COMPOSITIONS AND METHODS FOR IMPROVING PLASTID TRANSFORMATION EFFICIENCY IN HIGHER PLANTS

Abstract

Compositions and methods for improving plastid transformation in difficult to transform plants are disclosed.

Description

[0001] This application is a continuation of PCT/US2018/013034 filed Jan. 9, 2018 which claims priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional Patent Application No. 62/444,307, filed on Jan. 9, 2017. The foregoing application is incorporated by reference herein as though set forth in full.

FIELD OF THE INVENTION

[0002] The present invention relates the fields of plant biology and plastid transformation. More specifically, the invention pertains to molecular strategies for improving plastid transformation efficiency in recalcitrant plant species.

BACKGROUND OF THE INVENTION

[0003] Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.

[0004] Plastids are semi-autonomous plant organelles with thousands of copies of the .about.155-kb genome localized in 10 to 100 plastids per cell. The plastid genome of higher plants encodes about one hundred genes, the products of which assemble with .about.3,000 nucleus-encoded proteins to form the plastid transcription and translation machinery and carry out complex metabolic functions, including photosynthesis, and fatty acid and amino acid biosynthesis. Transformation of the plastid genome in flowering plants was first accomplished in tobacco (Nicotiana tabacum), the current model species of plastid engineering (Svab et al., 1990; Svab and Maliga, 1993).

[0005] Plastid transformation is routine only in tobacco, but reproducible protocols for plastid transformation have also been described in tomato (Ruf et al., 2001), potato (Valkov et al., 2011), lettuce (Kanamoto et al., 2006; Ruhlman et al., 2010) and soybean (Dufourmantel et al., 2004). Still, the technology is available in only a relatively small number of crops. Arabidopsis thaliana, the most widely used model plant is one of the species that is recalcitrant to plastid transformation. In Arabidopsis, only 2 transplastomic events were identified in 201 samples (Sikdar et al., 1998), a sample size that would have yielded .about.200 events in tobacco using the technology available in 1988 (Svab and Maliga, 1993). Until now the reasons for the low efficiency in Arabidopsis were not understood.

SUMMARY OF THE INVENTION

[0006] In accordance with the present invention, a method for increasing sensitivity to spectinomycin in plastids of higher plants for increasing plastid transformation efficiency is provided. An exemplary method comprises providing a plant having a nonfunctional ACC2 nuclear gene, introducing one or more plastid transformation vectors into plastids in cells from said plant, said one or more vectors comprising an aadA spectinomycin resistance marker sequence and a nucleic acid sequence encoding a protein of interest. The plant cells are then contacted with spectinomycin and spectinomycin resistant plant cells which accumulate the protein of interest in said plastids selected. The method also includes culturing said plant cells under conditions suitable to regenerate a transplastomic plant therefrom. In preferred embodiments, the plant is selected from the group consisting of Arabidopsis ssp., Brassica ssp., Camelina ssp., and Crambe spp. In a further aspect, the method entails excising the resistance marker from said plant. This can be achieved using the protocols provided in U.S. Pat. Nos. 8,841,511; 7,667,093 and 7,217,860.

[0007] Plants to be transformed can be naturally occurring ACC2 mutants which are defective in acc2 activity. Alternatively, desirable plant species can be identified and the ACC2 gene is inactivated in said plant using the CRISPR/Cas system and the appropriate guide strands.

[0008] In another embodiment, a method for seed-specific plastid expression is provided. An exemplary method comprises introducing a nuclear expression vector encoding a modified PPR10 binding protein driven by a seed-specific promoter and a plastid expression vector encoding a gene of interest linked to an upstream PPR10 binding site, wherein nuclear-expressed PPR10 is imported into plastids and binds said PPR10 binding site to drive expression of the gene of interest in seed plastids. In certain embodiments, the vector comprises a seed specific promoter selected from a napin or a phaseolin gene promoter. In other embodiments, the modified PPR10 binding protein is PPR10 and encoded by SEQ ID NO: 265. The PPR10 binding site may also be encoded by SEQ ID NO: 261. The vector may also comprise the aadA spectinomycin resistance gene. Additionally, in another aspect, the plastid expressed gene of interest is linked to an upstream sequence encoding a maize atpH gene and/or tRNA sequence in said plastid vector.

[0009] In another aspect of the invention, a method for increasing sensitivity to plastid translation inhibitors in plastids of higher plants for increasing plastid transformation efficiency is provided. An exemplary method comprises providing a plant comprising a nonfunctional ACC2 nuclear gene, introducing one or more plastid transformation vectors into the plastids in cells from said plant, said one or more vectors comprising a nucleic acid sequence conferring resistance to said plastid translation inhibitor, and a nucleic acid sequence encoding a protein of interest. The method further entails contacting said cells with said inhibitor and selecting plant cells which are resistant to said inhibitor and accumulate said protein of interest in said plastids; and culturing said plant cells under conditions suitable to regenerate a transplastomic plant therefrom. In certain embodiments, the plastid translation inhibitor is selected from kanamycin, chloramphenicol, tobramycin and gentamycin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIGS. 1A-1B. Defective ACC2 Gene Makes Chloroplasts More Sensitive to Spectinomycin. (FIG. 1A) In most accessions the heteromeric ACCase (hetACC) localizes in the chloroplast and is encoded by nuclear genes CAC1-A (At5g16390; Biotin Carboxyl Carrier Protein 1 (BCCP-1)), CAC1-B (At5g15530; Biotin Carboxyl Carrier Protein 2 (BCCP-2)) (not depicted in figure), CAC2 (At5g35360; Biotin Carboxylase (BC)), CAC3 (At2g38040, .alpha. subunit of Carboxyltransferase (.alpha.-CT) and the plastid encoded gene accD (AtCg00500; (.beta. subunit of Carboxyltransferase (.beta.-CT)). The homomeric ACC1 (At1g36160; homACCase) enzyme localizes in the cytoplasm and the ACC2 (At1g36180; homACCase) enzyme is imported into the chloroplast via the TIC/TOC membrane protein complex. If translation of the plastid accD mRNA is blocked by spectinomycin, the nuclear homomeric ACC2 gene supplies the cells with lipids so that cellular viability is not affected. (FIG. 1B) In ACC2 mutants, the absence of the homomeric ACCase makes the plants dependent on plastid translation to produce the heteromeric ACCase enzyme for fatty acid biosynthesis.

[0011] FIG. 2. Map of the Plastid Genome with the Integrated aadA-gfp Dicistronic Operon. The NruI-XbaI region is contained in the plastid transformation vector pATV1. P and T mark the positions of the PrrnL atpB promoter and the TpsbA terminator in the dicistronic vector. The black box at the aadA N terminus marks the atpB downstream box sequence (Kuroda and Maliga, 2001). The ribosome entry site is marked by black semi-ovals. The positions of the rrn16 and trnV plastid genes and relevant restriction enzyme sites are marked. Thick black and red lines indicate probes used for DNA and RNA gel-blot analyses, respectively.

[0012] FIGS. 3A-3F. Identification of Arabidopsis Transplastomic Clones. (FIG. 3A) Sterile Sav-0 plants grown in Petri dishes (diameter 10 cm) for six weeks. (FIG. 3B) Two days after bombardment (biolistic transformation) the leaves are incised and transferred to selective spectinomycin (100 mg/L) medium. (FIG. 3C) Sav-0 leaves on selective medium one month after bombardment. Note scanty callus formation and green cell cluster (arrow). (FIG. 3D) Culture shown in FIG. 3C, illuminated with UV light. Note green fluorescence indicating GFP accumulation in green cell cluster. (FIG. 3E) Sav-0 plant regenerated from a transplastomic clone #6. (FIG. 3F) Culture shown in FIG. 3E, illuminated with UV light. Inset--Sav-0 #3 seed progeny illuminated with UV light. Bar=1 mm.

[0013] FIG. 4. Green Fluorescent Protein (GFP) accumulates in chloroplasts. Shown are confocal images collected in the GFP, chlorophyll, and merged channels on a Leica TCS SP5II confocal microscope. Excitation wavelengths were at 488 and 568 nm, and detection was at 500 to 530 and 650 to 700 nm, respectively. Note the absence of GFP and chlorophyll in the wild-type Col-0 callus cells and mixed GFP-expressing transgenic and wild-type plastids in the Col-0 acc2-1 #1 and Sav-0 #6 lines. Note the absence of wild-type plastids in the leaves of Sav-0 #6 plants. Yellow color in the merged images indicates the colocalization of GFP and chlorophyll in plastids. Note that cells in the small green cell clusters are heteroplastomic. The only exception are cells in Sav-0 6 leaves, which are homoplastomic due to prolonged selection in tissue culture. Bars=10 .mu.m

[0014] FIGS. 5A-5B. Molecular Characterization of the Sav-0 Transplastomic Clones. (FIG. 5A) DNA gel blot using the rm16 probe (FIG. 2) indicates that the transplastomic Sav-0 calli and leaves are homoplastomic, carrying only the 4.7-kb EcoRI fragment and lacking the 2.7-kb wild type fragment. (FIG. 5B) The aadA and gfp probes recognize the same 2 kb dicistronic mRNA.

[0015] FIGS. 6A-6C. Alignment of homomeric ACCases in the Brassicaceae family. (FIG. 6A) Alignment of 200 the N-terminal amino acids of Arabidopsis thaliana ACC1 (At1g36160) and ACC2 (At1g36180) genes. (FIG. 6B) Alignment of 200 the N-terminal amino acids of Arabidopsis thaliana ACC1: At1g36160; Arabidopsis lyrata ACC1: XM_002891166.1; Camelina sativa ACC1-1: LOC104777496; Camelina sativa ACC1-2: LOC104743830; Capsella rubella ACC1: CARUB_v10011872 mg; Brassica oleracea ACC1: LOC106311006; Brassica napus ACC1-1: LOC106413885; Brassica napus ACC1-2: LOC106418889; Brassica rapa ACC1: LOC103833578. (FIG. 6C) Alignment of 300 the N-terminal amino acids of Arabidopsis thaliana ACC2: At1g36180; Arabidopsis lyrata ACC2: XM_002891167.1; Camelina sativa ACC2-1: LOC104777495; Camelina sativa ACC2-2: LOC104742086; Capsella rubella ACC2: CARUB_v10008063 mg; Brassica oleracea ACC2: LOC106301042; Brassica napus ACC2-1: Y10302; Brassica napus ACC2-2: X77576; Brassica rapa ACC2: LOC103871500.

[0016] FIG. 7. Design of sgRNAs for simultaneous mutagenesis of both B. napus ACC2 gene copies. Aligned are the first exons encoding the N-terminal plastid targeting regions. The GG of NGG of the PAM sequence is encircled; the 20 nucleotide forward guide sequence (5'-3') is marked with a horizontal line. The first nucleotide of the guide sequence should be changed to a G or an A, dependent on the use of U6 or U3 promoter, respectively (Belhaj et al., 2013). 9 of the 15 potential gRNA sequences are suitable for targeting both ACC2 copies (2-8 and 14,15). The reverse guide sequences are included in Table 3.

[0017] FIG. 8. Mutations generated by CRISPR/Cas9 mutagenesis in the Arabidopsis Wassiliewskija (Ws) and RLD ecotypes. Top--Columbia reference sequence and the parental Ws/RLD sequences. Note mutations that alter the reading frame yielding non-functional protein, such as a one nucleotide insertion in Ws-6-2 and RLD-6-2 lines. Bottom--oligonucleotide sequence used for construction of gRNA.

[0018] FIG. 9. Ws T3 seed germinated on 100 mg/L spectinomycin medium testing for hypersensitive response. After 2 weeks, the wild-type Ws seedlings bleach but develop primary leaves, in contrast to Ws-2-22 homozygous ACC2 knock-out seedlings which germinate, but do not develop shoot meristem outgrowths on spectinomycin.

[0019] FIG. 10. Schematic design of a Brassica napus plastid transformation vector is shown. The plastid targeting sequence comprises the rm16 targeting region (nucleotides 135473-137978 in GenBank accession KP161617). The vector carries a target site flanked selectable aadA marker gene. The recombinase target sites are marked with triangles. The marker gene and gene of interest have different promoters (P1, P2) and terminators (T1, T2) to avoid deletions by recombination via duplicates sequences.

[0020] FIG. 11. A schematic diagram depicting system for seed-specific expression of plastid genes from acc2 defective plants.

[0021] FIGS. 12A-12B. Transgenes for seed-specific expression in Brassica spp. (FIG. 12A) Plastid transgenes. P1 and T1 are the expression signals of the aadA marker gene. Preferred sequences are listed in text. P1 is the tobacco plastid Prrn sequence. The half circle is the maize sequence containing BS.sup.ZmGG sequence. gfp encodes green fluorescence protein. T1 is the rbcL gene terminator. Cloverleaf symbolizes tRNA gene. (FIG. 12B) The map of Agrobacterium binary vector pCAMBIA2300 with the PnpaA:Zm-PPR10GG:Tocs and selectable kanamycin resistance gene. P1 and T1 are Pnos/Tnos, the expression signals of kanamycin resistance (neo) gene. P2 is the PnpaA napin promoter; PPR10.sup.GG sequence is the mutant maize PPR10 protein coding sequence; T2 is Tocs octopine synthase transcription terminator. LB and RB are the T-DNA left and right border sequences.

[0022] FIG. 13. Alignment of the N-terminal nucleotides of Brassica napus cv Darmor-bzh ACC2-Br: BnaA06g04070D; ACC2-Bo: BnaC06g01580D; ACC1-Br: BnaA08g06180D; ACC1-Bo: BnaC08g06560D.

[0023] FIG. 14A-14C. FIG. 14A Functional ACC2 copies make B. napus plants tolerant to spectinomycin, permitting growth beyond the cotyledon stage. FIG. 14B and FIG. 14C. Flowchart to obtain Cas9-free spectinomycin hypersensitive acc2 Brassica napus. (14B) Selection of CRISPR/Cas9 transgenic plants by kanamycin resistance. (14C) Hypersensitivity bioassay identifies T1 families with putative knockouts in all ACC2 copies, leading to the isolation of Cas9-free acc2 individuals. Non-uniform hypersensitivity to spectinomycin will prompt an additional cycle of screening in the next seed generation.

DETAILED DESCRIPTION OF THE INVENTION

[0024] Spectinomycin, a preferred agent used for selecting for transplastomic events, binds to the 16S ribosomal RNA, blocking translation on the prokaryotic type 70S plastid ribosomes (Wirmer and Westhof, 2006; Wilson, 2014) inhibiting greening and shoot regeneration in tissue culture cells (Svab et al., 1990). When the plastid genome is transformed with the aadA gene encoding aminoglycoside-3''-adenylyltransferase, the modified antibiotic no longer binds to the 16S rRNA and translation proceeds, enabling greening. Tobacco, when cultured on a spectinomycin medium, bleaches and proliferates at a slow rate due to inhibition of plastid translation. Transplastomic tobacco cells are identified in tissue culture by the ability to green and regenerate shoots. In contrast, Arabidopsis bleaches but continues to proliferate on a spectinomycin medium in the absence of chloroplast ribosomes (Zubko and Day, 1998). Two major studies by Parker et al. (Parker et al., 2014, 2016) revealed the existence of rare Arabidopsis accessions, in which plastids are extremely sensitive to spectinomycin. Seeds of most accessions in the study germinated on spectinomycin and developed into albino plants.

[0025] However, in certain accessions, spectinomycin blocked plant development: the seeds germinated, but did not develop beyond the cotyledonary stage. Genetic analysis revealed that spectinomycin sensitivity in these accessions is due to mutations in the ACC2 nuclear gene. The ACC2 gene produces the homomeric acetyl-CoA-carboxylase (ACCase) that is imported into plastids, and duplicates the function of heteromeric ACCase, one subunit of which is encoded in the plastid accD gene (FIG. 1A). When plastid translation is blocked by spectinomycin and no heteromeric ACCase is made, the homomeric enzyme enables a limited amount of fatty acid biosynthesis and development of albino plants. In the absence of a functional ACC2 gene, fatty acid biosynthesis is dependent on the availability of heteromeric ACCase enzyme, the .beta.-Carboxylase subunit of which is translated on plastid ribosomes (FIG. 1B).

[0026] We hypothesized that the inefficiency of plastid transformation observed in our early efforts with Arabidopsis was due to the lack of the sensitivity to spectinomycin, and that transformation of mutants defective in ACC2 function should increase efficient recovery of transplastomic clones. We report here that the efficiency of plastid transformation in the acc2 background in Arabidopsis is comparable to that of tobacco, confirming our hypothesis. Antibiotics kanamycin, chloramphenicol, tobramycin and gentamycin are similar to spectinomycin in that they also act through inhibition of plastid translation. Kanamycin resistance is conferred by the neo (nptII) gene, encoding neomycin phosphotransferase or the aphA-6 gene encoding an aminoglycoside phosphotransferase. Chloramphenicol resistance is conferred by the cat gene encoding chloramphenicol acetyltransferase. Tobramycin/gentamycin resistance is conferred by the bifunctional aac(6')-Ie/aph(2'')-Ia gene, abbreviated as aac6-aph2 gene, encoding the bifunctional aminoglycoside phosphotransferase(6')-Ie/APH(2'')-Ia enzyme.

[0027] Thus, improved recovery of transplastomic events is expected in the acc2 defective background using these inhibitors of organellar translation as selective markers.

[0028] In view of this finding, we have expanded our efforts to create additional strains of acc2 defective plants in the Brassicaceae family. Herein below protocols and expression vectors are provided for both nuclear and plastid transformation in such plants, which include, without limitation, A. lyrata, C. sativa, C. ruella, B. oleracea, B. napus, B. rapa. The inventor also provides suitable guide strands for introducing mutations in ACCases via a CRISPR/CAS.

[0029] The definitions below are provided to facilitate an understanding of the invention.

[0030] Heteroplastomic refers to the presence of a mixed population of different plastid genomes within a single plastid or in a population of plastids contained in plant cells or tissues.

[0031] Homoplastomic refers to a pure population of plastid genomes, either within a plastid or within a population contained in plant cells and tissues. Homoplastomic plastids, cells or tissues are genetically stable because they contain only one type of plastid genome. Hence, they remain homoplastomic even after the selection pressure has been removed, and selfed progeny are also homoplastomic. For purposes of the present invention, heteroplastomic populations of genomes that are functionally homoplastomic (i.e., contain only minor populations of wild-type DNA or transformed genomes with sequence variations) may be referred to herein as "functionally homoplastomic" or "substantially homoplastomic." These types of cells or tissues can be readily purified to a homoplastomic state by continued selection.

[0032] Plastome refers to the genome of a plastid.

[0033] Transplastome refers to a transformed plastid genome.

[0034] Transformation of plastids refers to the stable integration of transforming DNA into the plastid genome that is transmitted to the seed progeny of plants containing the transformed plastids. Transient expression of heterologous DNA into the plastid or nuclear compartments can also be employed.

[0035] Selectable marker gene refers to a gene that upon expression confers a phenotype by which successfully transformed plastids or cells or tissues carrying the transformed plastid can be identified.

[0036] Transforming DNA refers to homologous DNA, or heterologous DNA flanked by homologous DNA, which when introduced into plastids becomes part of the plastid genome by homologous recombination.

[0037] "Operably linked" refers to two different regions or two separate genes spliced together in a construct such that both regions will function to promote gene expression and/or protein translation.

[0038] "Nucleic acid" or a "nucleic acid molecule" as used herein refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In discussing nucleic acid molecules, a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction. With reference to nucleic acids of the invention, the term "isolated nucleic acid" is sometimes used. This term, when applied to DNA, refers to a DNA molecule that is separated from sequences with which it is immediately contiguous in the naturally occurring genome of the organism in which it originated. For example, an "isolated nucleic acid" may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a prokaryotic or eukaryotic cell or host organism.

[0039] When applied to RNA, the term "isolated nucleic acid" refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.e., in cells or tissues). An isolated nucleic acid (either DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.

[0040] The term "functional" as used herein implies that the nucleic or amino acid sequence is functional for the recited assay or purpose.

[0041] Mao et al. provide detailed guidance for use of the CRISPR/Cas system in higher plants in Molecular Plant, 6: 2008-2011 (2013). The article entitled "Application of the CRISPR--Cas System for Efficient Genome Engineering in Plants" and its supplemental material is incorporated herein by reference as though set forth in full.

[0042] The terms "transform", "transfect", "transduce", shall refer to any method or means by which a nucleic acid is introduced into a cell or host organism and may be used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, PEG-fusion, biolistic bombardment and the like.

[0043] "Floral dip transformation" refers to Agrobacterium mediated DNA transfer, in which the flower is brought in contact with the Agrobacterium solution. Floral dip transformation has been described in Arabidopsis (Clough and Bent, 1998) and Brassica spp. (Verma et al., 2008; Tan et al., 2011).

[0044] "T-DNA" refers to the transferred-region of the Ti (tumor-inducing) plasmid of Agrobacterium tumefaciens. Ti plasmids are natural gene transfer systems for the introduction of heterologous nucleic acids into the nucleus of higher plants. Binary Agrobacterium vectors such pBIN20 and pPZP222 (GenBank Accession Number U10463.1) are known in the art.

[0045] A "plastid transit peptide" is a sequence which, when linked to the N-terminus of a protein, directs transport of the protein from the cytoplasm to the plastid.

[0046] A "clone" or "clonal cell population" is a population of cells derived from a single cell or common ancestor by mitosis.

[0047] A "cell line" is a clone of a primary cell or cell population that is capable of stable growth in vitro for many generations.

[0048] A "defective" and "nonfunctional" gene, such as acct, refers to a gene which does not encode a functional protein. For example, a one nucleotide insertion on deletion may alter the reading frame to creates an in-frame stop codon.

Methods for Creating Transplastomic Plants Using the Compositions of the Invention

[0049] Virtually all dicots have accD, an heteromeric ACCase subunit gene encoded in their plastid genome, but also have homomeric, plastid targeted nuclear ACC2 gene copies, which is the likely cause for the difficulty of extending the plastid transformation technology to all crops. Deletion of the nuclear ACC2 genes will enable plastid transformation in these dicot species and genetic lines.

[0050] The recognition that the plastid targeted ACCase in Arabidopsis is an impediment to plastid transformation provides a rational template to implement plastid transformation in recalcitrant crops. The accD gene is present on the plastid genome of most crops. The Arabidopsis thaliana ACC2 enzyme has an N-terminal extension relative to ACC1 that serves as an N-terminal plastid targeting sequence (Babijchuk et al., 2011). The ACC1 and ACC2 genes are present in all Brassicaceae species, including Arabidopsis lyrata, Camelina sativa, Camelina rubella, Brassica oleracea, Brassica napus and Brassica rapa. The homomeric ACC2 enzyme in these species has an N-terminal extension relative to ACC1. A targeted mutation in the N-terminal extension should selectively inactivate the ACC2 variant, expected to create a spectinomycin sensitive mutant similar to the Col-0 acct-1 mutant derivative (Parker et al., 2014). Plastid transformation has been achieved in cabbage (Brassica oleracea L. var. capitata L.), thus knockout of ACC2 is apparently not necessary to obtain transplastomic events in this crop, at least in the two cultivars tested (Liu et al., 2007; Liu et al., 2008). Plastid transformation in cauliflower (Brassica oleracea var. botrytis) has been obtained, but at a very low frequency (Nugent et al., 2006). Plastid transformation in oilseed rape (Brassica napus) has also been obtained, but no homoplastomic plants could be obtained (Hou et al., 2003; Cheng et al., 2010), or the transformation efficiency was low (Schneider et al., 2015). Plastid transformation in Lesquerella fendleri, another oilseed crop in the Brassicaceae, was feasible but inefficient (Skarjinskaia et al., 2003). Mutagenesis of ACC2 in the latter cases should significantly boost plastid transformation efficiency. Accordingly, a CRISPR/Cas approach for knocking out the ACC gene is provided in Example II.

[0051] Alternatively, desirable plant species could be screened for mutations in nuclear ACC genes and those strains harboring such mutations utilized in the plastid transformation methods disclosed herein. Such strains should inherently be more sensitive to spectinomycin.

[0052] The materials and methods set forth below were utilized in the performance of Example I.

Tissue Culture Media

[0053] The tissue culture media were adopted from Sikdar et al. (1998), originally described by Marton and Browse (1991). The culture media are based on Murashige and Skoog (MS) salts (Murashige and Skoog, 1962). ARM consists of MS salts, 3% (w/v) Suc, 0.8% (w/v) agar (A7921; Sigma), 200 mg of myoinositol, 0.1 mg of biotin (1 mL of 0.1 mg mL-1 stock), and 1 mL of vitamin solution (10 mg of vitamin B1, 1 mg of vitamin B6, 1 mg of nicotinic acid, and 1 mg of Gly per mL) per liter, pH 5.8. ARMS medium consists of ARM supplemented with 5% (w/v) Suc. ARMI medium consists of ARM containing 3 mg of IAA, 0.6 mg of benzyladenine, 0.15 mg of 2,4-D, and 0.3 mg of isopentenyladenine per liter. ARMIIr medium consists of ARM supplemented with 0.2 mg/L naphthaleneacetic acid and 0.4 mg of isopentenyladenine per liter. The stocks of filter-sterilized plant hormones and antibiotics (100 mg/L spectinomycin HCl) were added to media cooled to 45.degree. C. after autoclaving.

[0054] Shoot regeneration in the transplastomic Sav-0 clones was obtained on an ARM containing 2,4-D (0.5 mg/L), kinetin (0.05 mg/L), and spectinomycin (100 mg/L; 3 d) followed by incubation on an ARM containing IAA (0.15 mg/L), phenyladenine (1.6 mg/L), and spectinomycin (100 mg/L; Motte et al., 2013). Seed was obtained by growing shoots on MS salt medium containing 3% (w/v) Suc and 0.8% (w/v) agar (A7921; Sigma), pH 5.8.

