. 2:482, 1981; the global alignment algorithm of Needleman, S. B. and Wunsch, C. D. J. Mol. Biol. 48:443-453, 1970; the search-for-local alignment method of Pearson, W. R. and Lipman, D. J. Proc. Natl. Acad. Sci. 85:2444-2448, 1988; the algorithm of Karlin, S. and Altschul, S. F. Proc. Natl. Acad. Sci. USA 872264, 1990, modified as in Karlin, S. and Altschul, S. F. Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993.
[0072] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al., Gene 73:237-244, 1988; Higgins, D. G. et al., CABIOS 5:151-153, 1989; Corpet, F. et al., Nucleic Acids Res. 16:10881-90, 1988; Huang, X. et al., CABIOS 8:155-65, 1992; and Pearson, W. R. et al., Meth. Mol. Biol. 24:307-331, 1994. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul, S. F. et al., J. Mol. Biol. 215:403, 1990 are based on the algorithm of Karlin, S. and Altschul, S. F. (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul, S. F. et al., Nucleic Acids Res. 25:3389, 1997. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul, S. F. et al., (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.
[0073] Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
[0074] GAP uses the algorithm of Needleman, S. B. and Wunsch, C. D. J. Mol. Biol. 48:443-453, 1970 to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
[0075] GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff, S. and Henikoff, J. G. Proc. Natl. Acad. Sci. USA 89:10915, 1989).
[0076] (c) As used herein, "sequence identity" or "identity" in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
[0077] (d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
[0078] As used herein, the term "CYSTM region" in relation to nucleic acids (DNA, RNA) is a nucleotide segment extending to 52 and 52 nucleotides into both (5' and 3') directions, respectively, from the 2nd nucleotide of the stop codon of the "genes homologous to the Xcv-1 gene" or "genes homologous to the xcv-1 gene".
[0079] As used herein, the term "the desired 6-bp deletion" means a deletion of 6 base pairs in the DNA segment encoding the CYSTM region of genes homologous to the Xcv-1 gene, which results in resistance.
[0080] As used herein, the term "6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides" means a nucleotide segment extending 52 and 52 nucleotides into both directions from the 6-bp deletion created in the CYSTM region. The 6-bp deletion is created anywhere in the CYSTM region of the gene or in those nucleotides that encode the 5th and 6th amino acid from the C-terminus.
[0081] As used herein, the term "Type Three Secretion System" (TTSS in short) is a system by which certain pathogenic bacteria (e.g., Xanthomonas sp., Pseudomonas sp., Erwinia sp., Ralstonia sp., Escherichia sp., Yersinia sp. etc.) introduce effector molecules through the TTSS-specific pili into eukaryotic host organisms as described by Galan J. E. et al. (Nature 444:567-573, 2006).
[0082] As used herein, the term "genome editing" means a method in which a engineered nuclease or engineered nuclease pair performs double-strand breaks (DSB) in a predetermined specific DNA segment in which a DNA repair mechanism referred to as Non-homologous End Joining (NHEJ) creates short deletions or insertions as described by Gaj T. et al. (Trends Biotechnol. 31:397-405, 2013).
[0083] As used herein the term "Double Stranded Break" (DSB in short) means that a DNA sequence is cleaved by a specific nuclease or nuclease pair at both DNA-strands like a molecular pair of scissors. Nucleases performing double stranded breaks of the DNA include, among others, ZFN, TALEN and CRISPR/Cas.
[0084] As used herein, the term "Non-homologous End Joining" (NHEJ) means a method in which the DNA repair mechanism of the cells joins (ligates) two double-stranded DNA ends.
[0085] As used herein, the term "ZFN" means an artificial engineered nuclease recognising DNA sequences and cleaving both strands thereof (see below).
[0086] As used herein, the term "TALEN" means an artificial engineered nuclease recognising DNA sequences and cleaving both strands thereof (see below).
[0087] As used herein, "CRISPR/Cas" recognises complementary DNA sequences and cleaves both strands thereof with the help of the sgRNA and the Cas nuclease (see below).
[0088] As used herein, the term "an artificial nucleic acid molecule" is a non-naturally occurring nucleic acid molecule.
[0089] As used herein, the term "a gene" is a nucleotide sequence which comprises of promoter, exon(s), intron(s) in addition to 5'- and 3'-untranslated regions.
[0090] As used herein, the term "cDNA" is a nucleotide sequence of the copy of the mRNA of a gene.
[0091] As used herein, the term "an artificial nuclease" is an engineered nuclease.
[0092] As used herein, the term "engineered nucleases" are artificial restriction enzymes that can be programmed to cut a pre-determined nucleic acid sequence.
[0093] The Zinc Finger Nuclease (ZFN in short) is a fusion protein consisting of the part of the FokI restriction endonuclease protein responsible for DNA cleavage and a zinc finger protein which recognises specific, designed genomic sequences and cleaves the double-stranded DNS at those sequences, thereby producing free DNA ends (Urnov F. D. et al., Nat Rev Genet. 11:636-46, 2010; Carroll D., Genetics. 188:773-82, 2011).
[0094] The Transcription Activator-Like Effector Nuclease (TALEN in short) is a fusion protein consisting of the part of the FokI restriction endonuclease protein responsible for DNA cleavage, the part of the transcription activator-like effector (TALE) protein responsible for DNA binding, and an amino acid segment responsible for transfer into the nucleus (Nuclear Localization Signal, NLS in short). The DNA binding portion of the protein can be designed to be sequence-specific (Christian M. et al., Genetics 189:757-761, 2010; Mussolino C. et al., Nucleic Acids Res. 39:9283-9293, 2011; Miller J. C. et al., Nat. Biotechnol. 29:143-148, 2011; Cermak T. et al., Nucl. Acids Res. 39:e 82, 2011).
[0095] The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas in short) is an RNA-guided (simple guide RNA, sgRNA in short) DNA endonuclease system performing sequence-specific double-stranded breaks in a DNA segment homologous to the designed RNA. It is possible to design the specificity of the sequence (Cho S. W. et al., Nat. Biotechnol. 31:230-232, 2013; Cong L. et al., Science 339:819-823, 2013; Mali P. et al., Science 339:823-826, 2013; Feng Z. et al., Cell Research: 1-4, 2013).
[0096] One advantage of the above techniques is that the transgenes containing the TALEN or ZFN or CRISPR/Cas nuclease can be removed from the progeny of the Xe resistant plant by genetic segregation, that is, non-transgenic plants can be generated from them.
[0097] The mutant variants of the CYSTM region of the xcv-1 protein can be designed using computer programmes, and common features of them include their localisation at the C-terminus of the protein, the presence of at least 2 cysteines generally followed by an aspartic acid (D), glutamic acid (E) or asparagine (N) residue, and they are represented by the following sequences:
TABLE-US-00001 CXXXXCCCCD, XXXXXCCCCD, CXXXXXCCCD, CXXXXCXCCD, CXXXXCCXCD, CXXXXCCCXD, XXXXXXCCCD, XXXXXCXCCD CXXXXXXCCD, CXXXXCXCXD, CXXXXCCXXD, CXXXXCCCCE, XXXXXCCCCE, CXXXXXCCCE, CXXXXCXCCE, CXXXXCCXCE, CXXXXCCCXE, XXXXXXCCCE, XXXXXCXCCE, CXXXXXXCCE, CXXXXCXCXE, CXXXXCCXXE, CXXXXCCCCN, XXXXXCCCCN, CXXXXXCCCN, CXXXXCXCCN, CXXXXCCXCN, CXXXXCCCXN, XXXXXXCCCN, XXXXXCXCCN, CXXXXCXCCN, CXXXXCXCXN, CXXXXCCXXN
wherein D=aspartic acid, E=glutamic acid, N=asparagine, X=an amino acid residue compatible with the transmembrane character, mostly a hydrophobic or non-polar amino acid residue, e.g., glycine (G), cysteine (C), leucine (L), isoleucine (I), alanine (A), tryptophan (W), threonine (T), methionine (M), phenylalanine (F) or valine (V).
[0098] The xcv-1 gene providing recessive resistance was isolated from pepper using Capsicum annuum Gene Bank Accession No. PI163192. The isolation of the xcv-1 gene enables the designing of genetic markers using the sequence information of the gene and the linked DNA region, and the facilitation of traditional pepper breeding using these markers for Marker Assisted Selection, as well as the generation of resistant pepper and other plant varieties based on the sequence information of the gene by biotechnological methods involving the targeted modification or transforming the cells with a xcv-1 homologues gene prior to knocking-out the resident gene.
[0099] According to the invention, the xcv-1 gene was isolated from Capsicum annuum using the following method.
1. Genetic Mapping of the Xcv-1 Gene in Pepper
1.1. Generation of the F2 Segregating Population
[0100] For the mapping, a new population was created by intraspecies crossing (Capsicum annuum x Capsicum annuum) followed by the self-pollination of the F1 plants. For the crossing, Feherozon (FO), a commercially available Hungarian cultivated variety sensitive to Xanthomonas was used as the father parent and an Xe-resistant plant, Gene Bank Accession No. PI163192 (T1), was used as the mother parent. After the crossing, 45 seeds from the fruit of one of the mother plants were sown and the hybrid character of the resulting plants was confirmed by appropriate molecular DNA markers. Next, the 45 F1 individuals were grown, and the F2 seeds from the self-pollination were collected. The F2 individuals from the self-pollination of the F1 individuals were then used for the genetic mapping of the xcv-1 gene. The objective was to grow as many F2 individuals as possible to allow for the identification of individuals carrying recombination events as close to the xcv-1 gene as possible, and thereby for the narrowing of the genetic and--at the same time--the physical region comprising the xcv-1 gene. Until the identification of the xcv-1 gene, more than 3000 F2 individuals were generated, grown and subjected to xcv-1 phenotyping.
1.2. Xanthomonas Resistance Test
[0101] Phenotyping the segregating individuals as sensitive or resistant to Xanthomonas is indispensable and of key importance for localising the xcv-1 gene on the genetic map. An incorrect phenotyping makes genetic mapping impossible or extremely difficult. As a result of the biological tests, finally 765 and 2354 F2 plants proved to be resistant and sensitive, respectively.
1.3. Mapping of the Xcv-1 Locus
[0102] On the basis of the available genotypes and xcv-1 phenotypes, the locus of the xcv-1 gene was mapped to the third chromosome of pepper. For this, sequences available in gene banks or markers used by others were applied for the mapping of the xcv-1 gene. Specific primer pairs were designed, and the primer pairs were used for PCR amplification; the map location of the markers were determined on the basis of the genotype of the markers using the polymorphism data obtained upon electrophoresis. The mapping identified a genetic marker (CaCY), which mapped to the shortest distance from the xcv-1 locus. The CaCY marker can be genotyped using primers Pr_CaCYF1 (SEQ ID NO:1) and Pr_CaCYR1 (SEQ ID NO:2).
1.4. Chromosome Walking
[0103] The identified marker, CaCY, which is closely linked to the xcv-1 gene (located at a distance of 0.22 centimorgan from xcv-1) allowed the initiation of the chromosome walking. In the first step, the primary pepper BAC clone (Clone No. 279) was identified with the help of the CaCY marker using multiplex PCR. The terminal sequences of the primary BAC clone (No. 279) were determined and primer pairs specific to them were designed. With the help of the specific primer pairs, BAC clones overlapping with BAC Clone No. 279 were identified (Clones No. 632 and 1248), and another set of specific primers were designed for their terminal sequences and additional overlapping clones were identified, then additional clones were isolated in a similar manner. Using the specific primer pairs designed for BAC Clones No. 66, 1191, 50, 877 and 472, the BAC ends were back-mapped to the genetic map thereby verifying the correct direction of the contig building. With the specific primer pair designed for the -40 end of BAC Clone No. 50, we managed to pass a recombination towards the xcv-1 gene, therefore, contig building was only continued into this direction. From BAC Clone No. 50, the contig was extended by another overlapping BAC clone in the above manner, and upon back-mapping marker 472_op, further recombinant individuals delimiting the contig comprising the xcv-1 gene could be identified.
1.5. Sequencing of BAC Clones Overlapping the Xcv-1 Region: Subcloning, Sequencing of the Subclones and Solid Sequencing
[0104] In the next step, two BAC clones overlapping the xcv-1 region (No. 50 and 472) were sequenced. DNA sequencing was carried out in two ways: by subcloning and sequencing of the subclones from both sides, and by the new-generation Solid sequencing method developed by ABI.
2. Identification of the Xcv-1 Gene
2.1. Determination of the Gene Contents of the BAC Clones
[0105] The resulting DNA sequence data were handled and assembled into contigs giving overlapping segments by various computer programs. This "assembly" did not produce a sequence of the complete length of BAC but more than ten contigs, which were closed by the so-called primer walking technique. The sequences of the resulting partial BAC clones (No. 50 and 472) were determined.
[0106] The gene content of the two BAC clones were determined by the BLAST programmes (chiefly the blastn, blastx and blastp programmes) of NCBI (http://ncbi.nlm.nih.gov/BLAST/) and DFCI (http://compbio.dfci.harvard.edu/tgi/plant.html). On the basis of the gene content and gene order information, polymorph markers were prepared using specific primer pairs, and the genes were back-mapped. So far, a total of 13 protein-encoding genes were identified in the two BAC clones. On the basis of the mapping data, it was found that the xcv-1 gene is located between markers Pr6 and Pr4b as a single gene encoding more than 50 amino acids. The primer sequences of the markers are as follows: Pr6F1: SEQ ID NO:33, Pr6R1: SEQ ID NO:34, Pr4bF1: SEQ ID NO:35 and Pr4bR1: SEQ ID NO:36.
[0107] Next, the DNA sequence of both the xcv-1 gene and its wild-type counterpart (Xcv-1 gene) was determined in both parents using specific primer pairs; see SEQ ID NO:37 and SEQ ID NO:41, respectively.
[0108] Thus, the present invention relates to the xcv-1 gene isolated by the above method, which has the DNA sequence presented in SEQ ID NO:37. The cDNA sequence of the xcv-1 gene--which is the nucleotide sequence of SEQ ID NO:90--was generated, and is also covered by the scope of the invention.
[0109] The xcv-1 gene encodes a CYSTM protein, the xcv-1 protein, the amino acid sequence of which is that of SEQ ID NO:38, wherein the cysteine-rich transmembrane region (CYSTM region) of the protein carries a double Leu deletion at the locations corresponding to positions 87 and 88 of the wild-type Xcv-1 protein of SEQ ID NO:42. However, the invention also encompasses all those protein sequences which are proteins homologous to the xcv-1 protein and represent protein variants containing at least 53%, preferably 60% to 73%, more preferably 80% to 93% identical amino acids with respect to the last 13 amino acids of the C-terminus of the xcv-1 protein.
[0110] Furthermore, the present invention relates to vectors, preferably expression vectors, comprising the genes isolated and prepared according to the invention in a functional form. Preferred vectors useful for the purpose of the invention include the binary vectors of Agrobacterium tumefaciens, such as the pCAMBIA vector family (http://www.cambia.org/daisy/cambia/585).
[0111] Furthermore, the present invention relates to ZFN, TALEN and CRISPR/Cas nucleases specific to the interest sequence of the Xcv-1 gene, which are used to generate the 6-bp deletion or to inactivate (by knock-out) the genes homologous to the wild-type Xcv-1 DNA.
[0112] Furthermore, the present invention relates to host cells into which the vectors of the invention were introduced, e.g., by transformation or by genome editing techniques (ZFN, TALEN and CRISPR/Cas nuclease), and by means of which the desired 6-bp deletion was generated in the Xcv-1 homologous genes. Preferred host cells useful for the purpose of the invention include Solanaceae, Oryzae, Citroidieae etc. species.
[0113] The present invention also relates to a transformation method of generating a transgenic plant resistant to a biotic or abiotic factor, comprising the steps of: a) identifying one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in said plant, preparing in vitro the genomic and cDNA sequences of the identified gene in a DNA form, generating in vitro a 6-bp mutation corresponding to the deletion of two amino acids in the segments encoding the CYSTM region of the one or more identified genes and transforming the cells of a sensitive plant with the one or more mutant genes thus obtained in a manner ensuring the functional expression thereof, b) inactivating the one ore more resident genes identified in step a) or the mRNA or protein thereof in the transformant plant cells, and c) regenerating the plant from the transformants and selecting the resistant individuals.
[0114] In step a) of the above method, genetic engineering methods well-known to those skilled in the art were used to identify the gene(s), to prepare them in a DNA form, to generate the mutations and to transform the plant cells.
[0115] In step b) of the above method, the resident genes homologous to the Xcv-1 gene are inactivated (silenced). Inactivation (silencing) of the resident genes is necessary because the function of the wild-type protein (pl. Xcv-1) is dominant over the function of the mutant protein (e.g., xcv-1), i.e., the latter function is recessive. Well-known methods are also available for inactivating the gene, i.e., for eliminating the function of the gene. Examples include but are not limited to the following: inhibiting the expression of the protein products of the resident genes using natural, chemical or insertion mutagenesis, amiRNA (artificial miRNA), RNAi (RNA interference) or other techniques; inactivating the resident gene(s) using nuclease deletion methods generating knock-out mutants. Preferably, the above-described TALEN, ZFN or CRISPR/Cas nuclease technique is used. By expressing the gene of a monoclonal antibody specific to the protein, one can inactivate the Xcv-1, or the homologous proteins.
[0116] It is important to note that the amiRNA technique has been successfully used to create resistance against viruses in plants (Niu et al., Nat. Biotechnol. 24:1420-1428, 2006), however, resistance to pathogenic bacteria such as Xe has not been created in plants with the amiRNA technique yet.
[0117] In step b) of a preferred embodiment of the method of the invention, the mRNA products of the resident gene(s) are functionally inactivated (silenced) by the amiRNA technique as follows: b1) preparing an amiRNA gene construct for the ribonucleotide sequence complementary to the CYSTM region of the mRNA of one or more genes homologous to the wild-type Xcv-1 gene and identified in the transformant plant cells obtained in step a); b2) cloning the construct obtained in step b1) into an appropriate vector; b3) transforming the transformant plant cells with the vector obtained in step b2) in a manner ensuring the functional expression of the amiRNA and inactivation of the mRNA products of the wild-type CYSTM gene(s); and c) regenerating the transformants obtained in step b3) and selecting the resistant plant.
[0118] The nucleases of the invention (engineered nucleases) are used for two genome editing functions in the present invention: on the one hand, for inactivating the resident genes in the transformant plant cells; on the other hand, for creating the desired 6-bp deletion in the gene homologous to the Xcv-1 gene in the genome of a sensitive plant cell. The two functions may also be combined, for example, in tomato, where the desired 6-bp deletion appears in one of the two homologous wild-type genes (SlXcv-1A, SEQ ID NO:49 and SlXcv-1B, SEQ ID NO:51), and the gene is inactivated upon a deletion or insertion in the other.
[0119] In a manner obvious to a skilled person, the target sequences of the above nucleases are selected in a manner ensuring that they cover the 6-bp sequence to be deleted in order to prevent the recognition and cleavage of the DNA segment already comprising the deletion, and that the desired 6-bp deletion can be created.
[0120] In another preferred embodiment of the method of the invention, the resident gene(s) is/are inactivated by a nuclease deletion (genome editing) method as follows: b1) preparing DNA constructs encoding TALEN-L and TALEN-R or ZFN-L and ZFN-R proteins specific to the gene sequence(s) encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene and identified in the transformant plant cells obtained in step a), more specifically those specific to the DNA segments corresponding to the 6 nucleotides to be deleted and the surrounding nucleotides as target sequence in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence; b2) cloning the constructs obtained in step b1) into an appropriate vector; b3) transforming the plant cells with a vector comprising the constructs obtained in step b1) in a manner ensuring the functional expression of the transgenes; c) identifying knock-out deletion or insertion mutations in the genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41); d) selecting the plant cells containing the mutations identified in step c); e) regenerating the plant cells identified in step d) and selecting the resistant plants, and f) removing the transgenes comprising the TALEN or ZFN constructs or the CRISPR/Cas constructs containing the gene of the sgRNS protein by genetic segregation.
[0121] The present invention also relates to a method for generating a mutant plant showing an abiotic or biotic resistance using a genome editing method, comprising the steps of: a) identifying one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a sensitive plant; b) preparing DNA constructs encoding TALEN-L and TALEN-R or ZFN-L and ZFN-R proteins specific to the gene sequence encoding the CYSTM region of one or more genes homologous to the wild-type Xcv-1 gene and identified in step a), more specifically those specific to the 6 nucleotides to be deleted and the surrounding nucleotides as target sequence in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence; c) cloning the construct obtained in step b) into an appropriate vector; d) transforming sensitive plant cells with a vector comprising the construct obtained in step b) in a manner ensuring the functional expression of the transgenes and the generation of the 6-bp deletion in the CYSTM region by the nuclease; e) identifying the transformants carrying mutations showing the 6-bp deletion in the CYSTM region of the genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41); f) regenerating the plant cells having the mutation identified in step e) and selecting the resistant plants; and g) removing the transgenes comprising the TALEN or ZFN constructs or CRISPR/Cas constructs containing the gene of the sgRNS protein by genetic segregation.
[0122] Furthermore, the mutant plants showing biotic or abiotic resistance according to the invention can be generated by introducing TALEN or ZFN proteins specific to the CYSTM region into the plant using non-pathogenic bacteria, said method, comprising the steps of: a) identifying one or more genes homologous to the wild-type Xcv-1 gene (SEQ ID NO:41) in a sensitive plant; b) preparing DNA constructs encoding TALEN-L plus TALEN-R or ZFN-L plus ZFN-R proteins specific to the DNA segment encoding the CYSTM region of the gene identified in step a) in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs; c) cloning the constructs obtained in step b) into an appropriate bacterial vector and introducing them into non-pathogenic bacteria with active type three secretion system using said vector; d) infecting the sensitive plant with bacteria obtained step c); and e) regenerating a resistant plant from the infected plant tissue.
[0123] In the methods of the invention, not only the genomic sequences but also the cDNA sequences of the genes homologous to the wild-type Xcv-1 or the mutant xcv-1 gene can be used with identical results since the same protein is expressed from both.
[0124] Using the method of the invention, resistance can be created in plants which are sensitive to Xanthomonas species, such as Xanthomonas euvesicatoria, Xanthomonas perforans, Xanthomonas gardneri, Xanthomonas vesicatoria pv. oryzae, and other Xanthomonas species. One such preferred plant is tomato.
[0125] One of the most dangerous pathogens of tomato is Xanthomonas euvesicatoria (Xe), which also causes severe damage to pepper. Unlike pepper, tomato has not developed appropriate natural resistance, which would ensure sufficient protection. Thus, tomato production is still highly threatened by Xe infection (Hutton S. F. et al., Theor. Appl. Genet. 121:1275-87, 2010). The xcv-1 gene identified in pepper may also provide resistance to Xe infection in tomato by ensuring the introduction and functional expression of the mutant tomato genes Slxcv-1A and/or Slxcv-1B (SEQ ID NO:59 and/or SEQ ID NO:66), which are homologous to the xcv-1 gene, in the tomato genome followed by the inactivation of the dominant resident genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and SEQ ID NO:51) homologous to the wild-type Xcv-1 gene, i.e., by generating non-functional variants thereof.
[0126] The tomato plant resistant to Xanthomonas euvesicatoria can be generated using the following methods of the invention:
[0127] transforming the cells of the tomato plant with the mutant genes Slxcv-1A and/or Slxcv-1B carrying the double Leu deletion, followed by inhibiting the function of the SlXcv-1A and/or SlXcv-1B genes using the amiRNA technique; or
[0128] transforming the cells of the tomato plant with the mutant genes Slxcv-1A and/or Slxcv-1B carrying the double Leu deletion, followed by inactivating the resident genes SlXcv-1A and/or SLXcv-1B located there using the ZFN nuclease or TALEN nuclease or CRISPR/Cas nuclease technique; or
[0129] transforming the cells of the tomato plant with the mutant genes Slxcv-1A and/or Slxcv-1B carrying the double Leu deletion; followed by identifying the mutations inactivating the SlXcv-1A and/or SlXcv-1B gene using the TILLING or a similar technique upon or without mutagenesis; or
[0130] creating the desired 6-bp deletion in genes SlXcv-1A and/or SlXcv-1B in the genome of the sensitive tomato plant using a specific nuclease-based genome editing method, which provides resistance.
[0131] Thus, one object of the present invention is a method for generating a tomato plant resistant to Xanthomonas euvesicatoria, comprising the steps of:
[0132] a) identifying the resident genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and 51, respectively), which are homologous to the wild-type Xcv-1 gene in the tomato plant; b) preparing in vitro the genomic and cDNA sequences of the identified genes in the form of a DNA; c) creating in vitro the 6-bp deletion in the positions corresponding to the deletions in the xcv-1 gene thereby generating constructs carrying the mutant genes Slxcv-1A and Slxcv-1B (SEQ ID NO:59 and 66, respectively) or their cDNA sequences (SEQ ID NO:88 and 89, respectively); d) cloning the constructs obtained in step c) into an appropriate vector and transforming the cells of the tomato plant with the resulting vector in a manner ensuring the functional expression of the transgenes; e) preparing an amiRNA gene construct for silencing the wild-type genes SlXcv-1A and SlXcv-1B (SEQ ID NO:49 and/or SEQ ID NO:51); f) cloning the construct obtained in step e) into an appropriate vector; g) transforming the plant cells generated in step d) with the vector obtained in step f) in a manner ensuring the expression of the amiRNA and inactivation of the mRNA products of the wild-type CYSTM genes SlXcv-1A and SlXcv-1B; and h) regenerating the transformants obtained in step g) and selecting the resistant plant.