Plant Materials and Growth Conditions

[0055] The Arabidopsis (Arabidopsis thaliana) Sav-0 (CS28725) and Col-0 homozygous acc2-1 knockout line (SALK_148966C) seeds were obtained from the Arabidopsis Biological Resource Center. The Col-0 seeds were obtained from Juan Dong (Rutgers University). The RLD and Ler seeds were purchased form Lehle Seeds.

[0056] For surface sterilization, seeds (25 mg) were treated with 1.7% (w/v) sodium hypochlorite (5.times. diluted 8.5% (w/v) commercial bleach) in a 1.5-mL Eppendorf tube for 15 min with occasional mixing (vortex). The bleach was removed by pipetting and washed three times with sterile distilled water. Seeds were germinated on 50 mL of ARMS medium in deep petri dishes (20 mm high and 10 cm in diameter). The plates were illuminated for 8 h using cool-white fluorescent tubes (2,000 lx). The seeds germinated after 10 to 15 d of incubation at 24.degree. C. To grow plants with larger leaves, seedlings were transferred individually to ARMS plates (four plants per deep petri dish). The plates were illuminated for 8 h with cool-white fluorescent bulbs (2,000 lx) and incubated at 21.degree. C. during the day and 18.degree. C. during the night. One- to 2-cm-long, dark green leaves were harvested for bombardment after incubation for an additional 5 to 6 weeks.

Plastid Transformation Vector

[0057] The plastid transformation vector pATV1 targets insertion in the inverted repeat region of the plastid genome upstream of the trnV gene (FIG. 2). The targeting region is a 4.5 kb NruI/XbaI fragment derived from the Arabidopsis thaliana ptDNA (GenBank Accession No. NC_000932). The fragment was cloned in the KpnI-SacI site of a pBSKS+ BlueScript vector, ligating the vector KpnI site to the plastid NruI site and vector SacI site to the plastid XbaI site. The vector carries a dicistronic operon, in which the first open reading frame (ORF) encodes the aadA spectinomycin resistance gene and the second ORF encodes a green fluorescence protein (GFP). The operon is expressed from the PrrnLatpB promoter, obtained by fusing the plastid rRNA operon promoter (Prrn) with the atpB plastid gene leader (LatpB), originally described in the pHK30 plasmid (Kuroda and Maliga, 2001). The dicistronic aadA-gfp marker gene was excised as an EcoRI-HindIII fragment and cloned in the HincII site of the targeting region. In the dicistronic construct, 14 N-terminal amino acids of the ATP synthase beta subunit are translationally fused with the AAD N-terminus, as in plasmid pHK30 (Kuroda and Maliga, 2001). The intergenic region encodes the cry9Aa2 gene leader (Chakrabarti et al., 2006), followed by the gfp coding region and the 3'-UTR of the plastid psbA gene (TpsbA) for the stabilization of the mRNA. The DNA sequence of the EcoRI-HindIII fragment encoding the aadA-gfp dicistronic operon in plasmid pMRR13 is shown below.

TABLE-US-00001 (SEQ ID NO: 1) gagctcGCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGG GATTGACGTGAGGGGGCAGGGATGGCTATATTTCTGGGAGAATTAACCGA TCGACGTGCaAGCGGACATTTATTTTaAATTCGATAATTTTTGCAAAAAC ATTTCGACATATTTATTTATTTTATTATTATGAGAATCAATCCTACTACT TCTGGTTCTGGGGTTTCCACGgctactagcGAAGCGGTGATCGCCGAAGT ATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAAC CGACGTTGCTGGCCGTACATTTGTACGGCTCCGCAGTGGATGGCGGCCTG AAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGA TGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTCGGCTT CCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTG CACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATT TGGAGAATGGCAGCGCAATGACATTCTTGCAGGTATCTTCGAGCCAGCCA CGATCGACATTGATCTGGCTATCTTGCTGACAAAAGCAAGAGAACATAGC GTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGATCCGGTTCCTGA ACAGGATCTATTTGAGGCGCTAAATGAAACCTTAACGCTATGGAACTCGC CGCCCGACTGGGCTGGCGATGAGCGAAATGTAGTGCTTACGTTGTCCCGC ATTTGGTACAGCGCAGTAACCGGCAAAATCGCGCCGAAGGATGTCGCTGC CGACTGGGCAATGGAGCGCCTGCCGGCCCAGTATCAGCCCGTCATACTTG AAGCTAGACAGGCTTATCTTGGACAAGAAGAAGATCGCTTGGCCTCGCGC GCAGATCAGTTGGAAGAATTTGTCCACTACGTGAAAGGCGAGATCACCAA GGTAGTgGGCAAAgaaCAAAAACTCATTTCTGAAGAAGACTTGTAACTGC AGATAACCCAAATAATGTTTTAAAATTTTAAAAATAATGTAGGAGGAAAA ATTATGGCTAGCAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAAT TCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTG GAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATT TGCACTACTGGAAAACTACCTGTTCCtTGGCCAACACTTGTCACTACTTT CTCTTATGGTGTTCAATGCTTTTCAAGATACCCAGATCATATGAAGCGGC ACGACTTCTTCAAGAGCGCCATGCCTGAGGGATACGTGCAGGAGAGGACC ATCTCTTTCAAGGACGACGGGAACTACAAGACACGTGCTGAAGTCAAGTT TGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAAGGGAATCGATTTCA AGGAGGACGGAAACATCCTCGGCCACAAGTTGGAATACAACTACAACTCC CACAACGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAA CTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACC ATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGAC AACCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAA GAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACAC ATGGCATGGATGAACTATACAAATAAGctctagCTAGAGCgatcctggcc tagtctataggaggttttgaaaagaaaggagcaataatcattttcttgtt ctatcaagagggtgctattgctcctactattactattatttatttactag tatatacttacatagacttttttgtttacattatagaaaaagaaggagag gttattttcttgcatttattcatgGGGGATCAAAGCTT

Transformation and Selection of Transplastomic Lines

[0058] Plastid transformation in Arabidopsis was carried out using our 1998 protocol, as shown in FIG. 3A-3F (Sikdar et al., 1998). The leaves (10 to 20 mm) were harvested from aseptically grown plants and covered the surface of agar-solidified ARMI medium in a 10 cm petri dish. We used .about.100 leaves to cover the surface of the plate. The leaves were cultured for 4 days on ARMI medium, then bombarded with pATV1 vector DNA. Transforming DNA was coated on the surface of microscopic (0.6 .mu.m) gold particles, then introduced into chloroplasts by the biolistic process (1,100 psi) using a helium-driven PDS1000/He biolistic gun equipped with the Hepta-adaptor (Lutz et al., 2011). The plates were placed on the shelf at the lowest position for bombardment.

[0059] Following bombardment, the leaves were incubated for an additional 2 d on ARMI medium. After this time period, the leaves were stamped with a stack of 10 razor blades to create parallel incisions 1 mm apart. The stamped leaves were cut into smaller (1 cm2) pieces, transferred onto the same medium (ARMI) containing 100 mg/L spectinomycin, incubated at 28.degree. C., and illuminated for 16 h with fluorescent tubes (CXL F025/741). After 8 to 10 d, the leaf strips were transferred onto selective ARMIIr medium containing 100 mg/L spectinomycin for the selection of spectinomycin-resistant clones. The leaf strips were transferred to a fresh selective ARMIIr medium every 2 weeks until putative transplastomic clones were identified as resistant green calli.

Confocal Microscopy to Detect GFP in Plastids

[0060] Subcellular localization of GFP fluorescence was determined by a Leica TCS SP5II confocal microscope. To detect GFP and chlorophyll fluorescence, excitation wavelengths were at 488 nm and 568 nm, and the detection filters were set to 500-530 nm and 650-700 nm, respectively.

DNA and RNA Gel-Blot Analyses

[0061] Total leaf DNA was prepared by the cetyltrimethylammonium bromide protocol (Tungsuchat-Huang and Maliga, 2012). DNA gel-blot analyses was carried out as described (Svab and Maliga, 1993). Total cellular DNA was digested with the EcoRI restriction enzyme. The DNA probe was the ApaI-SphI ptDNA fragment encoding the plastid rm16 gene (FIG. 2).

[0062] Total cellular RNA was isolated from leaves frozen in liquid nitrogen using TRIzol (Ambion/Life Technologies) following the manufacturer's protocol. RNA gel-blot analyses were carried out as described (Kuroda and Maliga, 2001). The probes were as follows: for aadA, a 0.8-kb NcoI-XbaI fragment isolated from plasmid pHC1 (Caner et al., 1991); and for gfp, a fragment amplified from the gfp coding region using primers gfp-forward p1 (5'-TTTTCTGTCAGTGGAGAGGGTG-3') (SEQ ID NO: 2) and gfp-reverse p2 (5'-CCCAGCAGCTGTTACAAACT-3' (SEQ ID NO: 3) (FIG. 2).

Alignment of Homomeric ACCases

[0063] The alignment of homomeric ACCases in the Brassicaceae family was carried out with MultAlin software (Corpet, 1988).

Accession Numbers

[0064] The DNA sequence of the pATV1 Arabidopsis plastid transformation vector was deposited in GenBank under accession number MF461355.

[0065] The following examples are provided to illustrate certain embodiments of the invention. They are not intended to limit the invention in any way.

Example I

Dicistronic pATV1 Vector for Identification of Transplastomic Events

[0066] The plastid transformation vector pATV1 targets insertion upstream of the trnV gene in the inverted repeat region of the plastid genome (FIG. 2). Vector pATV1 carries a dicistronic operon, in which the first open reading frame (ORF) encodes the aadA spectinomycin resistance gene and the second ORF encodes a green fluorescence protein (GFP) (FIG. 2). Polycistronic mRNAs are not translated on the eukaryotic-type 80S ribosomes in the cytoplasm, thus accumulation of GFP in chloroplasts in spectinomycin-resistant clones indicates plastid transformation.

Plastid Transformation and Identification of Transplastomic Events

[0067] Plastid transformation was carried out in the Col-0 (Columbia) accession and the Columbia ACC2 T-DNA insertion line acct-1 (SALK_148966C) shown to be sensitive to spectinomycin in the Parker at el. study (Parker et al., 2014). We also evaluated plastid transformation efficiency in the Sav-0 (Slavice) accession that was the most sensitive to spectinomycin in the study (Parker et al., 2014). The Sav-0 ACC2 gene carries 15 missense mutations, but one variant alone (G135E) that alters a conserved residue immediately preceding the biotin carboxylase domain appears to be responsible for the hypersensitive phenotype (Parker et al., 2016). Plants were grown aseptically on ARMS medium (FIG. 3A); leaves for plastid transformation were harvested from sterile plants and placed on ARMI media. The leaf tissue was bombarded with gold particles coated with vector DNA. After two days, the leaves were stamped with a stack of razor blades to create a series of parallel incisions 1 mm apart. The mechanical wounds are essential to induce uniform callus formation in the leaf blades. The stamped leaves were transferred onto the same medium (ARMI) containing spectinomycin (100 mg/1; FIG. 3B) to facilitate preferential replication of plastids containing transformed ptDNA copies. The ARMI medium induces division of the leaf cells and formation of colorless, embryogenic callus. After 7-10 days of selection on ARMI medium, spectinomycin selection was continued on the ARMIIr medium, which induces greening. Since spectinomycin prevents greening of wild-type cells, only spectinomcyin-resistant cells formed green calli. Visible green cell clusters appeared within 21 to 40 days on the selective ARMIIr medium (FIG. 3C). Illumination of plates with UV light revealed intense fluorescence of GFP in the green calli (FIG. 3D).

[0068] In the wild-type Col-0 sample (four bombarded plates), no transplastomic event was found. We obtained eight events on five bombarded plates using leaf tissue in the acc2-1 mutant background and four events in four bombarded plates in the Sav-0 accession (Table 1). This transformation efficiency is comparable to the transformation efficiency obtained with current protocols in tobacco: four to five transplastomic events per bombardment (Maliga and Tungsuchat-Huang, 2014).

[0069] This is a significant advance, as high-frequency plastid transformation in Arabidopsis has been pursued since the publication of the original report (Sikdar et al., 1998) but has been largely unsuccessful. For example, bombardment of 26 plates of RLD and five plates of Landsberg erecta (Ler) leaf tissue did not yield a transplastomic event (Table 1). In contrast, nine bombardments of leaves with the acc2 null background yielded 12 transplastomic clones. Even though the technology improved significantly since 1998, no transplastomic clones were obtained until ACC2-defective leaf tissue was used for bombardments (Table 1), providing overwhelming support for the absence of ACC2 activity being critical for high-frequency plastid transformation in Arabidopsis.

TABLE-US-00002 TABLE 1 Identification of transplastomic events in Arabidopsis Au, Gold particles; Hepta, using the biolistic gun Hepta adaptor instead of a single flying disk; Tu, tungsten particles. No. of Left/Right Trans- Arm No. of plastomic Plasmid kb Marker Gene Accession Tissue Gun Plates Events Reference pGS31A 1.1/0.9 Prrn:LrbcL:aadA:TpsbA RLD Leaf Single, Tu/1 .mu.m 201 2 Sikdar et al. (1998) pAAK176 1.7/0.8 Prrn:LrbcL:aadA:TpsbA RLD Leaf Hepta, Au/0.6 .mu.m 10 0 Reported here Ler Leaf Hepta, Au/0.6 .mu.m 4 0 Reported here pTT626 1.7/0.8 Prrn:Lcry9:aadA-gfp:TpsbA RLD Leaf Hepta, Au/0.6 .mu.m 14 0 Reported here pATV1 1.7/0.8 PrrnLatpB:aadA:Lcry9:gfp:TpsbA RLD Leaf Hepta, Au/1 .mu.m 2 0 Reported here Ler Leaf Hepta, Au/1 .mu.m 1 0 Reported here Col-0 Leaf Hepta, Au/0.6 .mu.m 4 0 Reported here Col-0 acc2-1 Leaf Hepta, Au/0.6 .mu.m 5 8 Reported here Sav-0 Leaf Hepta, Au/0.6 .mu.m 4 4 Reported here

Confocal Microscopy to Confirm Transplastomic Events

[0070] Because GFP is encoded in the second ORF, GFP accumulation is expected only if the mRNA is translated in plastids on the prokaryotic type 60S ribosomes known to translate transgenic polycistronic mRNAs. Examples are the plastid psbE operon (Carrillo et al., 1986; Willey and Gray, 1989), the psaA/B transcript (Meng et al., 1988) and petA, which is not cleaved off the upstream ycf10 gene (Willey and Gray, 1990). Translation of polycistronic mRNAs created by operon extension has also been demonstrated (Staub and Maliga, 1995). Thus, GFP accumulation was anticipated only if the gfp gene is expressed in chloroplasts. The putative transplastomic lines identified as green cell clusters have subsequently been confirmed as transplastomic events by detecting localization of GFP to plastids by confocal microscopy. Overlay of the GFP and chlorophyll channels indicates that the clones are heteroplastomic, carrying transformed and wild type plastids in the same cells. A good example for mixed plastids is shown in the overlay of GFP and chlorophyll channels in Col-0 acct-1#3 in FIG. 4. The chloroplasts were not well developed in most tissue culture cells. Chlorophyll was detected in only a localized region of plastids in line with thylakoid biogenesis initiating from a localized center (Schottkowski et al., 2012). Good examples are overlays of Col-0 acc2-1#5 and Say-0 #1 in FIG. 4.

[0071] The heteroplastomic state detected in the cells of the green clusters was not maintained, and eventually, wild-type plastids (ptDNA) disappeared in the callus cells after continued cultivation on selective media. The homoplastomic state is confirmed by the uniform accumulation of GFP in the leaves of a Sav-0 #6 plant shown in FIG. 4 and by DNA gel-blot analyses of calli shown in FIG. 5B.

Regeneration of Transplastomic Sav-0 Plants and Transmission of GFP to Seed Progeny

[0072] After the bombardment of Col-0 and Sav-0 leaves, the selection of transplastomic events was carried out according to the published RLD protocol (Sikdar et al., 1998). However, when the transplastomic clones were transferred to the RLD shoot induction medium, the calli did not proliferate. Therefore, we transferred the transplastomic calli to media that were used successfully to regenerate plants from other accessions. We found that the two-step regeneration protocol described for shoot induction in the C24 background (Motte et al., 2013) triggered shoot regeneration in two surviving Sav-0 calli. Calli of Sav-0 transplastomic lines #3 and #6 were briefly (3 d) exposed to callus induction medium containing 0.5 mg/L 2,4-dichlorophenoxyactetic acid (2,4-D) and 0.05 mg/L kinetin and then transferred to a shoot regeneration medium containing 0.15 mg/L indole acetic acid (IAA) and 1.6 mg/L phenyladenine. Phenyladenine is a potent compound for shoot regeneration through the inhibition of cytokinin oxidase/dehydrogenase activity (Motte et al., 2013). Shoots from the calli developed in 45 to 60 days and flowered and formed siliques in sterile culture (FIG. 3E). The plants glowed intensely when illuminated with UV light, indicating high-level GFP accumulation (FIG. 3F-3G). Confocal microscopy suggests uniform transformation of plastid genomes in the leaves of Sav-0 #6 plants (FIG. 4) and was confirmed by molecular analyses (FIG. 5B).

[0073] The transplastomic shoots were transferred to larger 500-mL Erlenmeyer flasks containing ARM for seed set, where they continued to grow.

Molecular Analysis of Transplastomic Arabidopsis Clones

[0074] DNA and RNA gel blot analyses was carried out on the callus and shoots of the two Say-0 transplastomic lines #3 and #6. Wild-type plastids present in the cells of the green clusters were gradually lost by the time DNA gel-blot analyses were carried out, confirming uniform transformation of the plastid genomes in both calli and shoots (FIG. 5A). RNA gel blot analyses indicated the presence of a 2-kb dicistronic transcript detected by both the aadA and gfp probes (FIG. 5B).

DISCUSSION

[0075] Development of successful plastid transformation protocols takes multiple years, explaining the relative paucity of crops in which plastid transformation is routine (Maliga and Bock, 2011; Maliga, 2012; Bock, 2015). The expectation is to obtain transplastomic plants, which carry and transmit to the seed progeny a uniformly transformed plastid genome population. The time required to obtain a flowering transplastomic plants from seed takes about 5 to 6 months, as outlined in Table 2. This time frame can be broken up into discrete steps, each of which represents a milestone in developing a complete system. We report here a significant break-through: high frequency transformation of the Arabidopsis plastid genome in spectinomycin sensitive accessions and a marker system that enables rapid identification of transplastomic events by selective expression of a GFP gene in plastids. This step is a major advance towards developing a complete system of plastid engineering in Arabidopsis.

TABLE-US-00003 TABLE 2 An overview of the protocol for the construction of a transplastomic Arabidopsis Sav-0 plants. TIME OBJECTIVE CULTURE MEDIUM (No. of transfers) Step 1 Seed germination ARM5 Medium 14 days Step 2 Grow sterile plants ARM5 medium 42 days Step 3 Leaf callus, non ARMI medium 4 days selective Step 4 Leaf bombardment ARMI medium Step 5 Leaf callus, non ARMI medium 2 days selective Step 6 Leaf callus, ARMI medium + 14 days selective Spectinomycin (100 mg/L) Step 7 Leaf callus, ARMIIr + 21 days (2x) greening Spectinomycin (100 mg/L) Step 8 Shoot induction ARM medium + 2,4-D 3 days (0.5 mg/L), kinetin (0.05 mg/L), Spectinomycin (100 mg/L) Step 9 Shoot regeneration ARM medium + IAA 45-60 days (0.15 mg/L), Phe-Ade (1.6 mg/L), Spectinomycin (100 mg/L) Time to flowering plants: 145-160 days (~5 months)

Development of a Plastid Transformation Protocol in Arabidopsis

[0076] The steps of a complete system of plastid engineering in Arabidopsis consist of: (a) obtaining or generating sterile acc2 defective plants to provide a leaf source for transformation; (b) delivering DNA to plastids; (c) recovering transplastomic events; (d) regenerating shoots from transplastomic callus and (e) obtaining seed from the shoots.

[0077] We report here approximately 100-fold enhanced plastid transformation efficiency per bombardment in the acc2 null background: eight events in five bombarded samples in the Col-0 acc2-1 line and four events in four bombarded samples in the Sav-0 background. The increase from one event per approximately 100 bombardments to one event per one bombardment is due in part to technological advances. However, the lack of success with the latest technology in a large number of bombarded samples (Table 1) provides overwhelming evidence that the key to success was the choice of Arabidopsis lines lacking ACC2 activity.

[0078] Identification of transplastomic events in the RLD ecotype took 5 to 12 weeks in 1998 (Sikdar et al., 1998). The use of spectinomycin-sensitive acc2-knockout lines and the pATV1 dicistronic operon vector shortened the time period for identification of transplatomic events to 3 to 5 weeks. The use of the acc2 knockout lines shortened scoring because the proliferation of non-transformed cells growth was efficiently inhibited by spectinomycin, enabling identification of the spectinomycin-resistant green cell clusters. Spectinomycin resistance may be due to the integration of aadA in the plastid genome, and fortuitous expression from an upstream promoter or spontaneous mutations in the rrn16 gene (Svab and Maliga, 1993). GFP, encoded in the second ORF, is expressed only in chloroplasts, enabling the rapid identification of transplastomic clones in a small number of heteroplastomic cells by confocal microscopy.

[0079] Once transplastomic clones are identified, the next major step is plant regeneration. There is diversity for shoot regeneration potential in Arabidopsis accessions. Col-0 is well known for its recalcitrance to shoot regeneration from cultured cells. Therefore, no attempt was made to regenerate shoots from the Col-0 transplastomic callus tissue. There is no information about the tissue culture properties of the Sav-0 accession. Our first attempts at Sav-0 shoot regeneration from the transplastomic clones proved successful, yielding flowering shoots in culture (FIG. 3E). However, the seeds, with one exception, failed to germinate. Shoot regeneration protocols have been worked out from root (Marton and Browse, 1991) and leaf explants (Lutz et al., 2015) of the RLD ecotype; and from protoplasts (Chupeau et al., 2013), leaf explants (Zhao et al., 2014) and inflorescence stem explants (Zhao et al., 2013) of the Wassilewskya (Ws) ecotype. Thus, a routine protocol for plastid transformation in Arabidopsis can be obtained by the refinement of leaf regeneration protocol in the Sav-0 ecotype, or by developing ACC2 knockout mutations in the RLD (Marton and Browse, 1991) or Wassilewskya (Ws) (Chupeau et al., 2013; Zhao et al., 2014) nuclear backgrounds. Alternatively, the Col-0 acct-1 can be transformed with the steroid-inducible BABYBOOM gene to facilitate plant regeneration from transplastomic events (Lutz et al., 2015).

[0080] Seed from transplastomic tobacco is obtained by rooting shoots in tissue culture, then transferring the rooted cuttings to a greenhouse. Arabidopsis shoots obtained in tissue culture are notoriously difficult to root. Rather than making an effort to root the plants in culture and transfer them to the greenhouse, we obtained seed from plants in sterile culture, a two-three month process (Lutz et al., 2015).

Early Identification of Plastid Transformants

[0081] The dicistronic marker system is a developer's tool that enables early scoring, but severely burdens the developing plants due to the high level of AAD and GFP expression, .about.7% and .about.15% of total soluble cellular protein (TSP) in tobacco, respectively (unpublished). High-levels of AAD are not necessary to obtain transplastomic plants. We have found that a mutation in the promoter of the aadA gene reduced accumulation of AAD gene product below 1% without impact on the frequency of transplastomic events by spectinomycin selection (Sinagawa-Garcia et al., 2009). Therefore, the new Arabidopsis vectors expressing low levels of AAD described herein can be used to advantage as lowered expression levels of AAD do not compromise plant growth.

Plastid Transformation in Arabidopsis Provides Template for Recalcitrant Crops

[0082] The recognition that the duplicated ACCase in Arabidopsis is an impediment to plastid transformation provides the guidance necessary for implementation of plastid transformation in all Arabidopsis accessions and in crops having a plastid-encoded accD gene and a plastid-targeted ACC2 enzyme. The Arabidopsis thaliana ACC2 enzyme has an N-terminal extension compared to ACC1 (FIG. 6A). The N-terminal extension is a plastid targeting sequence shown by subcellular localization of a GFP fusion protein (Babiychuk et al., 2011). The ACC1 and ACC2 genes are present in most Brassicaceae species, including Arabidopsis lyrata, Camelina sativa, Camelina rubella, Brassica oleracea, Brassica napus and Brassica rapa. The homomeric ACC2 enzyme in these species has an N-terminal extension compared to ACC1 (FIGS. 6B and 6C). Thus, a targeted mutation in the N-terminal extension can selectively inactivate the ACC2 variant to create a spectinomycin hypersensitive variant similar to the Col-0 acc2-1 deletion derivative (Parker et al., 2014).

[0083] Crops recalcitrant to plastid transformation such as cotton (Gossypium raimondii), soybean (Glycine max) and alfalfa (Medicago truncatula) have a plastid accD gene and multiple homomeric nuclear ACC genes. Indeed, this method should prove effective in those plants having comparable ACC2 with an N-terminal extension. Moreover, further experimentation could be performed to determine how deletion of one or more of the homomeric ACCase genes enhances recovery of transplastomic events.

[0084] Mutations in genes other than ACC2 also made Arabidopsis sensitive to spectinomycin. The TIC20-IV gene, which is required for the import of proteins through the inner chloroplast membrane, appears to limit the import of ACC2 enzyme (Parker et al., 2014). Dicot plastid genomes have several essential genes, including accD, clpP, Ycf1 and Ycf2 (Scharff and Bock, 2014). Apparently, in photoheterotrophic cultures where sucrose in the medium eliminates the need for photosynthesis, only translation of the accD mRNA, hence fatty acid biosynthesis, is required to sustain plant life.

Conclusion

[0085] Boost of plastid transformation efficiency using ACC2 knockout lines in commercial species of Brassicaceae has obvious economic benefits. Genomic resources make Arabidopsis the favored model to study basic biological processes, and to explore new biotechnological applications (Weigel and Mott, 2009; Koornneef and Meinke, 2010; Stitt et al., 2010; Wallis and Browse, 2010). The exception is photosynthesis research and chloroplast biotechnology that utilizes tobacco (Nicotiana tabacum) because engineering of the plastid genome encoding key components of the photosynthetic machinery is routine in only this species (Hanson et al., 2016; Sharwood et al., 2016). If plastid transformation would be available in Arabidopsis, this research would be carried out in this model organism, in which a large mutant collection is available in virtually any nuclear gene contributing to photosynthesis. Recognizing the importance of plastid translation during selection of transplastomic events has identified a bottleneck of plastid transformation in Arabidopsis. High frequency plastid transformation in Arabidopsis thaliana will open up the unique resources of this model species to advance our understanding of plastid function and new biotechnological applications.

Example II

Deletion of a CC2 Genes in Brassicaceae Crops to Create Suitable Recipients for Plastid Transformation

[0086] As discussed above in Example I, crops in the Brassicaceae family encode homologs of the Arabidopsis ACC2 gene, characterized by an N-terminal extension as compared to ACC1. Manual inspection of the N-terminal region of ACC2 genes led to the identification of >20 suitable guide RNAs (see Table 3). The potential gRNAs targeting both stands (5' to 3' and 3' to 5') are identified as NNNNNNNNNNNNNNNNNNNN NGG sequence (20N+NGG, N=A/G/C/T) (SEQ ID NO: 4), where the only limitation is the presence of a GG sequence (Mali et al., 2013). More relaxed rules for sgRNA design can be used in plants, such as G(N).sub.19-22 for the U6 promoter and A(N).sub.19-22 for the U3 promoter and the 1.sup.st nucleotide does not have to match the genomic sequence (Belhaj et al., 2013).

[0087] Brassica napus L. (AACC, 2n=4x=38) is an amphidoploid species originating from spontaneous hybridization of Brassica rapa (AA, 2n=2x-20) and Brassica oleracea (CC, 2n=2x=18) (Song and Osborn, 1992; Howell et al., 2008). The Brassica napus genome encodes two ACC1 genes (Locus106413885; Locus106418889) and two ACC2 genes (GenBank accession numbers X77576, Y10302) (Schulte et al., 1997). Simultaneous mutation of two genomic sequences can be executed efficiently using CRISPR/Cas9, as described in the literature. A noteworthy example is simultaneous inactivation of 62 copies of a porcine endogenous retrovirus in pigs (Yang et al., 2015). Additionally, non-segregating seed progeny due to mutations in both genomic copies in the first generation of Arabidopsis and tomato plants (Feng et al., 2014) (Brooks et al., 2014). The alignment of 298 N-terminal nucleotides of the Brassica napus ACC2 genes reveals 7 mismatches. Still, 9 of the 15 potential forward sgRNAs are useful for simultaneously inducing mutations in both ACC2 gene copies (FIG. 7, Table 3).

[0088] To achieve targeted deletion in the ACC2 N-terminal region, the gRNAs are cloned into the CRISPR/Cas vector and introduced into different crops using a nuclear transformation system appropriate for the target species. For example, Camelina sativa plants will be transformed by the flower dip protocol (Liu et al., 2012). In the case of Brassica, introduction of the CRISPR/Cas vector system can be achieved using Agrobacterium-mediated transformation of hypocotyls (Cardoza and Stewart, 2003, 2006) or flower dip transformation (Tan et al., 2011; Verma et al., 2008) as described below.

Agrobacterium-Mediated Transformation of Hypocotyl Segments

[0089] Brassica napus L. cv. Westar is transformed with an Agrobacterium binary vector carrying kanamycin resistance as a plant marker. Seeds are surface-sterilized with 10% sodium hypochlorite with 0.1% Tween for 5 minutes, followed by a 1-min rinse with 95% ethanol and washing the seed 5.times. with sterile distilled water. The seeds are germinated in sterile culture on MS basal medium (Murashige and Skoog, 1962) containing 20 g/l sucrose and solidified with 2 g/l Gelrite. Hypocotyls for transformation are excised from 8 to 10-day-old seedlings and 1-cm pieces preconditioned for 48 h on MS medium supplemented with 1 mg/l 2,4-D (2,4-dichlorophenoxy acetic acid) and 30 g/l sucrose, solidified with 2 g/l Gelrite. The preconditioned hypocotyl segments were then inoculated with Agrobacterium grown overnight to an OD600=0.8 in liquid LB medium. The Agrobacterium cells are pelleted by centrifugation and re-suspended in liquid callus induction medium with 0.05 mM acetosyringone to induce T-DNA transfer.

[0090] Co-cultivation with Agrobacterium is performed for 48 h on MS medium with 1 mg/l 2,4-D. Following co-cultivation, the explants are transferred to the same medium with 400 mg/l timentin and 200 mg/l kanamycin to select for transformed cells. After 2 weeks, the explants are transferred to MS medium to promote organogenesis containing 4 mg/l BAP (6-benzylaminopurine), 2 mg/l zeatin, 5 mg/l silver nitrate, 400 mg/l timentin, 200 mg/l kanamycin and 30 g/l sucrose, solidified with 2 g/l Gelrite. After an additional 2 weeks, the tissue is transferred to MS medium containing 3 mg/l BAP, 2 mg/l zeatin, the same antibiotics, 30 g/l sucrose and 2 g/l Gelrite for shoot development. To encourage shoot elongation, the shoots are transferred to MS medium with 0.05 mg/l BAP, 30 g/l sucrose, antibiotics as above, solidified with 3 g/l Gelrite. The elongated shoots are rooted on a medium containing half-strength MS salts, 10 mg/l sucrose, 3 g/l Gelrite, 5 mg/l IBA, and 400 mg/l timentin and 200 mg/l kanamycin. The cultures are incubated at 25.+-.2.degree. C., 16/8-h (light/dark) photoperiod. The rooted shoots are transferred to soil and grown at 20.degree. C. 20, 16/8 h (light/dark) photoperiod. To prevent desiccation, the plants are initially covered with a plastic dome.

Floral Dip Transformation in Brassica Ssp. To Generate ACC2 Defective Plants

[0091] For Agrobacterium-mediated floral dip transformation of Brassica napus, for example cv. Westar, more recent protocols that do not require vacuum infiltration are preferred. Verma et al. (2008) and Tan et al. (2011) report such protocols. Verma et al. (2008) recommends growing up the Agrobacterium strain in a selective medium, harvesting the cells by centrifugation and then re-suspending them in transformation medium comprising half MS salts, 5% sucrose, 0.05% Silwet L-77 to obtain the desired density (OD600=0.8 to 2.0). Plants are inoculated by submerging inflorescences in the bacterial suspension for one minute and then the inflorescences are wrapped with Saran wrap for 24 h to maintain the humidity. Seeds are collected at maturity and germinated on a selective medium to identify T1 seedlings by the expression plant marker encoded in the T-DNA.

[0092] A variant of this protocol is described by (Tan et al., 2011). Agrobacterium cultures carrying a target construct are collected by centrifugation and then resuspended in a solution containing 0.53 MS salts, 3% Sucrose, 0.1% Silwet L-77, 2 mg/L 6-benzyladenine, and 8 mg/L acetosyringone. The inflorescence of flowering plants is dipped into a beaker containing the Agrobacterium culture for 1 to 2 min with gentle agitation, and the treated inflorescence is wrapped with Saran wrap to keep the flowers most. The plants are treated three times at two day intervals, then the plants are allowed to grow to maturation. Seeds harvested from the transformed plants were surface sterilized and sown on the MS medium containing the plant marker encoded in the T-DNA. If kanamycin resistance is the plant marker, 200 mg/L kanamycin is used to screen for putative transformants. The putative transformants are identified upon the initiation of the first pair of green true leaves. Additional protocols for floral dip transformation are listed in Table 3 below.

TABLE-US-00004 TABLE 3 Species Reference Brassica rapa L. ssp chinensis (Qing et al., 2000) Brassica campestris L. ssp (Liu et al., 1998) chinensis B. napus (Wang et al., 2003; Wang et al., 2005; Tan et al., 2011) B. napus, B. carinata, high (Verma et al., 2008) freq. Camelina sativa (Lu and Kang, 2008)

[0093] There are several Agrobacterium vector systems that have been described for CRISPR/Cas mutagenesis in plants (Belhaj et al., 2013; Li et al., 2014). We prefer the system described by Mao et al. for its simplicity (Mao et al., 2013). When a population of homozygous ACC2 knockout or biallelic mutant population is obtained, the seeds will be germinated on spectinomycin medium to identify the ACC2 defective plants by spectinomycin sensitivity (Parker et al., 2014). The type of knockout mutation will be verified by sequencing the target region and the progeny will be used as recipient in chloroplast transformation experiments. Brassica juncea is also an oilseed crop. The genomics of this crop is relatively undeveloped. However, guide RNAs to knockout the ACC2 gene can be designed using the principles outlined for the other Brassicaceae species as described herein above.

TABLE-US-00005 TABLE 3 Identification of guide RNAs in the N-terminal extension of the ACC2 gene in Brassicaceae species. Shown are the N-terminal protein sequence, the corresponding cDNA sequence and the potential gRNAs. >A. thaliana ACC2 (At1g36180) >MEMRALGSSCSTGNGGSAPITLTNISPWITTVFPSTVKLRSSLRTFKGVSSRVRTFKGVS STRVLSRTKQQFPLFCFLNPDPISFLENDVSEAERTVVLP (SEQ ID NO: 5) >ATGGAGATGAGAGCTTTGGGTTCTTCGTGTTCTACTGGTAATGGAGGTTCTGCTCC GATTACCCTCACGAATATATCTCCATGGATCACAACAGTTTTTCCGTCGACAGTGAA GCTGAGAAGTAGTTTGAGAACCTTCAAAGGAGTTTCGTCAAGAGTGAGAACCTTTA AAGGAGTTTCTTCGACAAGAGTTTTGTCTCGGACCAAACAACAGTTTCCTCTGTTTTG TTTCCTAAACCCTGATCCGATCTCCTTCTTGGAAAATGATGTATCTGAAGCTGAAAG GACAGTAGTTTTACCG (SEQ ID NO: 6) For potential gRNAs, see SEQ ID NO. 239-254 below. >A. lyrata ACC2 (XM_GG2891167.1) >MEMRALVSSCATGNGGSDPFSFTKVSPWITTVGGKDRDFPTTVKLRTSMRTFKGVSIR GRTFKGVSTRVLSRNKQQFPLFCFLNPDPTSFRDNDISEAQR (SEQ ID NO: 7) >5'-3' ATGGAGATGAGAGCTTTGGTTTCTTCGTGTGCTACCGGTAATGGAGGTTCTGATCCG TTTAGCTTCACGAAAGTTTCTCCATGGATCACAACAGTTGGTGGTAAGGACAGAGAT TTTCCAACGACAGTGAAGCTAAGAACTAGTATGAGAACCTTTAAAGGAGTTTCTATA AGAGGGAGAACCTTTAAAGGAGTTTCGACAAGAGTTTTGTCTCGGAACAAACAACA GTTTCCTCTGTTTTGTTTCCTAAACCCTGATCCGACCTCCTTCCGGGATAATGATATA TCTGAAGCTCAAAGG (SEQ ID NO: 8) TTGGTTTCTTCGTGTGCTAC CGG (SEQ ID NO: 9) TCTTCGTGTGCTACCGGTAA TGG (SEQ ID NO: 10) TCGTGTGCTACCGGTAATGG AGG (SEQ ID NO: 11) GCTTCACGAAAGTTTCTCCA TGG (SEQ ID NO: 12) TCTCCATGGATCACAACAGT TGG (SEQ ID NO: 13) CCATGGATCACAACAGTTGG TGG (SEQ ID NO: 14) GATCACAACAGTTGGTGGTA AGG (SEQ ID NO: 15) ACTAGTATGAGAACCTTTAA AGG (SEQ ID NO: 16) TTTAAAGGAGTTTCTATAAG AGG (SEQ ID NO: 17) ATAAGAGGGAGAACCTTTAA AGG (SEQ ID NO: 18) TTTCGACAAGAGTTTTGTCT CGG (SEQ ID NO: 19) ACCCTGATCCGACCTCCTTC CGG (SEQ ID NO: 20) ATGATATATCTGAAGCTCAA AGG (SEQ ID NO: 21) >3'-5' CCTTTGAGCTTCAGATATATCATTATCCCGGAAGGAGGTCGGATCAGGGTTTAGGAA ACAAAACAGAGGAAACTGTTGTTTGTTCCGAGACAAAACTCTTGTCGAAACTCCTTT AAAGGTTCTCCCTCTTATAGAAACTCCTTTAAAGGTTCTCATACTAGTTCTTAGCTTC ACTGTCGTTGGAAAATCTCTGTCCTTACCACCAACTGTTGTGATCCATGGAGAAACT TTCGTGAAGCTAAACGGATCAGAACCTCCATTACCGGTAGCACACGAAGAAACCAA AGCTCTCATCTCCAT (SEQ ID NO: 22) CTTCAGATATATCATTATCC CGG (SEQ ID NO: 23) AGATATATCATTATCCCGGA AGG (SEQ ID NO: 24) TATATCATTATCCCGGAAGG AGG (SEQ ID NO: 25) TCATTATCCCGGAAGGAGGT CGG (SEQ ID NO: 26) TCCCGGAAGGAGGTCGGATC AGG (SEQ ID NO: 27) CCCGGAAGGAGGTCGGATCA GGG (SEQ ID NO: 28) AGGAGGTCGGATCAGGGTTT AGG (SEQ ID NO: 29) GGGTTTAGGAAACAAAACAG AGG (SEQ ID NO: 30) TCTTGTCGAAACTCCTTTAA AGG (SEQ ID NO: 31) TCTTATAGAAACTCCTTTAA AGG (SEQ ID NO: 32) GTTCTTAGCTTCACTGTCGT TGG (SEQ ID NO: 33) CCACCAACTGTTGTGATCCA TGG (SEQ ID NO: 34) GAAACTTTCGTGAAGCTAAA CGG (SEQ ID NO: 35) CGGATCAGAACCTCCATTAC CGG (SEQ ID NO: 36) >C. sativa ACC2-1(LOC104777495) >MEMRALVSSYSTGNGGSDPISLTNGSPWITTVGGGASTMDREFPLTVKLGSSMRAFKG VSTTTVLSRTKQQFPLVCLARNNANSTDPTSFWENDISEVQR (SEQ ID NO: 37) >5'-3' ATGGAGATGAGAGCTTTGGTTTCTTCGTATTCTACCGGTAATGGAGGTTCTGATCCG ATCAGCCTCACGAATGGTTCTCCATGGATCACAACAGTTGGTGGTGGTGCAAGTACC ATGGACAGAGAGTTTCCATTGACTGTGAAGCTGGGAAGTAGTATGAGAGCCTTCAA AGGAGTAAGCACAACAACAGTTTTGTCTCGGACCAAACAACAGTTTCCTCTGGTATG CTTAGCAAGAAACAATGCGAACAGCACTGATCCGACCTCGTTCTGGGAGAATGATA TATCTGAAGTTCAAAGG (SEQ ID NO: 38) TTGGTTTCTTCGTATTCTAC CGG (SEQ ID NO: 39) TCTTCGTATTCTACCGGTAA TGG (SEQ ID NO: 40) TCGTATTCTACCGGTAATGG AGG (SEQ ID NO: 41) GATCCGATCAGCCTCACGAA TGG (SEQ ID NO: 42) GCCTCACGAATGGTTCTCCA TGG (SEQ ID NO: 43) TCTCCATGGATCACAACAGT TGG (SEQ ID NO: 44) CCATGGATCACAACAGTTGG TGG (SEQ ID NO: 45) TGGATCACAACAGTTGGTGG TGG (SEQ ID NO: 46) TGGTGGTGGTGCAAGTACCA TGG (SEQ ID NO: 47) GTTTCCATTGACTGTGAAGC TGG (SEQ ID NO: 48) AGTAGTATGAGAGCCTTCAA AGG (SEQ ID NO: 49) GCACAACAACAGTTTTGTCT CGG (SEQ ID NO: 50) GACCAAACAACAGTTTCCTC TGG (SEQ ID NO: 51) GCACTGATCCGACCTCGTTC TGG (SEQ ID NO: 52) ATGATATATCTGAAGTTCAA AGG (SEQ ID NO: 53) >3'-5' CCTTTGAACTTCAGATATATCATTCTCCCAGAACGAGGTCGGATCAGTGCTGTTCGC ATTGTTTCTTGCTAAGCATACCAGAGGAAACTGTTGTTTGGTCCGAGACAAAACTGT TGTTGTGCTTACTCCTTTGAAGGCTCTCATACTACTTCCCAGCTTCACAGTCAATGGA AACTCTCTGTCCATGGTACTTGCACCACCACCAACTGTTGTGATCCATGGAGAACCA TTCGTGAGGCTGATCGGATCAGAACCTCCATTACCGGTAGAATACGAAGAAACCAA AGCTCTCATCTCCAT (SEQ ID NO: 54) TATATCATTCTCCCAGAACG AGG (SEQ ID NO: 55) TCATTCTCCCAGAACGAGGT CGG (SEQ ID NO: 56) TTTCTTGCTAAGCATACCAG AGG (SEQ ID NO: 57) TACCAGAGGAAACTGTTGTT TGG (SEQ ID NO: 58) TGTTGTGCTTACTCCTTTGA AGG (SEQ ID NO: 59) CTTCCCAGCTTCACAGTCAA TGG (SEQ ID NO: 60) CAATGGAAACTCTCTGTCCA TGG (SEQ ID NO: 61) CCACCAACTGTTGTGATCCA TGG (SEQ ID NO: 62) TCCATGGAGAACCATTCGTG AGG (SEQ ID NO: 63) GAACCATTCGTGAGGCTGAT CGG (SEQ ID NO: 64) CGGATCAGAACCTCCATTAC CGG (SEQ ID NO: 65) >C. sativa ACC2-2 (LOC104742086) variant1 >MEMRALVSSCSTGNGGSDPISLTNGSPWITTVGGGASTMDREFPATVKLGSSMRAFKG VSTITVLSRTKQQFPLVCLARNNGNSTDPTSFWENDISETQR (SEQ ID NO: 66) >5'-3' ATGGAGATGAGAGCTTTGGTTTCTTCGTGTTCTACGGGGAATGGAGGGTCTGATCCG ATCAGCCTCACGAATGGTTCTCCATGGATCACAACAGTTGGTGGTGGTGCAAGTACC ATGGACAGAGAGTTTCCAGCGACTGTGAAGCTGGGAAGTAGTATGAGAGCCTTCAA AGGAGTAAGCACAATAACAGTTCTGTCTCGGACCAAACAACAGTTTCCTCTGGTATG CTTAGCAAGAAACAACGGAAACAGCACTGATCCGACCTCGTTCTGGGAGAACGATA TATCTGAAACTCAAAGG (SEQ ID NO: 67) TTTGGTTTCTTCGTGTTCTA CGG (SEQ ID NO: 68) TTGGTTTCTTCGTGTTCTAC GGG (SEQ ID NO: 69) TGGTTTCTTCGTGTTCTACG GGG (SEQ ID NO: 70) TCTTCGTGTTCTACGGGGAA TGG (SEQ ID NO: 71) TCGTGTTCTACGGGGAATGG AGG (SEQ ID NO: 72) GATCCGATCAGCCTCACGAA TGG (SEQ ID NO: 73) GCCTCACGAATGGTTCTCCA TGG (SEQ ID NO: 74) TCTCCATGGATCACAACAGT TGG (SEQ ID NO: 75) CCATGGATCACAACAGTTGG TGG (SEQ ID NO: 76) TGGATCACAACAGTTGGTGG TGG (SEQ ID NO: 77) TGGTGGTGGTGCAAGTACCA TGG (SEQ ID NO: 78) GTTTCCAGCGACTGTGAAGC TGG (SEQ ID NO: 79) AGTAGTATGAGAGCCTTCAA AGG (SEQ ID NO: 80) GCACAATAACAGTTCTGTCT CGG (SEQ ID NO: 81) GACCAAACAACAGTTTCCTC TGG (SEQ ID NO: 82) GTATGCTTAGCAAGAAACAA CGG (SEQ ID NO: 83) GCACTGATCCGACCTCGTTC TGG (SEQ ID NO: 84) ACGATATATCTGAAACTCAA AGG (SEQ ID NO: 85) >3'-5' CCTTTGAGTTTCAGATATATCGTTCTCCCAGAACGAGGTCGGATCAGTGCTGTTTCCG TTGTTTCTTGCTAAGCATACCAGAGGAAACTGTTGTTTGGTCCGAGACAGAACTGTT ATTGTGCTTACTCCTTTGAAGGCTCTCATACTACTTCCCAGCTTCACAGTCGCTGGAA ACTCTCTGTCCATGGTACTTGCACCACCACCAACTGTTGTGATCCATGGAGAACCAT TCGTGAGGCTGATCGGATCAGACCCTCCATTCCCCGTAGAACACGAAGAAACCAAA GCTCTCATCTCCAT (SEQ ID NO: 86) TATATCGTTCTCCCAGAACG AGG (SEQ ID NO: 87) TCGTTCTCCCAGAACGAGGT CGG (SEQ ID NO: 88) TTTCTTGCTAAGCATACCAG AGG (SEQ ID NO: 89) TACCAGAGGAAACTGTTGTT TGG (SEQ ID NO: 90) TATTGTGCTTACTCCTTTGA AGG (SEQ ID NO: 91) CTTCCCAGCTTCACAGTCGC TGG (SEQ ID NO: 92) CGCTGGAAACTCTCTGTCCA TGG (SEQ ID NO: 93) CCACCAACTGTTGTGATCCA TGG (SEQ ID NO: 94) TCCATGGAGAACCATTCGTG AGG (SEQ ID NO: 95) GAACCATTCGTGAGGCTGAT CGG (SEQ ID NO: 96) >C. rubella ACC2(CARUB_v1GG08063mg) >MEMRALVSSCSTGNGGSDPISLTNVSPWITTVGGGASSIDREFPTTVKLGSSLRTFKGVS STTVLSRTKQQFPLVCLARNNANSTDPTLFWENDISEAQS (SEQ ID NO: 97) >5'-3'