[0133] In a preferred embodiment the amiRNS gene construct is specific to the complementary ribonucleotide sequence corresponding to the CYSTM region of the wild type SlXcv-1A and/or SlXcv-1B genes (SEQ ID NO:49 and/or SEQ ID NO:51) more specifically an amiRNA gene construct specific to the 6 nucleotides to be deleted and the surrounding nucleotides (SlXe1-amiRNA, SEQ ID NO: 74). In a preferred embodiment the amiRNA gene construct comprises the nucleic acid molecule according to the invention.
[0134] The present invention also relates to another method for generating a tomato plant resistant to Xanthomonas euvesicatoria, comprising the steps of: repeating steps a) to d) above; e) preparing proteins TALEN-L (SEQ ID NO:78) and TALEN-R1 (SEQ ID NO:79) and TALEN-R2 (SEQ ID NO:80) specific to the target sequences (SEQ ID NO:75, 76, 77) specific to the CYSTM region of the wild-type SlXcv-1A and SlXcv-1B gene, or DNA constructs encoding the proteins ZFN-L and ZFN-R specific to SEQ ID NO: 86 and 87 in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence (SEQ ID NO: 81); f) cloning the construct obtained in step e) into an appropriate vector; g) transforming the plant cells generated in step d) with the vector obtained in step f) in a manner ensuring the functional expression of the above TALEN or ZFN or CRISPR/Cas nuclease transgenes and the functional inactivation of genes SlXcv-1A and SlXcv-1B by them; h) identifying deletion or insertion knock-out mutations in the transformant cells; i) regenerating the plant cells having the mutation identified in step h) and selecting the resistant plants; and f) removing the nuclease transgenes TALEN-L plus TALEN-R or ZFN-L plus ZFN-R or CRISPR/Cas by genetic segregation.
[0135] The present invention also relates to yet another method for generating a tomato plant resistant to Xanthomonas euvesicatoria, comprising the steps of: a) identifying the resident genes SlXcv-1A (SEQ ID NO:49) and SlXcv-1B (SEQ ID NO:51), which are homologous to the wild-type Xcv-1 gene in the tomato plant; b) preparing proteins TALEN-L (SEQ ID NO:78) and TALEN-R1 (SEQ ID NO:79) and TALEN-R2 (SEQ ID NO:80) specific to the target sequences (SEQ ID NO:75, 76, 77) specific to the CYSTM region of the wild-type SlXcv4A and SlXcv-1B gene, or DNA constructs encoding the proteins ZFN-L and ZFN-R specific to SEQ ID NO: 86 and 87 in a manner ensuring that the 6 nucleotides to be deleted from the wild-type gene are positioned in the middle of the spacer segment of the TALEN-L plus TALEN-R or ZFN-L plus ZFN-R pairs, or by preparing a CRISPR/Cas construct comprising the sgRNA gene sequence specific to the 6 nucleotides to be deleted from the wild-type gene and the surrounding nucleotides as target sequence (SEQ ID NO: 81); c) cloning the construct obtained in step b) into an appropriate vector; d) transforming the cells of the tomato plant with the vector obtained in step c) in a manner ensuring the functional expression of the above TALEN or ZFN or CRISPR/Cas nuclease transgenes and the creation of the 6-bp deletion in the CYSTM region of genes SlXcv-1A and SlXcv-1B by them; e) identifying the transformants carrying the 6-bp deletion in the CYSTM region of the wild-type genes SlXcv-1A and SlXcv-1B; f) regenerating the transformants having the mutation identified in step e) and selecting the resistant plant; and g) removing the nuclease transgenes TALEN-L plus TALEN-R or ZFN-L plus ZFN-R or CRISPR/Cas by genetic segregation.
[0136] The development of resistant cultivars has been the most effective, economical and environmental friendly strategy to control disease epidemic of cultivated plants. Out of many possibilities, pyramided resistance is far more durable than resistance that is controlled by a single dominant R genes (usually causing HR), because new races of pathogens could easily evolve to overcome or escape the resistance consequently plant resistant trait breaks down. Traditional breeding combined with molecular markers based marker assisted selection has made it possible to identify and pyramid valuable genes of agronomic importance in resistance. In addition to this strategy, transgenic approaches serve further possibility to pyramid resistant genes in plant cultivars. As mentioned above tomato, a close relative of pepper is highly susceptible to Xe. To fight against this pathogen and establish Xe resistant tomato, transgenic tomato plants expressing the Bs2 resistance gene from pepper was constructed recently (Horvath et al., PLoS One.; 7(8):e42036, 2012). In replicated multi-year field trials under commercial type growing conditions demonstrated improved resistance to bacterial spot disease caused by Xe. Taking into account the beneficial impact of pyramided gene configuration the above mentioned tomato Bs2 containing can be a starting material to produce double resistant derivatives by expressing the Slxcv-1A and/or Slxcv-1B gene carrying the beneficial 6 bp deletion as described in this invention. By this way highly resistant and durable Xe resistant cultivars of tomato can be breeded for commercial production. A skilled person would recognize that the resistance based on the expression of Slxcv-1A and/or Slxcv-1B gene can be combined not only with Bs2, but with other genes too, which may confer resistance to Xe in tomato including but not limited to Bs1, Bs3, Bs4, bs5, bs6.
[0137] In addition to pepper and tomato, several other plants are severely infected by Xanthomonas species causing disease on rice, potato, citrus, banana, grape, etc. (Dangle et al. Science 341: 746, 2013). The desired 6 bp deletion derivative can also be generated in the Xcv-1 homologous gene(s) of these plants and can be combined with other type of resistance genes against Xanthomonas. Accordingly, one can generated rice plants resistant against Xanthomonas oryzae, or citrus plants resistant against Xanthomonas citri pv. citri or Xanthomonas axonopodis pv. citri or banana plants resistant against Xanthomonas campestris pv. musacearum.
[0138] In addition, the method of the invention can be used to create resistance to an abiotic or biotic factor other than Xanthomonas sp. in plants.
[0139] The present invention further relates to mutant plants resistant to a biotic or abiotic factor, which carry a deletion of two amino acids in their CYSTM region in comparison with the wild-type plant. Preferably, the mutant plant is a pepper plant (Capsicum annuum), a tomato plant (Solanum lycopersicum), a plant from the Solanaceae family, e.g., potato, eggplant etc., a citrus (Citroideae), e.g., orange (Citrus aurantium), mandarin (Citrus reticulata), lemon (Citrus x medica L.), grapefruit (Citrus x paradisi), pomelo (Citrus maxima or grandis) etc., a plant from the Brassicaceae family, e.g., cabbage (Brassica oleracea convar. capitata var. alba), radish (Raphanus sativus), cauliflower (Brassica oleracea convar. botrytis var. botrytis), rape (Brassica napus) etc., a monocot plant (Monocotyledonae), e.g., rice (Oryzae sp.), maize (Zea mays), wheat (Triticum sp.), rye (Secale sp.), barley (Hordeum vulgare), millet (Panicum sp.), etc., a plant from the Fabaceae or Leguminosae families, e.g., alfafa (Medicago sp.), bean (Phaseolus sp.), pea (Pisum sp.), soy (Glycine sp.), horse bean (Faba sp.), lupine (Lupinus sp.), clover (Trifolium sp.), peanut (Arachis sp.), vicia (Vicia sp.), lathyrus (Lathyrus sp.), lentil (Lens sp.), chick-pea (Cicer sp.), mung bean (Vigna sp.), pigeon pea (Cajanus cajan) etc., a plant from the Cucurbitaceae family, e.g., pumpkin (Cucurbita sp.), cucumber (Cucumis sp.), melons (Citrullus sp.) etc., a plant form the Rosaceae family, e.g., apple (Malus sp.), pear (Pyrus communis), quince (Cydonia oblonga), cherry (Prunus subg. Cerasus), sour cherry (Prunus cerasus), plum (Prunus domestica subsp. domestica), apricot (Prunus armeniaca), peach (Prunus persica), grape (Vitis vinifera), etc., in which resistance has been created.
[0140] More preferably, the mutant plant is a mutant tomato plant (Solanum lycopersicum) resistant to Xe.
[0141] Another object of the present invention are the seeds and the products of the mutant plants generated by this invention, including but limited to fruits, juice, paste, etc., preferably the seeds and products of the mutant tomato plant and its progeny.
[0142] Furthermore, we can raise antibodies against the xcv-1 protein of the invention, which can be used as probes in in vitro methods performed in plant-derived cell lines in order to test whether a given plant is resistant to Xe or not. The methods of raising antibodies and such techniques are well known to those skilled in the art.
[0143] The present invention further relates to gene probes, which are specific to the xcv-1 gene or its homologous genes and hybridizing with them under stringent conditions.
[0144] Another objects of the present invention are primer pairs, which are specific to the xcv-1 gene or its homologous genes, especially to the Slxcv-1A and/or the SlXcv-1B, and can be used to genotype plants carrying the 6 bp deletion including but not limited to markere assisted selection.
[0145] The invention is described in more detail through the following examples without limiting the scope of the invention.
Example 1
Genetic Crosses and Analysis of the F2 Progeny of the Xcv Plant
[0146] For the generation of F1 individuals, commercially available C. annuum var. Feherozon sensitive to Xanthomonas euvesicatoria (Xe) was used as the father parent (marked as F0), and Capsicum annuum var. T1/1 carrying Xe resistance--an individual of Gene Bank Accession No. PI163192--was used as the mother parent (T1/1). After the crossing, 45 F1 seeds from the mother plant were sown and F2 plants were grown from them. When plants reached the 8-leaved age, a Xanthomonas euvesicatoria infection test was used to determine the sensitivity of the plants to Xe. Finally, 20 F2 individuals--8 resistant and 12 sensitive individuals (see Table 1)--were selected for the general mapping experiments; on the other hand, more than 3000 F2 individuals were used for the fine-mapping of the Xe resistance gene (xcv-1).
TABLE-US-00002 TABLE 1 Phenotypes of 20 F2 individuals of the segregating population after infection by Xanthomonas euvesicatoria (xcv phenotype); plant name xcv phenotype plant name xvc phenotype 1 S 11 R 2 S 12 R 3 S 13 R 4 S 14 R 5 S 15 R 6 S 16 R 7 S 17 S 8 S 18 S 9 R 19 S 10 R 20 S S = sensitive; R = resistant
Example 2
Identification of Markers Linked to the Xcv-1 Gene of the T1/1 Mutant Plant
[0147] Identification by genetic mapping of molecular markers mapping close to the mutated xcv gene, i.e., those linked to Xcv resistance, was carried out using the 20 F2 progeny mentioned in Example 1. Total DNA from fresh leaves was subjected to PCR amplification using specific primers designed on the basis of pepper sequences available in the databases, and the resulting fragments were subjected to electrophoresis on agarose gels or on so-called SSCP acrylamide gels. In order to visualise the DNA fragments, agarose gels and acrylamide gels were stained using ethidium bromide and silver, respectively. The linkage of markers showing polymorphism on the agarose or SSCP gels was determined by colour mapping (Kiss et al., Acta Biologica Hungarica 49:47-64, 1998) with respect to the xcv/Xcv phenotype after ascertaining the homozygote or heterozygote status. As a result of the systematic mapping, a single marker designated as CaCY showed a distance of 0.22 centimorgan.
[0148] The identifiers of the primers of the CaCY marker (Pr_CACY_F1 and Pr_CACY_R1) are SEQ ID NO:1 and SEQ ID NO:2, respectively.
[0149] Since other mapped markers were either unlinked to the xcv-1 gene or were located at much greater genetic distances; therefore the so-called chromosome walking was initiated using the CaCY marker to physically the xcv-1 gene.
Example 3
Isolation of BAC Clones Overlapping with the Xcv-1 Mutation, Contig Building
[0150] A primary BAC clone (No. 279) was isolated using the molecular marker showing the strongest linkage to the xcv-1 mutation, i.e., CaCY. The primary and other BAC clones are isolated using multiplex PCR from a BAC library comprising 380,000 BACs, which was prepared from an Xcv resistant pepper (Capsicum annuum) plant and ensures a 22-fold coverage of the pepper genome (Bukovinszki et al., VII. Hungarian Congress on Genetics, Abstract Book, p. 91, 2007).
[0151] Both ends of BAC Clone No. 279 were sequenced and additional two BAC clones were identified using primer pairs specific to these sequences: BAC Clone No. 1248 using primers Pr_279 op F1 (SEQ ID NO:3) and Pr_279 op R1 (SEQ ID NO:4), and BAC Clone No. 632 using primer pair Pr_279-40 F1 (SEQ ID NO:5) plus Pr_279-40 R1 (SEQ ID NO:6).
[0152] Primer pairs specific to the terminal sequences of BAC No. 1248 were used to identify BAC Clone No. 1191: the pair consisted of primers Pr_1248 op F1 (SEQ ID NO:7) and Pr_1248 op R1 (SEQ ID NO:8), and as control, BAC Clone No. 279 was reidentified using primer pair Pr_1248-40 F1 (SEQ ID NO:9) plus Pr_1248-40 R1 (SEQ ID NO:10).
[0153] Primer pairs specific to the terminal sequences of BAC No. 1191 were used to identify BAC Clone No. 877: the pair consisted of primers Pr_1191-40 F1 (SEQ ID NO:11) and Pr_1191-40 R1 (SEQ ID NO:12), and as control, BAC Clone No. 1248 was reidentified using primer pair Pr_1191 op F1 (SEQ ID NO:13) plus Pr_1191 op R1 (SEQ ID NO:14).
[0154] Primer pair Pr_877-40 F1 (SEQ ID NO:15) plus Pr_877-40 R1 (SEQ ID NO:16) was designed for the -40 terminal sequence of BAC No. 877, and was used for genetic back-mapping.
[0155] Primer pairs specific to the terminal sequences of BAC No. 632 were used to identify BAC Clone No. 66: the pair consisted of primers Pr_632-40 F1 (SEQ ID NO:17) and Pr_632-40 R1 (SEQ ID NO:18), and as control, BAC Clone No. 279 was reidentified using primer pair Pr_632 op F1 (SEQ ID NO:19) plus Pr_632 op R1 (SEQ ID NO:20).
[0156] Primer pairs specific to the terminal sequences of BAC No. 66 were used to identify BAC Clone No. 50: the pair consisted of primers Pr_66-40 F1 (SEQ ID NO:21) and Pr_66-40 R1 (SEQ ID NO:22), and as control, BAC Clone No. 632 was reidentified using primer pair Pr_66 op F1 (SEQ ID NO:23) plus Pr_66 op R1 (SEQ ID NO:24).
[0157] Primer pairs specific to the terminal sequences of BAC No. 50 were used to identify BAC Clone No. 472: the pair consisted of primers Pr_50 op F1 (SEQ ID NO:25) and Pr_50 op R1 (SEQ ID NO:26), and as control, BAC Clone No. 66 was reidentified using primer pair Pr_50-40 F1 (SEQ ID NO:27) plus Pr_50-40 R1 (SEQ ID NO:28).
[0158] On the one hand, primer pairs designed for the terminal sequences of BAC No. 472 were used to reidentify BAC Clone No. 66 as control: the pair consisted of primers Pr_472-40 F1 (SEQ ID NO:29) and Pr_472-40 R1 (SEQ ID NO:30), and primer pair Pr_472 op F1 (SEQ ID NO:31) plus Pr_472 op R1 (SEQ ID NO:32) was used for genetic back-mapping.
[0159] Thus, by classifying BAC Clones into contig groups, we could draft the so-called xcv contig (FIG. 1) characteristic of the xcv region, which physically covers the xcv-1 gene.
Example 4
More Precise Determination of the Location of the Xcv-1 Gene within the Contig by Identifying the Recombination Sites that are Nearest to the Mutation
[0160] Determining the location of the xcv-1 gene more precisely within the xcv contig is of key importance since the nearer the given molecular marker is, the lower number of BAC clones has to be sequenced, and this results in fewer candidate genes. More precise determination of the location of the xcv-1 gene within the xcv contig was performed as follows. On the one hand, the number of individuals in the F2 segregation population was increased to allow the analysis of as many recombination events as possible. Finally, a total of 3119 individuals were included in the genetic analysis. On the other hand, molecular markers were developed using terminal sequences of overlapping BACs and were back-mapped using the population segregating the xcv-1 gene. On the basis of the results, it was concluded that the position of the xcv-1 gene is between the -40 end of BAC No. 50 and the op end of BAC No. 472 separated by three and four recombination events, respectively.
Example 5
Subcloning of BAC Clone No. 50 and Sequencing of the Subclones
[0161] Subcloning of BAC Clone No. 50 was performed after cleavage by restriction enzymes BamHI, EcoRI and HindIII. Upon purification, the subclones were digested using BamHI, HindIII and EcoRI, and the resulting fragments are cloned into vectors digested with BamHI, HindIII es EcoRI and transformed into Escherichia coli cells. The sequences of the fluorescence-labelled amplificates of the recombinant clones were determined using ABI 373 and ABI 377 automated sequencers (Perkin Elmer Applied Biosystems; 850 Lincoln Centre Drive Foster City, Calif. 94404 USA).
[0162] The DNA sequence of BAC Clone No. 50 was also determined using second generation sequencing technologies (SOLID and Iontorrent, Applied Biosystems), as well as using the "primer walking" technique until the complete sequence was obtained.
Example 6
Fine-Mapping of the Xcv-1 Region
[0163] Partial sequence data were stored in a computer, and the analysis was started by determining their correct order on the basis of their overlapping sequences. In a manner obvious to those skilled in sequence alignment and sequence analysis, the overlapping terminal sequences of the BAC clones and their subclones generated by restriction digestion provide help for the assembly of the sequences and for the determination of the relative locations of the subclones generated by random and restriction digestion and of the BAC clones. Upon the assembly of the partial sequences, we succeeded in compiling the sequence of BAC Clone No. 50, which was then used to develop genetic markers at various distances from the BAC termini. When these genetic markers were back-mapped in the mapping population, it was revealed that the xcv-1 gene is located between PCR-based markers Pr6 and Pr4b. The primer sequences of markers Pr6 and Pr4b are as follows: Pr6F1: SEQ ID NO:33, Pr6R1: SEQ ID NO:34, and Pr4bF1: SEQ ID NO:35, Pr4bR1: SEQ ID NO:36.
Example 7
Sequence Analysis of the Xcv1 Region and Detailed Assessment of the Xcv-1 Gene
[0164] Upon obtaining the nucleotide sequence of the DNA segment between markers Pr4b and Pr6, the databases of NCBI (National Center for Biotechnology Information http://www.ncbi.nlm.nih.gov/BLAST/), DFCI (http://compbio.dfci.harvard.edu/tgi/plant.html), Medicago HapMap (http://www.medicagohapmap.org/?genome) and Arabidopsis (http://www.arabidopsis.org) were successfully searched for homologous genes. The sequences were evaluated with a view to the homology between the sequences, and the common general characteristics of the gene structures [consensus sequences such as start and stop codons; consensus sequences typical of open reading frames (ORFs), exons and introns such as the GT-AG rule, point of divergence etc.]. Sequence analysis was facilitated by the fact that only one gene encoding for a protein of more than 50 amino acids--which is the xcv-1 gene itself--is present between the two genetic markers located on the right and left side, respectively, of the xcv-1 gene responsible for the phenotype, which can be distinguished by single recombination events. The sequence of the DNA segment comprising the xcv-1 gene is represented by the nucleotide sequence of SEQ ID NO:37.
[0165] The databases were successfully searched for DNA sequences similar to the xcv-1 gene, and we noted that homologous cDNA sequences occur in the so-called EST (Expressed Sequence Tags) databanks in the case of C. annuum as well. These sequences are a result of random sequencing by laboratories into cDNA clones from cDNA libraries of various organs, tissues or cells or groups of cells (root, stem, leaves, fruit, flower, pistil, stamen, pollen etc.) without having any information regarding their functions. For example, these include sequence TC17947 (SEQ ID NO:39) from C. annuum found in the DFCI database (http://compbio.dfci.harvard.edu/tgi/plant.html), which is presented in the form of the so-called TC (Tentative Consensus), i.e., the DNA form of the mRNA of a gene. The base composition of the TCs are edited by aligning cDNA sequences of various lengths derived from a single gene and determining the consensus sequence. The protein encoded by sequence TC17947 is shown by the amino acid sequence of SEQ ID NO:40. The mutation responsible for the Xe resistance can be identified by searching for differences in the nucleotide sequence of the genome region (SEQ ID NO:37) comprising the xcv-1 gene from the mutant plant in comparison with the homologous regions of the sensitive pepper plant (SEQ ID NO:41). Such differences can be found by comparing the nucleotide sequence of the genomic region (SEQ ID NO:37) comprising the xcv-1 gene with the genomic (SEQ ID NO:41) sequence from the sensitive pepper (C. annuum var. Feherozon) plant. Upon aligning SEQ ID NO:37 and SEQ ID NO:41, it was found that the sequence from the resistant plant is shorter by 6 bp at a certain location. When the TC17947 sequence (SEQ ID NO:39) is aligned with the sequence (SEQ ID NO:37) from the resistant plant, then the cDNAs can be aligned to the genomic sequence at three different segments (these are the exons) (alignment of the genomic sequence of a gene to its cDNA sequence allows for the determination of the exact location of the exons and introns present in the gene). These three segments are separated by 2 introns of the genomic sequence (see SEQ ID NO:37). While the first and second segment of the TC17947 sequence shows 100% identity to the genomic sequence, the cDNA sequence of the third segment is longer by 6 bases at the very location where the two genomic sequences also show a difference of 6 bases. Since only one sequence typical of a gene encoding a protein [putative promoter segment, 5' UTR, exons, introns, 3'-UTR, start codon (ATG), stop codon (TGA), conserved exon/intron and intron/exon boundaries recognisable on the basis of the so-called GT-AG rule and by the point of divergence, Poly-A site etc.] is present the DNA segment in question, it is assumed that the protein variant carrying the 6-base deletion in the third exon of the genomic sequence is responsible for the resistance. Thus, as a consequence of this deletion, protein synthesis is normal but the resulting protein is shorter by 2 amino acids (see amino acid sequences SEQ ID NO:38 and SEQ ID NO:42).
[0166] The putative first codon of the wild-type Xcv-1 gene is the ATG start codon starting at nucleotide 917 of the genomic fragment, and the stop codon is the TAG stop codon starting at nucleotide 2076. The sequences typical of promoter regions are located in the putative promoter region at the 5'-end of the gene. In addition, a polyA site is present at the 3'-end of the gene.
[0167] The activity--i.e., the transcription--of genes Xcv-1 and xcv-1 in the cells is also confirmed by the fact that mRNAs corresponding to the two sequences were detected: mRNA segments lacking the 6-base deletion and mRNA segments containing the 6-base deletion were identified from the sensitive plant and the resistant plant, respectively, upon Solid type mRNA sequencing.
[0168] The amino acid sequence of the protein of the Xcv-1 gene can be deduced from the predicted cDNA sequence of the Xcv-1 (see features of SEQ ID NO:41). The longest amino acid sequence containing an open reading frame (ORF) deduced from the Xcv-1 cDNA is indicated by SEQ ID NO:42. The Xcv-1 protein consists of 92 amino acids. This protein contains characteristic sequence portions (e.g., the C-terminal transmembrane region), which can be determined using typical features accumulated in data banks and bioinformatics methods (e.g., TMpred and MPEx programmes). As a result of the bioinformatic analysis, it can be concluded that the intracellular part of the molecule starts with an N-terminus followed by a GYPQ-rich portion of 5 to 6 amino acids which contains a repeat sequence (GYPPE-GYPKD-SYPPP-GYPQQ-GYPQQ-GYPQQ-GYPPQ-GYPPQ-YAPQY), and a linker segment is attached to the a C-terminal portion, which is a so called Tail Anchored (TA) section extending into the membrane (amino acids 72 to 88). The transmembrane region is terminated by amino acids with a negative charge (aspartic acid, glutamic acid) or polar amino acids (asparagine). The transmembrane region of the Xcv-1 protein can be detected on the basis of hydrophobicity using programmes predicting transmembrane regions. Without limitation, these include, for example, the "DAS"--Transmembrane Prediction programme (http://www.sbc.su.se/˜miklos/DAS/), and the Tmpred software (http://www.ch.embnet.org/software/TMPRED_form.html). FIG. 2 shows the TM curve of the Xcv-1 protein (SEQ ID NO:42) predicted by the "DAS"-Transmembrane Prediction programme. The Xcv-1 protein belongs to the CYSTM family of proteins (Venancio T. M. and Aravind L. Bioinformatics 26:149-152, 2010).
[0169] Identification of cDNA sequences from various tissues supports the fact that the Xcv-1 gene is expressed in different pepper species and in their various tissues from root to flower [see the ESTs constituting the TC17497 sequence found in the DFCI data bank (SEQ ID NO:39); (http://compbio.dfci.harvard.edu/tgi/plant.html)].
[0170] In comparison with the wild-type gene, the protein product of the xcv-1 gene is shorter by 2 amino acids in the TM region. No sequences showing 100% percent identity with the xcv-1 protein were found in the data banks. Consequently, a new protein was identified to which the Xe resistance could be assigned on the basis of the data and experimental results.