ATGGAGATGAGAGCTTTGGTTTCTTCGTGTTCTACCGGTAATGGAGGTTCTGATCCG ATTAGCCTCACGAATGTTTCTCCATGGATCACAACAGTTGGTGGTGGTGCAAGTTCC ATTGACAGAGAGTTTCCAACGACTGTGAAGCTGGGAAGTAGTCTGAGAACTTTCAA AGGAGTAAGCTCTACGACAGTTTTGTCTCGGACCAAACAACAGTTTCCTCTGGTTTG TTTAGCAAGAAACAATGCCAACAGCACTGATCCAACCTTGTTCTGGGAAAATGACAT ATCTGAAGCTCAAAGC (SEQ ID NO: 98) TTGGTTTCTTCGTGTTCTAC CGG (SEQ ID NO: 99) TCTTCGTGTTCTACCGGTAA TGG (SEQ ID NO: 100) TCGTGTTCTACCGGTAATGG AGG (SEQ ID NO: 101) GCCTCACGAATGTTTCTCCA TGG (SEQ ID NO: 102) TCTCCATGGATCACAACAGT TGG (SEQ ID NO: 103) CCATGGATCACAACAGTTGG TGG (SEQ ID NO: 104) TGGATCACAACAGTTGGTGG TGG (SEQ ID NO: 101) GTTTCCAACGACTGTGAAGC TGG (SEQ ID NO: 105) AGTAGTCTGAGAACTTTCAA AGG (SEQ ID NO: 106) GCTCTACGACAGTTTTGTCT CGG (SEQ ID NO: 107) GACCAAACAACAGTTTCCTC TGG (SEQ ID NO: 108) GCACTGATCCAACCTTGTTC TGG (SEQ ID NO: 109) >3'-5' GCTTTGAGCTTCAGATATGTCATTTTCCCAGAACAAGGTTGGATCAGTGCTGTTGGC ATTGTTTCTTGCTAAACAAACCAGAGGAAACTGTTGTTTGGTCCGAGACAAAACTGT CGTAGAGCTTACTCCTTTGAAAGTTCTCAGACTACTTCCCAGCTTCACAGTCGTTGGA AACTCTCTGTCAATGGAACTTGCACCACCACCAACTGTTGTGATCCATGGAGAAACA TTCGTGAGGCTAATCGGATCAGAACCTCCATTACCGGTAGAACACGAAGAAACCAA AGCTCTCATCTCCAT (SEQ ID NO: 110) TATGTCATTTTCCCAGAACA AGG (SEQ ID NO: 111) TCATTTTCCCAGAACAAGGT TGG (SEQ ID NO: 112) CAAGGTTGGATCAGTGCTGT TGG (SEQ ID NO: 113) TTTCTTGCTAAACAAACCAG AGG (SEQ ID NO: 114) AACCAGAGGAAACTGTTGTT TGG (SEQ ID NO: 115) CTTCCCAGCTTCACAGTCGT TGG (SEQ ID NO: 116) CGTTGGAAACTCTCTGTCAA TGG (SEQ ID NO: 117) CCACCAACTGTTGTGATCCA TGG (SEQ ID NO: 118) TCCATGGAGAAACATTCGTG AGG (SEQ ID NO: 119) GAAACATTCGTGAGGCTAAT CGG (SEQ ID NO: 120) CGGATCAGAACCTCCATTAC CGG (SEQ ID NO: 121) >B. oleracea ACC2 (LOC106301042) >MEMRALVSCSAAGNGASDRFRLSNVSPWITSARGASGSDSPATVKLRSSSMIRAFKGV SIYKNKTRRNVLSQRNKQFRPMAYLGRKDLSSPDPTSFCDND (SEQ ID NO: 122) >5'-3' ATGGAGATGAGAGCTTTGGTTTCGTGTTCTGCTGCCGGAAATGGAGCTTCTGATCGG TTTAGACTCTCCAATGTTTCACCATGGATCACATCTGCTCGTGGTGCAAGTGGCAGT GACTCCCCAGCCACAGTGAAGCTGAGAAGCAGCTCTATGATTAGAGCTTTCAAAGG AGTTTCGATTTACAAAAACAAGACCAGAAGAAATGTTCTGTCTCAAAGGAACAAAC AGTTCCGTCCTATGGCCTACTTAGGAAGGAAGGACTTGAGCAGCCCTGATCCGACCT CCTTCTGCGATAATGAT (SEQ ID NO: 123) TTGGTTTCGTGTTCTGCTGC CGG (SEQ ID NO: 124) TCGTGTTCTGCTGCCGGAAA TGG (SEQ ID NO: 125) CCGGAAATGGAGCTTCTGAT CGG (SEQ ID NO: 126) GACTCTCCAATGTTTCACCA TGG (SEQ ID NO: 127) CCATGGATCACATCTGCTCG TGG (SEQ ID NO: 128) ACATCTGCTCGTGGTGCAAG TGG (SEQ ID NO: 129) TCTATGATTAGAGCTTTCAA AGG (SEQ ID NO: 130) GAAGAAATGTTCTGTCTCAA AGG (SEQ ID NO: 131) GAACAAACAGTTCCGTCCTA TGG (SEQ ID NO: 132) TTCCGTCCTATGGCCTACTT AGG (SEQ ID NO: 133) GTCCTATGGCCTACTTAGGA AGG (SEQ ID NO: 134) TATGGCCTACTTAGGAAGGA AGG (SEQ ID NO: 135) 3'-5' ATCATTATCGCAGAAGGAGGTCGGATCAGGGCTGCTCAAGTCCTTCCTTCCTAAGTA GGCCATAGGACGGAACTGTTTGTTCCTTTGAGACAGAACATTTCTTCTGGTCTTGTTT TTGTAAATCGAAACTCCTTTGAAAGCTCTAATCATAGAGCTGCTTCTCAGCTTCACTG TGGCTGGGGAGTCACTGCCACTTGCACCACGAGCAGATGTGATCCATGGTGAAACA TTGGAGAGTCTAAACCGATCAGAAGCTCCATTTCCGGCAGCAGAACACGAAACCAA AGCTCTCATCTCCAT (SEQ ID NO: 136) TCATTATCGCAGAAGGAGGT CGG (SEQ ID NO: 137) TCGCAGAAGGAGGTCGGATC AGG (SEQ ID NO: 138) CAAGTCCTTCCTTCCTAAGT AGG (SEQ ID NO: 139) TTCCTTCCTAAGTAGGCCAT AGG (SEQ ID NO: 140) TTCCTAAGTAGGCCATAGGA CGG (SEQ ID NO: 141) TTGAGACAGAACATTTCTTC TGG (SEQ ID NO: 143) GCTGCTTCTCAGCTTCACTG TGG (SEQ ID NO: 144) CTTCTCAGCTTCACTGTGGC TGG (SEQ ID NO: 145) TTCTCAGCTTCACTGTGGCT GGG (SEQ ID NO: 146) TCTCAGCTTCACTGTGGCTG GGG (SEQ ID NO: 147) CCACGAGCAGATGTGATCCA TGG (SEQ ID NO: 148) TGTGATCCATGGTGAAACAT TGG (SEQ ID NO: 149) CCGATCAGAAGCTCCATTTC CGG (SEQ ID NO: 150) >B. napus ACC2-1: Y10302 MEMRALVSCSAAGNGASDRFRLSNVSPWITSARGASGSDSPATVKLGSSSMIRAFKGVS IYKNKTRRNVLSQRNKQFRPMAYLGRKDLSSPDPTSFCDNDISEPQGTGSINGNDHSAV RVSQVDEFCKAHGGKRPIHSILVATNGMAAVKLIRSVRAWSYQTFGSEKSISLVAMATP EDMRINAEHIRIADQFMQVPGGTNNNNYANVHLIVEMAQATGVDAVWPGWGHASENP ELPDALKAKGVIFLGPTAASMLALGDKIGSSLIAQAADVPTLPWSGSHVKIPPGSSMVTIP EEMYRQACVYTTEEAVASCQVVGYPAMIKASWGGGGKGIREVHDDDEVRTLFKQVQG EVPGSPIFIMKVASQSRHL (SEQ ID NO: 151) >5'-3' 1.sup.ST EXON ATGGAGATGAGAGCTTTAGTTTCGTGTTCTGCTGCCGGAAATGGAGCTTCTGATCGG TTTAGACTCTCCAATGTTTCACCATGGATCACATCAGCTCGTGGTGCAAGTGGCAGT GACTCCCCAGCCACAGTGAAGCTGGGAAGCAGCTCTATGATTAGAGCTTTCAAAGG CGTTTCGATTTACAAAAACAAGACCAGAAGGAATGTTCTGTCTCAAAGGAACAAAC AGTTCCGTCCTATGGCCTACTTAGGAAGGAAGGACTTGAGCAGCCCTGATCCGACCT CCTTCTGCGATAATG (SEQ ID NO: 152) TTAGTTTCGTGTTCTGCTGC CGG (SEQ ID NO: 153) TCGTGTTCTGCTGCCGGAAA TGG (SEQ ID NO: 154) CCGGAAATGGAGCTTCTGAT CGG (SEQ ID NO: 155) GACTCTCCAATGTTTCACCA TGG (SEQ ID NO: 156) CCATGGATCACATCAGCTCG TGG (SEQ ID NO: 157) ACATCAGCTCGTGGTGCAAG TGG (SEQ ID NO: 158) CTCCCCAGCCACAGTGAAGC TGG (SEQ ID NO: 159) TCCCCAGCCACAGTGAAGCT GGG (SEQ ID NO: 160) TCTATGATTAGAGCTTTCAA AGG (SEQ ID NO: 161) TTTACAAAAACAAGACCAGA AGG (SEQ ID NO: 162) GAAGGAATGTTCTGTCTCAA AGG (SEQ ID NO: 163) GAACAAACAGTTCCGTCCTA TGG (SEQ ID NO: 164) TTCCGTCCTATGGCCTACTT AGG (SEQ ID NO: 165) GTCCTATGGCCTACTTAGGA AGG (SEQ ID NO: 166) TATGGCCTACTTAGGAAGGA AGG (SEQ ID NO: 167) >3'-5' CATTATCGCAGAAGGAGGTCGGATCAGGGCTGCTCAAGTCCTTCCTTCCTAAGTAGG CCATAGGACGGAACTGTTTGTTCCTTTGAGACAGAACATTCCTTCTGGTCTTGTTTTT GTAAATCGAAACGCCTTTGAAAGCTCTAATCATAGAGCTGCTTCCCAGCTTCACTGT GGCTGGGGAGTCACTGCCACTTGCACCACGAGCTGATGTGATCCATGGTGAAACATT GGAGAGTCTAAACCGATCAGAAGCTCCATTTCCGGCAGCAGAACACGAAACTAAAG CTCTCATCTCCAT (SEQ ID NO: 168) TCGCAGAAGGAGGTCGGATC AGG (SEQ ID NO: 169) CGCAGAAGGAGGTCGGATCA GGG (SEQ ID NO: 170) CAAGTCCTTCCTTCCTAAGT AGG (SEQ ID NO: 171) TTCCTTCCTAAGTAGGCCAT AGG (SEQ ID NO: 172) TTCCTAAGTAGGCCATAGGA CGG (SEQ ID NO: 173) TTGAGACAGAACATTCCTTC TGG (SEQ ID NO: 174) CTTCCCAGCTTCACTGTGGC TGG (SEQ ID NO: 175) TTCCCAGCTTCACTGTGGCTGGG (SEQ ID NO: 176) TCCCAGCTTCACTGTGGCTG GGG (SEQ ID NO: 177) CCACGAGCTGATGTGATCCA TGG (SEQ ID NO: 178) TGTGATCCATGGTGAAACAT TGG (SEQ ID NO: 179) CCGATCAGAAGCTCCATTTC CGG (SEQ ID NO: 180) >B. napus ACC2-2: X77576 MEMRALVSCSAAGNGASDRFRLSNVSPWITSARGASGSDSPATVKLGSSSMIRAFKGVS IYKNKTRRNVLSQRNKQFRPMAYLGRKDLSSPDPTSFCDNDISEPQGTGSINGNDHSAV RVSQVDEFCKAHGGKRPIHRILVATNGMAAVKFIRSVRAWSYQTFGSEKSISLVAMATP EDMRINAEHIRIADQFMQVPGGTNNNNYANVHLIVEMAEATGVDAVWPGWGHASENP ELPDALKAKGVIFLGPTAASMLALGDKIGSSLIAQAADVPTLPWSGSHVKIPPGSSLVTIP EEMYRQACVYTTEEAVASCQVVGYPAMIKASWGGGGKGIRKVHDDDEVRALFKQVQ GEVPGSPIFIMKVASQSRHLEVQLLCDQYGNVSALHSRDCSVQRRHQKIIEEGPITVAPR DTVKKLEQAARRLAKSVNYVGAATVEFLYSMDTGDYFFLELNPR (SEQ ID NO: 181) >5'-3' 1.sup.ST EXON ATGGAGATGAGAGCTTTGGTTTCGTGTTCTGCTGCCGGAAATGGAGCTTCTGATCGG TTTAGACTCTCCAATGTTTCACCATGGATCACATCAGCTCGTGGTGCAAGTGGCAGT GACTCCCCAGCCACAGTGAAGCTGGGAAGCAGCTCTATGATCAGAGCCTTCAAAGG AGTTTCGATTTACAAAAACAAGACCAGAAGAAATGTTTTGTCTCAAAGGAACAAAC AGTTTCGTCCTATGGCCTACTTAGGAAGGAAGGACTTGAGCAGCCCTGATCCGACCT CCTTCTGCGATAATG (SEQ ID NO: 182) TTGGTTTCGTGTTCTGCTGC CGG (SEQ ID NO: 183) TCGTGTTCTGCTGCCGGAAA TGG (SEQ ID NO: 184) CCGGAAATGGAGCTTCTGAT CGG (SEQ ID NO: 185) GACTCTCCAATGTTTCACCA TGG (SEQ ID NO: 186) CCATGGATCACATCAGCTCG TGG (SEQ ID NO: 187) ACATCAGCTCGTGGTGCAAG TGG (SEQ ID NO: 188) CTCCCCAGCCACAGTGAAGC TGG (SEQ ID NO: 189) TCCCCAGCCACAGTGAAGCT GGG (SEQ ID NO: 190) TCTATGATCAGAGCCTTCAA AGG (SEQ ID NO: 191) GAAGAAATGTTTTGTCTCAA AGG (SEQ ID NO: 192) GAACAAACAGTTTCGTCCTA TGG (SEQ ID NO: 193) TTTCGTCCTATGGCCTACTT AGG (SEQ ID NO: 194)

GTCCTATGGCCTACTTAGGA AGG (SEQ ID NO: 195) TATGGCCTACTTAGGAAGGA AGG (SEQ ID NO: 196) >3'-5' CATTATCGCAGAAGGAGGTCGGATCAGGGCTGCTCAAGTCCTTCCTTCCTAAGTAGG CCATAGGACGAAACTGTTTGTTCCTTTGAGACAAAACATTTCTTCTGGTCTTGTTTTT GTAAATCGAAACTCCTTTGAAGGCTCTGATCATAGAGCTGCTTCCCAGCTTCACTGT GGCTGGGGAGTCACTGCCACTTGCACCACGAGCTGATGTGATCCATGGTGAAACATT GGAGAGTCTAAACCGATCAGAAGCTCCATTTCCGGCAGCAGAACACGAAACCAAAG CTCTCATCTCCAT (SEQ ID NO: 197) TCGCAGAAGGAGGTCGGATC AGG (SEQ ID NO: 198) CGCAGAAGGAGGTCGGATCA GGG (SEQ ID NO: 199) CAAGTCCTTCCTTCCTAAGT AGG (SEQ ID NO: 200) TTCCTTCCTAAGTAGGCCAT AGG (SEQ ID NO: 201) TTGAGACAAAACATTTCTTC TGG (SEQ ID NO: 202) GTAAATCGAAACTCCTTTGA AGG (SEQ ID NO: 203) GCTGCTTCCCAGCTTCACTG TGG (SEQ ID NO: 204) CTTCCCAGCTTCACTGTGGC TGG (SEQ ID NO: 205) TTCCCAGCTTCACTGTGGCT GGG (SEQ ID NO: 206) TCCCAGCTTCACTGTGGCTG GGG (SEQ ID NO: 207) CCACGAGCTGATGTGATCCA TGG (SEQ ID NO: 208) TGTGATCCATGGTGAAACAT TGG (SEQ ID NO: 209) CCGATCAGAAGCTCCATTTC CGG (SEQ ID NO: 210) >B. rapa ACC2 (LOC1038715GG) >MEMRALVSCSAAGNGASDRFRLSNVSPWITSARGASGSDSPATVKLGSSSMIRAFKGV SIYKNKSRRNVLSQRNKQFRPMAYLGRKDLSSPDPTSFCDND (SEQ ID NO: 211) >5'-3' ATGGAGATGAGAGCTTTGGTTTCGTGTTCTGCTGCCGGAAATGGAGCTTCTGATCGG TTTAGACTCTCCAATGTTTCACCATGGATCACATCAGCTCGTGGTGCAAGTGGCAGT GACTCCCCAGCCACAGTGAAGCTGGGAAGCAGCTCTATGATCAGAGCCTTCAAAGG AGTTTCGATTTACAAAAACAAGAGCAGAAGAAATGTTCTGTCTCAAAGGAACAAAC AGTTTCGTCCTATGGCCTACTTAGGAAGGAAGGACTTGAGCAGCCCTGATCCGACCT CCTTCTGCGATAATGAT (SEQ ID NO: 212) TTGGTTTCGTGTTCTGCTGC CGG (SEQ ID NO: 213) TCGTGTTCTGCTGCCGGAAA TGG (SED ID NO: 214) CCGGAAATGGAGCTTCTGAT CGG (SED ID NO: 215) GACTCTCCAATGTTTCACCA TGG (SED ID NO: 216) CCATGGATCACATCAGCTCG TGG (SED ID NO: 217) ACATCAGCTCGTGGTGCAAG TGG (SED ID NO: 218) CTCCCCAGCCACAGTGAAGC TGG (SED ID NO: 219) TCTATGATCAGAGCCTTCAA AGG (SED ID NO: 220) GAAGAAATGTTCTGTCTCAA AGG (SED ID NO: 221) TTTCGTCCTATGGCCTACTT AGG (SED ID NO: 223) GTCCTATGGCCTACTTAGGA AGG (SED ID NO: 224) TATGGCCTACTTAGGAAGGA AGG (SED ID NO: 225) 3'-5' ATCATTATCGCAGAAGGAGGTCGGATCAGGGCTGCTCAAGTCCTTCCTTCCTAAGTA GGCCATAGGACGAAACTGTTTGTTCCTTTGAGACAGAACATTTCTTCTGCTCTTGTTT TTGTAAATCGAAACTCCTTTGAAGGCTCTGATCATAGAGCTGCTTCCCAGCTTCACT GTGGCTGGGGAGTCACTGCCACTTGCACCACGAGCTGATGTGATCCATGGTGAAAC ATTGGAGAGTCTAAACCGATCAGAAGCTCCATTTCCGGCAGCAGAACACGAAACCA AAGCTCTCATCTCCAT (SED ID NO: 226) TCATTATCGCAGAAGGAGGT CGG (SED ID NO: 227) TCGCAGAAGGAGGTCGGATC AGG (SED ID NO: 228) CAAGTCCTTCCTTCCTAAGT AGG (SED ID NO: 229) TTCCTTCCTAAGTAGGCCAT AGG (SED ID NO: 230) GTAAATCGAAACTCCTTTGA AGG (SED ID NO: 231) GCTGCTTCCCAGCTTCACTG TGG (SED ID NO: 232) CTTCCCAGCTTCACTGTGGC TGG (SED ID NO: 233) TTCCCAGCTTCACTGTGGCT GGG (SED ID NO: 234) TCCCAGCTTCACTGTGGCTG GGG (SED ID NO: 235) CCACGAGCTGATGTGATCCA TGG (SED ID NO: 236) TGTGATCCATGGTGAAACAT TGG (SED ID NO: 237) CCGATCAGAAGCTCCATTTC CGG (SED ID NO: 238)

Deletion of ACC2 Gene in Regenerable RLD and Ws Arabidopsis Ecotypes

[0094] The bacterial clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) defense system has been rapidly developed as a genome-engineering tool (Belhaj et al., 2013; Mali et al., 2013; Li et al., 2014). In this approach a small RNA guides the Cas9 nuclease to the target site. The nick is then repaired by non-homologous end joining, the process most often resulting in a one-nucleotide insertion or deletion in Arabidopsis thaliana (Feng et al., 2014). Because our objective is knocking out the ACC2 gene, we used the same system, an Agrobacterium binary transformation vector in which the sgRNA is transcribed under the control of Arabidopsis U6 snoRNA promoter (pAtU6) and Cas9 is expressed from the Arabidopsis ubiquitin promoter (pAtUBQ1) (Mao et al., 2013).

[0095] The 16 guide strands provided below are suitable for this approach.

TABLE-US-00006 sequence (SEQ ID NO: 239) TCCATGCAGATATATTCGTG AGG (SEQ ID NO: 240) CCCTCACGAATATATCTCCA TGG (SEQ ID NO: 241) GATATATTCGTGAGGGTAAT TGG (SEQ ID NO: 242) CTTCTCAGCTTCACTGTCGA CGG (SEQ ID NO: 243) CCATGGAGATATATTCGTGA GGG (SEQ ID NO: 244) CTTCGACAAGAGTTTTGTCT CGG (SEQ ID NO: 245) TCAAGAGTGAGAACCTTTAA AGG (SEQ ID NO: 246) TCTTCGTGTTCTACTGGTAA TGG (SEQ ID NO: 247) TTGGGTTCTTCGTGTTCTAC TGG (SEQ ID NO: 248) TGTCGAAGAAACTCCTTTAA AGG (SEQ ID NO: 249) TCTTGACGAAACTCCTTTGA AGG (SEQ ID NO: 250) AGTAGTTTGAGAACCTTCAA AGG (SEQ ID NO: 251) TCGTGTTCTACTGGTAATGG AGG (SEQ ID NO: 252) GGAAAAACTGTTGTGATCCA TGG (SEQ ID NO: 253) AAACAGAGGAAACTGTTGTT TGG (SEQ ID NO: 254) GGGTTTAGGAAACAAAACAG AGG

[0096] The target for mutagenesis was exon-1 of the ACC2 coding region encoding the chloroplast transit peptide. This N-terminal extension is absent in the ACC1 gene, which targets its product to the cytoplasm. To design the targeting region of the guide RNA, 240 nucleotides of ACC2 exon-1 were pasted into the guide RNA design at the MIT Optimized CRISPR Design website. The RLD and Ws sequence has a one-nucleotide (A instead of G) mismatch compared to Columbia, a sequence variation that was be considered when designing the sgRNA. The closest off target site in the Arabidopsis genome has three mismatches with this target site.

[0097] To target the ACC2 sequence CCCTCACGAATATATCTCCATGG (2.sup.nd target site in the list; SEQ ID NO: 240), we cloned two annealed oligonucleotides that form the target site in BbsI-digested CRISPR/Cas9 cassette psgR-Cas9-At (Mao et al., 2013). The oligonucleotides were gattgCCTCACGAATATATCTCCA (SEQ ID NO: 255), and aaacTGGAGATATATTCGTGAGGc (SEQ ID NO: 256). The CRISPR/Cas9 cassette was then cloned in a pCAMBIA2300 Agrobacterium binary vector and introduced into Arabidopsis by the flower dip protocol (Clough and Bent, 1998). Plants transformed with the CRISPR/Cas9 construct were selected by germinating seeds on kanamycin medium (100 mg/L).

[0098] Kanamycin resistant seedlings (T1 generation) were screened for a mutant ACC2 target site by the T7 exonuclease I (T7E1) assay (Xie and Yang, 2013). The T7 endonuclease recognizes and cleaves non-perfectly matched DNA. The ACC2 target region was PCR amplified using forward primer 5'-TCTCTTCCTCCTTAAAAAGCCACA-3' (SEQ ID NO: 257) and reverse primer 5'-CTAGGATTCGAAACCAGCGT-3' (SEQ ID NO: 258) using total cellular DNA as template, the amplicons were denatured, reannealed and treated with T7E1. Mismatch caused by CRISPR/Cas9 mutagenesis resulted in T7E1 cleaving the mismatched DNA, that was visualized by gel electrophoresis.

[0099] Plants carrying mutations in ACC2 gene copies were identified by T7E1 screening the heterozygous T1 seed progeny. Mutations in ACC2 genes were identified in the T2 generation by sequencing PCR amplicons (FIG. 8). The acc2 knockout mutants, in contrast to wild type, do not develop shoot meristem outgrowths when germinated on spectinomycin (Parker et al., 2014). Therefore, we collected seed from the T1 plants, and germinated a small sample on spectinomycin medium to identify non-segregating acc2 knockout populations by spectinomycin sensitivity. An example for seedling spectinomycin hypersensitive reaction is shown in FIG. 9. Note development of primary leaves on the seedlings of the parental Ws line, and the absence of any shoot meristem outgrowth on the hypersensitive Ws-2-22 mutant (Parker et al., 2014). Following this protocol uniform, non-segregating RLD and Ws seed was obtained. Such spectinomycin hypersensitive plants are the suitable recipients for plastid transformation.

Example III

Expression of Heterologous Genes in ACC2-Defective Brassica Spp.

[0100] Reproducible, high-frequency plastid transformation in the Brassicae oilseed and vegetable crops enables plastid genome engineering in spectinomycin hypersensitive Brassica spp. for a variety of biotechnological applications.

[0101] One application is replacement of part or the entire plastid genome with synthetic DNA. For example, the efficiency of sunlight to biomass conversion can be improved by introducing genes or groups of genes from other crop species, algae, and photosynthetic bacteria (Gimpel et al., 2016; Hanson et al., 2016; Sharwood et al., 2016).

[0102] Expression of plastid transgenes throughout the plant is desirable for some applications, for example tolerance to herbicides such as phosphinothricin (PPT) (Lutz et al., 2001; Ye et al., 2003), glyphosate (Ye et al., 2003), sulfonylurea, pyrimidinylcarboxylate (Shimizu et al., 2008) and diketonitrile (Dufourmantel et al., 2007). Equally useful are plastid expression of insecticidal protein genes (U.S. Pat. No. 5,545,818) and double-stranded RNAs that are toxic to insects (Zhang et al., 2015). The herbicide resistance and insecticidal genes are introduced by linkage to the selective spectinomycin resistance (aadA gene) marker. When uniform transformation of plastid genomes is obtained, the marker gene can be excised by a site-specific recombinase that targets sites flanking the marker gene. Various marker excision systems are suitable including the Cre/loxP or PhiC31/Int systems (as described in U.S. Pat. Nos. 7,217,860 and 8,841,511) or the BxB1 (Shao et al., 2014), ParA-MRS, and CinH-Rs2 (Shao et al., 2017) site-specific recombination systems.

[0103] Particularly effective for the recovery of transplastomic events are the PrrnLatpB/TrbcL, PrrnLatpB/TpsbA, PrrnLrbcL/TpsbA, PrrnLT7g10/TrbcL promoter/terminator cassettes (Kuroda and Maliga, 2001, 2001). Genes of interest may also be expressed using cassettes previously described in U.S. Pat. Nos. 5,977,402, 6,297,054, 6, 376, 744, 6,472,568, 6,624,296, 6,987,215, 7,176,355, 8,143,474. FIG. 10 shows a schematic design of a plastid transformation vector having a Brassica napus plastid targeting sequence containing the rm16 targeting region (nucleotides 135473-137978 in GenBank accession KP161617) and carrying a recombinase target site-flanked selectable aadA marker and a gene of interest.

[0104] Tissue-specific expression of plastid genes is desirable but thus far no practical system has been available to achieve this objective. We describe here seed-specific expression of proteins in plastids based on a transgene incorporated in the plastid genome that is regulated by a nuclear gene with a seed-specific promoter. The elements of the system are depicted in FIG. 11A. In a Brassica spp. the transgene encoding green fluorescent protein (or particular gene of interest) is present in the leaf cell, but is not translated in the absence of a modified PPR10 RNA binding protein. The engineered Zea mays PPR10GG protein gene that is required for expression is present in the nucleus, but is not active because it is under the control of a seed-specific Brassica napus napin gene promoter that is not transcribed in the nucleus of leaf cells (Ellerstrom et al., 1996). The native Brassica PPR10 protein (Bn-PPR10) stabilizes and facilitates translation of the atpH mRNA. However, Bn-PPR10 RNA binding protein does not recognize PBS.sup.ZmGG, the mutant maize PPR10 binding site because the 23-nucleotide Brassica binding site differs by 2 nucleotides from the wild-type maize PPR10 binding site and by 4 nucleotides from the mutant maize binding site.