Example 8
Intracellular Localisation of Polypeptides Xcv-1 and Xcv-1
[0171] To determine the intracellular localisation of polypeptides Xcv-1 and xcv-1, the coding sequence of the Green Fluorescent Protein (GFP) (Accession no: AF234298, nucleotides 2 to 757) is cloned in front of genes Xcv-1 and xcv-1, and is introduced into Nicotiana bentamiana, a plant of the Solanaceae family, through Agrobacterium tumefaciens-mediated gene transfer. Transformation is preferably carried out in a manner ensuring a so-called transient expression (Martin K. et al., Plant J. 59:150-62, 2010), as a result of which the GFP-Xcv-1/GFP-xcv-1 constructs are not stably incorporated into the genome of the plant but the transgene is expressed from a DNA in an extrachromosomal state. The experiment was carried out as follows:
[0172] First, the structural gene of GFP was cloned into a pGemT-Easy vector as follows: the coding sequence of GFP was amplified using the DNA of the pCambia1302 plasmid (www.cambia.org, Marker Gene Technologies, Inc. www.markergene.com) as template, and SEQ ID NO:43 and SEQ ID NO:44 as primers, and the resulting PCR product was cloned into a pGemT-Easy vector. The resulting plasmid was designated `pNcoGFPXba`. In the next step, leaf samples were taken from C. annuum var. T1/1 and C. annuum var. Feherozon plants in the six-leaved stage, and a QIAGEN RNA isolation kit (RNeasy Mini Kit, http://www.qiagen.com) was used to isolate RNA, and the SMART® cDNA Library Construction Kit (http://www.clontech.com) was used to synthesise double-stranded cDNA. The cDNAs of Xcv-1 and xcv-1 were amplified and cloned in a PCR reaction using the resulting cDNA preparation. To amplify the Xcv-1 allele, cDNA from C. annuum var. Feherozon and primers SEQ ID NO:45 and SEQ ID NO:46 were used for the amplification. The PCR reaction was performed as described in Example 2. The resulting fragment was cloned into a pGemT-Easy plasmid. The resulting plasmid was designated `pXcv-1CaFo`. Cloning of the xcv-1 allele was performed in a similar way except for using the cDNA of C. annuum var. T1/1 and the primers represented by SEQ ID NO:45 and SEQ ID NO:46. The resulting plasmid was designated `pxcv-1CaT1`. In the third phase, plasmids `pXcv-1CaFo` and `pxcv-1 CaT1` were digested with the enzymes XbaI and BcuI, and were cloned into a `pNcoGFPXba` plasmid digested with XbaI and BcuI. The resulting plasmids were designated `pGFP-Xcv-1CaFo` and `pGFP-xcv-1 CaT1`, respectively. In the last step, plasmids `pGFP-Xcv-1 CaFo` and `pGFP-xcv-1CaT1` were digested with SalI, and the plasmid pCambia 1302 was digested with BstEII, and then all three preparations were digested with Mung Bean Nuclease according to manufacturer's (New England Biolabs, Inc, www.neb.com) instructions. The blunt-ended linear molecules `pGFP-Xcv-1CaFo` and pCambia 1302, and `pGFP-xcv-1CaT1` and pCambia 1302 were mixed, and both preparations were digested with the enzyme NcoI, ligated and transformed into E. coli cells. The transformants were grown on kanamycin-containing plates, where only the pCambia1302 derivatives grow but not the pGemT-Easy derivatives. Among the transformant colonies, those comprising the fusion products GFP-Xcv-1 and GFP-xcv-1 were identified. The two products were designated "pCambia-GFP-Xcv-1CaFO" and `pCambia-GFP-xcv-1 CaT1`. The sequences of the two fusion products are shown in SEQ ID NO: 47 and SEQ ID NO:48, respectively.
[0173] The plasmids `pCambia-GFP-Xcv-1 CaFO` and `pCambia-GFP-xcv-1 CaT1` plasmids were transferred into A. tumefaciens C58 by triparental mating, followed by growing the A. tumefaciens strains comprising the two constructs (`pCambia-GFP-Xcv-1CaFO` and `pCambia-GFP-xcv-1CaT1`) on solid media and infiltrating into Nicotiana bentamiana leaves in accordance with the method described in the relevant literature. After 48 hours, protoplasts were prepared from the leaf areas giving green fluorescence under UV light, and serial photos were taken of the protoplasts with green fluorescence using confocal microscopy. The pictures clearly demonstrate that that the fusion product comprising the wild-type protein (`pCambia-GFP-Xcv-1CaFO`) occurs as islands (lipid rafts) in the plasma membrane of the cells, but the mutant protein carrying the double leucine deletion (`pCambia-GFP-xcv-1 CaT1`) does not form such islands and shows homogeneous distribution in the plasma membrane.
Example 9
Identification of Genes Homologous to the Xcv-1 Gene in Plants and Animals
[0174] Complete or partial nucleotide sequences of the genomes of several viruses, bacteria, fungi and animals were determined in the framework of various genome projects. In most cases, the determination of the DNA sequences did not involve the identification of the function of a given segment; thus, the function of the sequences remains unknown. In relation to the Xcv-1 gene, data bank searches revealed that several sequences encoding proteins with similar structures to the xcv-1 protein can be found in the data banks (Feng et al., Mol Biol Rep DOI 10.1007/s11033-010-0419-1, 2010; Lieber et al., Current Biology 21: 1009-1017, 2011; Li et al., Biotechnol. Lett 31:905-910, 2009; Venancio T. M. and Aravind L. Bioinformatics 26:149-152, 2010). Alignment of the amino acid sequences of these proteins clearly demonstrated their structural similarity. In most cases, the region immersed in the membrane is delimited by a negatively charged amino acid (aspartic acid or glutamic acid) or a polar amino acid (asparagine). Alignment of a part of the CYSTM proteins is shown in Venancio T. M. and Aravind L., Bioinformatics 26:149-152, 2010. For the purposes of the invention, "proteins homologous to the Xcv-1 protein" refer to protein variants which contain at least 53% identical amino acids at their C-termini with respect to the last 15 amino acids of the C-terminus of the Xcv-1 protein (CLAALCCCCLLDACF).
Example 10
Induction of Resistance to Xanthomonas euvesicatoria in Tomato by the amiRNA Technique
[0175] One of the most dangerous bacterial pathogens of tomato is Xanthomonas euvesicatoria (Xe), i.e., the same bacterium which also causes severe damage to pepper. Unlike pepper, tomato has not developed appropriate natural resistance, which would ensure acceptable protection. The bs4 gene identified in tomato does not provide sufficient protection, and therefore, tomato production is still highly threatened by Xe infection (Hutton et al., Theor. Appl. Genet. 121:1275-87, 2010). It was assumed that the xcv-1 gene identified in pepper could also provide resistance to Xe infection in tomato if the genes homologous to the Xcv-1 gene, i.e., genes SlXcv-1A and SlXcv-1B represented by SEQ ID NO:49 and SEQ ID NO:51, respectively, are inactivated in the tomato genome, and the inactivation is preceded by ensuring the functioning of the tomato genes homologous to the xcv-1 gene, i.e., genes Slxcv-1A and Slxcv-1B represented by a SEQ ID NO:59 and SEQ ID NO:66, respectively (see below). This strategy can be implemented in more than one ways including but not limited to:
1. Tomato is transformed with functional Slxcv-1A and Slxcv-1B genes followed by inactivating the resident genes SlXcv-1A and SlXcv-1B using the "amiRNA" technique. 2. Tomato is transformed with functional Slxcv-1A and Slxcv-1B genes followed by inactivating the resident genes SlXcv-1A and SlXcv-1B using the ZFN nuclease technique. 3. Tomato is transformed with functional Slxcv-1A and Slxcv-1B genes followed by identifying mutations--that inactivated SlXcv-1A and SlXcv-1B--using the TILLING or another similar technique, upon or without mutagenesis. 4. Tomato is transformed with functional Slxcv-1A and/or Slxcv-1B genes followed by inactivating the resident genes SlXcv-1A and SlXcv-1B present there using the TALEN technique. 5. Tomato is transformed with functional Slxcv-1A and/or Slxcv-1B genes followed by inactivating the resident genes SlXcv-1A and SlXcv-1B present there using the CRISPR/Cas technique.
[0176] A common feature of the above five strategies is the in vitro preparation of the Slxcv-1A and Slxcv-1B sequences and transformation into tomato in the first step. Since the functioning of the Slxcv-1A and Slxcv-1B genes is recessive in comparison with the wild-type genes, the second step involves inactivating the wild-type genes (SlXcv-1A and SlXcv-1B) with suitable methods--including but not limited to the above-listed three methods--in order to manifest the above functions.
Example 10A
Generation of Slxcv-1A and Slxcv-1B Sequences with Double Leucine Deletion
[0177] The preparation of the genes comprising the double leucine deletion (Slxcv-1A and Slxcv-1B) involved PCR amplification, cloning of the amplificates into pGemT-Easy vectors, and additional restriction digestions and reclonings--steps well-known to those skilled in genetic engineering. For the cloning, the sequences of two tomato genes, i.e., SlXcv-1A and SlXcv-1B, obtained from data banks were used. The sequences of genes SlXcv-1A and SlXcv-1B are SEQ ID NO:49 and SEQ ID NO:51, respectively.
[0178] Preparation of the Slxcv-1A Construct:
[0179] a PCR amplification was performed in the presence of a genomic DNA template from tomato using primers `SlProm1AF3` (SEQ ID NO:53) and `SlProm1AR3` (SEQ ID NO:54), and the 1074-bp DNA fragment was cloned into pGEM-T Easy vectors. The resulting plasmid (`p1A1`) was digested with the enzyme NsiI followed by ligation and transformation to generate plasmid `p1A2`. A PCR amplification was performed in the presence of a genomic DNA template from tomato using primers `SlTerm1AF3` (SEQ ID NO:55) and `SlTerm1AR3` (SEQ ID NO:56), and the 283-bp DNA fragment was cloned into pGEM-T Easy vectors to generate plasmid `p1A3`. The `p1A2` plasmid was digested with NsiI, and the `p1A3` plasmid was digested with NsiI and PstI; next, the two mixtures were combined and ligated, and--upon transformation--plasmid `p1A4`, in which the NsiI end of `p1A3` is positioned towards the genomic sequence in `p1A2`, was identified. A PCR amplification on tomato genomic DNA was performed using primers `SlMid1AF1` (SEQ ID NO:57) and `SlMid1ABR1` (SEQ ID NO:58), and the 1137-bp DNA fragment was cloned into pGEM-T Easy vectors to generate plasmid `p1A5`. Upon mixing `p1A4` and `p1A5`, the plasmids were digested with NsiI and ligated, and--upon transformation--plasmid `p1A6`, in which the 1094-bp NsiI fragment was cloned into the NsiI site of the `p1A4` plasmid in the correct orientation, was identified. Finally, this resulted in construct Slxcv-1A (SEQ ID NO:59), which encodes the Slxcv-1A protein (SEQ ID NO:60), a variant with the double leucine deletion.
[0180] Preparation of the Slxcv-1B Construct:
[0181] A PCR amplification was performed in the presence of a genomic DNA template from tomato using primers `SlProm1BF3` (SEQ ID NO:61) and `SlProm1BR3` (SEQ ID NO:62), and the 1450-bp DNA fragment was cloned into pGEM-T Easy vectors. The resulting plasmid (`p1B1`) was digested with the enzyme NsiI followed by ligation and transformation to generate plasmid `p1B2`. A PCR amplification was performed in the presence of a genomic DNA template from tomato using primers `SlTerm1 BF3` (SEQ ID NO:63) and `SlTerm1BR3` (SEQ ID NO:64), and the 787-bp DNA fragment was cloned into pGEM-T Easy vectors to generate plasmid `p1B3`. The `p1B2` plasmid was digested with NsiI, and the `p1B3` plasmid was digested with NsiI and PstI; next, the two mixtures were combined and ligated, and--upon transformation--plasmid `p1B4`, in which the NsiI end of `p1B3` is positioned towards the genomic sequence in `p1B2`, was identified. A PCR amplification on tomato genomic DNA was performed using primers `SlMid1BF1` (SEQ ID NO:65) and `SlMid1ABR1` (SEQ ID NO:58), and the 1273-bp DNA fragment was cloned into pGEM-T Easy vectors to generate plasmid `p1B5`. The `p1B4` plasmid was digested with NsiI but the `p1B5` plasmid was only partially digested with NsiI; next, the two samples were combined and ligated, and--upon transformation--plasmid `p1B6`, in which the 1236-bp fragment was cloned into the NsiI site of the `p1B4` plasmid in the correct orientation, was identified. Finally, this resulted in construct Slxcv-1B (see SEQ ID NO:66), which encodes the Slxcv-1B protein (SEQ ID NO:67), a variant with the double leucine deletion.
[0182] Upon preparing the two constructs, the Slxcv-1A and Slxcv-1B sequences were cloned head to head into an A. tumefaciens vector pCAMBIA2300 cut by XbaI from the pGemT-Easy vector using NotI-SpeI, and were transformed into E. coll. The resulting plasmid was designated `pDSlxcv-1AB`. From the E. coli host, the plasmid was introduced into the A. tumefaciens strain using triparental mating. The resulting strains were designated A. tumefaciens (pDSlxcv-1AB).
Example 10B
Transformation of the Slxcv-1A and Slxcv-1B Genes into Tomato Using Agrobacterium tumefaciens
[0183] A. tumefaciens transformation was carried out as follows: Tomato seeds were immersed in 70% ethanol for 1 minute, and were then transferred into a solution of 5.25% sodium perchlorate (NaClO) and 0.1% Tween 20 and shaken for 30 minutes. Next, the seeds were rinsed with distilled water 8 times and transferred to Petri dishes containing medium A, and were grown for 8 days at 25° C. with 16-hour light cycle. The cotyledons of the plants were cut at the apex and at the base, pricked, placed on medium B, and overlaid with MSO liquid medium containing A. tumefaciens (pDSlxcv-1AB). The MSO liquid medium containing A. tumefaciens (pDSlxcv-1AB) was prepared as follows: the A. tumefaciens (pDSlxcv-1AB) strain stored at -80° C. (prepared as described in Example 11A) was plated onto a medium containing YEP+100 μg/ml rifampicin and incubated at 30° C. One of the colonies was inoculated into 3 ml YEP liquid medium (in a 20-ml test tube) using an inoculation loop, and the bacteria were rotated in a roller to ensure aeration and cultured until reaching the stationary phase (24 hours). The bacteria were collected by centrifugation as previously described, the supernatants were discarded and the cells were suspended in 12 ml MSO liquid medium. The leaves were treated with the Agrobacterium suspension for 20 minutes, then the excess suspension was drawn off and the leaves were co-cultivated with the bacteria for 48 hours. After two days, the leaves were transferred to plates with medium C. The plants were transferred to fresh plates with medium C at two-week intervals. The developing calluses could be cut to smaller pieces, and were transferred to plates with medium D and then to fresh plates at two-week intervals. When the small growths appeared, they were transferred again to plates with medium D. When reaching a length of 2 to 4 cm, the growths were transferred to fresh plates with medium E, and they started to form roots. Plants of 5 cm could already be planted into potting soil. A total of 15 independent T0 transformant plants (T0/xcv1 to 15) were grown.
MSO medium (1000 ml): 4.3 g MS salts, 100 mg myo-inositol, 0.4 ml (1 mg/ml) thiamine-HCl, 20 g saccharose YEP medium (1000 ml): 10 g yeast extract, 10 g peptone, 5 g NaCl (pH adjusted to 7 with NaOH) Vitamin solution (per 1000 ml): 50 mg thiamine-HCl, 200 mg glycine, 500 mg nicotine aid, 50 mg pyridoxine-HCl, 50 mg folic acid, 5 mg biotin, 10 g myo-inositol
TABLE-US-00003 (per Substance/medium A B C D E 1000 ml) MS (Gibco) 4.3 4.3 4.3 4.3 2.15 g Saccharose 15 30 30 15 15 g Vitamin solution 1 1 1 1 1 ml NAA -- 2 -- -- -- ml BAP -- 2 -- -- -- ml Zeatin -- -- 2 -- -- mg IAA -- -- -- -- 5 mg GA -- -- 1 -- -- mg Km -- -- 100 100 50 mg Timentin -- -- 300 300 300 mg Agar -- -- -- -- 5 g (BAP = Benzyl-Aminopurine, NAA = Naphthalene Acetic Acid), IAA = Indol Acetic Acid, GA = Gibberelic Acid, Km = kanamycin)
[0184] When the stems and roots of the T0/xcv1-15 transgenic plants were strong enough, DNA was isolated from the leaves, and a PCR reaction was performed to detect the transformation events using the following primer pairs:
[0185] 1. Pr_Sl SlMid1AF1 primer (SEQ ID NO:57);
[0186] Pr_SlTerm1AR3 primer (SEQ ID NO:56);
[0187] length of the expected amplificate: 1400 bp;
[0188] 2. Pr_SlTerm1BF3 primer (SEQ ID NO:62);
[0189] Pr_SlTerm1BR3 primer (SEQ ID NO:63);
[0190] length of the expected amplificate: 787 bp. The transgene sequence between the primers used for the amplification could be detected in all cases.
Example 10C
Generation of the Gene Encoding the Prim-amiRNA Designated as Pri_SlXe1-amiRNA
[0191] The microRNAs (miRNAs) discovered in eukaryotic organisms inhibit the efficient expression of the corresponding genes. This gene inactivation allows for an alternative form of gene regulation through a specific mechanism resulting in the inhibition of the function of the gene, which is of great importance in terms of development and differentiation (Kidner C. A. es Martienssen R. A., Curr. Opin. Plant Biol. 8:38-44, 2005). miRNAs are ribonucleic acid molecules present in eukaryotic cells. The miRNAs are short molecules consisting of 21 to 24 nucleotides in contrast to the long RNA molecules fulfilling other functions (e.g., mRNA, ribosomal RNA). The miRNAs are post-transcriptional inhibitors of the functioning of mRNAs by physically inhibiting protein synthesis on complementary mRNAs, or by causing the degradation of complementary mRNAs upon binding to them (Bartel D. P., Cell 16:281-297, 2004).
[0192] Studies of the miRNAs and exploration of the biochemical processes on the molecular level made it possible to extend this specific inhibition mechanism to genes for which no natural miRNAs exist. Artificially prepared gene-specific miRNAs were designated amiRNAs (artificial miRNA) (Ossowski et al., Plant J. 53:674-690, 2008; Park et al., Plant Cell Rep. 28:469-480, 2009; Schwab et al., Methods Mol. Biol. 592:71, 2010; Sablok et al., Biochem. and Biophys. Res. Comm. 406:315-319, 2011). The amiRNA-based gene inactivation has been generated in a number of animal and plant systems (Schwab et al, Plant Cell 18:1121-1133, 2006), and in general terms, the target mRNAs can be inactivated, thereby eliminating the gene function in question, through carefully designed experiments.
[0193] An amiRNA gene coding for an amiRNA ribonucleotide consist of the following sequences: promoter, 5' stem extension, amiRNA*, loop region, the amiRNA and a 3' stem extension with polyA tail (Schwab et al., Methods Mol. Biol. 592:71, 2010).
[0194] Strategic Course of Inducing Resistance in Tomato Plants:
[0195] During the PCR amplification and sequencing of the tomato genomic DNA, two sequences homologous to the Xcv-1 gene of pepper (SlXcv-1A and SlXcv-1B) were identified: the nucleotide sequences and the deduced amino acid sequences are shown in the DNA sequences of SEQ ID NO:49 and SEQ ID NO:51, respectively, and the amino acid sequences of SEQ ID NO:50 and SEQ ID NO:52, respectively.
[0196] The target mRNA sequence--with which the amiRNA designated `SlXe1-amiRNS` will show partial complementarity (18 of 21 bases)--is the segment which precedes the stop codon of genes SlXcv-1A and SlXcv-1B and encodes the two leucines corresponding to the Xcv-1 gene (see FIG. 3). The SlXe1-amiRNA will not be complementary to the mRNAs of the genes comprising the double leucine deletion to be simultaneously expressed, and therefore will not inactivate them. The SlXe1-amiRNA (SEQ ID NO:74) is expressed with the help of pre-sly-MIR159miRNSpre-miDNA (SEQ ID NO:68), which is responsible for the expression of sly-MIR159 (Accession No. MI0009974) in tomato, and transcription generates preSlpre-slyM1159RNA (SEQ ID NO:69).
[0197] The coding segment of the pre-SlXe1-amiRNA (SEQ ID NO:73) sequence, i.e., the pre-SlXe1-amiDNA (SEQ ID NO:72) is prepared as follows. Amplification is carried out from tomato genomic DNA using the synthesised primers `Pri_SlXe1pre-amiRNA` (SEQ ID NO:70) and `Pr2_SlXe1pre-amiRNA` (SEQ ID NO:71), and the resulting double-stranded pre-SlXe1-amiDNA (SEQ ID NO:72) coding sequence is cloned into pGemT-Easy vectors, and--upon restriction by EcoRI-SpeI--pKSS vectors, and finally into pC61H vectors through KpnI and XbaI cleavage, as described in Example 3. The HindIII-EcorRI fragment of pCK61H was generated by cloning the EcoRI-HindIII fragment of BIN61S (Silhavy D. et al., EMBO J. 21:3070-3080, 2002) carrying the 35S promoter, polilinker and terminator sequences into the EcoRI-HindIII site of the pCAMBIA1300. The resulting plasmid was designated pC61H-pri-SlXe1-amiRNA and the strain containing the plasmid was designated A. tumefaciens (pC61H-pri-SlXe1-amiRNA). The HindIII-EcorRI fragment of pCK61H-SlXe1-amiRNA carrying the gene encoding SlXe1-ami RNA is shown in SEQ ID NO:92
Example 10D
Transformation of Pri-SlXe1-amiRNS Sequences into T0/1-15 Transgenic Plants Containing the Genes Slxcv-1A and Slxcv-1B
[0198] The transformation with A. tumefaciens was carried out as described in Example 10B, but the plants to be transformed were the T0/1-15 transgenic plants, the A. tumefaciens (pC61K-pri-SlXe1-amiRNA) strain was used for the transformation and the selection was for hygromycin. At the end of the transformation, seven independent plants (T0/ami1 to 7) were grown.
[0199] Pr1_SlXe1 pre-amiRNA (SEQ ID NO:70);
[0200] Pr2_SlXe1 pre-amiRNA (SEQ ID NO:71);
[0201] length of the expected amplificate: 178 bp.
[0202] In addition to DNA isolation, total RNA was isolated from the leaves of the control and transgenic plants using the RNeasy Mini Kit, and the total RNA was run on a 12% carbamide/acrylamide gel, transferred to a Hybond NX membrane (GE Healthcare Amersham) and hybridised with an alpha-32ATP-labelled LNA probe encoding the SlXe1-amiRNA. The autoradiogram obtained upon the hybridisation and the image of the total RNA loaded to the gel are shown in FIG. 4.
[0203] The transformant plants were grown until the 8-leaved stage, infected with the bacterium Xanthomonas euvesicatoria, and evaluated as described in Example 1. The results of the Xanthomonas euvesicatoria infection are summarised in Table 2.
TABLE-US-00004 TABLE 2 Detection of transgenes from transgenic and control plants after a PCR reaction using specific primer pairs, and plant phenotypes after infection with Xanthomonas euvesicatoria Appearance Appearance Appearance Phenotype after Plant of of of the infection with iden- amplificate amplificate 178-bp Xanthomonas tifier 1137 1273 amplificate euvesicatoria (Xe) C1 no no no Symptoms of Xe infection, tissue necrosis C2 no no no Symptoms of Xe infection, tissue necrosis C3 no no no Symptoms of Xe infection, tissue necrosis T1 yes yes yes healthy phenotype tissue oedema only T2 yes yes yes transitional phenotype slight tissue necrosis T3 yes yes yes healthy phenotype tissue oedema only T4 yes yes yes healthy phenotype tissue oedema only T5 yes yes yes transitional phenotype slight tissue necrosis T6 yes yes yes healthy phenotype tissue oedema only T7 yes yes yes healthy phenotype tissue oedema only
Example 11
Induction of Resistance to Xanthomonas euvesicatoria in Tomato Using the Engineered Nuclease Technique
[0204] In a certain prior reverse genetic approach, a plant was first mutagenised, and then the mutation was identified in the gene sought. In most cases, the mutation was identified using T_DNA and transposon insertion mutagenesis, and TILLING (Targeted Induced Local Lesions in Genomes) (Feldman, K. A. The Plant Journal 1:71-82, 1991; McCallum, C. M. et al., Nat. Biotech. 18455-457, 2000). However, this approach was troublesome, time-consuming and uncertain. The RNA interference (RNAi) and artificial microRNA (amiRNA) techniques mentioned in Example 10 are already specific to the desired gene, however, the expression of the gene is often impossible to eliminate completely, that is, null phenotype should be obtained by all means (Schwab et al, Plant Cell 18:1121-1133, 2006). Consequently, methods resulting in genes that are completely knocked out and thus guaranteeing a null phenotype are of vital importance. By now, three methods satisfying the above criteria have been disclosed. These are the above-mentioned ZFN, TALEN and CRISPR/Cas nuclease techniques, which generate gene specific c double stranded cuts in the DNA and following the activity of the repair mechanism of the cells insertions and deletions with the size of one to several tens of base pairs or more in a gene-specific manner [Urnov F. D. et al. Nat Rev Genet. 11:636-46, 2010; Carroll D. Genetics. 188:773-82, 2011; Christian M. et al., Genetics 189:757-761, 2010; Cermak, T. et al., Nucl. Acids Res. 39: e82, 2011; Mussolino C. et al., Nucleic Acids Res. 39:9283-9293, 2011; Miller J. C. et al., Nat. Biotechnol. 29:143-148, 2011; Christian M. et al., G3 (Genes,Genomes,Genetics, Bethesda), doi:10.1534/g3.113.007104, 2013; Cho S. W. et al., Nat Biotechnol. 31:230-232, 2013; Cong L. et al., Science 339:819-823, 2013; Mali P. et al., Science 339:823-826, 2013]. For the purpose of generating the 6-bp deletion in the tomato genes SlXcv-1A and SlXcv-1B, the TALEN, CRISPR/Cas nuclease and the ZFN technique can be equally used. From tomato cells only producing the double leucine deletion proteins Slxcv-1A and/or Slxcv-1B, Xe resistant tomato plants can be generated in the same way as in Example 10.
Example 11A
Induction of Resistance to Xanthomonas euvesicatoria in Tomato Using the TALEN Technique
[0205] The TALEN target sequence to which the TALEN pairs recognize (SlXcv-1AB_TALEN-L, SlXcv-1A_TALEN-R and SlXcv-1B_TALEN-R) should be selected and determined in view of the fact that the 6 bp deletions should located at positions 2277 to 2282 and 2640 to 2645, respectively. Since the applied TALEN nucleases quite often cut within the so-called "spacer" sequences, it is reasonable to chose a size of 18 base pairs for the spacer region, and to chose a TALEN target sequence extending into 17 and 17 base pairs both to the right and to the left in a manner ensuring that the target sequences are preceded by a T/A base pair in all cases [Cermak, T. et al., Nucl. Acids Res. 39:e82, 2011; Christian M. et al., G3 (Genes,Genomes,Genetics, Bethesda)]. Since the sequences of the two genes to the left side of the mutation are identical along an at least 36-bp segment from the left end of the desired deletion, the same left TALEN target sequence should be chosen for both genes (SlXcv-1AB_TAL-L, see SEQ ID NO:75 and FIG. 5). Counting from the right side of the desired deletion, differences between the two genes occur already after the 15th base, and therefore, the right TALEN target sequences will be different for SlXcv-1A and SlXcv-1B (SlXcv-1A_TAL-R, see SEQ ID NO:76; and SlXcv-1B_TAL-R, see SEQ ID NO:77 and FIG. 5). Each base of the target sequences are recognised by "repeat-variable di-residues" (RVDs in short)--that is, doublets of adjacent amino acids--present in the TAL effector proteins. The following RVD amino acids can be designed for each base: A is recognised by the NI amino acid doublet, C is recognised by the HD doublet, G is recognised by the NH doublet, and T is recognised by the NG doublet (A=adenosine, C=cytidine, G=guanosine, T=thymidine, NI=asparagine-isoleucine, HD=histidine-aspartic acid, NH=asparagine-histidine, NG=asparagine-glycine; see FIG. 5). The RVD sequences and the bordering repeat sequences can be synthesised and cloned in accordance with the relevant literature (pNI1-10, pHD1-10, pNH1-10, and pNG1-10, see Cermak, T. et al., Nucl. Acids Res. 39: e82, 2011, supplementary material). The sequences of the pNH series are identical with the pNN sequences except that the AAC CAT codon doublet, which encodes asparagine and histidine, should be used instead of the double asparagine codon (AAC AAT). The repeats containing RVDs can be cloned into plasmids pFUS_A and pFUS_B6 after BsaI cutting. Plasmids pFUS_A, pFUS_B6 and pLR-NG and pLR-NI, respectively, can be cut by Esp3I and cloned into the Esp3I site of pTAL3. (Cermak, T. et al., Nucl. Acids Res. 39: e82, 2011). Concerning the SlXcv-1A and the SlXcv-1B genes, specific TALEN sequences comprising of TAL-N', SlXcv-1A, and SlXcv-1B specific TAL sequences containing the 17 repeats and RVDs (SlXcv-1AB_TAL-L, SlXcv-1A_TAL-R and SlXcv-1B_TAL-R), the TAL-C in which the NLS sequence is present, and finally the catalitic domain of the FokI nuclease. These sequences (TALEN-L es TALEN_R, see FIG. 6.) can be reclonded, first the FokI domain on a SacI (the end are made blunt ended) BamHI fragment is cloned into the BamHI-MlyI site of BIN61S vector (Silhavy D. et al., EMBO J. 21:3070-3080, 2002), then this derivative is cut by BamHI and the SlXcv-1AB_TALEN-, (SEQ ID NO:78), SlXcv-1A_TALEN-R (SEQ ID NO:79), and SlXcv-1B_TALEN-, (SEQ ID NO:80) sequences, respectively are cloned in pairs (see FIG. 6.) on a HindIII-EcoRI fragment into the EcoRI site of pCAMBIA1300 and/or pCAMBIA2300.--followed by introducing into Agrobacterium tumefaciens by transformation, and finally transformed into suitable tomato plants as described in Example 10. The tomato species should be selected in a manner ensuring the functional expression of the TALEN gene. FIG. 6 shows the functional map of the sequences between the left border (LB) and right border (RB) sequences in the vectors used for transformation. Transformation should be performed with the four vectors shown in FIG. 6 (SlXcv-1_TALEN 1AH, SlXcv-1_TALEN 1BH, SlXcv-1_TALEN 1AK, SlXcv-1_TALEN 1BK), and hygromycin and kanamycin resistant calluses should be selected and regenerated in the presence of hygromycin (50 μg/ml) and kanamycin (100 μg/ml). The SlXcv-1A and SlXcv-1B genes can be detected from the calluses using PCR between bases 2277 to 2282 and 2640 to 2645, respectively (see FIGS. 5 and 7). The Slxcv-1A and Slxcv-1B genotypes carrying the 6-bp deletion, as well as other deletion/insertion derivatives can be identified in both selections. Plants are regenerated from the Slxcv-1A and Slxcv-1B calluses, and the hygromycin resistant plants--which carry the Slxcv-1A gene--are transformed with the SlXcv-1_TALEN 1 BK vector, and the kanamycin resistant plants--which carry the Slxcv-1B gene--are transformed with the SlXcv-1_TALEN 1AH vector, and we proceed as described above, that is, kanamycin and hygromycin resistant calluses are grown, and PCR techniques are used to identify the deletions in the Slxcv-1B and Slxcv-1A genes. During the genotyping of the resistant calluses, deletions are sought between base pairs 2277 to 2282 and 2640 to 2645. Three of the deletions thus identifiable are mentioned below.
[0206] In Variant 1, the desired 6-bp deletion is present in both genes; thus, these plants carry the genes Slxcv-1A and Slxcv-1B, which encode the Slxcv-1A (SEQ ID NO:60) and the Slxcv-1B (SEQ ID NO:67) amino acid sequences (see FIG. 7), respectively.
[0207] In Variant 2, the plants carry the Slxcv-1A gene, which encodes the Slxcv-1A (SEQ ID NO:60) amino acid sequence. In Variant 2, the derivative of the SlXcv-1B gene contains a deletion/insertion which changes the open reading frame (out of frame deletion) (see FIG. 7).
[0208] In Variant 3, the plants carry the Slxcv-1B gene, which encodes the Slxcv-1B (SEQ ID NO:67) amino acid sequence. In Variant 3, the derivative of the SlXcv-1A gene contains a deletion/insertion which changes the open reading frame (out of frame deletion) consequently the gene is inactivated (see FIG. 7).
[0209] The above three transformant derivatives (Variants 1, 2 and 3) can be vegetatively propagated and grown to the 8-leaved stage, then infected with Xanthomonas euvesicatoria, and the products resistant to Xanthomonas euvesicatoria can be selected. For a skilled person, it is obvious that the transgenes (T-DNAs) can be removed from the genome of the selected plants resistant to Xanthomonas euvesicatoria by crossing, because the transgenes will be segregated from a part of the progeny, that is, non-transgenic plants can be generated from them.
Example 11B
Induction of Resistance to Xanthomonas euvesicatoria in Tomato Using the CRISPR/Cas Nuclease Technique
[0210] The above-described CRISPR/Cas technology (Cho S. W. et al., Nat. Biotechnol. 31:230-232, 2013; Cong L. et al., Science 339:819-823, 2013; Mali P. et al., Science 339:823-826, 2013; Feng Z. et al., Cell Research: 1-4, 2013) is also useful for generating the desired 6-bp deletion(s), or gene mutations resulting in null phenotypes, in tomato and in other plants. In the first case, `single guide RNAs` (sgRNAs in short) were designed for the wild-type tomato sequences corresponding to the desired 6-bp deletion (see FIG. 5A) as target sequence. The procedure is as follows: after reannealing, the oligonucleotides (sgRNA_SlXcv-1F, 5'-GATTTCTGTGCTGTTGCTGTCTCT, SEQ ID NO:82 and sgRNA_SlXcv-1R, 5'-AAACAGACACGACAACGACAGAGA, SEQ ID NO:83) designed and synthesised for the 20-bp target sequence (CRISPR_SlXcv-1, 5'-TCTGTGCTGTTGCTGTCTCT, see SEQ ID NO:81) are cloned into a BsaI/BsaI site of a suitable vector after the tomato-specific U6 promoter and before the sgRNA sequence. This construct is followed by a double 35S promoter, an NLS sequence, the hspCAS9 gene (Feng Z. et al., Cell Research:1-4, 2013), another NLS sequence and the NOS terminator (Cong L., et al. Science 339:819-823, 2013). The resulting construct is cloned into pCAMBIA plant-derived transformation vectors carrying a hygromycin and a kanamycin resistance gene, respectively (see above), and is used to transform tomato cells. It is important to note, that the CRISPR_SlXcv-1 (SEQ ID NO:81) sequence may not target and cut Slxcv-1A or Slxcv-1B sequences containing the 6 bp deletion.
[0211] Upon preparing the vectors, the tomato cells are transformed as described in Example 10, the hygromycin and kanamycin resistant calluses are selected as described in Example 11, and the gene derivatives of SlXcv-1A and SlXcv-1B are evaluated as described in Example 11. As a result of the experiments and tests, similar results are expected as in the case of the three variants described in Example 11, that is, the desired 6-bp deletion is present in both SlXcv-1 genes (SlXcv-1A and SlXcv-1B), which encode the Slxcv-1A (SEQ ID NO:60) and Slxcv-1B (SEQ ID NO:67) amino acid sequences, respectively; or the desired 6-bp deletion is only present in SlXcv-1A and SlXcv-1B, and early stop codon, or an out of frame deletion/insertion giving null phenotype is generated in the other gene.
[0212] Upon treatment with the CRISPR/Cas nuclease, the transformant derivatives having the advantageous feature (Variants 1, 2 and 3, see Example 11A.) can be vegetatively propagated and grown to the 8-leaved stage, then infected with Xanthomonas euvesicatoria, and the progeny resistant to Xanthomonas euvesicatoria can be selected. For a skilled person, it is obvious that--in this case too--the transgenes (T-DNAs) can be removed from the genome of the selected plants resistant to Xanthomonas euvesicatoria by crossing, because the transgenes will be segregated from a part of the progeny, that is, non-transgenic plants can be generated from them.
Example 11C
Induction of Resistance to Xanthomonas euvesicatoria in Tomato Using the ZFN Technique
[0213] The above-mentioned ZFN technology (Urnov F. D. et al. Nat Rev Genet. 11:636-46, 2010, Carroll D. Genetics. 188:773-82, 2011) is also useful for generating the desired 6-bp deletion(s), or gene mutations resulting in null phenotypes, in tomato and in other plants. In case of generating the desired 6-bp deletion, the procedure is as follows: the target sequences to be recognised by the two zinc finger (ZF) proteins are selected within the segment from bases 2253 to 2306 (SEQ ID NO:84) of the SlXcv-1A gene sequence (SEQ ID NO:49) and within the segment from bases 2616 to 2669 (SEQ ID NO:85) of the SlXcv-1B gene sequence (SEQ ID NO:51)--as shown in FIG. 5--in a manner ensuring a distance of 5 to 7 bp from each other. It is obvious that more than one arrangements can be selected as the target sequence along the above the DNA segments (SEQ ID NO:84, SEQ ID NO:85) and the specific ZF proteins consisting of ZF domains, which are specific to the left and right side of the target sequences. Possible examples for the SlXcv-1A gene (SEQ ID NO:49) and SlXcv-1B gene (SEQ ID NO:51) include but are not limited to 6-bp target sequences exactly covering the sequences of the desired 6-bp deletion in the above two genes. In both cases, the target sequence is 5'-CTCTTG, which encodes two leucines, and the target sequences of the left and right specific ZF proteins [5'-TGTGCTGTTGCT (SEQ ID NO:86) and 5'-TTTCGTACGTAG (SEQ ID NO:87), respectively] are identical in both genes because they show 100% identity in this region. The genes of the ZF proteins recognising the specific left and right target sequences are cloned into pCAMBIA plant-derived transformation vectors carrying a hygromycin and a kanamycin resistance gene, respectively (see above), and are used to transform tomato cells.
[0214] Upon preparing the vectors, the tomato cells are transformed as described in Example 10, and the procedures described in Examples 11A and 11B are followed thereafter.
[0215] The deletion/insertion procedure using the ZNF, TALEN and CRIPSR/Cas nucleases can be performed in the transgenic tomato plants carrying one of the genes (i.e., either Slxcv-1A or Slxcv-1B), or both (Slxcv-1A and Slxcv-1B), wherein knock-out insertion or deletion derivatives are sought in the resident genes SlXcv-1A and SlXcv-1B among the plants treated with the ZFN, TALEN or CRIPSR/Cas nucleases. In fortunate cases, the double null allele variant can also be obtained after performing the transformation, and the second transformation is unnecessary.
[0216] For the skilled person, it is obvious and understandable that knock-out null mutants in the SlXcv-1A and SlXcv-1B genes may not only be generated within the segment from bases 2244 to 2318 (SEQ ID NO:84) of the SlXcv-1A gene sequence (SEQ ID NO:49) and within the segment from bases 2607 to 2681 (SEQ ID NO:85) of the SlXcv-1B gene sequence (SEQ ID NO:51)--as shown in FIG. 5--with the above-mentioned techniques (TALEN and CRIPSR/Cas nuclease and ZFN) and other mutagenesis techniques (mutagenesis, ECOTILLING, Comai L. et al., Plant J. 37:778-786, 2004 etc.), but they can also be designed for the entire sequence of SlXcv-1A (SEQ ID NO:49) and SlXcv-1B (SEQ ID NO:51), that is, for all those sequences that are responsible for the expression and manifestation of the above genes and their products (functional proteins) (e.g., the promoter, the 3' and 5' UTR, exon, intron etc. sequences).
Sequence CWU
1
1
92122DNACapsicum annuumsource1..22mol type=unassigned DNA note=Primer
CaCY F1 organism=Capsicum annuum 1gtggctcatg ctgtggattt ct
22222DNACapsicum annuumsource1..22mol
type=unassigned DNA note=Primer CaCY R1 organism=Capsicum
annuum 2ccaggagtgc aggggtaggt ta
22322DNACapsicum annuumsource1..22mol type=unassigned DNA
note=Primer BAC 279 op F1 organism=Capsicum annuum 3gctggtctat
cttgatcctt ca
22422DNACapsicum annuumsource1..22mol type=unassigned DNA
note=Primer BAC 279 op R1 organism=Capsicum annuum 4atgtccctcc
ctgtcattct at
22520DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 279 -40 F1 organism=Capsicum annuum 5tgggactaat
aaggaaagaa
20620DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 279 -40 R1 organism=Capsicum annuum 6gaagtgatga
aagtgggttg
20718DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer BAC 1248 op F1 organism=Capsicum annuum 7acgagcttga
gatactga
18818DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer BAC 1248 op R1 organism=Capsicum annuum 8ctcttgggaa
aggtcata
18918DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer BAC 1248 -40 F1 organism=Capsicum annuum 9gtcttacatg
ccccaaat
181018DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer BAC 1248 -40 R1 organism=Capsicum annuum 10catcacgagc
actacctg
181120DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 1191 -40 F1 organism=Capsicum annuum 11aaaactgggt
taatgttggg
201220DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 1191- 40 R1 organism=Capsicum annuum 12cgtggcggct
gtattgtctc
201321DNACapsicum annuumsource1..21mol type=unassigned DNA
note=Primer BAC 1191 op F1 organism=Capsicum annuum 13acgagcaaat
agaaggcaat g
211420DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 1191 op R1 organism=Capsicum annuum 14caccctctac
aagaaactct
201521DNACapsicum annuumsource1..21mol type=unassigned DNA
note=Primer BAC 877 -40 F1 organism=Capsicum annuum 15atgtcaagaa
tcacaaccgt a
211621DNACapsicum annuumsource1..21mol type=unassigned DNA
note=Primer BAC 877 -40 R1 organism=Capsicum annuum 16gtaagatggc
cgattaatat g
211720DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 632 -40 F1 organism=Capsicum annuum 17ttgccagaag
ttgtcctatt
201820DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 632 -40 R1 organism=Capsicum annuum 18attgtcttgt
tgtgcgttat
201920DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 632 op F1 organism=Capsicum annuum 19tcaacaaagg
cagcagaatg
202020DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 632 op R1 organism=Capsicum annuum 20ttctgctctt
ttcccctgaa
202118DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer BAC 66 -40 F1 organism=Capsicum annuum 21gcttagaggg
caggtagt
182218DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer 66 -40 R1 organism=Capsicum annuum 22ttctcagagc taggcaca
182320DNACapsicum
annuumsource1..20mol type=unassigned DNA note=Primer 66 op F1
organism=Capsicum annuum 23tatgcaaagc acatgaaatg
202420DNACapsicum annuumsource1..20mol
type=unassigned DNA note=Primer BAC 66 op R1 organism=Capsicum
annuum 24cttatgacac cccaccaaat
202518DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer BAC 50 op F1 organism=Capsicum annuum 25accaactaga
atccaaat
182618DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer BAC 50 op R1 organism=Capsicum annuum 26tgaacttaaa
gatgctga
182720DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 50 -40 F1 organism=Capsicum annuum 27atgatttcta
tgatggctag
202819DNACapsicum annuumsource1..19mol type=unassigned DNA
note=Primer BAC 50 -40 R1 organism=Capsicum annuum 28gttggaagta
ttgggttaa
192920DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 472 -40 F1 organism=Capsicum annuum 29cttgcttcta
gttttgatcc
203020DNACapsicum annuumsource1..20mol type=unassigned DNA
note=Primer BAC 472 -40 R1 organism=Capsicum annuum 30ctatctggca
agtaaccacc
203117DNACapsicum annuumsource1..17mol type=unassigned DNA
note=Primer BAC 472 op F1 organism=Capsicum annuum 31cttcaatccc
tttctca
173218DNACapsicum annuumsource1..18mol type=unassigned DNA
note=Primer BAC 472 op R1 organism=Capsicum annuum 32tatcatgctc
atccctat
183324DNACapsicum annuumsource1..24mol type=unassigned DNA
note=Primer, a Pr6 marker F1 primere organism=Capsicum annuum
33tttcgtgagt attattcctt ttta
243419DNACapsicum annuumsource1..19mol type=unassigned DNA
note=Preimer, a Pr6 marker R1 primere organism=Capsicum annuum
34cgctgctttt tcgctatgt
193519DNACapsicum annuumsource1..19mol type=unassigned DNA
note=Primer, a Pr4b marker F1 primere organism=Capsicum annuum
35cgctgctttt tcgctatgt
193619DNACapsicum annuumsource1..19mol type=unassigned DNA
note=Primer, a Pr4b marker R1 primere organism=Capsicum annuum
36tacgacaaac caccgactc
19372433DNACapsicum annuumsource1..2433mol type=unassigned DNA
note=Caxcv-1 genomi DNS organism=Capsicum annuum 37acttctaatt
gtggataaaa atagtttaaa atttaatact tccttcgttt caaaataatt 60gaattgttga
gtatttttta gggttcaaaa taattaaatt gttcattatt caagatatat 120gttgaatttt
tttatttttt ttttaaattt acttttatta attaaatttt caagattgag 180ttccaatggt
cattattaat gttttagaat ttgaaaagga caaaaatgaa aaaacatgac 240taatttatat
ttttatcttt ttttcttaaa agtgtgtcat attttaataa ttcaattatt 300ttgaaacgag
gagagtaatt ttttttaatc aactaaaacc ataattttat actctcttcg 360tcccaaattt
tctaatttgt ttttccatgt gtttaccctt tgcattattt cttttttctt 420caaattaaaa
tgtaaacatg atttaatagg gatattatgg taaactagac atgttattaa 480ttatttttct
taatcaatgt gtcatctcaa tctgaaacgg agggagtatc tttatctttt 540ttttcttaaa
actgtgtcat atttcaacaa ttcaattatt ttaaaacgga ggaattaatt 600tttttcaatc
aaataaaacc ataattttat aataattctt taaaaaaaaa taaaaaagaa 660tttccgcgca
ttggacgcgg gtacgtatta aagctaccta tgacaacatg ggaaaagatt 720acattataaa
aaaaacaaaa ataagagttt cttggaatgt gcaatcgtct ttgttttccc 780ctttgacttt
actctataaa aacttcacaa atatcacctc ttcactgtac cccattatct 840ttctttgtgg
ttaagcaaat acacaaaata aataaatata actctcctct tagattaaac 900tagtagatcc
atcaaca atg agt tac tac aat caa caa caa cct cct gtt 950
Met Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val 1
5 10 ggt gta cct cca cca caa g