TABLE-US-00007 Zm-PPR10 wt Binding site: (SEQ ID NO: 259) ATTGTATCcTTAACcATTTCTTT Bn-PPR10 wt Binding site: (SEQ ID NO: 260) ATTGTATCATTAACTATTTCTTT Zm-PPR10.sup.GG mut Binding site: (SEQ ID NO: 261) ATTGTAggcTTAACcATTTCTTT

[0105] In the Brassica ssp. seed (embryo) cell, the napin seed storage protein gene promoter is turned on, the mRNA is translated in the cytoplasm and the PPR10.sup.GG protein is imported into chloroplasts where it binds to its cognate binding site upstream of the gfp AUG translation initiation codon. Binding of the Zm-PPR10.sup.GG stabilizes the gfp mRNA and facilitates its translation. The result is high-level GFP protein accumulation in the plastids of embryo cells in oilseed crops.

[0106] To construct the regulated plastid transgenes, the tobacco Prrn promoter is linked up with the 100 nt sequence directly upstream of the maize atpH gene. The two sequences together constitute the 5' regulatory region driving GFP expression. The gfp coding region is followed by the rbcL gene terminator (TrbcL). Prrn-PPR10.sup.GG-GFP-TrbcL corresponds to SEQ ID NO. 262. The transgene is cloned adjacent to an aadA gene in the B. napus-specific plastid transformation vector shown in FIG. 12A. Also shown in FIG. 12A is a variant, where a T-RNA (symbolized with a cloverleaf; SEQ ID NO. 263) is cloned between the promoter and the 100 nt maize sequence. The tRNA is efficiently processed to create a processed end that is more sensitive to degradation in the absence of the protecting Zm-PPR10.sup.GG protein, reducing background in the absence of the PPR10.sup.GG protein. This construct can be engineered to express a protein of interest in the place of GFP, or a protein of interest can be operably linked to GFP via cleavable protein linker.

[0107] Likewise, a Brassica napus seed-specific PnpaA:PPR10:Tocs nuclear transgene can be cloned into a pCAMBIA2300 Agrobacterium binary vector with a plant-selectable kanamycin resistance gene for transformation of the B. napus nucleus (FIG. 12B). The modified Zea mays PPR10 gene sequence that results in selective recognition of the modified GG RNA binding site is described (Barkan et al., 2012) (SEQ ID NO: 265). For reference, the wild-type maize PPR10 sequence is also listed (SEQ ID NO: 264). The PPR10 protein is naturally targeted to chloroplasts, thus it requires only a tissue-specific promoter and a eukaryotic transcription terminator, such as octopine synthase 3' UTR (Tocs) (GenBank accession no. AJ311872.1). The napin gene is encoded in a small gene family. A suitable promoter for the PnpaA:PPR10.sup.GG:Tocs gene was characterized experimentally (Ellerstrom et al., 1996) (GenBank accession J02798), and additional B. napus promoters are available (Sohrabi et al., 2015). The promoter of a legume storage protein gene, phaseolin, (SEQ ID NO: 266) is known to be very efficient for the expression of recombinant proteins in Arabidopsis thaliana (De Jaegert et al., 2002). The promoter sequence is available in US patent application 2003/0159183.

TABLE-US-00008 >Prrn-PPR10GG-GFP-TrbcL SEQ ID NO: 262 >GagctcGCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGGGATTGAC GTGAGGGGGCAGGGATGGCTATATTTCTGGGAGTTACTTCTACCCGATAGAGCTTAG AAGTTGGAAGTAATAATTTCTTGGTTGATTGTAGGCTTAACCATTTCTTTTTTTTTGA CACGAGGAACTCATCATGgctagcAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCC AATTCTTGTTGAATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGA GGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTGG AAAACTACCTGTTCCtTGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCT TTTCAAGATACCCAGATCATATGAAGCGGCACGACTTCTTCAAGAGCGCCATGCCTG AGGGATACGTGCAGGAGAGGACCATCTCTTTCAAGGACGACGGGAACTACAAGACA CGTGCTGAAGTCAAGTTTGAGGGAGACACCCTCGTCAACAGGATCGAGCTTAAGGG AATCGATTTCAAGGAGGACGGAAACATCCTCGGCCACAAGTTGGAATACAACTACA ACTCCCACAACGTATACATCACGGCAGACAAACAAAAGAATGGAATCAAAGCTAAC TTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCA ACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGTC CACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCT TGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTgTACAAATAAAtc tagaAAACAGTAGACATTAGCAGATAAATTAGCAGGAAATAAAGAAGGATAAGGAGA AAGAACTCAAGTAATTATCCTTCGTTCTCTTAATTGAATTGCAATTAAACTCGGCCC AATCTTTTACTAAAAGGATTGAGCCGAATACAACAAAGATTCTATTGCATATATTTT GACTAAGTATATACTTACCTAGATATACAAGATTTGAAATACAAAATCTAaagctt >tRNA (trnP, 147 bp) SEQ ID NO: 263 AAGTCTTTACAATGACAATGGAAACCGATGTAAAGGGATGTAGCGCAGCTTGGTAG CGCGTTTGTTTTGGGTACAAAATGTCACAGGTTCAAATCCTGTCATCCCTATCCCTAA CTtGTAGTTATCGTATCAGCAGTAACAATAGAT (SEQ ID NO: 263) >Zm_PPR10 WT SEQ ID NO: 264 ATGGAGGCCACCGGCAGGGGGCTGTTCCCGAACAAGCCCACCCTCCCGGCGGGGCC GAGGAAACGGGGCCCGCTCCTCCCGGCCGCGCCCCCGCCACCGTCCCCCTCCTCGCT CCCGCTCGACTCGCTCCTGCTCCACCTCACCGCGCCCGCCCCCGCGCCGGCCCCCGC GCCGCGGCGGTCGCACCAGACGCCGACGCCGCCGCACTCCTTCCTCTCCCCCGACGC GCAGGTGCTGGTGCTCGCCATCTCCTCGCACCCGCTCCCCACGCTGGCGGCCTTCCT GGCCTCCCGCCGCGACGAGCTCCTCCGCGCGGACATCACGTCCCTGCTCAAGGCGCT GGAGCTCTCGGGGCACTGGGAGTGGGCGCTCGCGCTCCTCCGGTGGGCAGGCAAGG AGGGTGCCGCCGACGCGTCGGCGCTCGAGATGGTCGTCCGCGCGCTGGGCCGCGAG GGCCAGCACGACGCCGTCTGCGCGCTGCTCGACGAAACGCCGCTCCCGCCGGGCTC CCGCCTCGACGTCCGCGCCTACACCACCGTGCTGCACGCGCTCTCCCGCGCGGGCCG GTACGAGCGCGCGCTCGAGCTCTTCGCCGAGCTCCGGCGCCAGGGGGTGGCGCCCA CGCTCGTCACCTACAACGTCGTGCTGGACGTGTACGGGCGGATGGGCCGGTCGTGG CCGCGGATCGTCGCCCTCCTCGATGAGATGCGCGCCGCCGGGGTCGAGCCCGACGG CTTCACCGCCAGCACGGTGATCGCCGCGTGCTGCCGCGACGGGCTGGTTGACGAGG CGGTGGCGTTCTTCGAGGACCTCAAGGCCCGCGGCCACGCCCCGTGCGTCGTCACGT ACaacGCGTTGCTCCAGGTGTTCGGCAAGGCCGGGAACTACACGGAGGCGCTGCGCG TGCTCGGGGAGATGGAGCAGAACGGCTGCCAGCCAGATGCTGTGACGTACaacGAGC TCGCCGGAACGTACGCCCGGGCTGGGTTCTTCGAGGAGGCTGCCAGGTGCCTGGAC ACAATGGCATCCAAGGGTCTGTTGCCAAACGCATTCACGTACAACACCGTGATGAC AGCCTATGGGAATGTTGGGAAGGTGGATGAGGCGCTCGCTCTGTTTGACCAGATGA AGAAGACCGGGTTCGTGCCGAACGTGAACACGTACAATCTTGTCCTTGGCATGCTTG GCAAGAAGTCAAGGTTCACGGTGATGCTAGAGATGCTTGGAGAGATGTCGAGGAGC GGATGCACACCGAACCGGGTAACATGGAACACAATGCTTGCAGTCTGTGGGAAGCG TGGCATGGAGGACTACGTCACCCGGGTTCTGGAGGGGATGAGGTCTTGCGGGGTTG AACTGAGCCGAGACACCTACAACACCCTGATAGCTGCGTACGGCCGGTGTGGCTCG AGGACTAATGCCTTCAAGATGTACAACGAGATGACCAGCGCTGGATTCACCCCCTG CATCACCACGTACAACGCGTTGCTGAACGTGCTGTCGCGGCAGGGCGACTGGTCCA CCGCCCAGTCGATCGTAAGCAAAATGAGGACCAAGGGGTTCAAGCCGAACGAGCAG TCGTATTCGCTGCTGCTCCAGTGCTACGCGAAGGGGGGCAACGTGGCAGGGATAGC CGCGATCGAGAACGAGGTGTACGGATCAGGTGCCGTTTTCCCAAGCTGGGTGATCCT GAGGACCCTTGTCATCGCCAATTTCAAGTGCCGGCGACTGGATGGCATGGAGACGG CGTTTCAAGAGGTGAAGGCCAGAGGCTACAACCCGGACCTCGTGATATTCAACTCC ATGCTGTCCATCTACGCGAAGAACGGGATGTACAGCAAGGCCACCGAGGTCTTCGA CTCCATCAAGCGGAGCGGGCTGAGCCCCGACCTCATCACCTACAACAGCCTGATGG ACATGTACGCCAAGTGCAGCGAGTCGTGGGAGGCCGAGAAGATACTGAACCAGCTC AAGTGCTCCCAGACGATGAAGCCCGACGTGGTGTCCTACAACACGGTCATAAACGG GTTCTGCAAGCAGGGGCTGGTGAAAGAGGCCCAGAGGGTCCTCTCGGAGATGGTCG CCGACGGCATGGCCCCCTGCGCCGTGACCTACCACACGCTCGTCGGGGGTTACTCCA GCCTGGAGATGTTCAGCGAGGCCAGGGAGGTCATCGGCTACATGGTCCAGCACGGC CTCAAGCCTATGGAGCTGACCTACAGGAGAGTCGTCGAGAGCTACTGCAGAGCGAA GCGGTTCGAGGAGGCTCGCGGCTTCCTGTCCGAGGTCTCGGAGACCGACCTGGATTT TGACAAGAAGGCGCTCGAAGCCTATATAGAGGATGCGCAGTTTGGAAGGTAG (SEQ >Zm_PPR10 GG SEQ ID NO: 265 ATGGAGGCCACCGGCAGGGGGCTGTTCCCGAACAAGCCCACCCTCCCGGCGGGGCC GAGGAAACGGGGCCCGCTCCTCCCGGCCGCGCCCCCGCCACCGTCCCCCTCCTCGCT CCCGCTCGACTCGCTCCTGCTCCACCTCACCGCGCCCGCCCCCGCGCCGGCCCCCGC GCCGCGGCGGTCGCACCAGACGCCGACGCCGCCGCACTCCTTCCTCTCCCCCGACGC GCAGGTGCTGGTGCTCGCCATCTCCTCGCACCCGCTCCCCACGCTGGCGGCCTTCCT GGCCTCCCGCCGCGACGAGCTCCTCCGCGCGGACATCACGTCCCTGCTCAAGGCGCT GGAGCTCTCGGGGCACTGGGAGTGGGCGCTCGCGCTCCTCCGGTGGGCAGGCAAGG AGGGTGCCGCCGACGCGTCGGCGCTCGAGATGGTCGTCCGCGCGCTGGGCCGCGAG GGCCAGCACGACGCCGTCTGCGCGCTGCTCGACGAAACGCCGCTCCCGCCGGGCTC CCGCCTCGACGTCCGCGCCTACACCACCGTGCTGCACGCGCTCTCCCGCGCGGGCCG GTACGAGCGCGCGCTCGAGCTCTTCGCCGAGCTCCGGCGCCAGGGGGTGGCGCCCA CGCTCGTCACCTACAACGTCGTGCTGGACGTGTACGGGCGGATGGGCCGGTCGTGG CCGCGGATCGTCGCCCTCCTCGATGAGATGCGCGCCGCCGGGGTCGAGCCCGACGG CTTCACCGCCAGCACGGTGATCGCCGCGTGCTGCCGCGACGGGCTGGTTGACGAGG CGGTGGCGTTCTTCGAGGACCTCAAGGCCCGCGGCCACGCCCCGTGCGTCGTCACGT ACacaGCGTTGCTCCAGGTGTTCGGCAAGGCCGGGAACTACACGGAGGCGCTGCGCG TGCTCGGGGAGATGGAGCAGAACGGCTGCCAGCCAGATGCTGTGACGTACaccGAGC TCGCCGGAACGTACGCCCGGGCTGGGTTCTTCGAGGAGGCTGCCAGGTGCCTGGAC ACAATGGCATCCAAGGGTCTGTTGCCAAACGCATTCACGTACAACACCGTGATGAC AGCCTATGGGAATGTTGGGAAGGTGGATGAGGCGCTCGCTCTGTTTGACCAGATGA AGAAGACCGGGTTCGTGCCGAACGTGAACACGTACAATCTTGTCCTTGGCATGCTTG GCAAGAAGTCAAGGTTCACGGTGATGCTAGAGATGCTTGGAGAGATGTCGAGGAGC GGATGCACACCGAACCGGGTAACATGGAACACAATGCTTGCAGTCTGTGGGAAGCG TGGCATGGAGGACTACGTCACCCGGGTTCTGGAGGGGATGAGGTCTTGCGGGGTTG AACTGAGCCGAGACACCTACAACACCCTGATAGCTGCGTACGGCCGGTGTGGCTCG AGGACTAATGCCTTCAAGATGTACAACGAGATGACCAGCGCTGGATTCACCCCCTG CATCACCACGTACAACGCGTTGCTGAACGTGCTGTCGCGGCAGGGCGACTGGTCCA CCGCCCAGTCGATCGTAAGCAAAATGAGGACCAAGGGGTTCAAGCCGAACGAGCAG TCGTATTCGCTGCTGCTCCAGTGCTACGCGAAGGGGGGCAACGTGGCAGGGATAGC CGCGATCGAGAACGAGGTGTACGGATCAGGTGCCGTTTTCCCAAGCTGGGTGATCCT GAGGACCCTTGTCATCGCCAATTTCAAGTGCCGGCGACTGGATGGCATGGAGACGG CGTTTCAAGAGGTGAAGGCCAGAGGCTACAACCCGGACCTCGTGATATTCAACTCC ATGCTGTCCATCTACGCGAAGAACGGGATGTACAGCAAGGCCACCGAGGTCTTCGA CTCCATCAAGCGGAGCGGGCTGAGCCCCGACCTCATCACCTACAACAGCCTGATGG ACATGTACGCCAAGTGCAGCGAGTCGTGGGAGGCCGAGAAGATACTGAACCAGCTC AAGTGCTCCCAGACGATGAAGCCCGACGTGGTGTCCTACAACACGGTCATAAACGG GTTCTGCAAGCAGGGGCTGGTGAAAGAGGCCCAGAGGGTCCTCTCGGAGATGGTCG CCGACGGCATGGCCCCCTGCGCCGTGACCTACCACACGCTCGTCGGGGGTTACTCCA GCCTGGAGATGTTCAGCGAGGCCAGGGAGGTCATCGGCTACATGGTCCAGCACGGC CTCAAGCCTATGGAGCTGACCTACAGGAGAGTCGTCGAGAGCTACTGCAGAGCGAA GCGGTTCGAGGAGGCTCGCGGCTTCCTGTCCGAGGTCTCGGAGACCGACCTGGATTT TGACAAGAAGGCGCTCGAAGCCTATATAGAGGATGCGCAGTTTGGAAGGTAG (SEQ Phaseolin promoter SEQ ID NO: 266 ggtcgacggtatcgataagcttgatatcgaattcctgcagcccaattcattgtactc ccagtatcattatagtgaaagttttggctctctcgccggtggttttttacctctatt taaaggggattccacctaaaaattctggtatcattctcacatacttgaacataattt ctcataatcatggttgaaattatcacgcttccgcacacgatatccctacaaatttat tatttgttaaacattttcaaaccgcataaaattttatgaagtcccgtctatctttaa tgtagtctaacattttcatattgaaatatataatttacttaatatagcgaggtagaa agcataatgatttattcttattcacttcatataaatgtttaatatacaatataaaca aattctttaccttaagaaggatttcccattttatattttaaaaatatatttatcaaa tatttttcaaccacgtaaatctcataataataagttgtttcaaaagtaataaaattt aactccataatttttttattcgactgatcttaaagcaacacccagtgacacaactag ccatttttttctttggataaaaaaatccaattatcattgtattttttttatacaatg aaaatttcaccaaacaatcatttgtggtatttctgaagcaagtcatgttatgcaaaa ttctataattcccatttgacactacggaagtaactgaagatctgcttttacatgcga

gacacatcttctaaagtaattttaataatagttactatattcaagatttcatatatc aaatactcaatattacttctaaaaaattaattagatataattaaaatattacttttt aattttaatttaattgttgaatttgtgactattgatttattattctactatgtttaa attgttttatagatagtttaaagtaaatataagtaatgtagtagagtgttagagtgt taccctaaaccataaactataacatttatggtggactaattttcatatatttcttat tgcattaccttttcaggtatgtaagtccgtaactagaattactgtgggttgccatgg catctgtggtcttttggttcatgcatggatgcttgcgcaagaaaaagacaaagaaca aagaaaaaagacaaaacagagagacaaaacgcaatcacacaaccaactcaaattagt cactggctgatcaagatcgccgcgtccatgtatgtctaaatgccatgcaaagcaaca cgtgcttaacatgcactttaaatggctcacccatctcaacccacacacaaacacatt gcctattcttcatcatcaccacaaccacctgatatattcattctatccgccacacaa tttcttcacttcaacacacgtcaacctgca

[0108] The Arabidopsis nuclear genome encodes >400 Pentatricopeptide Repeat Proteins (PPRs), of which PPR10 is a member (Barkan and Small, 2014). Other P-type proteins that function similar to PPR10 are the Arabidopsis HCF152 and PGR3 proteins which is required for the accumulation of transcripts cleaved in the psbH-petB intergenic region and petL operon, respectively (Meierhoff et al., 2003; Yamazaki et al., 2004). Zea maize CRP1 is involved in the processing and translation of the chloroplast petD and petA RNAs (Fisk et al., 1999). HCF107, a member in the half-a-tetratricopeptide (HAT) family, also defines the processed end of psbH and enhance its translation by remodeling its 5' UTR (Hammani et al., 2012). These proteins with their cognate binding site can be engineered to test and establish similar chloroplast transgene regulation system as PPR10.

Targeted Mutagenesis of Brassica napas ACC2 Genes to Obtain Spectinomycin Hypersensitive Plants

[0109] Chloroplast genome engineering in crops enables many applications, including improvement of photosynthetic efficiency, incorporation of novel metabolic pathways and delivery of vaccines in veterinary applications. This platform technology is absent in oilseed rape (Brassica napus or canola) due to its tolerance to spectinomycin, the selective agent used to obtain plants with transformed chloroplast genomes. We delete the ACC2 gene copies in the nuclear genome of oilseed rape to obtain spectinomycin hypersensitive, chloroplast transformation competent lines.

[0110] Brassica napus is a recent amphiploid hybrid of Brassica rapa and Brassica oleracea, and therefore carries at least one copy of each gene from the parental species. Because the common ancestor of the parental species underwent a genome triplication, this number may be as high as six. The B. napus cv Darmor-bzh darft genome available at the Genoscope website has only a single annotated ACC2 gene copy for each of the parental genomes: the Brassica rapa-like ACC2-Br BnaA06g04070D gene encoded in chromosome A6 and the Brassica oleracea-like ACC2-Bo BnaC06g01580D on chromosome C6. If multiple ACC2 gene copies are present, we hypothesized that over evolutionary time single nucleotide polymorphic mutations must have accumulated unique to each gene. To obtain information about the actual number of ACC2 gene copies and facilitate the design of gRNAs that, simultaneously target each nuclear ACC2 gene copies, we cloned and sequenced PCR products of the N-terminal regions. Analyses of the data indicates that there are at least three B. rapa-like copies and two B. oleracea-like copies present in the B. napus cv. Westar nuclear genome. Inspection of the N-terminal extension lead to the identification of 28 potential sgRNAs with a GGN PAM sequence (Table 4). SgRNA3 was selected to target a single site and sgRNA1 and sgRNA2 to target two sites in the ACC2 N-terminal extension (FIG. 13). The benefit of targeting two sites is a deletion of DNA segment between the two sites, that may be used for tracking the mutant alleles by PC R.

TABLE-US-00009 TABLE 4 Genomic Sequence Target (5'-3') Strand Forward Oligo Reverse Oligo sgRNA1 ggtttagactctccaatgtttc + GATTGctttgtaacctctcagatt AAACaatctgagaggttacaaagC sgRNA2 ggaaggaaggacttgagcagcc + GATTGccgacgagttcaggaagga AAACtccttcctgaactcgtcggC sgRNA3 ggtgaaacattggagagtctaa - GATTGaatctgagaggttacaaag AAACctttgtaacctctcagattC 4 ggagcttctgatcggtttagac + 5 ggtgcaagtggcagtgactccc + 6 gacaccgacccctcagtgacgg + 7 ggagtttcgatttacaaaaaca + 8 ggcctacttaggaaggaaggac + 9 ggaaggacttgagcagccctga + 10 gtcctatggcctacttaggaagg + 11 ggacttgagcagccctgatccg + 12 atggcctacttaggaaggaagg + 13 cgacctccttctgcgataatgg + 14 ggagagtctaaaccgatcagaa - 15 ggagtcactgccacttgcacca - 16 ggggagtcactgccacttgcacc - 17 ggctggggagtcactgccacttg - 18 ggtcttgtttttgtaaatcgaa - 19 tccttcctaagtaggccatagg - 20 aagtccttccttcctaagtagg - 21 ggctgctcaagtccttccttcc - 22 gcagaaggaggtcggatcaggg - 23 gggctgctcaagtccttccttc - 24 cgcagaaggaggtcggatcagg - 25 cattatcgcagaaggaggtcgg - 26 ggtcggatcagggctgctcaag - 27 ggaggtcggatcagggctgctc - 28 agcaaaccattatcgcagaagg -

[0111] CRSPR/Cas9-mediated gene ACC2 gene editing in Brassica napus is carried out using the vector system developed in the Jiang-Kang Zhu laboratory (Mao et al., 2013; Liu et al., 2015). Single-stranded oligonucleotides were designed to fit the BbsI-digested p998/psgR-cas9-At vector, a pCAMBIA2300 vector derivative (Table 4). To accommodate the Arabidopsis U6 promoter, a G nucleotide was added at the end opposite to the PAM sequence. Agrobacterium vectors carrying two sgRNAs were obtained following the detailed protocol of Liu et al. (2015). Agrobacterium vectors carrying the sgRNAs were then introduced into Agrobacterium strain EHA105 or GV3101, and transformed into B. napus cotyledons following the protocol of Bates at all. (Bates et al., 2017). Progress in losing ACC2 activity is tracked by the absence of leaf formation on germinating seedlings. A tolerant B. napus seedling with well-developed leaves is shown in FIG. 14A. FIGS. 14B and 14C show a flowchart to obtain Cas9-free spectinomycin hypersensitive acc2 Brassica napus. (14B) Selection of CRISPR/Cas9 transgenic plants by kanamycin resistance. (14C) Hypersensitivity bioassay identifies T1 families with putative knockouts in all ACC2 copies, leading to the isolation of Cas9-free acc2 individuals. In certain instances, hypersensitivity will be uniform in the plant. Non-uniform hypersensitivity to spectinomycin will prompt an additional cycle of screening in the next seed generation.