gtaaaaaaaa aaaaaggaag aaacaactct 999Gly Val Pro Pro Pro Gln
15
gactttgttg tatgtgaatt gttttagtta ttagatctga ttgattttta
tttttttggg 1059ggtatttttt gtgatttag gg tat cca cca gaa ggt tac cca aaa
gat tca 1110 Gly Tyr Pro Pro Glu Gly Tyr Pro Lys
Asp Ser 20 25
tac cca cca cct gga tat cca cag caa ggg tac cct caa caa ggg tat
1158Tyr Pro Pro Pro Gly Tyr Pro Gln Gln Gly Tyr Pro Gln Gln Gly Tyr
30 35 40 cca cca caa
ggg tac cct cca cag tat gca cct cag tat ggt gca cca 1206Pro Pro Gln
Gly Tyr Pro Pro Gln Tyr Ala Pro Gln Tyr Gly Ala Pro 45
50 55 60 cct cct caa caa caa cat caa
tca tct agt agt act gga tta ttg caa 1254Pro Pro Gln Gln Gln His Gln
Ser Ser Ser Ser Thr Gly Leu Leu Gln 65
70 75 gga tg gtatgtacac cttattagga ctctattttt
ctatatgttg acttgtatgg 1309Gly Cys
atgtgttatg taggatctga ggtttatagg attgagttga
ccatgtcttg tttgtgtgat 1369aagatatgaa ttgatgtgaa cttgattcat caagatctat
atgacctcag gttttgttga 1429ctgagttgcc tgaatttttt atcaaaatat gtattaggtt
ttaaaaagat ttaacggatc 1489aaagcggtgt taaaactaga aatagtttat gacagtaacg
ttaacaagac tctgcaactc 1549tgccacatca tactatttgt cagagttgca gagtctctga
agttctaagt caactctgca 1609acatcagact cacaaagttg atttggcaga attgcagtgt
ctcttcatta ttggtgaggt 1669tttaagtcaa aaacgttatt gtcagtaacg attcctgctt
tgacgtcgtc cattgttcat 1729tttgtgtccg ttaatttttt taaaacttaa taacgtttat
actggttttg gaaagttttc 1789taaaaacata ctactctggt gaattgattt aaaaaataat
ttattttggt caaaacttca 1849agttgcttgg tgaagctgac ctcgtgtcta accgggaggt
actggctgaa aatagcctca 1909tgcaagataa ggctaggtta taataaacct ttgtggttcg
gttcttccta cacaccgagt 1969ctcatgctgt ttccaattat tggtcggatt aataatcgat
tttttttatt ttattttttt 2029tcag t ttg gct gct ctt tgc tgt tgc tgt gat
gca tgc ttt tgatgctgta 2080 Leu Ala Ala Leu Cys Cys Cys Cys Asp
Ala Cys Phe 80 85 90
aatgatctgt acgcaaagtg ttgatgacaa aagatgattg aaatccatta
tcatagtcta 2140gattattttc cttgaacgtg ttttgtcctt gttgtcctgt catttataaa
taatttgatc 2200ttgctatggt gtctatttgc caaattatac gtttatgtac aacgtgagag
attgtatttt 2260attttttatg ttttggacct caatatgtga atcaatgcac cttgatttgg
ttaaacaatt 2320tatcgcctca tgtgtgtcta taatccaagc gcttggatag tggcggattc
aggatttact 2380ttgagagggt tcagaagtat atatacgaga attaatcaaa ggggtttaat
atc 24333890PRTCapsicum
annuum[CDS]join(918..969,1079..1259,2034..2070) from SEQ ID NO 37
38Met Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val Gly Val Pro Pro Pro 1
5 10 15 Gln Gly Tyr Pro
Pro Glu Gly Tyr Pro Lys Asp Ser Tyr Pro Pro Pro 20
25 30 Gly Tyr Pro Gln Gln Gly Tyr Pro Gln
Gln Gly Tyr Pro Pro Gln Gly 35 40
45 Tyr Pro Pro Gln Tyr Ala Pro Gln Tyr Gly Ala Pro Pro Pro
Gln Gln 50 55 60
Gln His Gln Ser Ser Ser Ser Thr Gly Leu Leu Gln Gly Cys Leu Ala 65
70 75 80 Ala Leu Cys Cys Cys
Cys Asp Ala Cys Phe 85 90 39649DNACapsicum
annuumsource1..649mol type=unassigned DNA note=TC14947
organism=Capsicum annuum 39ctttactcta taaaaacttc acaaatatca cctcttcact
gtaccccatt atctttcttt 60gtggttaagc aaatacacaa aataaataaa tataactctc
ctcttagatt aaactagtag 120atccatcaac a atg agt tac tac aat caa caa caa
cct cct gtt ggt gta 170 Met Ser Tyr Tyr Asn Gln Gln Gln
Pro Pro Val Gly Val 1 5 10
cct cca cca caa ggg tat cca cca gaa ggt tac cca aaa gat tca
tac 218Pro Pro Pro Gln Gly Tyr Pro Pro Glu Gly Tyr Pro Lys Asp Ser
Tyr 15 20 25 cca
cca cct gga tat cca cag caa ggg tac cct caa caa ggg tat cca 266Pro
Pro Pro Gly Tyr Pro Gln Gln Gly Tyr Pro Gln Gln Gly Tyr Pro 30
35 40 45 cca caa ggg tac cct cca
cag tat gca cct cag tat ggt gca cca cct 314Pro Gln Gly Tyr Pro Pro
Gln Tyr Ala Pro Gln Tyr Gly Ala Pro Pro 50
55 60 cct caa caa caa cat caa tca tct agt agt act
gga tta ttg caa gga 362Pro Gln Gln Gln His Gln Ser Ser Ser Ser Thr
Gly Leu Leu Gln Gly 65 70
75 tgt ttg gct gct ctt tgc tgt tgc tgt ctc ttg gat gca tgc ttt
407Cys Leu Ala Ala Leu Cys Cys Cys Cys Leu Leu Asp Ala Cys Phe
80 85 90 tgatgctgta
aatgatctgt acgcaaagtg ttgatgacaa aagatgattg aaatccatta 467tcatagtcta
gattattttc cttgaacgtg ttttgtcctt gttgtcctgt catttataaa 527taatttgatc
ttgctatggt gtctatttgc caaattatag gtttatgtac aacgtgagag 587attgtatttt
attttttatg ttttggacct caatatgtga atcaatgcac cttgatttgg 647tt
6494092PRTCapsicum annuum[CDS]132..407 from SEQ ID NO 39 40Met Ser Tyr
Tyr Asn Gln Gln Gln Pro Pro Val Gly Val Pro Pro Pro 1 5
10 15 Gln Gly Tyr Pro Pro Glu Gly Tyr
Pro Lys Asp Ser Tyr Pro Pro Pro 20 25
30 Gly Tyr Pro Gln Gln Gly Tyr Pro Gln Gln Gly Tyr Pro
Pro Gln Gly 35 40 45
Tyr Pro Pro Gln Tyr Ala Pro Gln Tyr Gly Ala Pro Pro Pro Gln Gln 50
55 60 Gln His Gln Ser
Ser Ser Ser Thr Gly Leu Leu Gln Gly Cys Leu Ala 65 70
75 80 Ala Leu Cys Cys Cys Cys Leu Leu Asp
Ala Cys Phe 85 90
412436DNACapsicum annuumsource1..2436mol type=unassigned DNA
note=Xcv-1 organism=Capsicum annuum 41acttctaatt gtggataaaa
atagtttaaa atttaatact tccttcgttt caaaataatt 60gaattgttga gtatttttta
gggttcaaaa taattaaatt gttcattatt caagatatat 120gttgaatttt tttatttttt
tttaaattta cttttattaa ttaaattttc aagattgagt 180tccaatggtc attattaatg
ttttagaatt tgaaaaggac aaaaatgaaa aaacatgact 240aatttatatt tttatctttt
tttcttaaaa gtgtgtcata ttttaataat tcaattattt 300tgaaacgagg agagtaattt
tttttaatca actaaaacca taattttata ctctcttcgt 360cccaaatttt ctaatttgtt
tttccatgtg tttacccttt gcattatttc ttttttcttc 420aaattaaaat gtaaacatga
tttaataggg atattatggt aaactagaca tgttattaat 480tatttttctt aatcaatgtg
tcatctcaat ctgaaacgga gggagtatct ttatcttttt 540tttcttaaaa ctgtgtcata
tttcaacaat tcaattattt taaaacggag gaattaattt 600ttttcaatca aataaaacca
taattttata ataattcttt aaaaaaaaat aaaaaagaat 660ttccgcgcat tggacgcggg
tacgtattaa agctacctat gacaacatgg gaaaagatta 720cattataaaa aaaacaaaaa
taagagtttc ttggaatgtg caatcgtctt tgttttcccc 780tttgacttta ctctataaaa
acttcacaaa tatcacctct tcactgtacc ccattatctt 840tctttgtggt taagcaaata
cacaaaataa ataaatataa ctctcctctt agattaaact 900agtagatcca tcaaca atg
agt tac tac aat caa caa caa cct cct gtt ggt 952 Met
Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val Gly 1
5 10 gta cct cca cca caa g gtaaaaaaaa
aaaaaggaag aaacaactct gactttgttg 1008Val Pro Pro Pro Gln
15
tatgtgaatt gttttagtta ttagatctga ttgattttta
tttttttggg ggtatttttt 1068gtgatttag gg tat cca cca gaa ggt tac cca aaa
gat tca tac cca cca 1118 Gly Tyr Pro Pro Glu Gly Tyr Pro Lys
Asp Ser Tyr Pro Pro 20 25 30
cct gga tat cca cag caa ggg tac cct caa caa ggg tat cca cca caa
1166Pro Gly Tyr Pro Gln Gln Gly Tyr Pro Gln Gln Gly Tyr Pro Pro Gln
35 40 45 ggg tac cct cca cag
tat gca cct cag tat ggt gca cca cct cct caa 1214Gly Tyr Pro Pro Gln
Tyr Ala Pro Gln Tyr Gly Ala Pro Pro Pro Gln 50 55
60 caa caa cat caa tca tct agt agt act gga tta
ttg caa gga tg 1258Gln Gln His Gln Ser Ser Ser Ser Thr Gly Leu
Leu Gln Gly Cys 65 70 75
gtatgtacac cttattagga ctctattttt ctatatgttg acttgtatgg atgtgttatg
1318taggatctga ggtttatagg attgagttga ccatgtcttg tttgtgtgat aagatatgaa
1378ttgatgtgaa cttgattcat caagatctat atgacctcag gttttgttga ctgagttgcc
1438tgaatttttt atcaaaatat gtattaggtt ttaaaaagat ttaacggatc aaagcggtgt
1498taaaactaga aatagtttat gacagtaacg ttaacaagac tctgcaactc tgccacatca
1558tactatttgt cagagttgca gagtctctga agttctaagt caactctgca acatcagact
1618cacaaagttg atttggcaga attgcagtgt ctcttcatta ttggtgaggt tttaagtcaa
1678aaacgttatt gtcagtaacg attcctgctt tgacgtcgtc cattgttcat tttgtgtccg
1738ttaatttttt taaaacttaa taacgtttat actggttttg gaaagttttc taaaaacata
1798ctactctggt gaattgattt aaaaaataat ttattttggt caaaacttca agttgcttgg
1858tgaagctgac ctcgtgtcta accgggaggt actggctgaa aatagcctca tgcaagataa
1918ggctaggtta taataaacct ttgtggttcg gttcttccta cacaccgagt ctcatgctgt
1978ttccaattat cggtcggatt aataatcgat tttttttatt ttattttttt tcag t ttg
2036 Leu gct gct ctt
tgc tgt tgc tgt ctc ttg gat gca tgc ttt tgatgctgta 2085Ala Ala Leu
Cys Cys Cys Cys Leu Leu Asp Ala Cys Phe 80 85
90 aatgatctgt acgcaaagtg ttgatgacaa
aagatgattg aaatccatta tcatagtcta 2145gattattttc cttgaacgtg ttttgtcctt
gttgtcctgt catttataaa taatttgatc 2205ttgctatggt gtctatttgc caaattatag
gtttatgtac aacgtgagag attgtatttt 2265attttttatg ttttggacct caatatgtga
atcaatgcac cttgatttgg ttaaacaatt 2325tatcgcctca tgtgtctata atccaagcgc
ttggatagtg gcggattcag gatttacttt 2385gagagggttc agaagtatat atacgagaat
taatcaaagg ggtttaatat c 24364292PRTCapsicum
annuum[CDS]join(917..968,1078..1258,2033..2075) from SEQ ID NO 41
42Met Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val Gly Val Pro Pro Pro 1
5 10 15 Gln Gly Tyr Pro
Pro Glu Gly Tyr Pro Lys Asp Ser Tyr Pro Pro Pro 20
25 30 Gly Tyr Pro Gln Gln Gly Tyr Pro Gln
Gln Gly Tyr Pro Pro Gln Gly 35 40
45 Tyr Pro Pro Gln Tyr Ala Pro Gln Tyr Gly Ala Pro Pro Pro
Gln Gln 50 55 60
Gln His Gln Ser Ser Ser Ser Thr Gly Leu Leu Gln Gly Cys Leu Ala 65
70 75 80 Ala Leu Cys Cys Cys
Cys Leu Leu Asp Ala Cys Phe 85 90
4322DNAEscherichia colisource1..22mol type=unassigned DNA
note=Primer Pr mGFP NcoI F for cloning GFP in pGemT-Easy
organism=Escherichia coli 43ccatggtaag taaaggagaa ga
224424DNAEscherichia colisource1..24mol
type=unassigned DNA note=Primer Pr mGFP XbaI R for cloing GFP into
pGemT-Easy organism=Escherichia coli 44tctagaagct ttgtatagtt catc
244526DNAEscherichia
colisource1..26mol type=unassigned DNA note=Primer Pr Xcv-1 XbaI F
for cloning both alleles organism=Escherichia coli 45tctagaatga
gttactacaa tcaaca
264621DNAEscherichia colisource1..21mol type=unassigned DNA
note=Primer Pr Xcv-1 BcuI R for cloning both alleles
organism=Escherichia coli 46actagttcaa aagcatgcat c
214710817DNAEscherichia colisource1..10817mol
type=unassigned DNA note=pCambia GFP-Xcv-1 organism=Escherichia
coli 47ccatggtaag taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag
60atggtgatgt taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat
120acggaaaact tacccttaaa tttatttgca ctactggaaa actacctgtt ccgtggccaa
180cacttgtcac tactttctct tatggtgttc aatgcttttc aagataccca gatcatatga
240agcggcacga cttcttcaag agcgccatgc ctgagggata cgtgcaggag aggaccatct
300tcttcaagga cgacgggaac tacaagacac gtgctgaagt caagtttgag ggagacaccc
360tcgtcaacag gatcgagctt aagggaatcg atttcaagga ggacggaaac atcctcggcc
420acaagttgga atacaactac aactcccaca acgtatacat catggccgac aagcaaaaga
480acggcatcaa agccaacttc aagacccgcc acaacatcga agacggcggc gtgcaactcg
540ctgatcatta tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc
600attacctgtc cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg
660tccttcttga gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaag
720cttctagaat gagttactac aatcaacaac aacctcctgt tggtgtacct ccaccacaag
780ggtatccacc agaaggttac ccaaaagatt catacccacc acctggatat ccacagcaag
840ggtaccctca acaagggtat ccaccacaag ggtaccctcc acagtatgca cctcagtatg
900gtgcaccacc tcctcaacaa caacatcaat catctagtag tactggatta ttgcaaggat
960gtttggctgc tctttgctgt tgctgtctct tggatgcatg cttttgaact agtgaattcg
1020cggccgcctg caggcagctc gaatttcccc gatcgttcaa acatttggca ataaagtttc
1080ttaagattga atcctgttgc cggtcttgcg atgattatca tataatttct gttgaattac
1140gttaagcatg taataattaa catgtaatgc atgacgttat ttatgagatg ggtttttatg
1200attagagtcc cgcaattata catttaatac gcgatagaaa acaaaatata gcgcgcaaac
1260taggataaat tatcgcgcgc ggtgtcatct atgttactag atcgggaatt aaactatcag
1320tgtttgacag gatatattgg cgggtaaacc taagagaaaa gagcgtttat tagaataacg
1380gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca tttgtatgtg catgccaacc
1440acagggttcc cctcgggatc aaagtacttt gatccaaccc ctccgctgct atagtgcagt
1500cggcttctga cgttcagtgc agccgtcttc tgaaaacgac atgtcgcaca agtcctaagt
1560tacgcgacag gctgccgccc tgcccttttc ctggcgtttt cttgtcgcgt gttttagtcg
1620cataaagtag aatacttgcg actagaaccg gagacattac gccatgaaca agagcgccgc
1680cgctggcctg ctgggctatg cccgcgtcag caccgacgac caggacttga ccaaccaacg
1740ggccgaactg cacgcggccg gctgcaccaa gctgttttcc gagaagatca ccggcaccag
1800gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta cgccctggcg acgttgtgac
1860agtgaccagg ctagaccgcc tggcccgcag cacccgcgac ctactggaca ttgccgagcg
1920catccaggag gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg acaccaccac
1980gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg agcgttccct
2040aatcatcgac cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg
2100cccccgccct accctcaccc cggcacagat cgcgcacgcc cgcgagctga tcgaccagga
2160aggccgcacc gtgaaagagg cggctgcact gcttggcgtg catcgctcga ccctgtaccg
2220cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg
2280tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac gccaagagga
2340acaagcatga aaccgcacca ggacggccag gacgaaccgt ttttcattac cgaagagatc
2400gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg
2460cggctgcatg aaatcctggc cggtttgtct gatgccaagc tggcggcctg gccggccagc
2520ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac
2580agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag taaataaaca aatacgcaag
2640gggaacgcat gaaggttatc gctgtactta accagaaagg cgggtcaggc aagacgacca
2700tcgcaaccca tctagcccgc gccctgcaac tcgccggggc cgatgttctg ttagtcgatt
2760ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg
2820ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc cggcgcgact
2880tcgtagtgat cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag
2940ccgacttcgt gctgattccg gtgcagccaa gcccttacga catatgggcc accgccgacc
3000tggtggagct ggttaagcag cgcattgagg tcacggatgg aaggctacaa gcggcctttg
3060tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag gcgctggccg
3120ggtacgagct gcccattctt gagtcccgta tcacgcagcg cgtgagctac ccaggcactg
3180ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc
3240aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt taatgaggta aagagaaaat
3300gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc
3360aacgttggcc agcctggcag acacgccagc catgaagcgg gtcaactttc agttgccggc
3420ggaggatcac accaagctga agatgtacgc ggtacgccaa ggcaagacca ttaccgagct
3480gctatctgaa tacatcgcgc agctaccaga gtaaatgagc aaatgaataa atgagtagat
3540gaattttagc ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc accgacgccg
3600tggaatgccc catgtgtgga ggaacgggcg gttggccagg cgtaagcggc tgggttgtct
3660gccggccctg caatggcact ggaaccccca agcccgagga atcggcgtga cggtcgcaaa
3720ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg acctggtgga gaagttgaag
3780gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg tgaatcgtgg
3840caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg
3900tcgattagga agccgcccaa gggcgacgag caaccagatt ttttcgttcc gatgctctat
3960gacgtgggca cccgcgatag tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag
4020cgtgaccgac gagctggcga ggtgatccgc tacgagcttc cagacgggca cgtagaggtt
4080tccgcagggc cggccggcat ggccagtgtg tgggattacg acctggtact gatggcggtt
4140tcccatctaa ccgaatccat gaaccgatac cgggaaggga agggagacaa gcccggccgc
4200gtgttccgtc cacacgttgc ggacgtactc aagttctgcc ggcgagccga tggcggaaag
4260cagaaagacg acctggtaga aacctgcatt cggttaaaca ccacgcacgt tgccatgcag
4320cgtacgaaga aggccaagaa cggccgcctg gtgacggtat ccgagggtga agccttgatt
4380agccgctaca agatcgtaaa gagcgaaacc gggcggccgg agtacatcga gatcgagcta
4440gctgattgga tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct gacggttcac
4500cccgattact ttttgatcga tcccggcatc ggccgttttc tctaccgcct ggcacgccgc
4560gccgcaggca aggcagaagc cagatggttg ttcaagacga tctacgaacg cagtggcagc
4620gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg
4680ccggagtacg atttgaagga ggaggcgggg caggctggcc cgatcctagt catgcgctac
4740cgcaacctga tcgagggcga agcatccgcc ggttcctaat gtacggagca gatgctaggg
4800caaattgccc tagcagggga aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac
4860attgggaacc caaagccgta cattgggaac cggaacccgt acattgggaa cccaaagccg
4920tacattggga accggtcaca catgtaagtg actgatataa aagagaaaaa aggcgatttt
4980tccgcctaaa actctttaaa acttattaaa actcttaaaa cccgcctggc ctgtgcataa
5040ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc
5100tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc aaaaatggct
5160ggcctacggc caggcaatct accagggcgc ggacaagccg cgccgtcgcc actcgaccgc
5220cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg
5280acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca
5340agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc
5400acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg
5460agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc
5520aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
5580gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
5640ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
5700ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
5760cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
5820ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
5880tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
5940gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
6000tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
6060gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
6120tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag
6180ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
6240agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
6300gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
6360attttggtca tgcattctag gtactaaaac aattcatcca gtaaaatata atattttatt
6420ttctcccaat caggcttgat ccccagtaag tcaaaaaata gctcgacata ctgttcttcc
6480ccgatatcct ccctgatcga ccggacgcag aaggcaatgt cataccactt gtccgccctg
6540ccgcttctcc caagatcaat aaagccactt actttgccat ctttcacaaa gatgttgctg
6600tctcccaggt cgccgtggga aaagacaagt tcctcttcgg gcttttccgt ctttaaaaaa
6660tcatacagct cgcgcggatc tttaaatgga gtgtcttctt cccagttttc gcaatccaca
6720tcggccagat cgttattcag taagtaatcc aattcggcta agcggctgtc taagctattc
6780gtatagggac aatccgatat gtcgatggag tgaaagagcc tgatgcactc cgcatacagc
6840tcgataatct tttcagggct ttgttcatct tcatactctt ccgagcaaag gacgccatcg
6900gcctcactca tgagcagatt gctccagcca tcatgccgtt caaagtgcag gacctttgga
6960acaggcagct ttccttccag ccatagcatc atgtcctttt cccgttccac atcataggtg
7020gtccctttat accggctgtc cgtcattttt aaatataggt tttcattttc tcccaccagc
7080ttatatacct tagcaggaga cattccttcc gtatctttta cgcagcggta tttttcgatc
7140agttttttca attccggtga tattctcatt ttagccattt attatttcct tcctcttttc
7200tacagtattt aaagataccc caagaagcta attataacaa gacgaactcc aattcactgt
7260tccttgcatt ctaaaacctt aaataccaga aaacagcttt ttcaaagttg ttttcaaagt
7320tggcgtataa catagtatcg acggagccga ttttgaaacc gcggtgatca caggcagcaa
7380cgctctgtca tcgttacaat caacatgcta ccctccgcga gatcatccgt gtttcaaacc
7440cggcagctta gttgccgttc ttccgaatag catcggtaac atgagcaaag tctgccgcct
7500tacaacggct ctcccgctga cgccgtcccg gactgatggg ctgcctgtat cgagtggtga
7560ttttgtgccg agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg
7620tgtaaacaaa ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg
7680aattaacgcc gaattaattc gggggatctg gattttagta ctggattttg gttttaggaa
7740ttagaaattt tattgataga agtattttac aaatacaaat acatactaag ggtttcttat
7800atgctcaaca catgagcgaa accctatagg aaccctaatt cccttatctg ggaactactc
7860acacattatt atggagaaac tcgagcttgt cgatcgacag atccggtcgg catctactct
7920atttctttgc cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta
7980cacagccatc ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc
8040cggctccgga tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat
8100tgccgtcaac caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga
8160gtcgtggcga tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca
8220tacaagccaa ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga
8280acatcgcctc gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt
8340tggagccgaa atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca
8400tcagctcatc gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc
8460agtgatacac atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac
8520cgattccttg cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga
8580tcgcatccat agcctccgcg accggttgta gaacagcggg cagttcggtt tcaggcaggt
8640cttgcaacgt gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctaaact
8700ccccaatgtc aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat
8760aacgatcttt gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc
8820ctacatcgaa gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga
8880cgctgtcgaa cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt
8940tcatatctca ttgccccccg ggatctgcga aagctcgaga gagatagatt tgtagagaga
9000gactggtgat ttcagcgtgt cctctccaaa tgaaatgaac ttccttatat agaggaaggt
9060cttgcgaagg atagtgggat tgtgcgtcat cccttacgtc agtggagata tcacatcaat
9120ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtg
9180ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc tttcctttat
9240cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac
9300agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct
9360caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt
9420cgtgctccac catgttatca catcaatcca cttgctttga agacgtggtt ggaacgtctt
9480ctttttccac gatgctcctc gtgggtgggg gtccatcttt gggaccactg tcggcagagg
9540catcttgaac gatagccttt cctttatcgc aatgatggca tttgtaggtg ccaccttcct
9600tttctactgt ccttttgatg aagtgacaga tagctgggca atggaatccg aggaggtttc
9660ccgatattac cctttgttga aaagtctcaa tagccctttg gtcttctgag actgtatctt
9720tgatattctt ggagtagacg agagtgtcgt gctccaccat gttggcaagc tgctctagcc
9780aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
9840gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
9900ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
9960cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg agctcggtac
10020ccggggatcc tctagagtcg acctgcaggc atgcaagctt ggcactggcc gtcgttttac
10080aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc
10140ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc
10200gcagcctgaa tggcgaatgc tagagcagct tgagcttgga tcagattgtc gtttcccgcc
10260ttcagtttag cttcatggag tcaaagattc aaatagagga cctaacagaa ctcgccgtaa
10320agactggcga acagttcata cagagtctct tacgactcaa tgacaagaag aaaatcttcg
10380tcaacatggt ggagcacgac acacttgtct actccaaaaa tatcaaagat acagtctcag
10440aagaccaaag ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat
10500tccattgccc agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct
10560acaaatgcca tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg
10620gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca
10680cgtcttcaaa gcaagtggat tgatgtgata tctccactga cgtaagggat gacgcacaat
10740cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat ttggagagaa
10800cacgggggac tcttgac
108174810811DNAEscherichia colisource1..10811mol type=unassigned DNA
note=pCambia- GFP-xcv-1 organism=Escherichia coli 48ccatggtaag
taaaggagaa gaacttttca ctggagttgt cccaattctt gttgaattag 60atggtgatgt
taatgggcac aaattttctg tcagtggaga gggtgaaggt gatgcaacat 120acggaaaact
tacccttaaa tttatttgca ctactggaaa actacctgtt ccgtggccaa 180cacttgtcac
tactttctct tatggtgttc aatgcttttc aagataccca gatcatatga 240agcggcacga
cttcttcaag agcgccatgc ctgagggata cgtgcaggag aggaccatct 300tcttcaagga
cgacgggaac tacaagacac gtgctgaagt caagtttgag ggagacaccc 360tcgtcaacag
gatcgagctt aagggaatcg atttcaagga ggacggaaac atcctcggcc 420acaagttgga
atacaactac aactcccaca acgtatacat catggccgac aagcaaaaga 480acggcatcaa
agccaacttc aagacccgcc acaacatcga agacggcggc gtgcaactcg 540ctgatcatta
tcaacaaaat actccaattg gcgatggccc tgtcctttta ccagacaacc 600attacctgtc
cacacaatct gccctttcga aagatcccaa cgaaaagaga gaccacatgg 660tccttcttga
gtttgtaaca gctgctggga ttacacatgg catggatgaa ctatacaaag 720cttctagaat
gagttactac aatcaacaac aacctcctgt tggtgtacct ccaccacaag 780ggtatccacc
agaaggttac ccaaaagatt catacccacc acctggatat ccacagcaag 840ggtaccctca
acaagggtat ccaccacaag ggtaccctcc acagtatgca cctcagtatg 900gtgcaccacc
tcctcaacaa caacatcaat catctagtag tactggatta ttgcaaggat 960gtttggctgc
tctttgctgt tgctgtgatg catgcttttg aactagtgaa ttcgcggccg 1020cctgcaggca
gctcgaattt ccccgatcgt tcaaacattt ggcaataaag tttcttaaga 1080ttgaatcctg
ttgccggtct tgcgatgatt atcatataat ttctgttgaa ttacgttaag 1140catgtaataa
ttaacatgta atgcatgacg ttatttatga gatgggtttt tatgattaga 1200gtcccgcaat
tatacattta atacgcgata gaaaacaaaa tatagcgcgc aaactaggat 1260aaattatcgc
gcgcggtgtc atctatgtta ctagatcggg aattaaacta tcagtgtttg 1320acaggatata
ttggcgggta aacctaagag aaaagagcgt ttattagaat aacggatatt 1380taaaagggcg
tgaaaaggtt tatccgttcg tccatttgta tgtgcatgcc aaccacaggg 1440ttcccctcgg
gatcaaagta ctttgatcca acccctccgc tgctatagtg cagtcggctt 1500ctgacgttca
gtgcagccgt cttctgaaaa cgacatgtcg cacaagtcct aagttacgcg 1560acaggctgcc
gccctgccct tttcctggcg ttttcttgtc gcgtgtttta gtcgcataaa 1620gtagaatact
tgcgactaga accggagaca ttacgccatg aacaagagcg ccgccgctgg 1680cctgctgggc
tatgcccgcg tcagcaccga cgaccaggac ttgaccaacc aacgggccga 1740actgcacgcg
gccggctgca ccaagctgtt ttccgagaag atcaccggca ccaggcgcga 1800ccgcccggag
ctggccagga tgcttgacca cctacgccct ggcgacgttg tgacagtgac 1860caggctagac
cgcctggccc gcagcacccg cgacctactg gacattgccg agcgcatcca 1920ggaggccggc
gcgggcctgc gtagcctggc agagccgtgg gccgacacca ccacgccggc 1980cggccgcatg
gtgttgaccg tgttcgccgg cattgccgag ttcgagcgtt ccctaatcat 2040cgaccgcacc
cggagcgggc gcgaggccgc caaggcccga ggcgtgaagt ttggcccccg 2100ccctaccctc
accccggcac agatcgcgca cgcccgcgag ctgatcgacc aggaaggccg 2160caccgtgaaa
gaggcggctg cactgcttgg cgtgcatcgc tcgaccctgt accgcgcact 2220tgagcgcagc
gaggaagtga cgcccaccga ggccaggcgg cgcggtgcct tccgtgagga 2280cgcattgacc
gaggccgacg ccctggcggc cgccgagaat gaacgccaag aggaacaagc 2340atgaaaccgc
accaggacgg ccaggacgaa ccgtttttca ttaccgaaga gatcgaggcg 2400gagatgatcg
cggccgggta cgtgttcgag ccgcccgcgc acgtctcaac cgtgcggctg 2460catgaaatcc
tggccggttt gtctgatgcc aagctggcgg cctggccggc cagcttggcc 2520gctgaagaaa
ccgagcgccg ccgtctaaaa aggtgatgtg tatttgagta aaacagcttg 2580cgtcatgcgg
tcgctgcgta tatgatgcga tgagtaaata aacaaatacg caaggggaac 2640gcatgaaggt
tatcgctgta cttaaccaga aaggcgggtc aggcaagacg accatcgcaa 2700cccatctagc
ccgcgccctg caactcgccg gggccgatgt tctgttagtc gattccgatc 2760cccagggcag
tgcccgcgat tgggcggccg tgcgggaaga tcaaccgcta accgttgtcg 2820gcatcgaccg
cccgacgatt gaccgcgacg tgaaggccat cggccggcgc gacttcgtag 2880tgatcgacgg
agcgccccag gcggcggact tggctgtgtc cgcgatcaag gcagccgact 2940tcgtgctgat
tccggtgcag ccaagccctt acgacatatg ggccaccgcc gacctggtgg 3000agctggttaa
gcagcgcatt gaggtcacgg atggaaggct acaagcggcc tttgtcgtgt 3060cgcgggcgat
caaaggcacg cgcatcggcg gtgaggttgc cgaggcgctg gccgggtacg 3120agctgcccat
tcttgagtcc cgtatcacgc agcgcgtgag ctacccaggc actgccgccg 3180ccggcacaac
cgttcttgaa tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc 3240tggccgctga
aattaaatca aaactcattt gagttaatga ggtaaagaga aaatgagcaa 3300aagcacaaac
acgctaagtg ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt 3360ggccagcctg
gcagacacgc cagccatgaa gcgggtcaac tttcagttgc cggcggagga 3420tcacaccaag
ctgaagatgt acgcggtacg ccaaggcaag accattaccg agctgctatc 3480tgaatacatc
gcgcagctac cagagtaaat gagcaaatga ataaatgagt agatgaattt 3540tagcggctaa
aggaggcggc atggaaaatc aagaacaacc aggcaccgac gccgtggaat 3600gccccatgtg
tggaggaacg ggcggttggc caggcgtaag cggctgggtt gtctgccggc 3660cctgcaatgg
cactggaacc cccaagcccg aggaatcggc gtgacggtcg caaaccatcc 3720ggcccggtac
aaatcggcgc ggcgctgggt gatgacctgg tggagaagtt gaaggccgcg 3780caggccgccc
agcggcaacg catcgaggca gaagcacgcc ccggtgaatc gtggcaagcg 3840gccgctgatc
gaatccgcaa agaatcccgg caaccgccgg cagccggtgc gccgtcgatt 3900aggaagccgc
ccaagggcga cgagcaacca gattttttcg ttccgatgct ctatgacgtg 3960ggcacccgcg
atagtcgcag catcatggac gtggccgttt tccgtctgtc gaagcgtgac 4020cgacgagctg
gcgaggtgat ccgctacgag cttccagacg ggcacgtaga ggtttccgca 4080gggccggccg
gcatggccag tgtgtgggat tacgacctgg tactgatggc ggtttcccat 4140ctaaccgaat
ccatgaaccg ataccgggaa gggaagggag acaagcccgg ccgcgtgttc 4200cgtccacacg
ttgcggacgt actcaagttc tgccggcgag ccgatggcgg aaagcagaaa 4260gacgacctgg
tagaaacctg cattcggtta aacaccacgc acgttgccat gcagcgtacg 4320aagaaggcca
agaacggccg cctggtgacg gtatccgagg gtgaagcctt gattagccgc 4380tacaagatcg
taaagagcga aaccgggcgg ccggagtaca tcgagatcga gctagctgat 4440tggatgtacc
gcgagatcac agaaggcaag aacccggacg tgctgacggt tcaccccgat 4500tactttttga
tcgatcccgg catcggccgt tttctctacc gcctggcacg ccgcgccgca 4560ggcaaggcag
aagccagatg gttgttcaag acgatctacg aacgcagtgg cagcgccgga 4620gagttcaaga
agttctgttt caccgtgcgc aagctgatcg ggtcaaatga cctgccggag 4680tacgatttga
aggaggaggc ggggcaggct ggcccgatcc tagtcatgcg ctaccgcaac 4740ctgatcgagg
gcgaagcatc cgccggttcc taatgtacgg agcagatgct agggcaaatt 4800gccctagcag
gggaaaaagg tcgaaaaggt ctctttcctg tggatagcac gtacattggg 4860aacccaaagc
cgtacattgg gaaccggaac ccgtacattg ggaacccaaa gccgtacatt 4920gggaaccggt
cacacatgta agtgactgat ataaaagaga aaaaaggcga tttttccgcc 4980taaaactctt
taaaacttat taaaactctt aaaacccgcc tggcctgtgc ataactgtct 5040ggccagcgca
cagccgaaga gctgcaaaaa gcgcctaccc ttcggtcgct gcgctcccta 5100cgccccgccg
cttcgcgtcg gcctatcgcg gccgctggcc gctcaaaaat ggctggccta 5160cggccaggca
atctaccagg gcgcggacaa gccgcgccgt cgccactcga ccgccggcgc 5220ccacatcaag
gcaccctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat 5280gcagctcccg
gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg 5340tcagggcgcg
tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag 5400cgatagcgga
gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg 5460caccatatgc
ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc 5520tcttccgctt
cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 5580tcagctcact
caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 5640aacatgtgag
caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 5700tttttccata
ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 5760tggcgaaacc
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 5820cgctctcctg
ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 5880agcgtggcgc
tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 5940tccaagctgg
gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 6000aactatcgtc
ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 6060ggtaacagga
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 6120cctaactacg
gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 6180accttcggaa
aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 6240ggtttttttg
tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 6300ttgatctttt
ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 6360gtcatgcatt
ctaggtacta aaacaattca tccagtaaaa tataatattt tattttctcc 6420caatcaggct
tgatccccag taagtcaaaa aatagctcga catactgttc ttccccgata 6480tcctccctga
tcgaccggac gcagaaggca atgtcatacc acttgtccgc cctgccgctt 6540ctcccaagat
caataaagcc acttactttg ccatctttca caaagatgtt gctgtctccc 6600aggtcgccgt
gggaaaagac aagttcctct tcgggctttt ccgtctttaa aaaatcatac 6660agctcgcgcg
gatctttaaa tggagtgtct tcttcccagt tttcgcaatc cacatcggcc 6720agatcgttat
tcagtaagta atccaattcg gctaagcggc tgtctaagct attcgtatag 6780ggacaatccg
atatgtcgat ggagtgaaag agcctgatgc actccgcata cagctcgata 6840atcttttcag
ggctttgttc atcttcatac tcttccgagc aaaggacgcc atcggcctca 6900ctcatgagca
gattgctcca gccatcatgc cgttcaaagt gcaggacctt tggaacaggc 6960agctttcctt
ccagccatag catcatgtcc ttttcccgtt ccacatcata ggtggtccct 7020ttataccggc
tgtccgtcat ttttaaatat aggttttcat tttctcccac cagcttatat 7080accttagcag
gagacattcc ttccgtatct tttacgcagc ggtatttttc gatcagtttt 7140ttcaattccg
gtgatattct cattttagcc atttattatt tccttcctct tttctacagt 7200atttaaagat
accccaagaa gctaattata acaagacgaa ctccaattca ctgttccttg 7260cattctaaaa
ccttaaatac cagaaaacag ctttttcaaa gttgttttca aagttggcgt 7320ataacatagt
atcgacggag ccgattttga aaccgcggtg atcacaggca gcaacgctct 7380gtcatcgtta
caatcaacat gctaccctcc gcgagatcat ccgtgtttca aacccggcag 7440cttagttgcc
gttcttccga atagcatcgg taacatgagc aaagtctgcc gccttacaac 7500ggctctcccg
ctgacgccgt cccggactga tgggctgcct gtatcgagtg gtgattttgt 7560gccgagctgc
cggtcgggga gctgttggct ggctggtggc aggatatatt gtggtgtaaa 7620caaattgacg
cttagacaac ttaataacac attgcggacg tttttaatgt actgaattaa 7680cgccgaatta
attcggggga tctggatttt agtactggat tttggtttta ggaattagaa 7740attttattga
tagaagtatt ttacaaatac aaatacatac taagggtttc ttatatgctc 7800aacacatgag
cgaaacccta taggaaccct aattccctta tctgggaact actcacacat 7860tattatggag
aaactcgagc ttgtcgatcg acagatccgg tcggcatcta ctctatttct 7920ttgccctcgg
acgagtgctg gggcgtcggt ttccactatc ggcgagtact tctacacagc 7980catcggtcca
gacggccgcg cttctgcggg cgatttgtgt acgcccgaca gtcccggctc 8040cggatcggac
gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg aaattgccgt 8100caaccaagct
ctgatagagt tggtcaagac caatgcggag catatacgcc cggagtcgtg 8160gcgatcctgc
aagctccgga tgcctccgct cgaagtagcg cgtctgctgc tccatacaag 8220ccaaccacgg
cctccagaag aagatgttgg cgacctcgta ttgggaatcc ccgaacatcg 8280cctcgctcca
gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca ttgttggagc 8340cgaaatccgc
gtgcacgagg tgccggactt cggggcagtc ctcggcccaa agcatcagct 8400catcgagagc
ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt tgccagtgat 8460acacatgggg
atcagcaatc gcgcatatga aatcacgcca tgtagtgtat tgaccgattc 8520cttgcggtcc
gaatgggccg aacccgctcg tctggctaag atcggccgca gcgatcgcat 8580ccatagcctc
cgcgaccggt tgtagaacag cgggcagttc ggtttcaggc aggtcttgca 8640acgtgacacc
ctgtgcacgg cgggagatgc aataggtcag gctctcgcta aactccccaa 8700tgtcaagcac
ttccggaatc gggagcgcgg ccgatgcaaa gtgccgataa acataacgat 8760ctttgtagaa
accatcggcg cagctattta cccgcaggac atatccacgc cctcctacat 8820cgaagctgaa
agcacgagat tcttcgccct ccgagagctg catcaggtcg gagacgctgt 8880cgaacttttc
gatcagaaac ttctcgacag acgtcgcggt gagttcaggc tttttcatat 8940ctcattgccc
cccgggatct gcgaaagctc gagagagata gatttgtaga gagagactgg 9000tgatttcagc
gtgtcctctc caaatgaaat gaacttcctt atatagagga aggtcttgcg 9060aaggatagtg
ggattgtgcg tcatccctta cgtcagtgga gatatcacat caatccactt 9120gctttgaaga
cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc 9180catctttggg
accactgtcg gcagaggcat cttgaacgat agcctttcct ttatcgcaat 9240gatggcattt
gtaggtgcca ccttcctttt ctactgtcct tttgatgaag tgacagatag 9300ctgggcaatg
gaatccgagg aggtttcccg atattaccct ttgttgaaaa gtctcaatag 9360ccctttggtc
ttctgagact gtatctttga tattcttgga gtagacgaga gtgtcgtgct 9420ccaccatgtt
atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt 9480ccacgatgct
cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt 9540gaacgatagc
ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta 9600ctgtcctttt
gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata 9660ttaccctttg
ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat 9720tcttggagta
gacgagagtg tcgtgctcca ccatgttggc aagctgctct agccaatacg 9780caaaccgcct
ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 9840cgactggaaa
gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc 9900accccaggct
ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 9960acaatttcac
acaggaaaca gctatgacca tgattacgaa ttcgagctcg gtacccgggg 10020atcctctaga
gtcgacctgc aggcatgcaa gcttggcact ggccgtcgtt ttacaacgtc 10080gtgactggga
aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg 10140ccagctggcg
taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 10200tgaatggcga
atgctagagc agcttgagct tggatcagat tgtcgtttcc cgccttcagt 10260ttagcttcat
ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg 10320gcgaacagtt
catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca 10380tggtggagca
cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc 10440aaagggcaat
tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt 10500gcccagctat
ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat 10560gccatcattg
cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca 10620aagatggacc
cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt 10680caaagcaagt
ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact 10740atccttcgca
agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg 10800ggactcttga c
10811493297DNASolanum lycopersicumsource1..3297mol type=unassigned DNA
note=SlXcv-1A organism=Solanum lycopersicum 49gagcatctga
atatttaagt atgaaaaatc aaaatctcat taataagata aaagtttaaa 60taatcaactc
attgaagcta ttaaaattct cgtcaaattc ttactaatac ataaacaaat 120atatatttaa
aatcacaaat tctaaaagta attttcatcc atctcaaaag cattatttcg 180ttgtgatttt
tttttccttc ccaaaggcgt tgtccactaa gatttgaagt ccttagacaa 240agcaaaagaa
atagcattat gaaagaggat gagatataaa gtgaatttct ttttctctat 300ttcataaaaa
aaacttagat atacaaaaga agaaaatgtt tatattttta aaatctgttt 360gtccataaat
tatgcctttt taagtgtgta gttatacaga tccacccact atagcatccc 420aacaaaactc
atgacttcat caatcactat taaaaaattt taataaatta tggagattta 480tttatatggt
cataaaaaaa aacatttcaa taaagacagg ttaaacatta attcttctag 540ataaatcgtt
gatcaacttt tctttggaag aactaataat acagtgtagg tcatatctaa 600attaaaaaat
catcaaaaga tgtaaacagt ttgtcaataa ctaatgaaaa taacaaatta 660aaataacatg
aaaagttaaa tagctgaaaa aaattatgaa atattcaatt tcttttttac 720aaacataaca
ctgaaaaaaa aattcaattt cttttcgata tttatataac agctcgatta 780aaattgaata
aaatttgaga aaaagattcg gcagtaaaat caaattagga tttcttggaa 840tgcgcttttt
tgctggtcct cgttgtgtcc actttgattt gccttataaa tatcccttct 900ccactgtgca
gcccattatc tttctttgtg tctaagcaaa agcaccaaca gaaactctct 960ctacagttag
atccaaccca aattcttcat taattcaaca atg agt tac tac aat 1015
Met Ser Tyr Tyr Asn
1 5 caa caa caa ccc cct gtt ggt gtg
cca cca cca caa g gtaaatcact 1062Gln Gln Gln Pro Pro Val Gly Val
Pro Pro Pro Gln 10 15
tcaattcctt tttttctgat tttattttgg taaagttgat gtttttatat
gtttttttga 1122tgactagatc tgatggggtt ttgaattttt gtag ga tat ccg cca
gaa ggt tac 1176 Gly Tyr Pro Pro
Glu Gly Tyr 20
cca aaa gat gca tac cca cca cca ggg tac cca cag cag ggt tac cct
1224Pro Lys Asp Ala Tyr Pro Pro Pro Gly Tyr Pro Gln Gln Gly Tyr Pro 25
30 35 40 caa caa ggt tac
cca cct caa ggg tac cct cca cag tat gca cct cag 1272Gln Gln Gly Tyr
Pro Pro Gln Gly Tyr Pro Pro Gln Tyr Ala Pro Gln 45
50 55 tat ggt gct cca cct cct cat caa caa
caa cag caa tct ggt act ggt 1320Tyr Gly Ala Pro Pro Pro His Gln Gln
Gln Gln Gln Ser Gly Thr Gly 60 65
70 ttc atg gaa gga tg gtatgaacct taaagactca atttttatgt
tcatatgtta 1374Phe Met Glu Gly Cys
75
tgattcagtt acagtaattt gtatgatttt gtttgagaga gatctgtatt gatattggtt
1434ttttgtagag cggttgagga gattgtttat ctgtcttgtc tgttactatg tgtgtgtttc
1494ttgttgattt ggtgtgtctt gtcaaaacat gatttctcga attattattg gtctgtttga
1554tgtttacttt gtgagcactt gtatttaata actatggttc tggacgagtt ctggacgcga
1614actagaaggg gtccttatca tttgtatgtc tagttgaaca ttaacgcgag caggagagac
1674aattagaatg cctgaatgat tctctatgtg caaacatagt ccaggccttg ccacaccttt
1734gattttatct ctgactgctg aaatcgcgcc cctatcatac ttcagactgg aagggggtgc
1794atggactaga cataacttgt gttgctttgt ctatagctat aggttttcca ctgttgctgc
1854tgcaataaga caataatgac ctcttatcaa agaaatctac actgtcacgt taattgtttc
1914ttcgttgtaa tagactatgg gagtatcctt gttgatgctc cattccactg taagataagt
1974agttaactga tctttcaaaa caatcacata attgtcatat taatcacacg tttaagttat
2034cgcagtgcca gtgaaccaat tagacaatgg acgtggtctt ggctacccta aattttcata
2094gaatatagtg gtggtctttc acatttcaac agctttgtga ttttctgttt taggcgttcc
2154ttctttcttg tagcctctcc tcattttatt tgagcttgct gtatttgtta tttccggtgg
2214gggactaatc attggttgtt atatttttgt tttgcag t ttg gct gct ctg tgc
2267 Leu Ala Ala Leu Cys
80 tgt tgc tgt ctc
ttg gat gca tgc ttt tgatgctgta aatgatctgt 2314Cys Cys Cys Leu
Leu Asp Ala Cys Phe 85 90
gccatgtgtt ggtggcaaaa gtttattaaa ccaattctat
catagtctag actttctctt 2374tttgtgtttg tctttggtgt cctgtactgt ccttgataaa
taatttgata ttaaatatga 2434cgatgcacct tgttatggtg ggagaattca agtgtctatt
tgccaattta tacattattg 2494tgcaatgcaa gagattctat ttttttaaaa tcttggttgt
ctatatgtga atgcgacctt 2554gctcttaggt tatatatgct gctgatttct atttactgtg
tgggcgatca ccattttttg 2614ggtttaaagt ttattatact gacttgttag acgcccaaag
tataaccaat tgagtggtga 2674aatacttcaa caagttggca agttgtaaaa tcttctgggt
gttagtatat cctacaacag 2734gcttgtgggg tgcctagtat gtttcttaat ttagaccata
atgaaagtcc gccaaaacct 2794ttaatccttc atcttatgct ttgttggtgg gcaggtgaca
tatattttgt agaacacaac 2854cagacttgga ctcagccgtt ggttcttgat ccttgtgctt
atgcaaaccg aataggtaat 2914agaaataggt tcttttgtgc catagattca tatataaagg
actttactaa aaacaatcaa 2974aggttataaa atataacgac aagcaaaact caaaagacca
ccccaaaagt aggcccattt 3034atgtgaatgc ctttgagatt ttcttcagca gatgacccat