REFERENCES

[0112] Babiychuk E, Vandepoele K, Wissing J, Garcia-Diaz M, De Rycke R, Akbari H, Joubes J, Beeckman T, Jansch L, Frentzen M, Van Montagu M C, Kushnir S (2011) Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family Proc Natl Acad Sci USA 108: 6674-6679

[0113] Barkan A, Rojas M, Fujii S, Yap A, Chong Y S, Bond C S, Small I (2012) A Combinatorial Amino Acid Code for RNA Recognition by Pentatricopeptide Repeat Proteins. PLOS Genetics 8

[0114] Barkan A, Small I (2014) Pentatricopeptide repeat proteins in plants. Annu Rev Plant Biol 65: 415-442

[0115] Belhaj K, Chaparro-Garcia A, Kamoun S, Nekrasov V (2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system. Plant Methods 9: 39

[0116] Bock R (2015) Engineering Plastid Genomes: Methods, Tools, and Applications in Basic Research and Biotechnology. Annu Rev Plant Biol 66: 211-241

[0117] Brooks C, Nekrasov V, Lippman Z B, Van Eck J (2014) Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPR-associated9 system. Plant Physiol 166: 1292-1297

[0118] Carrer H, Staub J M, Maliga P (1991) Gentamycin resistance in Nicotiana conferred by AAC(3)-I, a narrow substrate specificity acetyl transferase. Plant Mol Biol 17: 301-303

[0119] Carrillo N, Seyer P, Tyagi A, Herrmann R G (1986) Cytochrome b-559 genes from Oenothera hookeri and Nicotiana tabacum show a remarkably high degree of conservation as compared to spinach. The enigma of cytochrome b-559: highly conserved genes and proteins but no known function. Curr Genet 10: 619-624

[0120] Chakrabarti S K, Lutz K A, Lertwirijawong B, Svab Z, Maliga P (2006) Expression of the cry9Aa2 B.t. gene in the tobacco chlroplasts confers resistance to potato tuber moth. Transgenic Res 15: 481-488

[0121] Cheng L, Li H P, Qu B, Huang T, Tu J X, Fu T D, Liao Y C (2010) Chloroplast transformation of rapeseed (Brassica napus) by particle bombardment of cotyledons. Plant Cell Rep 29: 371-381

[0122] Chupeau M C, Granier F, Pichon O, Renou J P, Gaudin V, Chupeau Y (2013) Characterization of the early events leading to totipotency in an Arabidopsis protoplast liquid culture by temporal transcript profiling. Plant Cell 25: 2444-2463

[0123] Clough S J, Bent A F (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743

[0124] Corpet F (1988) Multiple sequence alignment with hierarchical clustering. Nucleic Acids Res 16: 10881-10890

[0125] De Jaegert G, Scheffer S, Jacobs A, Zambre M, Zobell O, Goossens A, Depicker A, Angenon G (2002) Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolis vulgaris regulatory asequences. Nat Biotechnol 20: 1265-1268

[0126] Dufourmantel N, Dubald M, Matringe M, Canard H, Garcon F, Job C, Kay E, Wisniewski J P, Ferullo J M, Pelissier B, Sailland A, Tissot G (2007) Generation and characterization of soybean and marker-free tobacco plastid transformants over-expressing a bacterial 4-hydroxyphenylpyruvate dioxygenase which provides strong herbicide tolerance. Plant Biotechnol J 5: 118-133

[0127] Dufourmantel N, Pelissier B, Garcon F, Peltier G, Ferullo J M, Tissot G (2004) Generation of fertile transplastomic soybean. Plant Mol Biol 55: 479-489

[0128] Ellerstrom M, Stalberg K, Ezcurra I, Rask L (1996) Functional dissection of a napin gene promoter: Identification of promoter elements required for embryo and endosperm-specific transcription. Plant Mol Biol 32: 1019-1027

[0129] Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang D L, Wang Z, Zhang Z, Zheng R, Yang L, Zeng L, Liu X, Zhu J K (2014) Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proc Natl Acad Sci USA 111: 4632-4637

[0130] Fisk D G, Walker M B, Barkan A (1999) Molecular cloning of the maize gene crpl reveals similarity between regulators of mitochondrial and chloroplast gene expression. EMBO J 18: 2621-2630

[0131] Gimpel J A, Nour-Eldin H H, Scranton M A, Li D, Mayfield S P (2016) Refactoring the Six-Gene Photosystem II Core in the Chloroplast of the Green Algae Chlamydomonas reinhardtii. ACS Synth Biol 5: 589-596

[0132] Hammani K, Cook W B, Barkan A (2012) RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression. Proc Natl Acad Sci USA 109: 5651-5656

[0133] Hanson M R, Lin M T, Carmo-Silva A E, Parry M A (2016) Towards engineering carboxysomes into C3 plants. Plant J 87: 38-50

[0134] Hou B K, Zhou Y H, Wan L H, Zhang Z L, Shen G F, Chen Z H, Hu Z M (2003) Chloroplast transformation in oilseed rape. Transgenic Res 12: 111-114

[0135] Howell E C, Kearsey M J, Jones G H, King G J, Armstrong S J (2008) A and C genome distinction and chromosome identification in Brassica napus by sequential fluorescence in situ hybridization and genomic in situ hybridization. Genetics 180: 1849-1857

[0136] Kanamoto H, Yamashita A, Asao H, Okumura S, Takase H, Hattori M, Yokota A, Tomizawa K (2006) Efficient and stable transformation of Lactuca sativa L. cv. Cisco (lettuce) plastids. Transgenic Res 15: 205-217

[0137] Koornneef M, Meinke D (2010) The development of Arabidopsis as a model plant. Plant J 61: 909-921

[0138] Kuroda H, Maliga P (2001) Complementarity of the 16S rRNA penultimate stem with sequences downstream of the AUG destabilizes the plastid mRNAs. Nucleic Acids Res 29: 970-975

[0139] Kuroda H, Maliga P (2001) Sequences downstream of the translation initiation codon are important determinants of translation efficiency in chloroplasts. Plant Physiol 125: 430-436

[0140] Li J F, Zhang D, Sheen J (2014) Cas9-based genome editing in Arabidopsis and tobacco. Methods Enzymol 546: 459-472

[0141] Liu C W, Lin C C, Chen I L Tseng M J (2007) Stable chloroplast transformation in cabbage (Brassica oleracea L. var. capitata L.) by particle bombardment. Plant Cell Rep 26: 1733-1744

[0142] Liu C W, Lin C C, Yiu J C, Chen J J, Tseng M J (2008) Expression of a Bacillus thuringiensis toxin (crylAb) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella. Theor Appl Genet 117: 75-88

[0143] Liu F, Cao M, Yao L, Li Y, Robaglia C, Tourneur C (1998) In planta transformation of pakchoi (Brassica campestris 1. ssp. chinensis) by infiltration of adult plants with Agrobacterium. Acta Horticult 467: 187

[0144] Liu X, Brost J, Hutcheon C, Guilfoil R, Wilson A K, Leung S, Shewmaker C K, Rooke S, Nguyen T, Kiser J, De Rocher J (2012) Transformation of the oilseed crop Camelina sativa by Agrobacterium-mediated floral dip and simple large-scale screening of transformants In Vitro Cellular & Developmental Biology--Plant 48: 462-468

[0145] Lu C F, Kang J L (2008) Generation of transgenic plants of a potential oilseed crop Camelina sativa by Agrobacterium-mediated transformation. Plant Cell Rep 27: 273-278

[0146] Lutz K A, Azhagiri A, Maliga P (2011) Transplastomics in Arabidopsis: Progress towards developing an efficient method. In RP Jarvis, ed, Chloroplast research in Arabidopsis, Vol 774. Springer Science+Business Media, LLC, New York, pp 133-147.

[0147] Lutz K A, Knapp J E, Maliga P (2001) Expression of bar in the plastid genome confers herbicide resistance. Plant Physiol 125: 1585-1590

[0148] Lutz K A, Martin C, Khairzada S, Maliga P (2015) Steroid-inducible BABY BOOM system for development of fertile Arabidopsis thaliana plants after prolonged tissue culture. Plant Cell Rep 34: 1849-1856

[0149] Mali P, Esvelt K M, Church G M (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10: 957-963

[0150] Maliga P (2012) Plastid transformation in flowering plants. In R Bock, V Knoop, eds, Genomics of Chloroplasts and Mitochondria, Vol 35. Springer, pp 393-414

[0151] Maliga P, Bock R (2011) Plastid biotechnology: food, fuel and medicine for the 21st century. Plant Physiol 155: 1501-1510

[0152] Maliga P, Tungsuchat-Huang T (2014) Plastid transformation in Nicotiana tabacum and Nicotiana sylvestris by biolistic DNA delivery to leaves. In P Maliga, ed, Chloroplast Biotechnology: Methods and Protocols, Vol 1132. Springer Science+Business Media, New York, pp 147-163

[0153] Mao Y, Zhang H, Xu N, Zhang B, Gou F, Zhu J K (2013) Application of the CRISPR-Cas system for efficient genome engineering in plants. Mol Plant 6: 2008-2011

[0154] Marton L, Browse J (1991) Facile transformation of Arabidopsis. Plant Cell Rep 10: 235-239 Meierhoff K, Felder S, Nakamura T, Bechtold N, Schuster G (2003) HCF152, an Arabidopsis RNA binding pentatricopeptide repeat protein involved in the processing of chloroplast psbB-psbT-psbH-petB-petD RNAs. Plant Cell 15: 1480-1495

[0155] Meng B Y, Tanaka M, Wakasugi T, Ohme M, Shinozaki K, Sugiura M (1988) Cotranscription of the genes encoding two P700 chlorophyll a apoproteins with the gene for ribosomal protein CS14: determination of the transcriptional initiation site by in vitro capping. Curr Genet 14: 395-400

[0156] Motte H, Galuszka P, Spichal L, Tarkowski P, Plihal O, Smehilova M, Jaworek P, Vereecke D, Werbrouck S, Geelen D (2013) Phenyl-adenine, identified in a LIGHT-DEPENDENT SHORT HYPOCOTYLS4-assisted chemical screen, is a potent compound for shoot regeneration through the inhibition of CYTOKININ OXIDASE/DEHYDROGENASE activity. Plant Physiol 161: 1229-1241

[0157] Nugent G D, Coyne S, Nguyen T T, Kavanagh T A, Dix P J (2006) Nuclear and plastid transformation of Brassica oleracea var. botrytis (cauliflower) using PEG-mediated uptake into protoplasts. Plant Sci 170: 135-142

[0158] Parker N, Wang Y, Meinke D (2014) Natural variation in sensitivity to a loss of chloroplast translation in Arabidopsis. Plant Physiol 166: 2013-2027

[0159] Parker N, Wang Y, Meinke D (2016) Analysis of Arabidopsis Accessions Hypersensitive to a Loss of Chloroplast Translation. Plant Physiol 172: 1862-1875

[0160] Qing C M, Fan L, Lei Y, Bouchez D, Tourneur C, Yan L, Robaglia C (2000) Transformation of Pakchoi (Brassica rapa L. ssp chinensis) by Agrobacterium infiltration. Molecular Breeding 6: 67-72

[0161] Ruf S, Hermann M, Berger L I, Carrer H, Bock R (2001) Stable genetic transformation of tomato plastids: foreign protein expression in fruit. Nat Biotechnol 19: 870-875

[0162] Ruhlman T, Verma D, Samson N, Daniell H (2010) The role of heterologous chloroplast sequence elements in transgene integration and expression. Plant Physiol 152: 2088-2104

[0163] Scharff L B, Bock R (2014) Synthetic biology in plastids. Plant J 78: 783-798

[0164] Schneider A, Stelljes C, Adams C, Kirchner S, Burkhard G, Jarzombski S, Broer I, Horn P,

[0165] Elsayed A, Hagl P, Leister D, Koop H U (2015) Low frequency paternal transmission of plastid genes in Brassicaceae. Transgenic Res 24: 267-277

[0166] Schottkowski M, Peters M, Zhan Y, Rifai O, Zhang Y, Zerges W (2012) Biogenic membranes of the chloroplast in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 109: 19286-19291

[0167] Schulte W, Topfer R, Stracke R, Schell J, Martini N (1997) Multi-functional acetyl-CoA carboxylase from Brassica napus is encoded by a multi-gene family: indication for plastidic localization of at least one isoform. Proc Natl Acad Sci USA 94: 3465-3470

[0168] Shao M, Blechl A, Thomson J G (2017) Small serine recombination systems ParA-MRS and CinH-RS2 perform precise excision of plastid DNA. Plant Biotechnol J 15: 1577-1589

[0169] Shao M, Kumar S, Thomson J G (2014) Precise excision of plastid DNA by the large serine recombinase Bxbl. Plant Biotechnol J 12: 322-329

[0170] Sharwood R E, Ghannoum O, Whitney S M (2016) Prospects for improving CO2 fixation in C3-crops through understanding C4-Rubisco biogenesis and catalytic diversity. Curr Opin Plant Biol 31: 135-142

[0171] Shimizu M, Goto M, Hanai M, Shimizu T, Izawa N, Kanamoto H, Tomizawa K, Yokota A, Kobayashi H (2008) Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco. Plant Physiol 147: 1976-1983

[0172] Sikdar S R, Serino G, Chaudhuri S, Maliga P (1998) Plastid transformation in Arabidopsis thaliana. Plant Cell Rep 18: 20-24

[0173] Sinagawa-Garcia S R, Tungsuchat-Huang T, Paredes-Lopez O, Maliga P (2009) Next generation synthetic vectors for transformation of the plastid genome of higher plants. Plant Mol Biol 70: 487-498

[0174] Skarjinskaia M, Svab Z, Maliga P (2003) Plastid transformation in Lesquerella fendleri, an oilseed Brassicacea. Transgenic Res 12: 115-122

[0175] Sohrabi M, Zebarjadi A, Najaphy A, Kahrizi D (2015) Isolation and sequence analysis of napin seed specific promoter from Iranian Rapeseed (Brassica napus L.). Gene 563: 160-164

[0176] Song K, Osborn T C (1992) Polyphyletic Origins of Brassica-Napus--New Evidence Based on Organelle and Nuclear Rflp Analyses. Genome 35: 992-1001

[0177] Staub J M, Maliga P (1995) Expression of a chimeric uidA gene indicates that polycistronic mRNAs are efficiently translated in tobacco plastids. Plant J 7: 845-848

[0178] Stitt M, Lunn J, Usadel B (2010) Arabidopsis and primary photosynthetic metabolism--more than the icing on the cake. Plant J 61: 1067-1091

[0179] Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87: 8526-8530

[0180] Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90: 913-917

[0181] Tan H L, Yang X H, Zhang F X, Zheng X, Qu C M, Mu J Y, Fu F Y, Li J A, Guan R Z, Zhang H S, Wang G D, Zuo J R (2011) Enhanced Seed Oil Production in Canola by Conditional Expression of Brassica napus LEAFY COTYLEDON1 and LEC1-LIKE in Developing Seeds. Plant Physiol 156: 1577-1588

[0182] Tungsuchat-Huang T, Maliga P (2012) Visual marker and Agrobacterium-delivered recombinase enable the manipulation of the plastid genome in greenhouse-grown tobacco plants. Plant J 70: 717-725

[0183] Valkov V T, Gargano D, Manna C, Formisano G, Dix P J, Gray J C, Scotti N, Cardi T (2011) High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5' and 3' regulatory sequences. Transgenic Res 20: 137-151

[0184] Verma S, Chinnusamy V, Bansal K (2008) A Simplified Floral Dip Method for Transformation of Brassica napus and B. carinata. J Plant Biochemistry and Biotechnology 17: 197-200

[0185] Wallis J G, Browse J (2010) Lipid biochemists salute the genome. Plant J 61: 1092-1106

[0186] Wang T W, Wu W, Zhang C G, Nowack L M, Liu Z D, Thompson J E (2005) Antisense suppression of deoxyhypusine synthase by vacuum-infiltration of Agrobacterium enhances growth and seed yield of canola. Physiol Plant 124: 493-503

[0187] Wang W C, Menon G, Hansen G (2003) Development of a novel Agrobacterium-mediated transformation method to recover transgenic Brassica napus plants. Plant Cell Rep 22: 274-281



[0188] Weigel D, Mott R (2009) The 1001 genomes project for Arabidopsis thaliana. Genome biology 10: 107

[0189] Willey D L, Gray J C (1989) Two small open reading frames are co-transcribed with the pea chloroplast genes for the polypeptides of cytochrome b-559. Curr Genet 15: 213-220

[0190] Willey D L, Gray J C (1990) An open reading frame encoding a putative haem-binding polypeptide is cotranscribed with the pea chloroplast gene for apocytochrome f. Plant Mol Biol 15: 347-356

[0191] Wilson D N (2014) Ribosome-targeting antibiotics and mechanisms of bacterial resistance. Nat Rev Microbiol 12: 35-48

[0192] Wirmer J, Westhof E (2006) Molecular contacts between antibiotics and the 30S ribosomal particle. Methods Enzymol 415: 180-202

[0193] Xie K, Yang Y (2013) RNA-guided genome editing in plants using a CRISPR-Cas system. Mol Plant 6: 1975-1983

[0194] Yamazaki H, Tasaka M, Shikanai T (2004) PPR motifs of the nucleus-encoded factor, PGR3, function in the selective and distinct steps of chloroplast gene expression in Arabidopsis. Plant J 38: 152-163

[0195] Yang L, Guell M, Niu D, George H, Lesha E, Grishin D, Aach J, Shrock E, Xu W, Poci J, Cortazio R, Wilkinson R A, Fishman J A, Church G (2015) Genome-wide inactivation of porcine endogenous retroviruses (PERVs). Science 350: 1101-1104

[0196] Ye G N, Colburn S, Xu C W, Hajdukiewicz P T J, Staub J M (2003) Persistance of unselected transgenic DNA during a plastid transformation and segregation approach to herbicide resistance. Plant Physiol 133: 402-410

[0197] Zhang J, Khan S A, Hasse C, Ruf S, Heckel D G, Bock R (2015) Pest control. Full crop protection from an insect pest by expression of long double-stranded RNAs in plastids. Science 347: 991-994

[0198] Zhao X, Liang G, Li X, Zhang X (2014) Hormones regulate in vitro organ regeneration from leaf-derived explants in Arabidopsis. Am J Plant Sci 5: 3535-3550

[0199] Zhao X Y, Su Y H, Zhang C L, Wang L, Li X, Zhang X S (2013) Differences in capacities of in vitro organ regeneration between two Arabidopsis ecotypes Wassilewskija and Columbia. Plant Cell Tissue Organ Cult. 112: 65-74

[0200] Zubko M K, Day A (1998) Stable albinism induced without mutagenesis: a model for ribosome-free plastid inheritance. Plant J 15: 265-271

[0201] While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Sequence CWU 1

1

35312043DNAArtificial SequenceEcoRI-HindIII fragment 1gagctcgctc ccccgccgtc gttcaatgag aatggataag aggctcgtgg gattgacgtg 60agggggcagg gatggctata tttctgggag aattaaccga tcgacgtgca agcggacatt 120tattttaaat tcgataattt ttgcaaaaac atttcgacat atttatttat tttattatta 180tgagaatcaa tcctactact tctggttctg gggtttccac ggctactagc gaagcggtga 240tcgccgaagt atcgactcaa ctatcagagg tagttggcgt catcgagcgc catctcgaac 300cgacgttgct ggccgtacat ttgtacggct ccgcagtgga tggcggcctg aagccacaca 360gtgatattga tttgctggtt acggtgaccg taaggcttga tgaaacaacg cggcgagctt 420tgatcaacga ccttttggaa acttcggctt cccctggaga gagcgagatt ctccgcgctg 480tagaagtcac cattgttgtg cacgacgaca tcattccgtg gcgttatcca gctaagcgcg 540aactgcaatt tggagaatgg cagcgcaatg acattcttgc aggtatcttc gagccagcca 600cgatcgacat tgatctggct atcttgctga caaaagcaag agaacatagc gttgccttgg 660taggtccagc ggcggaggaa ctctttgatc cggttcctga acaggatcta tttgaggcgc 720taaatgaaac cttaacgcta tggaactcgc cgcccgactg ggctggcgat gagcgaaatg 780tagtgcttac gttgtcccgc atttggtaca gcgcagtaac cggcaaaatc gcgccgaagg 840atgtcgctgc cgactgggca atggagcgcc tgccggccca gtatcagccc gtcatacttg 900aagctagaca ggcttatctt ggacaagaag aagatcgctt ggcctcgcgc gcagatcagt 960tggaagaatt tgtccactac gtgaaaggcg agatcaccaa ggtagtgggc aaagaacaaa 1020aactcatttc tgaagaagac ttgtaactgc agataaccca aataatgttt taaaatttta 1080aaaataatgt aggaggaaaa attatggcta gcagtaaagg agaagaactt ttcactggag 1140ttgtcccaat tcttgttgaa ttagatggtg atgttaatgg gcacaaattt tctgtcagtg 1200gagagggtga aggtgatgca acatacggaa aacttaccct taaatttatt tgcactactg 1260gaaaactacc tgttccttgg ccaacacttg tcactacttt ctcttatggt gttcaatgct 1320tttcaagata cccagatcat atgaagcggc acgacttctt caagagcgcc atgcctgagg 1380gatacgtgca ggagaggacc atctctttca aggacgacgg gaactacaag acacgtgctg 1440aagtcaagtt tgagggagac accctcgtca acaggatcga gcttaaggga atcgatttca 1500aggaggacgg aaacatcctc ggccacaagt tggaatacaa ctacaactcc cacaacgtat 1560acatcacggc agacaaacaa aagaatggaa tcaaagctaa cttcaaaatt agacacaaca 1620ttgaagatgg aagcgttcaa ctagcagacc attatcaaca aaatactcca attggcgatg 1680gccctgtcct tttaccagac aaccattacc tgtccacaca atctgccctt tcgaaagatc 1740ccaacgaaaa gagagaccac atggtccttc ttgagtttgt aacagctgct gggattacac 1800atggcatgga tgaactatac aaataagctc tagctagagc gatcctggcc tagtctatag 1860gaggttttga aaagaaagga gcaataatca ttttcttgtt ctatcaagag ggtgctattg 1920ctcctttctt tttttctttt tatttattta ctagtatttt acttacatag acttttttgt 1980ttacattata gaaaaagaag gagaggttat tttcttgcat ttattcatgg gggatcaaag 2040ctt 2043222DNAArtificial Sequenceprimer 2ttttctgtca gtggagaggg tg 22320DNAArtificial Sequenceprimer 3cccagcagct gttacaaact 20423DNAArtificial SequencegRNA designmisc_feature(1)..(21)n is a, c, g, or t 4nnnnnnnnnn nnnnnnnnnn ngg 235100PRTArabidopsis thaliana 5Met Glu Met Arg Ala Leu Gly Ser Ser Cys Ser Thr Gly Asn Gly Gly1 5 10 15Ser Ala Pro Ile Thr Leu Thr Asn Ile Ser Pro Trp Ile Thr Thr Val 20 25 30Phe Pro Ser Thr Val Lys Leu Arg Ser Ser Leu Arg Thr Phe Lys Gly 35 40 45Val Ser Ser Arg Val Arg Thr Phe Lys Gly Val Ser Ser Thr Arg Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Phe Cys Phe Leu Asn Pro65 70 75 80Asp Pro Ile Ser Phe Leu Glu Asn Asp Val Ser Glu Ala Glu Arg Thr 85 90 95Val Val Leu Pro 1006300DNAArabidopsis thaliana 6atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccgatt 60accctcacga atatatctcc atggatcaca acagtttttc cgtcgacagt gaagctgaga 120agtagtttga gaaccttcaa aggagtttcg tcaagagtga gaacctttaa aggagtttct 180tcgacaagag ttttgtctcg gaccaaacaa cagtttcctc tgttttgttt cctaaaccct 240gatccgatct ccttcttgga aaatgatgta tctgaagctg aaaggacagt agttttaccg 3007100PRTArabidopsis lyrata 7Met Glu Met Arg Ala Leu Val Ser Ser Cys Ala Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Phe Ser Phe Thr Lys Val Ser Pro Trp Ile Thr Thr Val 20 25 30Gly Gly Lys Asp Arg Asp Phe Pro Thr Thr Val Lys Leu Arg Thr Ser 35 40 45Met Arg Thr Phe Lys Gly Val Ser Ile Arg Gly Arg Thr Phe Lys Gly 50 55 60Val Ser Thr Arg Val Leu Ser Arg Asn Lys Gln Gln Phe Pro Leu Phe65 70 75 80Cys Phe Leu Asn Pro Asp Pro Thr Ser Phe Arg Asp Asn Asp Ile Ser 85 90 95Glu Ala Gln Arg 1008300DNAArabidopsis lyrata 8atggagatga gagctttggt ttcttcgtgt gctaccggta atggaggttc tgatccgttt 60agcttcacga aagtttctcc atggatcaca acagttggtg gtaaggacag agattttcca 120acgacagtga agctaagaac tagtatgaga acctttaaag gagtttctat aagagggaga 180acctttaaag gagtttcgac aagagttttg tctcggaaca aacaacagtt tcctctgttt 240tgtttcctaa accctgatcc gacctccttc cgggataatg atatatctga agctcaaagg 300923DNAArtificial SequencegRNA 9ttggtttctt cgtgtgctac cgg 231023DNAArtificial SequencegRNA 10tcttcgtgtg ctaccggtaa tgg 231123DNAArtificial SequencegRNA 11tcgtgtgcta ccggtaatgg agg 231223DNAArtificial SequencegRNA 12gcttcacgaa agtttctcca tgg 231323DNAArtificial SequencegRNA 13tctccatgga tcacaacagt tgg 231423DNAArtificial SequencegRNA 14ccatggatca caacagttgg tgg 231523DNAArtificial SequencegRNA 15gatcacaaca gttggtggta agg 231623DNAArtificial SequencegRNA 16actagtatga gaacctttaa agg 231723DNAArtificial SequencegRNA 17tttaaaggag tttctataag agg 231823DNAArtificial SequencegRNA 18ataagaggga gaacctttaa agg 231923DNAArtificial SequencegRNA 19tttcgacaag agttttgtct cgg 232023DNAArtificial SequencegRNA 20accctgatcc gacctccttc cgg 232123DNAArtificial SequencegRNA 21atgatatatc tgaagctcaa agg 2322300DNAArabidopsis lyrata 22cctttgagct tcagatatat cattatcccg gaaggaggtc ggatcagggt ttaggaaaca 60aaacagagga aactgttgtt tgttccgaga caaaactctt gtcgaaactc ctttaaaggt 120tctccctctt atagaaactc ctttaaaggt tctcatacta gttcttagct tcactgtcgt 180tggaaaatct ctgtccttac caccaactgt tgtgatccat ggagaaactt tcgtgaagct 240aaacggatca gaacctccat taccggtagc acacgaagaa accaaagctc tcatctccat 3002323DNAArtificial SequencegRNA 23cttcagatat atcattatcc cgg 232423DNAArtificial SequencegRNA 24agatatatca ttatcccgga agg 232523DNAArtificial SequencegRNA 25tatatcatta tcccggaagg agg 232623DNAArtificial SequencegRNA 26tcattatccc ggaaggaggt cgg 232723DNAArtificial SequencegRNA 27tcccggaagg aggtcggatc agg 232823DNAArtificial SequencegRNA 28cccggaagga ggtcggatca ggg 232923DNAArtificial SequencegRNA 29aggaggtcgg atcagggttt agg 233023DNAArtificial SequencegRNA 30gggtttagga aacaaaacag agg 233123DNAArtificial SequencegRNA 31tcttgtcgaa actcctttaa agg 233223DNAArtificial SequencegRNA 32tcttatagaa actcctttaa agg 233323DNAArtificial SequencegRNA 33gttcttagct tcactgtcgt tgg 233423DNAArtificial SequencegRNA 34ccaccaactg ttgtgatcca tgg 233523DNAArtificial SequencegRNA 35gaaactttcg tgaagctaaa cgg 233623DNAArtificial SequencegRNA 36cggatcagaa cctccattac cgg 2337100PRTCannabis sativa 37Met Glu Met Arg Ala Leu Val Ser Ser Tyr Ser Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Ile Ser Leu Thr Asn Gly Ser Pro Trp Ile Thr Thr Val 20 25 30Gly Gly Gly Ala Ser Thr Met Asp Arg Glu Phe Pro Leu Thr Val Lys 35 40 45Leu Gly Ser Ser Met Arg Ala Phe Lys Gly Val Ser Thr Thr Thr Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Val Cys Leu Ala Arg Asn65 70 75 80Asn Ala Asn Ser Thr Asp Pro Thr Ser Phe Trp Glu Asn Asp Ile Ser 85 90 95Glu Val Gln Arg 10038300DNACannabis sativa 38atggagatga gagctttggt ttcttcgtat tctaccggta atggaggttc tgatccgatc 60agcctcacga atggttctcc atggatcaca acagttggtg gtggtgcaag taccatggac 120agagagtttc cattgactgt gaagctggga agtagtatga gagccttcaa aggagtaagc 180acaacaacag ttttgtctcg gaccaaacaa cagtttcctc tggtatgctt agcaagaaac 240aatgcgaaca gcactgatcc gacctcgttc tgggagaatg atatatctga agttcaaagg 3003923DNAArtificial SequencegRNA 39ttggtttctt cgtattctac cgg 234023DNAArtificial SequencegRNA 40tcttcgtatt ctaccggtaa tgg 234123DNAArtificial SequencegRNA 41tcgtattcta ccggtaatgg agg 234223DNAArtificial SequencegRNA 42gatccgatca gcctcacgaa tgg 234323DNAArtificial SequencegRNA 43gcctcacgaa tggttctcca tgg 234423DNAArtificial SequencegRNA 44tctccatgga tcacaacagt tgg 234523DNAArtificial SequencegRNA 45ccatggatca caacagttgg tgg 234623DNAArtificial SequencegRNA 46tggatcacaa cagttggtgg tgg 234723DNAArtificial SequencegRNA 47tggtggtggt gcaagtacca tgg 234823DNAArtificial SequencegRNA 48gtttccattg actgtgaagc tgg 234923DNAArtificial SequencegRNA 49agtagtatga gagccttcaa agg 235023DNAArtificial SequencegRNA 50gcacaacaac agttttgtct cgg 235123DNAArtificial SequencegRNA 51gaccaaacaa cagtttcctc tgg 235223DNAArtificial SequencegRNA 52gcactgatcc gacctcgttc tgg 235323DNAArtificial SequencegRNA 53atgatatatc tgaagttcaa agg 2354300DNACannabis sativa 54cctttgaact tcagatatat cattctccca gaacgaggtc ggatcagtgc tgttcgcatt 60gtttcttgct aagcatacca gaggaaactg ttgtttggtc cgagacaaaa ctgttgttgt 120gcttactcct ttgaaggctc tcatactact tcccagcttc acagtcaatg gaaactctct 180gtccatggta cttgcaccac caccaactgt tgtgatccat ggagaaccat tcgtgaggct 240gatcggatca gaacctccat taccggtaga atacgaagaa accaaagctc tcatctccat 3005523DNAArtificial SequencegRNA 55tatatcattc tcccagaacg agg 235623DNAArtificial SequencegRNA 56tcattctccc agaacgaggt cgg 235723DNAArtificial SequencegRNA 57tttcttgcta agcataccag agg 235823DNAArtificial SequencegRNA 58taccagagga aactgttgtt tgg 235923DNAArtificial SequencegRNA 59tgttgtgctt actcctttga agg 236023DNAArtificial SequencegRNA 60cttcccagct tcacagtcaa tgg 236123DNAArtificial SequencegRNA 61caatggaaac tctctgtcca tgg 236223DNAArtificial SequencegRNA 62ccaccaactg ttgtgatcca tgg 236323DNAArtificial SequencegRNA 63tccatggaga accattcgtg agg 236423DNAArtificial SequencegRNA 64gaaccattcg tgaggctgat cgg 236523DNAArtificial SequencegRNA 65cggatcagaa cctccattac cgg 2366100PRTCannabis sativa 66Met Glu Met Arg Ala Leu Val Ser Ser Cys Ser Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Ile Ser Leu Thr Asn Gly Ser Pro Trp Ile Thr Thr Val 20 25 30Gly Gly Gly Ala Ser Thr Met Asp Arg Glu Phe Pro Ala Thr Val Lys 35 40 45Leu Gly Ser Ser Met Arg Ala Phe Lys Gly Val Ser Thr Ile Thr Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Val Cys Leu Ala Arg Asn65 70 75 80Asn Gly Asn Ser Thr Asp Pro Thr Ser Phe Trp Glu Asn Asp Ile Ser 85 90 95Glu Thr Gln Arg 10067300DNACannabis sativa 67atggagatga gagctttggt ttcttcgtgt tctacgggga atggagggtc tgatccgatc 60agcctcacga atggttctcc atggatcaca acagttggtg gtggtgcaag taccatggac 120agagagtttc cagcgactgt gaagctggga agtagtatga gagccttcaa aggagtaagc 180acaataacag ttctgtctcg gaccaaacaa cagtttcctc tggtatgctt agcaagaaac 240aacggaaaca gcactgatcc gacctcgttc tgggagaacg atatatctga aactcaaagg 3006823DNAArtificial SequencegRNA 68tttggtttct tcgtgttcta cgg 236923DNAArtificial SequencegRNA 69ttggtttctt cgtgttctac ggg 237023DNAArtificial SequencegRNA 70tggtttcttc gtgttctacg ggg 237123DNAArtificial SequencegRNA 71tcttcgtgtt ctacggggaa tgg 237223DNAArtificial SequencegRNA 72tcgtgttcta cggggaatgg agg 237323DNAArtificial SequencegRNA 73gatccgatca gcctcacgaa tgg 237423DNAArtificial SequencegRNA 74gcctcacgaa tggttctcca tgg 237523DNAArtificial SequencegRNA 75tctccatgga tcacaacagt tgg 237623DNAArtificial SequencegRNA 76ccatggatca caacagttgg tgg 237723DNAArtificial SequencegRNA 77tggatcacaa cagttggtgg tgg 237823DNAArtificial SequencegRNA 78tggtggtggt gcaagtacca tgg 237923DNAArtificial SequencegRNA 79gtttccagcg actgtgaagc tgg 238023DNAArtificial SequencegRNA 80agtagtatga gagccttcaa agg 238123DNAArtificial SequencegRNA 81gcacaataac agttctgtct cgg 238223DNAArtificial SequencegRNA 82gaccaaacaa cagtttcctc tgg 238323DNAArtificial SequencegRNA 83gtatgcttag caagaaacaa cgg 238423DNAArtificial SequencegRNA 84gcactgatcc gacctcgttc tgg 238523DNAArtificial SequencegRNA 85acgatatatc tgaaactcaa agg 2386300DNACannabis sativa 86cctttgagtt tcagatatat cgttctccca gaacgaggtc ggatcagtgc tgtttccgtt 60gtttcttgct aagcatacca gaggaaactg ttgtttggtc cgagacagaa ctgttattgt 120gcttactcct ttgaaggctc tcatactact tcccagcttc acagtcgctg gaaactctct 180gtccatggta cttgcaccac caccaactgt tgtgatccat ggagaaccat tcgtgaggct 240gatcggatca gaccctccat tccccgtaga acacgaagaa accaaagctc tcatctccat 3008723DNAArtificial SequencegRNA 87tatatcgttc tcccagaacg agg 238823DNAArtificial SequencegRNA 88tcgttctccc agaacgaggt cgg 238923DNAArtificial SequencegRNA 89tttcttgcta agcataccag agg 239023DNAArtificial SequencegRNA 90taccagagga aactgttgtt tgg 239123DNAArtificial SequencegRNA 91tattgtgctt actcctttga agg 239223DNAArtificial SequencegRNA 92cttcccagct tcacagtcgc tgg 239323DNAArtificial SequencegRNA 93cgctggaaac tctctgtcca tgg 239423DNAArtificial SequencegRNA 94ccaccaactg ttgtgatcca tgg 239523DNAArtificial SequencegRNA 95tccatggaga accattcgtg agg 239623DNAArtificial SequencegRNA 96gaaccattcg tgaggctgat cgg 2397100PRTCapsella rubella 97Met Glu Met Arg Ala Leu Val Ser Ser Cys Ser Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Ile Ser Leu Thr Asn Val Ser Pro Trp Ile Thr Thr Val 20 25 30Gly Gly Gly Ala Ser Ser Ile Asp Arg Glu Phe Pro Thr Thr Val Lys 35 40 45Leu Gly Ser Ser Leu Arg Thr Phe Lys Gly Val Ser Ser Thr Thr Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Val Cys Leu Ala Arg Asn65 70