taggatgttg ggttgggccc 3094ttaaaatcta gaaacctttt cctctttgct tcctaaattg
catcaaagct cttgttcgaa 3154tttcacttgc tatttgaaca agtggaaaat catttcatat
ttcacaatag agaagggtag 3214aacctctttg tacgctttaa taacgtgcaa tagtgaatta
ttattcatcc gataataatg 3274atttatttgg aaaaactttt aac
32975091PRTSolanum
lycopersicum[CDS]join(1001..1052,1157..1334,2252..2294) from SEQ ID
NO 49 50Met Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val Gly Val Pro Pro Pro 1
5 10 15 Gln Gly Tyr
Pro Pro Glu Gly Tyr Pro Lys Asp Ala Tyr Pro Pro Pro 20
25 30 Gly Tyr Pro Gln Gln Gly Tyr Pro
Gln Gln Gly Tyr Pro Pro Gln Gly 35 40
45 Tyr Pro Pro Gln Tyr Ala Pro Gln Tyr Gly Ala Pro Pro
Pro His Gln 50 55 60
Gln Gln Gln Gln Ser Gly Thr Gly Phe Met Glu Gly Cys Leu Ala Ala 65
70 75 80 Leu Cys Cys Cys
Cys Leu Leu Asp Ala Cys Phe 85 90
513638DNASolanum lycopersicumsource1..3638mol type=unassigned DNA
note=SlXcv-1B organism=Solanum lycopersicum 51cataagctat tggagtggga
atgcataaga atgaaaaaac atgtcatggt gctccacccc 60tctccataac tcaacagtga
tatcgggtgg agtcagaatt tttattaata aacttataat 120ataattaaat aacaagcgaa
tgaataagct tacaaaaatt aagcacctac tatatgttat 180atatttaaca atttggttaa
aacttttact cttatcctaa taagtggttt cataaatcgt 240caataaactc taaaacatac
tatataacac aaaatcctaa aaataacata taatggagga 300aactcttttt ctcttccttt
tcgatcgtga atcaaataat ttaaaattta attaaatcta 360taaaatttta cttcctttct
caataatact ataaatagat agttcatttt tatttatttt 420aaataattaa aaataattta
attactcatc tataatatat ttttaactat agaattctct 480aaatattaaa tttattgctc
cttttatact ttataaatag attttcactt tcattatcat 540ttttatcata ttaagaaaaa
cttttatttt tatttttatt tcatcctcaa tattaattaa 600ttattcttca aatcattaat
tataatttaa gtctatgcac tatttaatat agaaaattat 660tatttttatt taggagtact
tatttaggtg aacatagcca gtcaatatat gataaataaa 720ataaaataaa cataagagta
atatatatat atagaaaaat aatattctga cactttacgc 780gcatcacaca cggggtactt
ttctagtacc cttggaaaag atcacataat aaattaataa 840attactaagg ccttgttgga
tttaacaaat tatctctgaa ataaatttta aaattaatta 900tttcacaatt actggatata
agataaaaaa aatagaataa ttaatttcta actatataaa 960taaattttga ttttaattat
ttcttatccg tctgtagcaa cagagtcgga gagtttcttg 1020gaatatgctg ggcttagtcc
ttgtttgact tgctctataa aacctaacaa ataaacccca 1080tttctttgtg cataagcaaa
aactcaaaac tctcttcaac aagaaatttc aatt atg 1137
Met
1 agt tac tac aat cag cag caa ccc cct gtt ggt
gta cca cca cca caa g 1186Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val Gly
Val Pro Pro Pro Gln 5 10
15 gtaaattgct tcaatttcat tttttttttg atgatttatg gtaaagttca
tatttttata 1246tattgttttg atgatttatg gtgaagttga tgtttttata tgttgttttg
atgatttgtg 1306gtaaagttga tgtttttata tgttattttg atgattagat ctgatggggt
tttggggaat 1366tttgtaatta g gt tat cca cca gaa ggt tac tct aaa gat gca
tac cca 1415 Gly Tyr Pro Pro Glu Gly Tyr Ser Lys Asp Ala
Tyr Pro 20 25 30
cca cca ggg tat cct cag caa ggg tat cca cca cag ggt tat cct caa
1463Pro Pro Gly Tyr Pro Gln Gln Gly Tyr Pro Pro Gln Gly Tyr Pro Gln
35 40 45 caa ggg tat cca
cct cca cag tat gca cct cag tat ggt gct cca cct 1511Gln Gly Tyr Pro
Pro Pro Gln Tyr Ala Pro Gln Tyr Gly Ala Pro Pro 50
55 60 cct caa caa cat caa cag caa tct agt
agc act gga tta atg caa gga 1559Pro Gln Gln His Gln Gln Gln Ser Ser
Ser Thr Gly Leu Met Gln Gly 65 70
75 tg gtatgcatct ttacactcgt gatgttttgt tatgatttag
ttgtgttgtt 1611Cys
gaattgtatg atctgagatt ttttttggat tgagttgact atggatgata
aggtgtgtaa 1671tgatttgatt ttgtcaactg aattgggtga ggttagtatg atctcgggtt
ttgttgacta 1731agttgccgag agacttgttc atatttgtgt gtctttcctt gtttgtctcg
aaaagatctt 1791ttgatcatca aagggggaaa gtagaagcaa ggggggctag acctctagac
cttaattgct 1851gcatagattc atcatgttgc tattagttga ttttctttga atattttagc
aacctgtgga 1911ttcgatataa gcaatctagg tttttttttg gatgaatttc ttgaccagtt
ttagtcgaga 1971gaagatcaaa ggaatttgga gaagaaatgt ccttttgggg cagaagctcg
agcaatggaa 2031tatccctcgt gtgttacaca ttaacacgag ttggagatcc aaccgattct
aatgcgtgaa 2091tattttttat gtgccaccat agtccggtcc tttccattac tgttatcaac
gcccttcatt 2151ttgtctcttg ttttcattgt atctactgat tcatcaccag tagcatgcaa
tggaattttc 2211aacttgctgt acaatatcat gtttctcatt aaaattctgc cctgaataca
ctgctcaaat 2271tgccggttta gacttaactt gctttgtcta tagctattgt tttccaactg
ctgctcctat 2331agataaaata atgacctctt agccaaatca gtctagagct aagtttggca
aagtaatcca 2391cactgccgcc ttagttgttg tttctttgtt tctacctcca atatcctatg
ttgatgctcc 2451ttcccattga tcaaaccttc aagttgtcgc agtgccaatg aactagtcag
acaatgattc 2511ttgattccgt gatgtttcag tagccctttt ttcttttagc actttgtcat
attatttcga 2571atttccagtg ggactaataa tggtttgtgg tattgttttg cag t ttg
gct gct ctg 2627 Leu
Ala Ala Leu 80
tgc tgt tgc tgt ctc ttg gat gca tgc ttt tgagggtgta aatgatctgt
2677Cys Cys Cys Cys Leu Leu Asp Ala Cys Phe
85 90 gccatgtgtt
gatggcaaaa gtttattgaa tcaattatat catagtctag acttttttct 2737ttctgtgttt
tgtcctatac ttactttgat aaataatttg atctttgatg tgctcaagat 2797tccaaagtgt
ttgttttgcc aaattatagg tggttggtta tgtacaatgt gacagattct 2857atttttattt
ttttcaatgt tttggacctc aaaatattat atgtgaatca atgcaccttg 2917ctctggttac
aaaatttatg ctcctgttgt attaaatgtg ttagcgaaca caacgtttgg 2977gttaaagccc
cgatggtttc gaataagagt gtacactagg cactattatt ttcccttaaa 3037gtgtaagtca
gtttggatat aaaagaataa aatgaaataa gacattaaaa ccccctttct 3097ttaaactact
agtaaaaatg tgtatgcaca tggtgtgttt taacttgttt acttactagg 3157taattagtga
aggggtaaat aataggggag aaagtaagtt aatttaagat tttcgcatgt 3217aattaacaat
tttattatat tttagatgtg tttctatgcc taaaacctga aaatcaaaaa 3277tgattttttt
taaatagagt agtgatgcca ccaatgacaa ggaacagatc atataataac 3337actccacagc
cacatgctat tgaatattat gctcaaagag ccacatgcta ttgaatatta 3397tactcgatat
acactattaa catatcggag tatacctaat gtttgaagcc atcatgttat 3457gttattcatg
cttcaaatgt tcagacttaa ttatttatta atagagtttt ttagaaaaag 3517tgtaatgtat
ataatattgt gtacttgcta agtagtgtat atatattgtg tagtacataa 3577ttgaacaatt
ttttaatatt gaagtcaacg ataggagtga tgaatatggt gggagcatag 3637a
36385293PRTSolanum
lycopersicum[CDS]join(1135..1186,1378..1561,2615..2657) from SEQ ID
NO 51 52Met Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val Gly Val Pro Pro Pro 1
5 10 15 Gln Gly Tyr
Pro Pro Glu Gly Tyr Ser Lys Asp Ala Tyr Pro Pro Pro 20
25 30 Gly Tyr Pro Gln Gln Gly Tyr Pro
Pro Gln Gly Tyr Pro Gln Gln Gly 35 40
45 Tyr Pro Pro Pro Gln Tyr Ala Pro Gln Tyr Gly Ala Pro
Pro Pro Gln 50 55 60
Gln His Gln Gln Gln Ser Ser Ser Thr Gly Leu Met Gln Gly Cys Leu 65
70 75 80 Ala Ala Leu Cys
Cys Cys Cys Leu Leu Asp Ala Cys Phe 85
90 5322DNASolanum lycopersicumsource1..22mol type=unassigned
DNA note=Pr SlProm1AF3 organism=Solanum lycopersicum
53aaaggcgttg tccactaaga tt
225423DNASolanum lycopersicumsource1..23mol type=unassigned DNA
note=Pr SlProm1AR3 organism=Solanum lycopersicum 54catactgagg
tgcatactgt gga
235520DNASolanum lycopersicumsource1..20mol type=unassigned DNA
note=Pr SlTerm1AF3 organism=Solanum lycopersicum 55tcttggatgc
atgcttttga
205620DNASolanum lycopersicumsource1..20mol type=unassigned DNA
note=Pr SlTerm1AR3 organism=Solanum lycopersicum 56aagagcaagg
tcgcattcac
205720DNASolanum lycopersicumsource1..20mol type=unassigned DNA
note=Pr SlMid1AF1 organism=Solanum lycopersicum 57aggatatccg
ccagaaggtt
205831DNASolanum lycopersicumsource1..31mol type=unassigned DNA
note=Pr SlMid1ABR1 organism=Solanum lycopersicum 58gcatgcatca
cagcaacagc acagagcagc c
31592352DNASolanum lycopersicumsource1..2352mol type=unassigned DNA
note=Slxcv-1A organism=Solanum lycopersicum 59aaaggcgttg tccactaaga
tttgaagtcc ttagacaaag caaaagaaat agcattatga 60aagaggatga gatataaagt
gaatttcttt ttctctattt cataaaaaaa acttagatat 120acaaaagaag aaaatgttta
tatttttaaa atctgtttgt ccataaatta tgccttttta 180agtgtgtagt tatacagatc
cacccactat agcatcccaa caaaactcat gacttcatca 240atcactatta aaaaatttta
ataaattatg gagatttatt tatatggtca taaaaaaaaa 300catttcaata aagacaggtt
aaacattaat tcttctagat aaatcgttga tcaacttttc 360tttggaagaa ctaataatac
agtgtaggtc atatctaaat taaaaaatca tcaaaagatg 420taaacagttt gtcaataact
aatgaaaata acaaattaaa ataacatgaa aagttaaata 480gctgaaaaaa attatgaaat
attcaatttc ttttttacaa acataacact gaaaaaaaaa 540ttcaatttct tttcgatatt
tatataacag ctcgattaaa attgaataaa atttgagaaa 600aagattcggc agtaaaatca
aattaggatt tcttggaatg cgcttttttg ctggtcctcg 660ttgtgtccac tttgatttgc
cttataaata tcccttctcc actgtgcagc ccattatctt 720tctttgtgtc taagcaaaag
caccaacaga aactctctct acagttagat ccaacccaaa 780ttcttcatta attcaaca atg
agt tac tac aat caa caa caa ccc cct gtt 831 Met
Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val 1
5 10 ggt gtg cca cca cca caa g gtaaatcact
tcaattcctt tttttctgat 880Gly Val Pro Pro Pro Gln
15
tttattttgg taaagttgat gtttttatat gtttttttga tgactagatc tgatggggtt
940ttgaattttt gtag ga tat ccg cca gaa ggt tac cca aaa gat gca tac
989 Gly Tyr Pro Pro Glu Gly Tyr Pro Lys Asp Ala Tyr
20 25 cca cca cca ggg
tac cca cag cag ggt tac cct caa caa ggt tac cca 1037Pro Pro Pro Gly
Tyr Pro Gln Gln Gly Tyr Pro Gln Gln Gly Tyr Pro 30 35
40 45 cct caa ggg tac cct cca cag tat gca
cct cag tat ggt gct cca cct 1085Pro Gln Gly Tyr Pro Pro Gln Tyr Ala
Pro Gln Tyr Gly Ala Pro Pro 50 55
60 cct cat caa caa caa cag caa tct ggt act ggt ttc atg gaa
gga tg 1132Pro His Gln Gln Gln Gln Gln Ser Gly Thr Gly Phe Met Glu
Gly Cys 65 70 75
gtatgaacct taaagactca atttttatgt tcatatgtta tgattcagtt acagtaattt
1192gtatgatttt gtttgagaga gatctgtatt gatattggtt ttttgtagag cggttgagga
1252gattgtttat ctgtcttgtc tgttactatg tgtgtgtttc ttgttgattt ggtgtgtctt
1312gtcaaaacat gatttctcga attattattg gtctgtttga tgtttacttt gtgagcactt
1372gtatttaata actatggttc tggacgagtt ctggacgcga actagaaggg gtccttatca
1432tttgtatgtc tagttgaaca ttaacgcgag caggagagac aattagaatg cctgaatgat
1492tctctatgtg caaacatagt ccaggccttg ccacaccttt gattttatct ctgactgctg
1552aaatcgcgcc cctatcatac ttcagactgg aagggggtgc atggactaga cataacttgt
1612gttgctttgt ctatagctat aggttttcca ctgttgctgc tgcaataaga caataatgac
1672ctcttatcaa agaaatctac actgtcacgt taattgtttc ttcgttgtaa tagactatgg
1732gagtatcctt gttgatgctc cattccactg taagataagt agttaactga tctttcaaaa
1792caatcacata attgtcatat taatcacacg tttaagttat cgcagtgcca gtgaaccaat
1852tagacaatgg acgtggtctt ggctacccta aattttcata gaatatagtg gtggtctttc
1912acatttcaac agctttgtga ttttctgttt taggcgttcc ttctttcttg tagcctctcc
1972tcattttatt tgagcttgct gtatttgtta tttccggtgg gggactaatc attggttgtt
2032atatttttgt tttgcag t ttg gct gct ctg tgc tgt tgc tgt gat gca tgc
2083 Leu Ala Ala Leu Cys Cys Cys Cys Asp Ala Cys
80 85 ttt
tgatgctgta aatgatctgt gccatgtgtt ggtggcaaaa gtttattaaa 2136Phe
ccaattctat
catagtctag actttctctt tttgtgtttg tctttggtgt cctgtactgt 2196ccttgataaa
taatttgata ttaaatatga cgatgcacct tgttatggtg ggagaattca 2256agtgtctatt
tgccaattta tacattattg tgcaatgcaa gagattctat ttttttaaaa 2316tcttggttgt
ctatatgtga atgcgacctt gctctt
23526089PRTSolanum lycopersicum[CDS]join(799..850,955..1132,2050..2086)
from SEQ ID NO 59 60Met Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val Gly
Val Pro Pro Pro 1 5 10
15 Gln Gly Tyr Pro Pro Glu Gly Tyr Pro Lys Asp Ala Tyr Pro Pro Pro
20 25 30 Gly Tyr Pro
Gln Gln Gly Tyr Pro Gln Gln Gly Tyr Pro Pro Gln Gly 35
40 45 Tyr Pro Pro Gln Tyr Ala Pro Gln
Tyr Gly Ala Pro Pro Pro His Gln 50 55
60 Gln Gln Gln Gln Ser Gly Thr Gly Phe Met Glu Gly Cys
Leu Ala Ala 65 70 75
80 Leu Cys Cys Cys Cys Asp Ala Cys Phe 85
6120DNASolanum lycopersicumsource1..20mol type=unassigned DNA
note=Pr SlProm1BF3 organism=Solanum lycopersicum 61ctccacccct
ctccataact
206222DNASolanum lycopersicumsource1..22mol type=unassigned DNA
note=Pr-SlProm1BR3 organism=Solanum lycopersicum 62ccatactgag
gtgcatactg tg
226319DNASolanum lycopersicumsource1..19mol type=unassigned DNA
note=Pr SlTerm1BF3 organism=Solanum lycopersicum 63ctgccgcctt
agttgttgt
196423DNASolanum lycopersicumsource1..23mol type=unassigned DNA
note=Pr SlTerm1BR3 organism=Solanum lycopersicum 64tcccctatta
tttacccctt cac
236520DNASolanum lycopersicumsource1..20mol type=unassigned DNA
note=Pr SlMid1BF1 organism=Solanum lycopersicum 65aggttatcca
ccagaaggtt
20663128DNASolanum lycopersicumsource1..3128mol type=unassigned DNA
note=Slxcv-1B organism=Solanum lycopersicum 66ctccacccct ctccataact
caacagtgat atcgggtgga gtcagaattt ttattaataa 60acttataata taattaaata
acaagcgaat gaataagctt acaaaaatta agcacctact 120atatgttata tatttaacaa
tttggttaaa acttttactc ttatcctaat aagtggtttc 180ataaatcgtc aataaactct
aaaacatact atataacaca aaatcctaaa aataacatat 240aatggaggaa actctttttc
tcttcctttt cgatcgtgaa tcaaataatt taaaatttaa 300ttaaatctat aaaattttac
ttcctttctc aataatacta taaatagata gttcattttt 360atttatttta aataattaaa
aataatttaa ttactcatct ataatatatt tttaactata 420gaattctcta aatattaaat
ttattgctcc ttttatactt tataaataga ttttcacttt 480cattatcatt tttatcatat
taagaaaaac ttttattttt atttttattt catcctcaat 540attaattaat tattcttcaa
atcattaatt ataatttaag tctatgcact atttaatata 600gaaaattatt atttttattt
aggagtactt atttaggtga acatagccag tcaatatatg 660ataaataaaa taaaataaac
ataagagtaa tatatatata tagaaaaata atattctgac 720actttacgcg catcacacac
ggggtacttt tctagtaccc ttggaaaaga tcacataata 780aattaataaa ttactaaggc
cttgttggat ttaacaaatt atctctgaaa taaattttaa 840aattaattat ttcacaatta
ctggatataa gataaaaaaa atagaataat taatttctaa 900ctatataaat aaattttgat
tttaattatt tcttatccgt ctgtagcaac agagtcggag 960agtttcttgg aatatgctgg
gcttagtcct tgtttgactt gctctataaa acctaacaaa 1020taaaccccat ttctttgtgc
ataagcaaaa actcaaaact ctcttcaaca agaaatttca 1080att atg agt tac tac aat
cag cag caa ccc cct gtt ggt gta cca cca 1128Met Ser Tyr Tyr Asn Gln
Gln Gln Pro Pro Val Gly Val Pro Pro 1 5
10 15 cca caa g gtaaattgct tcaatttcat tttttttttg
atgatttatg gtaaagttca 1185Pro Gln
tatttttata tattgttttg atgatttatg gtgaagttga
tgtttttata tgttgttttg 1245atgatttgtg gtaaagttga tgtttttata tgttattttg
atgattagat ctgatggggt 1305tttggggaat tttgtaatta g gt tat cca cca gaa
ggt tac tct aaa gat 1355 Gly Tyr Pro Pro Glu
Gly Tyr Ser Lys Asp 20
25 gca tac cca cca cca ggg tat cct cag caa ggg tat cca cca cag
ggt 1403Ala Tyr Pro Pro Pro Gly Tyr Pro Gln Gln Gly Tyr Pro Pro Gln
Gly 30 35 40 tat
cct caa caa ggg tat cca cct cca cag tat gca cct cag tat ggt 1451Tyr
Pro Gln Gln Gly Tyr Pro Pro Pro Gln Tyr Ala Pro Gln Tyr Gly 45
50 55 gct cca cct cct caa caa
cat caa cag caa tct agt agc act gga tta 1499Ala Pro Pro Pro Gln Gln
His Gln Gln Gln Ser Ser Ser Thr Gly Leu 60 65
70 75 atg caa gga tg gtatgcatct ttacactcgt
gatgttttgt tatgatttag 1550Met Gln Gly Cys
ttgtgttgtt gaattgtatg atctgagatt ttttttggat
tgagttgact atggatgata 1610aggtgtgtaa tgatttgatt ttgtcaactg aattgggtga
ggttagtatg atctcgggtt 1670ttgttgacta agttgccgag agacttgttc atatttgtgt
gtctttcctt gtttgtctcg 1730aaaagatctt ttgatcatca aagggggaaa gtagaagcaa
ggggggctag acctctagac 1790cttaattgct gcatagattc atcatgttgc tattagttga
ttttctttga atattttagc 1850aacctgtgga ttcgatataa gcaatctagg tttttttttg
gatgaatttc ttgaccagtt 1910ttagtcgaga gaagatcaaa ggaatttgga gaagaaatgt
ccttttgggg cagaagctcg 1970agcaatggaa tatccctcgt gtgttacaca ttaacacgag
ttggagatcc aaccgattct 2030aatgcgtgaa tattttttat gtgccaccat agtccggtcc
tttccattac tgttatcaac 2090gcccttcatt ttgtctcttg ttttcattgt atctactgat
tcatcaccag tagcatgcaa 2150tggaattttc aacttgctgt acaatatcat gtttctcatt
aaaattctgc cctgaataca 2210ctgctcaaat tgccggttta gacttaactt gctttgtcta
tagctattgt tttccaactg 2270ctgctcctat agataaaata atgacctctt agccaaatca
gtctagagct aagtttggca 2330aagtaatcca cactgccgcc ttagttgttg tttctttgtt
tctacctcca atatcctatg 2390ttgatgctcc ttcccattga tcaaaccttc aagttgtcgc
agtgccaatg aactagtcag 2450acaatgattc ttgattccgt gatgtttcag tagccctttt
ttcttttagc actttgtcat 2510attatttcga atttccagtg ggactaataa tggtttgtgg
tattgttttg cag t ttg 2567
Leu
80 gct gct ctg tgc tgt tgc tgt gat gca tgc ttt tgagggtgta
aatgatctgt 2620Ala Ala Leu Cys Cys Cys Cys Asp Ala Cys Phe
85 90
gccatgtgtt gatggcaaaa gtttattgaa tcaattatat catagtctag acttttttct
2680ttctgtgttt tgtcctatac ttactttgat aaataatttg atctttgatg tgctcaagat
2740tccaaagtgt ttgttttgcc aaattatagg tggttggtta tgtacaatgt gacagattct
2800atttttattt ttttcaatgt tttggacctc aaaatattat atgtgaatca atgcaccttg
2860ctctggttac aaatttatgc tcctgttgta ttaaatgtgt tagcgaacac aacgtttggg
2920ttaaagcccc gatggtttcg aataagagtg tacactaggc actattattt tcccttaaag
2980tgtaagtcag tttggatata aaagaataaa atgaaataag acattaaaac cccctttctt
3040taaactacta gtaaaaatgt gtatgcacat ggtgtgtttt aacttgttta cttactaggt
3100aattagtgaa ggggtaaata atagggga
31286791PRTSolanum
lycopersicum[CDS]join(1084..1135,1327..1510,2564..2600) from SEQ ID
NO 66 67Met Ser Tyr Tyr Asn Gln Gln Gln Pro Pro Val Gly Val Pro Pro Pro 1
5 10 15 Gln Gly Tyr
Pro Pro Glu Gly Tyr Ser Lys Asp Ala Tyr Pro Pro Pro 20
25 30 Gly Tyr Pro Gln Gln Gly Tyr Pro
Pro Gln Gly Tyr Pro Gln Gln Gly 35 40
45 Tyr Pro Pro Pro Gln Tyr Ala Pro Gln Tyr Gly Ala Pro
Pro Pro Gln 50 55 60
Gln His Gln Gln Gln Ser Ser Ser Thr Gly Leu Met Gln Gly Cys Leu 65
70 75 80 Ala Ala Leu Cys
Cys Cys Cys Asp Ala Cys Phe 85 90
68177DNASolanum lycopersicumsource1..177mol type=unassigned DNA
note=pre-sly-MIR159miRNSpre-miDNA organism=Solanum lycopersicum
68tggagctcct tgaagtccaa caaaaaatct aacaggttaa attgagctgc tgacctatgg
60attcctcagc cctatctatt tatgatttca aacatataga taggttttgg gtttgcatat
120gtcaggagct ttattttacc ctttgtttga tcattttttg gattgaaggg agctcta
17769177RNASolanum lycopersicumsource1..177mol type=unassigned RNA
note=preSlpre-slyMI159RNA organism=Solanum lycopersicum 69uggagcuccu
ugaaguccaa caaaaaaucu aacagguuaa auugagcugc ugaccuaugg 60auuccucagc
ccuaucuauu uaugauuuca aacauauaga uagguuuugg guuugcauau 120gucaggagcu
uuauuuuacc cuuuguuuga ucauuuuuug gauugaaggg agcucua
1777046DNASolanum lycopersicumsource1..46mol type=unassigned DNA
note=Pr1 SlXe1 pre-amiRNS organism=Solanum lycopersicum 70gtgttgatgt
cgattggatg caaaaaatct aacaggttaa attgag
467145DNASolanum lycopersicumsource1..45mol type=unassigned DNA
note=Pr2 SlXe1 pre-amiRNS organism=Solanum lycopersicum 71gtgttgatgt
ctcttggatg aaaatgatca aacaaagggt aaaat
4572178DNASolanum lycopersicumsource1..178mol type=unassigned DNA
note=pre-SlXe1-amiDNA organism=Solanum lycopersicum 72gtgttgatgt
cctttggatg ccaaaaaatc taacaggtta aattgagctg ctgacctatg 60gattcctcag
ccctatctat ttatgatttc aaacatatag ataggttttg ggtttgcata 120tgtcaggagc
tttattttac cctttgtttg atcattttca tccaagagac atcaacac
17873178RNASolanum lycopersicumsource1..178mol type=unassigned RNA
note=pre-SlXe1-amiRNA organism=Solanum lycopersicum 73guguugaugu
ccuuuggaug ccaaaaaauc uaacagguua aauugagcug cugaccuaug 60gauuccucag
cccuaucuau uuaugauuuc aaacauauag auagguuuug gguuugcaua 120ugucaggagc
uuuauuuuac ccuuuguuug aucauuuuca uccaagagac aucaacac
1787421RNASolanum lycopersicumsource1..21mol type=unassigned RNA
note=SlXe1-amiRNA organism=Solanum lycopersicum 74ucauccaaga
gacaucaaca c
217517DNASolanum lycopersicumsource1..17mol type=unassigned DNA
note=SlXcv-1AB TAL-L organism=Solanum lycopersicum 75tggctgctct
gtgctgt
177617DNASolanum lycopersicumsource1..17mol type=unassigned DNA
note=SlXcv-1A TAL-R organism=Solanum lycopersicum 76ttacagcatc
aaaagca
177717DNASolanum lycopersicumsource1..17mol type=unassigned DNA
note=SlXcv-1B TAL-R organism=Solanum lycopersicum 77ttacaccctc
aaaagca
17783859DNASolanum lycopersicumsource1..3859mol type=unassigned DNA
note=SlXcv-1AB TALEN-L organism=Solanum lycopersicum 78ggatcccatt
cgtccgcgca ggccaagtcc tgcccgcgag cttctgcccg gaccccaacc 60ggatagggtt
cagccgactg cagatcgtgg ggtgtctgcg cctgctggca gccctctgga 120tggcttgccc
gctcggcgga cggtgtcccg gacccggctg ccatctcccc ctgcgccctc 180acctgcgttc
tcggcgggca gcttcagcga tctgctccgt ccgttcgatc cgtcgcttct 240tgatacatcg
cttcttgatt cgatgcctgc cgtcggcacg ccgcatacag cggctgcccc 300agcagagtgg
gatgaggcgc aatcggctct gcgtgcagcc gatgacccgc cacccaccgt 360gcgtgtcgct
gtcactgccg cgcggccgcc gcgcgccaag ccggccccgc gacggcgtgc 420tgcgcaaccc
tccgacgctt cgccggccgc gcaggtggat ctacgcacgc tcggctacag 480tcagcagcag
caagagaaga tcaaaccgaa ggtgcgttcg acagtggcgc agcaccacga 540ggcactggtg
ggccatgggt ttacacacgc gcacatcgtt gcgctcagcc aacacccggc 600agcgttaggg
accgtcgctg tcacgtatca gcacataatc acggcgttgc cagaggcgac 660acacgaagac
atcgttggcg tcggcaaaca gtggtccggc gcacgcgccc tggaggcctt 720gctcacggat
gcgggggagt tgagaggtcc gccgttacag ttggacacag gccaacttgt 780gaagattgca
aaacgtggcg gcgtgaccgc aatggaggca gtgcatgcat cgcgcaatgc 840actgacgggt
gcccccctga acctgacccc ggaccaagtg gtggctatcg ccagcaacgg 900tggcggcaag
caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca 960tggcctgacc
ccggaccaag tggtggctat cgccagcaac catggcggca agcaagcgct 1020cgaaacggtg
cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca 1080agtggtggct
atcgccagca accatggcgg caagcaagcg ctcgaaacgg tgcagcggct 1140gttgccggtg
ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag 1200ccacgatggc
ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca 1260ggaccatggc
ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca 1320agcgctcgaa
acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc 1380ggaccaagtg
gtggctatcg ccagcaacca tggcggcaag caagcgctcg aaacggtgca 1440gcggctgttg
ccggtgctgt gccaggacca tggcctgact ccggaccaag tggtggctat 1500cgccagccac
gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct 1560gtgccaggac
catggcctga ccccggacca agtggtggct atcgccagca acggtggcgg 1620caagcaagcg
ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct 1680gactccggac
caagtggtgg ctatcgccag ccacgatggc ggcaagcaag cgctcgaaac 1740ggtgcagcgg
ctgttgccgg tgctgtgcca ggaccatggc ctgaccccgg accaagtggt 1800ggctatcgcc
agcaacggtg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc 1860ggtgctgtgc
caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacca 1920tggcggcaag
caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca 1980tggcctgacc
ccggaccaag tggtggctat cgccagcaac ggtggcggca agcaagcgct 2040cgaaacggtg
cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca 2100agtggtggct
atcgccagca accatggcgg caagcaagcg ctcgaaacgg tgcagcggct 2160gttgccggtg
ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag 2220ccacgatggc
ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca 2280ggaccatggc
ctgaccccgg accaagtggt ggctatcgcc agcaacggtg gcggcaagca 2340agcgctcgaa
acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc 2400ggaccaagtg
gtggctatcg ccagcaacca tggcggcaag caagcgctcg aaacggtgca 2460gcggctgttg
ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat 2520cgccagcaac
ggtggcggca agcaagcgct cgaaagcatt gtggcccagc tgagccggcc 2580tgatccggcg
ttggccgcgt tgaccaacga ccacctcgtc gccttggcct gcctcggcgg 2640acgtcctgcc
atggatgcag tgaaaaaggg attgccgcac gcgccggaat tgatcagaag 2700agtcaatcgc
cgtattggcg aacgcacgtc ccatcgcgtt gccgactacg cgcaagtggt 2760tcgcgtgctg
gagtttttcc agtgccactc ccacccagcg tacgcatttg atgaggccat 2820gacgcagttc
gggatgagca ggaacgggtt ggtacagctc tttcgcagag tgggcgtcac 2880cgaactcgaa
gcccgcggtg gaacgctccc cccagcctcg cagcgttggg accgtatcct 2940ccaggcatca
gggatgaaaa gggccaaacc gtcccctact tcagctcaaa caccggatca 3000ggcgtctttg
catgcattcg ccgattcgct ggagcgtgac cttgatgcgc ccagcccaat 3060gcacgaggga
gatcagacgc gggcaagcag ccgtaaacgg tcccgatcgg atcgtgctgt 3120caccggcccc
tccgcacagc aggctgtcga ggtgcgcgtt cccgaacagc gcgatgcgct 3180gcatttgccc
ctcagctgga gggtaaaacg cccgcgtacc aggatctggg gcggcctccc 3240ggatccgata
tctagatccc agctagtgaa atctgaattg gaagagaaga aatctgaact 3300tagacataaa
ttgaaatatg tgccacatga atatattgaa ttgattgaaa tcgcaagaaa 3360ttcaactcag
gatagaatcc ttgaaatgaa ggtgatggag ttctttatga aggtttatgg 3420ttatcgtggt
aaacatttgg gtggatcaag gaaaccagac ggagcaattt atactgtcgg 3480atctcctatt
gattacggtg tgatcgttga tactaaggca tattcaggag gttataatct 3540tccaattggt
caagcagatg aaatgcaaag atatgtcgaa gagaatcaaa caagaaacaa 3600gcatatcaac
cctaatgaat ggtggaaagt ctatccatct tcagtaacag aatttaagtt 3660cttgtttgtg
agtggtcatt tcaaaggaaa ctacaaagct cagcttacaa gattgaatca 3720tatcactaat
tgtaatggag ctgttcttag tgtagaagag cttttgattg gtggagaaat 3780gattaaagct
ggtacattga cacttgagga agtgagaagg aaatttaata acggtgagat 3840aaacttttaa
taggagctc
3859793859DNASolanum lycopersicumsource1..3859mol type=unassigned DNA
note=SlXcv-1A TALEN-R organism=Solanum lycopersicum 79ggatcccatt
cgtccgcgca ggccaagtcc tgcccgcgag cttctgcccg gaccccaacc 60ggatagggtt
cagccgactg cagatcgtgg ggtgtctgcg cctgctggca gccctctgga 120tggcttgccc
gctcggcgga cggtgtcccg gacccggctg ccatctcccc ctgcgccctc 180acctgcgttc
tcggcgggca gcttcagcga tctgctccgt ccgttcgatc cgtcgcttct 240tgatacatcg
cttcttgatt cgatgcctgc cgtcggcacg ccgcatacag cggctgcccc 300agcagagtgg
gatgaggcgc aatcggctct gcgtgcagcc gatgacccgc cacccaccgt 360gcgtgtcgct
gtcactgccg cgcggccgcc gcgcgccaag ccggccccgc gacggcgtgc 420tgcgcaaccc
tccgacgctt cgccggccgc gcaggtggat ctacgcacgc tcggctacag 480tcagcagcag
caagagaaga tcaaaccgaa ggtgcgttcg acagtggcgc agcaccacga 540ggcactggtg
ggccatgggt ttacacacgc gcacatcgtt gcgctcagcc aacacccggc 600agcgttaggg
accgtcgctg tcacgtatca gcacataatc acggcgttgc cagaggcgac 660acacgaagac
atcgttggcg tcggcaaaca gtggtccggc gcacgcgccc tggaggcctt 720gctcacggat
gcgggggagt tgagaggtcc gccgttacag ttggacacag gccaacttgt 780gaagattgca
aaacgtggcg gcgtgaccgc aatggaggca gtgcatgcat cgcgcaatgc 840actgacgggt
gcccccctga acctgacccc ggaccaagtg gtggctatcg ccagcaacgg 900tggcggcaag
caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca 960tggcctgacc
ccggaccaag tggtggctat cgccagcaac ggtggcggca agcaagcgct 1020cgaaacggtg
cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca 1080agtggtggct
atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct 1140gttgccggtg
ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag 1200ccacgatggc
ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca 1260ggaccatggc
ctgaccccgg accaagtggt ggctatcgcc agcaacattg gcggcaagca 1320agcgctcgaa
acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgacccc 1380ggaccaagtg
gtggctatcg ccagcaacca tggcggcaag caagcgctcg aaacggtgca 1440gcggctgttg
ccggtgctgt gccaggacca tggcctgact ccggaccaag tggtggctat 1500cgccagccac
gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct 1560gtgccaggac
catggcctga ccccggacca agtggtggct atcgccagca acattggcgg 1620caagcaagcg
ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct 1680gaccccggac
caagtggtgg ctatcgccag caacggtggc ggcaagcaag cgctcgaaac 1740ggtgcagcgg
ctgttgccgg tgctgtgcca ggaccatggc ctgactccgg accaagtggt 1800ggctatcgcc
agccacgatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc 1860ggtgctgtgc
caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacat 1920tggcggcaag
caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca 1980tggcctgacc
ccggaccaag tggtggctat cgccagcaac attggcggca agcaagcgct 2040cgaaacggtg
cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca 2100agtggtggct
atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct 2160gttgccggtg
ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag 2220caacattggc
ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca 2280ggaccatggc
ctgaccccgg accaagtggt ggctatcgcc agcaaccatg gcggcaagca 2340agcgctcgaa
acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc 2400ggaccaagtg
gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca 2460gcggctgttg
ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat 2520cgccagcaac
attggcggca agcaagcgct cgaaagcatt gtggcccagc tgagccggcc 2580tgatccggcg
ttggccgcgt tgaccaacga ccacctcgtc gccttggcct gcctcggcgg 2640acgtcctgcc
atggatgcag tgaaaaaggg attgccgcac gcgccggaat tgatcagaag 2700agtcaatcgc
cgtattggcg aacgcacgtc ccatcgcgtt gccgactacg cgcaagtggt 2760tcgcgtgctg
gagtttttcc agtgccactc ccacccagcg tacgcatttg atgaggccat 2820gacgcagttc
gggatgagca ggaacgggtt ggtacagctc tttcgcagag tgggcgtcac 2880cgaactcgaa
gcccgcggtg gaacgctccc cccagcctcg cagcgttggg accgtatcct 2940ccaggcatca
gggatgaaaa gggccaaacc gtcccctact tcagctcaaa caccggatca 3000ggcgtctttg
catgcattcg ccgattcgct ggagcgtgac cttgatgcgc ccagcccaat 3060gcacgaggga
gatcagacgc gggcaagcag ccgtaaacgg tcccgatcgg atcgtgctgt 3120caccggcccc
tccgcacagc aggctgtcga ggtgcgcgtt cccgaacagc gcgatgcgct 3180gcatttgccc
ctcagctgga gggtaaaacg cccgcgtacc aggatctggg gcggcctccc 3240ggatccgata
tctagatccc agctagtgaa atctgaattg gaagagaaga aatctgaact 3300tagacataaa
ttgaaatatg tgccacatga atatattgaa ttgattgaaa tcgcaagaaa 3360ttcaactcag
gatagaatcc ttgaaatgaa ggtgatggag ttctttatga aggtttatgg 3420ttatcgtggt
aaacatttgg gtggatcaag gaaaccagac ggagcaattt atactgtcgg 3480atctcctatt
gattacggtg tgatcgttga tactaaggca tattcaggag gttataatct 3540tccaattggt
caagcagatg aaatgcaaag atatgtcgaa gagaatcaaa caagaaacaa 3600gcatatcaac
cctaatgaat ggtggaaagt ctatccatct tcagtaacag aatttaagtt 3660cttgtttgtg
agtggtcatt tcaaaggaaa ctacaaagct cagcttacaa gattgaatca 3720tatcactaat
tgtaatggag ctgttcttag tgtagaagag cttttgattg gtggagaaat 3780gattaaagct
ggtacattga cacttgagga agtgagaagg aaatttaata acggtgagat 3840aaacttttaa
taggagctc
3859803859DNASolanum lycopersicumsource1..3859mol type=unassigned DNA
note=SlXcv-1B TALEN-R organism=Solanum lycopersicum 80ggatcccatt
cgtccgcgca ggccaagtcc tgcccgcgag cttctgcccg gaccccaacc 60ggatagggtt
cagccgactg cagatcgtgg ggtgtctgcg cctgctggca gccctctgga 120tggcttgccc
gctcggcgga cggtgtcccg gacccggctg ccatctcccc ctgcgccctc 180acctgcgttc
tcggcgggca gcttcagcga tctgctccgt ccgttcgatc cgtcgcttct 240tgatacatcg
cttcttgatt cgatgcctgc cgtcggcacg ccgcatacag cggctgcccc 300agcagagtgg
gatgaggcgc aatcggctct gcgtgcagcc gatgacccgc cacccaccgt 360gcgtgtcgct
gtcactgccg cgcggccgcc gcgcgccaag ccggccccgc gacggcgtgc 420tgcgcaaccc
tccgacgctt cgccggccgc gcaggtggat ctacgcacgc tcggctacag 480tcagcagcag
caagagaaga tcaaaccgaa ggtgcgttcg acagtggcgc agcaccacga 540ggcactggtg
ggccatgggt ttacacacgc gcacatcgtt gcgctcagcc aacacccggc 600agcgttaggg
accgtcgctg tcacgtatca gcacataatc acggcgttgc cagaggcgac 660acacgaagac
atcgttggcg tcggcaaaca gtggtccggc gcacgcgccc tggaggcctt 720gctcacggat
gcgggggagt tgagaggtcc gccgttacag ttggacacag gccaacttgt 780gaagattgca
aaacgtggcg gcgtgaccgc aatggaggca gtgcatgcat cgcgcaatgc 840actgacgggt
gcccccctga acctgacccc ggaccaagtg gtggctatcg ccagcaacgg 900tggcggcaag
caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca 960tggcctgacc
ccggaccaag tggtggctat cgccagcaac ggtggcggca agcaagcgct 1020cgaaacggtg
cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca 1080agtggtggct
atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct 1140gttgccggtg
ctgtgccagg accatggcct gactccggac caagtggtgg ctatcgccag 1200ccacgatggc
ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca 1260ggaccatggc
ctgaccccgg accaagtggt ggctatcgcc agcaacattg gcggcaagca 1320agcgctcgaa
acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc 1380ggaccaagtg
gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca 1440gcggctgttg
ccggtgctgt gccaggacca tggcctgact ccggaccaag tggtggctat 1500cgccagccac
gatggcggca agcaagcgct cgaaacggtg cagcggctgt tgccggtgct 1560gtgccaggac
catggcctga ctccggacca agtggtggct atcgccagcc acgatggcgg 1620caagcaagcg
ctcgaaacgg tgcagcggct gttgccggtg ctgtgccagg accatggcct 1680gaccccggac
caagtggtgg ctatcgccag caacggtggc ggcaagcaag cgctcgaaac 1740ggtgcagcgg
ctgttgccgg tgctgtgcca ggaccatggc ctgactccgg accaagtggt 1800ggctatcgcc
agccacgatg gcggcaagca agcgctcgaa acggtgcagc ggctgttgcc 1860ggtgctgtgc
caggaccatg gcctgacccc ggaccaagtg gtggctatcg ccagcaacat 1920tggcggcaag
caagcgctcg aaacggtgca gcggctgttg ccggtgctgt gccaggacca 1980tggcctgacc
ccggaccaag tggtggctat cgccagcaac attggcggca agcaagcgct 2040cgaaacggtg
cagcggctgt tgccggtgct gtgccaggac catggcctga ccccggacca 2100agtggtggct
atcgccagca acattggcgg caagcaagcg ctcgaaacgg tgcagcggct 2160gttgccggtg
ctgtgccagg accatggcct gaccccggac caagtggtgg ctatcgccag 2220caacattggc
ggcaagcaag cgctcgaaac ggtgcagcgg ctgttgccgg tgctgtgcca 2280ggaccatggc
ctgaccccgg accaagtggt ggctatcgcc agcaaccatg gcggcaagca 2340agcgctcgaa
acggtgcagc ggctgttgcc ggtgctgtgc caggaccatg gcctgactcc 2400ggaccaagtg
gtggctatcg ccagccacga tggcggcaag caagcgctcg aaacggtgca 2460gcggctgttg
ccggtgctgt gccaggacca tggcctgacc ccggaccaag tggtggctat 2520cgccagcaac
attggcggca agcaagcgct cgaaagcatt gtggcccagc tgagccggcc 2580tgatccggcg
ttggccgcgt tgaccaacga ccacctcgtc gccttggcct gcctcggcgg 2640acgtcctgcc
atggatgcag tgaaaaaggg attgccgcac gcgccggaat tgatcagaag 2700agtcaatcgc
cgtattggcg aacgcacgtc ccatcgcgtt gccgactacg cgcaagtggt 2760tcgcgtgctg
gagtttttcc agtgccactc ccacccagcg tacgcatttg atgaggccat 2820gacgcagttc
gggatgagca ggaacgggtt ggtacagctc tttcgcagag tgggcgtcac 2880cgaactcgaa
gcccgcggtg gaacgctccc cccagcctcg cagcgttggg accgtatcct 2940ccaggcatca
gggatgaaaa gggccaaacc gtcccctact tcagctcaaa caccggatca 3000ggcgtctttg
catgcattcg ccgattcgct ggagcgtgac cttgatgcgc ccagcccaat 3060gcacgaggga
gatcagacgc gggcaagcag ccgtaaacgg tcccgatcgg atcgtgctgt 3120caccggcccc
tccgcacagc aggctgtcga ggtgcgcgtt cccgaacagc gcgatgcgct 3180gcatttgccc
ctcagctgga gggtaaaacg cccgcgtacc aggatctggg gcggcctccc 3240ggatccgata
tctagatccc agctagtgaa atctgaattg gaagagaaga aatctgaact 3300tagacataaa
ttgaaatatg tgccacatga atatattgaa ttgattgaaa tcgcaagaaa 3360ttcaactcag
gatagaatcc ttgaaatgaa ggtgatggag ttctttatga aggtttatgg 3420ttatcgtggt
aaacatttgg gtggatcaag gaaaccagac ggagcaattt atactgtcgg 3480atctcctatt
gattacggtg tgatcgttga tactaaggca tattcaggag gttataatct 3540tccaattggt
caagcagatg aaatgcaaag atatgtcgaa gagaatcaaa caagaaacaa 3600gcatatcaac
cctaatgaat ggtggaaagt ctatccatct tcagtaacag aatttaagtt 3660cttgtttgtg
agtggtcatt tcaaaggaaa ctacaaagct cagcttacaa gattgaatca 3720tatcactaat
tgtaatggag ctgttcttag tgtagaagag cttttgattg gtggagaaat 3780gattaaagct
ggtacattga cacttgagga agtgagaagg aaatttaata acggtgagat 3840aaacttttaa
taggagctc
38598120DNASolanum lycopersicumsource1..20mol type=unassigned DNA
note=CRISPR SlXcv-1 organism=Solanum lycopersicum 81tctgtgctgt
tgctgtctct
208224DNASolanum lycopersicumsource1..24mol type=unassigned DNA
note=sgRNA SlXcv-1F organism=Solanum lycopersicum 82gatttctgtg
ctgttgctgt ctct
248324DNASolanum lycopersicumsource1..24mol type=unassigned DNA
note=sgRNA SlXcv-1R organism=Solanum lycopersicum 83aaacagacac
gacaacgaca gaga
248454DNASolanum lycopersicumsource1..54mol type=unassigned DNA
note=SEQ ID NO 49 szekvencia reszlet 2253-2306 bp organism=Solanum
lycopersicum 84ttggctgctc tgtgctgttg ctgtctcttg gatgcatgct tttgatgctg
taaa 548554DNASolanum lycopersicumsource1..54mol
type=unassigned DNA note=SEQ ID NO51 szekvencia reszlet 2616 - 2669
bp organism=Solanum lycopersicum 85ttggctgctc tgtgctgttg ctgtctcttg
gatgcatgct tttgagggtg taaa 548612DNASolanum
lycopersicumsource1..12mol type=unassigned DNA note=Bal oldali ZF
feherje celszekvenciaja az SLXcv-1AB genben organism=Solanum
lycopersicum 86tgtgctgttg ct
128712DNASolanum lycopersicumsource1..12mol type=unassigned
DNA note=Jobb oldali ZF feherje celszekvenciaja az SlXcv-1AB genben
organism=Solanum lycopersicum 87tttcgtacgt ag
1288575DNASolanum
lycopersicumsource1..575mol type=unassigned DNA note=Slxcv-1A cDNS
organism=Solanum lycopersicum 88tctttctttg tgtctaagca aaagcaccaa
cagaaactct ctctacagtt agatccaacc 60caaattcttc attaattcaa caatgagtta
ctacaatcaa caacaacccc ctgttggtgt 120gccaccacca caaggatatc cgccagaagg
ttacccaaaa gatgcatacc caccaccagg 180gtacccacag cagggttacc ctcaacaagg
ttacccacct caagggtacc ctccacagta 240tgcacctcag tatggtgctc cacctcctca
tcaacaacaa cagcaatctg gtactggttt 300catggaagga tgtttggctg ctctgtgctg
ttgctgtgat gcatgctttt gatgctgtaa 360atgatctgtg ccatgtgttg gtggcaaaag
tttattaaac caattctatc atagtctaga 420ctttctcttt ttgtgtttgt ctttggtgtc
ctgtactgtc cttgataaat aatttgatat 480taaatatgac gatgcacctt gttatggtgg
gagaattcaa gtgtctattt gccaatttat 540acattattgt gcaatgcaag agattctatt
ttttt 57589640DNASolanum
lycopersicumsource1..640mol type=unassigned DNA note=Slxcv-1B
organism=Solanum lycopersicum 89cttagtcctt gtttgacttg ctctataaaa
cctaacaaat aaaccccatt tctttgtgca 60taagcaaaaa ctcaaaactc tcttcaacaa
gaaatttcaa ttatgagtta ctacaatcag 120cagcaacccc ctgttggtgt accaccacca
caaggttatc caccagaagg ttactctaaa 180gatgcatacc caccaccagg gtatcctcag
caagggtatc caccacaggg ttatcctcaa 240caagggtatc cacctccaca gtatgcacct
cagtatggtg ctccacctcc tcaacaacat 300caacagcaat ctagtagcac tggattaatg
caaggatgtt tggctgctct gtgctgttgc 360tgtgatgcat gcttttgagg gtgtaaatga
tctgtgccat gtgttgatgg caaaagttta 420ttgaatcaat tatatcatag tctagacttt
tttctttctg tgttttgtcc tatacttact 480ttgataaata atttgatctt tgatgtgctc
aagattccaa agtgtttgtt ttgccaaatt 540ataggtggtt ggttatgtac aatgtgacag
attctatttt tatttttttc aatgttttgg 600acctcaaata ttatatgtga atcaatgcac
cttgctctgg 64090644DNACapsicum
annuumsource1..644mol type=unassigned DNA note=xcv-1 cDNA
organism=Capsicum annuum 90ctttactcta taaaaacttc acaaatatca cctcttcact
gtaccccatt atctttcttt 60gtggttaagc aaatacacaa aataaataaa tataactctc
ctcttagatt aaactagtag 120atccatcaac aatgagttac tacaatcaac aacaacctcc
tgttggtgta cctccaccac 180aagggtatcc accagaaggt tacccaaaag attcataccc
accacctgga tatccacagc 240aagggtaccc tcaacaaggg tatccaccac aagggtaccc
tccacagtat gcacctcagt 300atggtgcacc acctcctcaa caacaacatc aatcatctag
tagtactgga ttattgcaag 360gatgtttggc tgctctttgc tgttgctgtg atgcatgctt
ttgatgctgt aaatgatctg 420tacgcaaagt gttgatgaca aaagatgatt gaaatccatt
atcatagtct agattatttt 480ccttgaacgt gttttgtcct tgttgtcctg tcatttataa
ataatttgat cttgctatgg 540tgtctatttg ccaaattata ggtttatgta caacgtgaga
gattgtattt tattttttat 600gttttggacc tcaatatgtg aatcaatgca ccttgatttg
gtta 64491652DNACapsicum annuumsource1..652mol
type=unassigned DNA note=Xcv-1 cDNS organism=Capsicum annuum
91ctttgacttt actctataaa aacttcacaa atatcacctc ttcactgtac cccattatct
60ttctttgtgg ttaagcaaat acacaaaata aataaatata actctcctct tagattaaac
120tagtagatcc atcaacaatg agttactaca atcaacaaca acctcctgtt ggtgtacctc
180caccacaagg gtatccacca gaaggttacc caaaagattc atacccacca cctggatatc
240cacagcaagg gtaccctcaa caagggtatc caccacaagg gtaccctcca cagtatgcac
300ctcagtatgg tgcaccacct cctcaacaac aacatcaatc atctagtagt actggattat
360tgcaaggatg tttggctgct ctttgctgtt gctgtctctt ggatgcatgc ttttgatgct
420gtaaatgatc tgtacgcaaa gtgttgatga caaaagatga ttgaaatcca ttatcatagt
480ctagattatt ttccttgaac gtgttttgtc cttgttgtcc tgtcatttat aaataatttg
540atcttgctat ggtgtctatt tgccaaatta taggtttatg tacaacgtga gagattgtat
600tttatttttt atgttttgga cctcaatatg tgaatcaatg caccttgatt tg
652921271DNASolanum lycopersicumsource1..1271mol type=unassigned DNA
note=HinIII 35Spromoter az SlXe1-amiRNS-t kodolo gen terminator
szekvencia EcoRI organism=Solanum lycopersicum 92aagcttgcca
acatggtgga gcacgacact ctcgtctact ccaagaatat caaagataca 60gtctcagaag
accaaagggc tattgagact tttcaacaaa gggtaatatc gggaaacctc 120ctcggattcc
attgcccagc tatctgtcac ttcatcaaaa ggacagtaga aaaggaaggt 180ggcacctaca
aatgccatca ttgcgataaa ggaaaggcta tcgttcaaga tgcctctgcc 240gacagtggtc
ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt 300ccaaccacgt
cttcaaagca agtggattga tgtgaacatg gtggagcacg acactctcgt 360ctactccaag
aatatcaaag atacagtctc agaagaccaa agggctattg agacttttca 420acaaagggta
atatcgggaa acctcctcgg attccattgc ccagctatct gtcacttcat 480caaaaggaca
gtagaaaagg aaggtggcac ctacaaatgc catcattgcg ataaaggaaa 540ggctatcgtt
caagatgcct ctgccgacag tggtcccaaa gatggacccc cacccacgag 600gagcatcgtg
gaaaaagaag acgttccaac cacgtcttca aagcaagtgg attgatgtga 660tatctccact
gacgtaaggg atgacgcaca atcccactat ccttcgcaag acccttcctc 720tatataagga
agttcatttc atttggagag gacacgctga aatcaccagt ctctctctac 780aaatctatct
cttctagaac tagtgattgt gttgatgtcc tttggatgcc aaaaaatcta 840acaggttaaa
ttgagctgct gacctatgga ttcctcagcc ctatctattt atgatttcaa 900acatatagat
aggttttggg tttgcatatg tcaggagctt tattttaccc tttgtttgat 960cattttcatc
caagagacat caacacaatc gaattcgata tcaagcttat cgataccgtc 1020gacctcgagg
gggggcccgg tacccgggga tcctctagag tcgacctgca ggcatgcaag 1080ctcgagtttc
tccataataa tgtgtgagta gttcccagat aagggaatta gggttcctat 1140agggtttcgc
tcatgtgttg agcatataag aaacccttag tatgtatttg tatttgtaaa 1200atacttctat
caataaaatt tctaattcct aaaaccaaaa tccagtacta aaatccagat 1260cccccgaatt c
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