75 80Asn Ala Asn Ser Thr Asp Pro Thr Leu Phe Trp Glu Asn Asp Ile Ser 85 90 95Glu Ala Gln Ser 10098300DNACapsella rubella 98atggagatga gagctttggt ttcttcgtgt tctaccggta atggaggttc tgatccgatt 60agcctcacga atgtttctcc atggatcaca acagttggtg gtggtgcaag ttccattgac 120agagagtttc caacgactgt gaagctggga agtagtctga gaactttcaa aggagtaagc 180tctacgacag ttttgtctcg gaccaaacaa cagtttcctc tggtttgttt agcaagaaac 240aatgccaaca gcactgatcc aaccttgttc tgggaaaatg acatatctga agctcaaagc 3009923DNAArtificial SequencegRNA 99ttggtttctt cgtgttctac cgg 2310023DNAArtificial SequencegRNA 100tcttcgtgtt ctaccggtaa tgg 2310123DNAArtificial SequencegRNA 101tcgtgttcta ccggtaatgg agg 2310223DNAArtificial SequencegRNA 102gcctcacgaa tgtttctcca tgg 2310323DNAArtificial SequencegRNA 103tctccatgga tcacaacagt tgg 2310423DNAArtificial SequencegRNA 104ccatggatca caacagttgg tgg 2310523DNAArtificial SequencegRNA 105gtttccaacg actgtgaagc tgg 2310623DNAArtificial SequencegRNA 106agtagtctga gaactttcaa agg 2310723DNAArtificial SequencegRNA 107gctctacgac agttttgtct cgg 2310823DNAArtificial SequencegRNA 108gaccaaacaa cagtttcctc tgg 2310923DNAArtificial SequencegRNA 109gcactgatcc aaccttgttc tgg 23110300DNACapsella rubella 110gctttgagct tcagatatgt cattttccca gaacaaggtt ggatcagtgc tgttggcatt 60gtttcttgct aaacaaacca gaggaaactg ttgtttggtc cgagacaaaa ctgtcgtaga 120gcttactcct ttgaaagttc tcagactact tcccagcttc acagtcgttg gaaactctct 180gtcaatggaa cttgcaccac caccaactgt tgtgatccat ggagaaacat tcgtgaggct 240aatcggatca gaacctccat taccggtaga acacgaagaa accaaagctc tcatctccat 30011123DNAArtificial SequencegRNA 111tatgtcattt tcccagaaca agg 2311223DNAArtificial SequencegRNA 112tcattttccc agaacaaggt tgg 2311323DNAArtificial SequencegRNA 113caaggttgga tcagtgctgt tgg 2311423DNAArtificial SequencegRNA 114tttcttgcta aacaaaccag agg 2311523DNAArtificial SequencegRNA 115aaccagagga aactgttgtt tgg 2311623DNAArtificial SequencegRNA 116cttcccagct tcacagtcgt tgg 2311723DNAArtificial SequencegRNA 117cgttggaaac tctctgtcaa tgg 2311823DNAArtificial SequencegRNA 118ccaccaactg ttgtgatcca tgg 2311923DNAArtificial SequencegRNA 119tccatggaga aacattcgtg agg 2312023DNAArtificial SequencegRNA 120gaaacattcg tgaggctaat cgg 2312123DNAArtificial SequencegRNA 121cggatcagaa cctccattac cgg 23122100PRTBrassica oleracea 122Met Glu Met Arg Ala Leu Val Ser Cys Ser Ala Ala Gly Asn Gly Ala1 5 10 15Ser Asp Arg Phe Arg Leu Ser Asn Val Ser Pro Trp Ile Thr Ser Ala 20 25 30Arg Gly Ala Ser Gly Ser Asp Ser Pro Ala Thr Val Lys Leu Arg Ser 35 40 45Ser Ser Met Ile Arg Ala Phe Lys Gly Val Ser Ile Tyr Lys Asn Lys 50 55 60Thr Arg Arg Asn Val Leu Ser Gln Arg Asn Lys Gln Phe Arg Pro Met65 70 75 80Ala Tyr Leu Gly Arg Lys Asp Leu Ser Ser Pro Asp Pro Thr Ser Phe 85 90 95Cys Asp Asn Asp 100123300DNABrassica oleracea 123atggagatga gagctttggt ttcgtgttct gctgccggaa atggagcttc tgatcggttt 60agactctcca atgtttcacc atggatcaca tctgctcgtg gtgcaagtgg cagtgactcc 120ccagccacag tgaagctgag aagcagctct atgattagag ctttcaaagg agtttcgatt 180tacaaaaaca agaccagaag aaatgttctg tctcaaagga acaaacagtt ccgtcctatg 240gcctacttag gaaggaagga cttgagcagc cctgatccga cctccttctg cgataatgat 30012423DNAArtificial SequencegRNA 124ttggtttcgt gttctgctgc cgg 2312523DNAArtificial SequencegRNA 125tcgtgttctg ctgccggaaa tgg 2312623DNAArtificial SequencegRNA 126ccggaaatgg agcttctgat cgg 2312723DNAArtificial SequencegRNA 127gactctccaa tgtttcacca tgg 2312823DNAArtificial SequencegRNA 128ccatggatca catctgctcg tgg 2312923DNAArtificial SequencegRNA 129acatctgctc gtggtgcaag tgg 2313023DNAArtificial SequencegRNA 130tctatgatta gagctttcaa agg 2313123DNAArtificial SequencegRNA 131gaagaaatgt tctgtctcaa agg 2313223DNAArtificial SequencegRNA 132gaacaaacag ttccgtccta tgg 2313323DNAArtificial SequencegRNA 133ttccgtccta tggcctactt agg 2313423DNAArtificial SequencegRNA 134gtcctatggc ctacttagga agg 2313523DNAArtificial SequencegRNA 135tatggcctac ttaggaagga agg 23136300DNABrassica oleracea 136atcattatcg cagaaggagg tcggatcagg gctgctcaag tccttccttc ctaagtaggc 60cataggacgg aactgtttgt tcctttgaga cagaacattt cttctggtct tgtttttgta 120aatcgaaact cctttgaaag ctctaatcat agagctgctt ctcagcttca ctgtggctgg 180ggagtcactg ccacttgcac cacgagcaga tgtgatccat ggtgaaacat tggagagtct 240aaaccgatca gaagctccat ttccggcagc agaacacgaa accaaagctc tcatctccat 30013723DNAArtificial SequencegRNA 137tcattatcgc agaaggaggt cgg 2313823DNAArtificial SequencegRNA 138tcgcagaagg aggtcggatc agg 2313923DNAArtificial SequencegRNA 139caagtccttc cttcctaagt agg 2314023DNAArtificial SequencegRNA 140ttccttccta agtaggccat agg 2314123DNAArtificial SequencegRNA 141ttcctaagta ggccatagga cgg 2314223DNAArtificial SequencegRNA 142tggatcacaa cagttggtgg tgg 2314323DNAArtificial SequencegRNA 143ttgagacaga acatttcttc tgg 2314423DNAArtificial SequencegRNA 144gctgcttctc agcttcactg tgg 2314523DNAArtificial SequencegRNA 145cttctcagct tcactgtggc tgg 2314623DNAArtificial SequencegRNA 146ttctcagctt cactgtggct ggg 2314723DNAArtificial SequencegRNA 147tctcagcttc actgtggctg ggg 2314823DNAArtificial SequencegRNA 148ccacgagcag atgtgatcca tgg 2314923DNAArtificial SequencegRNA 149tgtgatccat ggtgaaacat tgg 2315023DNAArtificial SequencegRNA 150ccgatcagaa gctccatttc cgg 23151371PRTBrassica napus 151Met Glu Met Arg Ala Leu Val Ser Cys Ser Ala Ala Gly Asn Gly Ala1 5 10 15Ser Asp Arg Phe Arg Leu Ser Asn Val Ser Pro Trp Ile Thr Ser Ala 20 25 30Arg Gly Ala Ser Gly Ser Asp Ser Pro Ala Thr Val Lys Leu Gly Ser 35 40 45Ser Ser Met Ile Arg Ala Phe Lys Gly Val Ser Ile Tyr Lys Asn Lys 50 55 60Thr Arg Arg Asn Val Leu Ser Gln Arg Asn Lys Gln Phe Arg Pro Met65 70 75 80Ala Tyr Leu Gly Arg Lys Asp Leu Ser Ser Pro Asp Pro Thr Ser Phe 85 90 95Cys Asp Asn Asp Ile Ser Glu Pro Gln Gly Thr Gly Ser Ile Asn Gly 100 105 110Asn Asp His Ser Ala Val Arg Val Ser Gln Val Asp Glu Phe Cys Lys 115 120 125Ala His Gly Gly Lys Arg Pro Ile His Ser Ile Leu Val Ala Thr Asn 130 135 140Gly Met Ala Ala Val Lys Leu Ile Arg Ser Val Arg Ala Trp Ser Tyr145 150 155 160Gln Thr Phe Gly Ser Glu Lys Ser Ile Ser Leu Val Ala Met Ala Thr 165 170 175Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln 180 185 190Phe Met Gln Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val 195 200 205His Leu Ile Val Glu Met Ala Gln Ala Thr Gly Val Asp Ala Val Trp 210 215 220Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu225 230 235 240Lys Ala Lys Gly Val Ile Phe Leu Gly Pro Thr Ala Ala Ser Met Leu 245 250 255Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp 260 265 270Val Pro Thr Leu Pro Trp Ser Gly Ser His Val Lys Ile Pro Pro Gly 275 280 285Ser Ser Met Val Thr Ile Pro Glu Glu Met Tyr Arg Gln Ala Cys Val 290 295 300Tyr Thr Thr Glu Glu Ala Val Ala Ser Cys Gln Val Val Gly Tyr Pro305 310 315 320Ala Met Ile Lys Ala Ser Trp Gly Gly Gly Gly Lys Gly Ile Arg Glu 325 330 335Val His Asp Asp Asp Glu Val Arg Thr Leu Phe Lys Gln Val Gln Gly 340 345 350Glu Val Pro Gly Ser Pro Ile Phe Ile Met Lys Val Ala Ser Gln Ser 355 360 365Arg His Leu 370152298DNABrassica napus 152atggagatga gagctttagt ttcgtgttct gctgccggaa atggagcttc tgatcggttt 60agactctcca atgtttcacc atggatcaca tcagctcgtg gtgcaagtgg cagtgactcc 120ccagccacag tgaagctggg aagcagctct atgattagag ctttcaaagg cgtttcgatt 180tacaaaaaca agaccagaag gaatgttctg tctcaaagga acaaacagtt ccgtcctatg 240gcctacttag gaaggaagga cttgagcagc cctgatccga cctccttctg cgataatg 29815323DNAArtificial SequencegRNA 153ttagtttcgt gttctgctgc cgg 2315423DNAArtificial SequencegRNA 154tcgtgttctg ctgccggaaa tgg 2315523DNAArtificial SequencegRNA 155ccggaaatgg agcttctgat cgg 2315623DNAArtificial SequencegRNA 156gactctccaa tgtttcacca tgg 2315723DNAArtificial SequencegRNA 157ccatggatca catcagctcg tgg 2315823DNAArtificial SequencegRNA 158acatcagctc gtggtgcaag tgg 2315923DNAArtificial SequencegRNA 159ctccccagcc acagtgaagc tgg 2316023DNAArtificial SequencegRNA 160tccccagcca cagtgaagct ggg 2316123DNAArtificial SequencegRNA 161tctatgatta gagctttcaa agg 2316223DNAArtificial SequencegRNA 162tttacaaaaa caagaccaga agg 2316323DNAArtificial SequencegRNA 163gaaggaatgt tctgtctcaa agg 2316423DNAArtificial SequencegRNA 164gaacaaacag ttccgtccta tgg 2316523DNAArtificial SequencegRNA 165ttccgtccta tggcctactt agg 2316623DNAArtificial SequencegRNA 166gtcctatggc ctacttagga agg 2316723DNAArtificial SequencegRNA 167tatggcctac ttaggaagga agg 23168298DNABrassica napus 168cattatcgca gaaggaggtc ggatcagggc tgctcaagtc cttccttcct aagtaggcca 60taggacggaa ctgtttgttc ctttgagaca gaacattcct tctggtcttg tttttgtaaa 120tcgaaacgcc tttgaaagct ctaatcatag agctgcttcc cagcttcact gtggctgggg 180agtcactgcc acttgcacca cgagctgatg tgatccatgg tgaaacattg gagagtctaa 240accgatcaga agctccattt ccggcagcag aacacgaaac taaagctctc atctccat 29816923DNAArtificial SequencegRNA 169tcgcagaagg aggtcggatc agg 2317023DNAArtificial SequencegRNA 170cgcagaagga ggtcggatca ggg 2317123DNAArtificial SequencegRNA 171caagtccttc cttcctaagt agg 2317223DNAArtificial SequencegRNA 172ttccttccta agtaggccat agg 2317323DNAArtificial SequencegRNA 173ttcctaagta ggccatagga cgg 2317423DNAArtificial SequencegRNA 174ttgagacaga acattccttc tgg 2317523DNAArtificial SequencegRNA 175cttcccagct tcactgtggc tgg 2317623DNAArtificial SequencegRNA 176ttcccagctt cactgtggct ggg 2317723DNAArtificial SequencegRNA 177tcccagcttc actgtggctg ggg 2317823DNAArtificial SequencegRNA 178ccacgagctg atgtgatcca tgg 2317923DNAArtificial SequencegRNA 179tgtgatccat ggtgaaacat tgg 2318023DNAArtificial SequencegRNA 180ccgatcagaa gctccatttc cgg 23181455PRTBrassica napus 181Met Glu Met Arg Ala Leu Val Ser Cys Ser Ala Ala Gly Asn Gly Ala1 5 10 15Ser Asp Arg Phe Arg Leu Ser Asn Val Ser Pro Trp Ile Thr Ser Ala 20 25 30Arg Gly Ala Ser Gly Ser Asp Ser Pro Ala Thr Val Lys Leu Gly Ser 35 40 45Ser Ser Met Ile Arg Ala Phe Lys Gly Val Ser Ile Tyr Lys Asn Lys 50 55 60Thr Arg Arg Asn Val Leu Ser Gln Arg Asn Lys Gln Phe Arg Pro Met65 70 75 80Ala Tyr Leu Gly Arg Lys Asp Leu Ser Ser Pro Asp Pro Thr Ser Phe 85 90 95Cys Asp Asn Asp Ile Ser Glu Pro Gln Gly Thr Gly Ser Ile Asn Gly 100 105 110Asn Asp His Ser Ala Val Arg Val Ser Gln Val Asp Glu Phe Cys Lys 115 120 125Ala His Gly Gly Lys Arg Pro Ile His Arg Ile Leu Val Ala Thr Asn 130 135 140Gly Met Ala Ala Val Lys Phe Ile Arg Ser Val Arg Ala Trp Ser Tyr145 150 155 160Gln Thr Phe Gly Ser Glu Lys Ser Ile Ser Leu Val Ala Met Ala Thr 165 170 175Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln 180 185 190Phe Met Gln Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val 195 200 205His Leu Ile Val Glu Met Ala Glu Ala Thr Gly Val Asp Ala Val Trp 210 215 220Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu225 230 235 240Lys Ala Lys Gly Val Ile Phe Leu Gly Pro Thr Ala Ala Ser Met Leu 245 250 255Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp 260 265 270Val Pro Thr Leu Pro Trp Ser Gly Ser His Val Lys Ile Pro Pro Gly 275 280 285Ser Ser Leu Val Thr Ile Pro Glu Glu Met Tyr Arg Gln Ala Cys Val 290 295 300Tyr Thr Thr Glu Glu Ala Val Ala Ser Cys Gln Val Val Gly Tyr Pro305 310 315 320Ala Met Ile Lys Ala Ser Trp Gly Gly Gly Gly Lys Gly Ile Arg Lys 325 330 335Val His Asp Asp Asp Glu Val Arg Ala Leu Phe Lys Gln Val Gln Gly 340 345 350Glu Val Pro Gly Ser Pro Ile Phe Ile Met Lys Val Ala Ser Gln Ser 355 360 365Arg His Leu Glu Val Gln Leu Leu Cys Asp Gln Tyr Gly Asn Val Ser 370 375 380Ala Leu His Ser Arg Asp Cys Ser Val Gln Arg Arg His Gln Lys Ile385 390 395 400Ile Glu Glu Gly Pro Ile Thr Val Ala Pro Arg Asp Thr Val Lys Lys 405 410 415Leu Glu Gln Ala Ala Arg Arg Leu Ala Lys Ser Val Asn Tyr Val Gly 420 425 430Ala Ala Thr Val Glu Phe Leu Tyr Ser Met Asp Thr Gly Asp Tyr Phe 435 440 445Phe Leu Glu Leu Asn Pro Arg 450 455182298DNABrassica napus 182atggagatga gagctttggt ttcgtgttct gctgccggaa atggagcttc tgatcggttt 60agactctcca atgtttcacc atggatcaca tcagctcgtg gtgcaagtgg cagtgactcc 120ccagccacag tgaagctggg aagcagctct atgatcagag ccttcaaagg agtttcgatt 180tacaaaaaca agaccagaag aaatgttttg tctcaaagga acaaacagtt tcgtcctatg 240gcctacttag gaaggaagga cttgagcagc cctgatccga cctccttctg cgataatg 29818323DNAArtificial SequencegRNA 183ttggtttcgt gttctgctgc cgg 2318423DNAArtificial SequencegRNA 184tcgtgttctg ctgccggaaa tgg

2318523DNAArtificial SequencegRNA 185ccggaaatgg agcttctgat cgg 2318623DNAArtificial SequencegRNA 186gactctccaa tgtttcacca tgg 2318723DNAArtificial SequencegRNA 187ccatggatca catcagctcg tgg 2318823DNAArtificial SequencegRNA 188acatcagctc gtggtgcaag tgg 2318923DNAArtificial SequencegRNA 189ctccccagcc acagtgaagc tgg 2319023DNAArtificial SequencegRNA 190tccccagcca cagtgaagct ggg 2319123DNAArtificial SequencegRNA 191tctatgatca gagccttcaa agg 2319223DNAArtificial SequencegRNA 192gaagaaatgt tttgtctcaa agg 2319323DNAArtificial SequencegRNA 193gaacaaacag tttcgtccta tgg 2319423DNAArtificial SequencegRNA 194tttcgtccta tggcctactt agg 2319523DNAArtificial SequencegRNA 195gtcctatggc ctacttagga agg 2319623DNAArtificial SequencegRNA 196tatggcctac ttaggaagga agg 23197298DNABrassica napus 197cattatcgca gaaggaggtc ggatcagggc tgctcaagtc cttccttcct aagtaggcca 60taggacgaaa ctgtttgttc ctttgagaca aaacatttct tctggtcttg tttttgtaaa 120tcgaaactcc tttgaaggct ctgatcatag agctgcttcc cagcttcact gtggctgggg 180agtcactgcc acttgcacca cgagctgatg tgatccatgg tgaaacattg gagagtctaa 240accgatcaga agctccattt ccggcagcag aacacgaaac caaagctctc atctccat 29819823DNAArtificial SequencegRNA 198tcgcagaagg aggtcggatc agg 2319923DNAArtificial SequencegRNA 199cgcagaagga ggtcggatca ggg 2320023DNAArtificial SequencegRNA 200caagtccttc cttcctaagt agg 2320123DNAArtificial SequencegRNA 201ttccttccta agtaggccat agg 2320223DNAArtificial SequencegRNA 202ttgagacaaa acatttcttc tgg 2320323DNAArtificial SequencegRNA 203gtaaatcgaa actcctttga agg 2320423DNAArtificial SequencegRNA 204gctgcttccc agcttcactg tgg 2320523DNAArtificial SequencegRNA 205cttcccagct tcactgtggc tgg 2320623DNAArtificial SequencegRNA 206ttcccagctt cactgtggct ggg 2320723DNAArtificial SequencegRNA 207tcccagcttc actgtggctg ggg 2320823DNAArtificial SequencegRNA 208ccacgagctg atgtgatcca tgg 2320923DNAArtificial SequencegRNA 209tgtgatccat ggtgaaacat tgg 2321023DNAArtificial SequencegRNA 210ccgatcagaa gctccatttc cgg 23211100PRTBrassica rapa 211Met Glu Met Arg Ala Leu Val Ser Cys Ser Ala Ala Gly Asn Gly Ala1 5 10 15Ser Asp Arg Phe Arg Leu Ser Asn Val Ser Pro Trp Ile Thr Ser Ala 20 25 30Arg Gly Ala Ser Gly Ser Asp Ser Pro Ala Thr Val Lys Leu Gly Ser 35 40 45Ser Ser Met Ile Arg Ala Phe Lys Gly Val Ser Ile Tyr Lys Asn Lys 50 55 60Ser Arg Arg Asn Val Leu Ser Gln Arg Asn Lys Gln Phe Arg Pro Met65 70 75 80Ala Tyr Leu Gly Arg Lys Asp Leu Ser Ser Pro Asp Pro Thr Ser Phe 85 90 95Cys Asp Asn Asp 100212300DNABrassica rapa 212atggagatga gagctttggt ttcgtgttct gctgccggaa atggagcttc tgatcggttt 60agactctcca atgtttcacc atggatcaca tcagctcgtg gtgcaagtgg cagtgactcc 120ccagccacag tgaagctggg aagcagctct atgatcagag ccttcaaagg agtttcgatt 180tacaaaaaca agagcagaag aaatgttctg tctcaaagga acaaacagtt tcgtcctatg 240gcctacttag gaaggaagga cttgagcagc cctgatccga cctccttctg cgataatgat 30021323DNAArtificial SequencegRNA 213ttggtttcgt gttctgctgc cgg 2321423DNAArtificial SequencegRNA 214tcgtgttctg ctgccggaaa tgg 2321523DNAArtificial SequencegRNA 215ccggaaatgg agcttctgat cgg 2321623DNAArtificial SequencegRNA 216gactctccaa tgtttcacca tgg 2321723DNAArtificial SequencegRNA 217ccatggatca catcagctcg tgg 2321823DNAArtificial SequencegRNA 218acatcagctc gtggtgcaag tgg 2321923DNAArtificial SequencegRNA 219ctccccagcc acagtgaagc tgg 2322023DNAArtificial SequencegRNA 220tctatgatca gagccttcaa agg 2322123DNAArtificial SequencegRNA 221gaagaaatgt tctgtctcaa agg 2322295PRTArabidopsis thaliana 222Met Ala Gly Ser Val Asn Gly Asn His Ser Ala Val Gly Pro Gly Ile1 5 10 15Asn Tyr Glu Thr Val Ser Gln Val Asp Glu Phe Cys Lys Ala Leu Arg 20 25 30Gly Lys Arg Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala 35 40 45Ala Val Lys Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe 50 55 60Gly Thr Glu Lys Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp65 70 75 80Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val 85 90 9522323DNAArtificial SequencegRNA 223tttcgtccta tggcctactt agg 2322423DNAArtificial SequencegRNA 224gtcctatggc ctacttagga agg 2322523DNAArtificial SequencegRNA 225tatggcctac ttaggaagga agg 23226300DNABrassica napus 226atcattatcg cagaaggagg tcggatcagg gctgctcaag tccttccttc ctaagtaggc 60cataggacga aactgtttgt tcctttgaga cagaacattt cttctgctct tgtttttgta 120aatcgaaact cctttgaagg ctctgatcat agagctgctt cccagcttca ctgtggctgg 180ggagtcactg ccacttgcac cacgagctga tgtgatccat ggtgaaacat tggagagtct 240aaaccgatca gaagctccat ttccggcagc agaacacgaa accaaagctc tcatctccat 30022723DNAArtificial SequencegRNA 227tcattatcgc agaaggaggt cgg 2322823DNAArtificial SequencegRNA 228tcgcagaagg aggtcggatc agg 2322923DNAArtificial SequencegRNA 229caagtccttc cttcctaagt agg 2323023DNAArtificial SequencegRNA 230ttccttccta agtaggccat agg 2323123DNAArtificial SequencegRNA 231gtaaatcgaa actcctttga agg 2323223DNAArtificial SequencegRNA 232gctgcttccc agcttcactg tgg 2323323DNAArtificial SequencegRNA 233cttcccagct tcactgtggc tgg 2323423DNAArtificial SequencegRNA 234ttcccagctt cactgtggct ggg 2323523DNAArtificial SequencegRNA 235tcccagcttc actgtggctg ggg 2323623DNAArtificial SequencegRNA 236ccacgagctg atgtgatcca tgg 2323723DNAArtificial SequencegRNA 237tgtgatccat ggtgaaacat tgg 2323823DNAArtificial SequencegRNA 238ccgatcagaa gctccatttc cgg 2323923DNAArtificial SequencegRNA 239tccatggaga tatattcgtg agg 2324023DNAArtificial SequencegRNA 240ccctcacgaa tatatctcca tgg 2324123DNAArtificial SequencegRNA 241gatatattcg tgagggtaat tgg 2324223DNAArtificial SequencegRNA 242cttctcagct tcactgtcga cgg 2324323DNAArtificial SequencegRNA 243ccatggagat atattcgtga ggg 2324423DNAArtificial SequencegRNA 244cttcgacaag agttttgtct cgg 2324523DNAArtificial SequencegRNA 245tcaagagtga gaacctttaa agg 2324623DNAArtificial SequencegRNA 246tcttcgtgtt ctactggtaa tgg 2324723DNAArtificial SequencegRNA 247ttgggttctt cgtgttctac tgg 2324823DNAArtificial SequencegRNA 248tgtcgaagaa actcctttaa agg 2324923DNAArtificial SequencegRNA 249tcttgacgaa actcctttga agg 2325023DNAArtificial SequencegRNA 250agtagtttga gaaccttcaa agg 2325123DNAArtificial SequencegRNA 251tcgtgttcta ctggtaatgg agg 2325223DNAArtificial SequencegRNA 252ggaaaaactg ttgtgatcca tgg 2325323DNAArtificial SequencegRNA 253aaacagagga aactgttgtt tgg 2325423DNAArtificial SequencegRNA 254gggtttagga aacaaaacag agg 2325524DNAArtificial SequenceACC2 target 255gattgcctca cgaatatatc tcca 2425624DNAArtificial SequenceACC2 target 256aaactggaga tatattcgtg aggc 2425724DNAArtificial Sequenceprimer 257tctcttcctc cttaaaaagc caca 2425820DNAArtificial Sequenceprimer 258ctaggattcg aaaccagcgt 2025923DNAArtificial SequenceZm-PPR10 wt binding site 259attgtatcct taaccatttc ttt 2326023DNAArtificial SequenceBn-PPR10 wt binding site 260attgtatcat taactatttc ttt 2326123DNAArtificial SequenceZm-PPR10GG mut binding site 261attgtaggct taaccatttc ttt 232621140DNAArtificial SequencePrrn-PPR10GG-GFP-TrbcL 262gagctcgctc ccccgccgtc gttcaatgag aatggataag aggctcgtgg gattgacgtg 60agggggcagg gatggctata tttctgggag ttacttctac ccgatagagc ttagaagttg 120gaagtaataa tttcttggtt gattgtaggc ttaaccattt cttttttttt gacacgagga 180actcatcatg gctagcagta aaggagaaga acttttcact ggagttgtcc caattcttgt 240tgaattagat ggtgatgtta atgggcacaa attttctgtc agtggagagg gtgaaggtga 300tgcaacatac ggaaaactta cccttaaatt tatttgcact actggaaaac tacctgttcc 360ttggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttttcaa gatacccaga 420tcatatgaag cggcacgact tcttcaagag cgccatgcct gagggatacg tgcaggagag 480gaccatctct ttcaaggacg acgggaacta caagacacgt gctgaagtca agtttgaggg 540agacaccctc gtcaacagga tcgagcttaa gggaatcgat ttcaaggagg acggaaacat 600cctcggccac aagttggaat acaactacaa ctcccacaac gtatacatca cggcagacaa 660acaaaagaat ggaatcaaag ctaacttcaa aattagacac aacattgaag atggaagcgt 720tcaactagca gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc 780agacaaccat tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga 840ccacatggtc cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact 900gtacaaataa atctagaaaa cagtagacat tagcagataa attagcagga aataaagaag 960gataaggaga aagaactcaa gtaattatcc ttcgttctct taattgaatt gcaattaaac 1020tcggcccaat cttttactaa aaggattgag ccgaatacaa caaagattct attgcatata 1080ttttgactaa gtatatactt acctagatat acaagatttg aaatacaaaa tctaaagctt 1140263147DNAArtificial SequencetRNA 263aagtctttac aatgacaatg gaaaccgatg taaagggatg tagcgcagct tggtagcgcg 60tttgttttgg gtacaaaatg tcacaggttc aaatcctgtc atccctatcc ctaacttgta 120gttatcgtat cagcagtaac aatagat 1472642361DNAZea mays 264atggaggcca ccggcagggg gctgttcccg aacaagccca ccctcccggc ggggccgagg 60aaacggggcc cgctcctccc ggccgcgccc ccgccaccgt ccccctcctc gctcccgctc 120gactcgctcc tgctccacct caccgcgccc gcccccgcgc cggcccccgc gccgcggcgg 180tcgcaccaga cgccgacgcc gccgcactcc ttcctctccc ccgacgcgca ggtgctggtg 240ctcgccatct cctcgcaccc gctccccacg ctggcggcct tcctggcctc ccgccgcgac 300gagctcctcc gcgcggacat cacgtccctg ctcaaggcgc tggagctctc ggggcactgg 360gagtgggcgc tcgcgctcct ccggtgggca ggcaaggagg gtgccgccga cgcgtcggcg 420ctcgagatgg tcgtccgcgc gctgggccgc gagggccagc acgacgccgt ctgcgcgctg 480ctcgacgaaa cgccgctccc gccgggctcc cgcctcgacg tccgcgccta caccaccgtg 540ctgcacgcgc tctcccgcgc gggccggtac gagcgcgcgc tcgagctctt cgccgagctc 600cggcgccagg gggtggcgcc cacgctcgtc acctacaacg tcgtgctgga cgtgtacggg 660cggatgggcc ggtcgtggcc gcggatcgtc gccctcctcg atgagatgcg cgccgccggg 720gtcgagcccg acggcttcac cgccagcacg gtgatcgccg cgtgctgccg cgacgggctg 780gttgacgagg cggtggcgtt cttcgaggac ctcaaggccc gcggccacgc cccgtgcgtc 840gtcacgtaca acgcgttgct ccaggtgttc ggcaaggccg ggaactacac ggaggcgctg 900cgcgtgctcg gggagatgga gcagaacggc tgccagccag atgctgtgac gtacaacgag 960ctcgccggaa cgtacgcccg ggctgggttc ttcgaggagg ctgccaggtg cctggacaca 1020atggcatcca agggtctgtt gccaaacgca ttcacgtaca acaccgtgat gacagcctat 1080gggaatgttg ggaaggtgga tgaggcgctc gctctgtttg accagatgaa gaagaccggg 1140ttcgtgccga acgtgaacac gtacaatctt gtccttggca tgcttggcaa gaagtcaagg 1200ttcacggtga tgctagagat gcttggagag atgtcgagga gcggatgcac accgaaccgg 1260gtaacatgga acacaatgct tgcagtctgt gggaagcgtg gcatggagga ctacgtcacc 1320cgggttctgg aggggatgag gtcttgcggg gttgaactga gccgagacac ctacaacacc 1380ctgatagctg cgtacggccg gtgtggctcg aggactaatg ccttcaagat gtacaacgag 1440atgaccagcg ctggattcac cccctgcatc accacgtaca acgcgttgct gaacgtgctg 1500tcgcggcagg gcgactggtc caccgcccag tcgatcgtaa gcaaaatgag gaccaagggg 1560ttcaagccga acgagcagtc gtattcgctg ctgctccagt gctacgcgaa ggggggcaac 1620gtggcaggga tagccgcgat cgagaacgag gtgtacggat caggtgccgt tttcccaagc 1680tgggtgatcc tgaggaccct tgtcatcgcc aatttcaagt gccggcgact ggatggcatg 1740gagacggcgt ttcaagaggt gaaggccaga ggctacaacc cggacctcgt gatattcaac 1800tccatgctgt ccatctacgc gaagaacggg atgtacagca aggccaccga ggtcttcgac 1860tccatcaagc ggagcgggct gagccccgac ctcatcacct acaacagcct gatggacatg 1920tacgccaagt gcagcgagtc gtgggaggcc gagaagatac tgaaccagct caagtgctcc 1980cagacgatga agcccgacgt ggtgtcctac aacacggtca taaacgggtt ctgcaagcag 2040gggctggtga aagaggccca gagggtcctc tcggagatgg tcgccgacgg catggccccc 2100tgcgccgtga cctaccacac gctcgtcggg ggttactcca gcctggagat gttcagcgag 2160gccagggagg tcatcggcta catggtccag cacggcctca agcctatgga gctgacctac 2220aggagagtcg tcgagagcta ctgcagagcg aagcggttcg aggaggctcg cggcttcctg 2280tccgaggtct cggagaccga cctggatttt gacaagaagg cgctcgaagc ctatatagag 2340gatgcgcagt ttggaaggta g 23612652361DNAArtificial SequenceZm_PPR10 GG 265atggaggcca ccggcagggg gctgttcccg aacaagccca ccctcccggc ggggccgagg 60aaacggggcc cgctcctccc ggccgcgccc ccgccaccgt ccccctcctc gctcccgctc 120gactcgctcc tgctccacct caccgcgccc gcccccgcgc cggcccccgc gccgcggcgg 180tcgcaccaga cgccgacgcc gccgcactcc ttcctctccc ccgacgcgca ggtgctggtg 240ctcgccatct cctcgcaccc gctccccacg ctggcggcct tcctggcctc ccgccgcgac 300gagctcctcc gcgcggacat cacgtccctg ctcaaggcgc tggagctctc ggggcactgg 360gagtgggcgc tcgcgctcct ccggtgggca ggcaaggagg gtgccgccga cgcgtcggcg 420ctcgagatgg tcgtccgcgc gctgggccgc gagggccagc acgacgccgt ctgcgcgctg 480ctcgacgaaa cgccgctccc gccgggctcc cgcctcgacg tccgcgccta caccaccgtg 540ctgcacgcgc tctcccgcgc gggccggtac gagcgcgcgc tcgagctctt cgccgagctc 600cggcgccagg gggtggcgcc cacgctcgtc acctacaacg tcgtgctgga cgtgtacggg 660cggatgggcc ggtcgtggcc gcggatcgtc gccctcctcg atgagatgcg cgccgccggg 720gtcgagcccg acggcttcac cgccagcacg gtgatcgccg cgtgctgccg cgacgggctg 780gttgacgagg cggtggcgtt cttcgaggac ctcaaggccc gcggccacgc cccgtgcgtc 840gtcacgtaca cagcgttgct ccaggtgttc ggcaaggccg ggaactacac ggaggcgctg 900cgcgtgctcg gggagatgga gcagaacggc tgccagccag atgctgtgac gtacaccgag 960ctcgccggaa cgtacgcccg ggctgggttc ttcgaggagg ctgccaggtg cctggacaca 1020atggcatcca agggtctgtt gccaaacgca ttcacgtaca acaccgtgat gacagcctat 1080gggaatgttg ggaaggtgga tgaggcgctc gctctgtttg accagatgaa gaagaccggg 1140ttcgtgccga acgtgaacac gtacaatctt gtccttggca tgcttggcaa gaagtcaagg 1200ttcacggtga tgctagagat gcttggagag atgtcgagga gcggatgcac accgaaccgg 1260gtaacatgga acacaatgct tgcagtctgt gggaagcgtg gcatggagga ctacgtcacc 1320cgggttctgg aggggatgag gtcttgcggg gttgaactga gccgagacac ctacaacacc 1380ctgatagctg cgtacggccg gtgtggctcg aggactaatg ccttcaagat gtacaacgag 1440atgaccagcg ctggattcac cccctgcatc accacgtaca acgcgttgct gaacgtgctg 1500tcgcggcagg gcgactggtc caccgcccag tcgatcgtaa gcaaaatgag gaccaagggg 1560ttcaagccga acgagcagtc gtattcgctg ctgctccagt gctacgcgaa ggggggcaac 1620gtggcaggga tagccgcgat cgagaacgag gtgtacggat caggtgccgt tttcccaagc 1680tgggtgatcc tgaggaccct tgtcatcgcc aatttcaagt gccggcgact ggatggcatg 1740gagacggcgt ttcaagaggt gaaggccaga ggctacaacc cggacctcgt gatattcaac 1800tccatgctgt ccatctacgc gaagaacggg atgtacagca aggccaccga ggtcttcgac 1860tccatcaagc ggagcgggct gagccccgac ctcatcacct acaacagcct gatggacatg 1920tacgccaagt gcagcgagtc gtgggaggcc gagaagatac tgaaccagct caagtgctcc 1980cagacgatga agcccgacgt ggtgtcctac aacacggtca taaacgggtt ctgcaagcag 2040gggctggtga aagaggccca gagggtcctc tcggagatgg tcgccgacgg catggccccc 2100tgcgccgtga cctaccacac gctcgtcggg ggttactcca gcctggagat gttcagcgag 2160gccagggagg tcatcggcta catggtccag cacggcctca agcctatgga gctgacctac 2220aggagagtcg tcgagagcta ctgcagagcg aagcggttcg aggaggctcg cggcttcctg 2280tccgaggtct cggagaccga cctggatttt gacaagaagg cgctcgaagc ctatatagag 2340gatgcgcagt ttggaaggta g

23612661415DNAArtificial SequencePhaseolin promoter 266ggtcgacggt atcgataagc ttgatatcga attcctgcag cccaattcat tgtactccca 60gtatcattat agtgaaagtt ttggctctct cgccggtggt tttttacctc tatttaaagg 120ggttttccac ctaaaaattc tggtatcatt ctcactttac ttgttacttt aatttctcat 180aatctttggt tgaaattatc acgcttccgc acacgatatc cctacaaatt tattatttgt 240taaacatttt caaaccgcat aaaattttat gaagtcccgt ctatctttaa tgtagtctaa 300cattttcata ttgaaatata taatttactt aattttagcg ttggtagaaa gcataatgat 360ttattcttat tcttcttcat ataaatgttt aatatacaat ataaacaaat tctttacctt 420aagaaggatt tcccatttta tattttaaaa atatatttat caaatatttt tcaaccacgt 480aaatctcata ataataagtt gtttcaaaag taataaaatt taactccata atttttttat 540tcgactgatc ttaaagcaac acccagtgac acaactagcc atttttttct ttggataaaa 600aaatccaatt atcattgtat tttttttata caatgaaaat ttcaccaaac aatcatttgt 660ggtatttctg aagcaagtca tgttatgcaa aattctataa ttcccatttg acactacgga 720agtaactgaa gatctgcttt tacatgcgag acacatcttc taaagtaatt ttaataatag 780ttactatatt caagatttca tatatcaaat actcaatatt acttctaaaa aattaattag 840atataattaa aatattactt ttttaatttt aagtttaatt gttgaatttg tgactattga 900tttattattc tactatgttt aaattgtttt atagatagtt taaagtaaat ataagtaatg 960tagtagagtg ttagagtgtt accctaaacc ataaactata acatttatgg tggactaatt 1020ttcatatatt tcttattgct tttacctttt cttggtatgt aagtccgtaa ctagaattac 1080tgtgggttgc catggcactc tgtggtcttt tggttcatgc atggatgctt gcgcaagaaa 1140aagacaaaga acaaagaaaa aagacaaaac agagagacaa aacgcaatca cacaaccaac 1200tcaaattagt cactggctga tcaagatcgc cgcgtccatg tatgtctaaa tgccatgcaa 1260agcaacacgt gcttaacatg cactttaaat ggctcaccca tctcaaccca cacacaaaca 1320cattgccttt ttcttcatca tcaccacaac cacctgtata tattcattct cttccgccac 1380ctcaatttct tcacttcaac acacgtcaac ctgca 141526722DNABrassica napus 267ggtttagact ctccaatgtt tc 2226824DNAArtificial SequenceForward Oligo 268gattgctttg taacctctca gatt 2426924DNAArtificial SequenceReverse Oligo 269aaacaatctg agaggttaca aagc 2427022DNABrassica napus 270ggaaggaagg acttgagcag cc 2227124DNAArtificial SequenceForward Oligo 271gattgccgac gagttcagga agga 2427224DNAArtificial SequenceReverse Oligo 272aaactccttc ctgaactcgt cggc 2427322DNABrassica napus 273ggtgaaacat tggagagtct aa 2227424DNAArtificial SequenceForward Oligo 274gattgaatct gagaggttac aaag 2427524DNAArtificial SequenceReverse Oligo 275aaacctttgt aacctctcag attc 2427622DNABrassica napus 276ggagcttctg atcggtttag ac 2227722DNABrassica napus 277ggtgcaagtg gcagtgactc cc 2227822DNABrassica napus 278gacaccgacc cctcagtgac gg 2227922DNABrassica napus 279ggagtttcga tttacaaaaa ca 2228022DNABrassica napus 280ggcctactta ggaaggaagg ac 2228122DNABrassica napus 281ggaaggactt gagcagccct ga 2228223DNABrassica napus 282gtcctatggc ctacttagga agg 2328322DNABrassica napus 283ggacttgagc agccctgatc cg 2228422DNABrassica napus 284atggcctact taggaaggaa gg 2228522DNABrassica napus 285cgacctcctt ctgcgataat gg 2228622DNABrassica napus 286ggagagtcta aaccgatcag aa 2228722DNABrassica napus 287ggagtcactg ccacttgcac ca 2228823DNABrassica napus 288ggggagtcac tgccacttgc acc 2328923DNABrassica napus 289ggctggggag tcactgccac ttg 2329022DNABrassica napus 290ggtcttgttt ttgtaaatcg aa 2229122DNABrassica napus 291tccttcctaa gtaggccata gg 2229222DNABrassica napus 292aagtccttcc ttcctaagta gg 2229322DNABrassica napus 293ggctgctcaa gtccttcctt cc 2229422DNABrassica napus 294gcagaaggag gtcggatcag gg 2229522DNABrassica napus 295gggctgctca agtccttcct tc 2229622DNABrassica napus 296cgcagaagga ggtcggatca gg 2229722DNABrassica napus 297cattatcgca gaaggaggtc gg 2229822DNABrassica napus 298ggtcggatca gggctgctca ag 2229922DNABrassica napus 299ggaggtcgga tcagggctgc tc 2230022DNABrassica napus 300agcaaaccat tatcgcagaa gg 22301197PRTArabidopsis thaliana 301Met Glu Met Arg Ala Leu Gly Ser Ser Cys Ser Thr Gly Asn Gly Gly1 5 10 15Ser Ala Pro Ile Thr Leu Thr Asn Ile Ser Pro Trp Ile Thr Thr Val 20 25 30Phe Pro Ser Thr Val Lys Leu Arg Ser Ser Leu Arg Thr Phe Lys Gly 35 40 45Val Ser Ser Arg Val Arg Thr Phe Lys Gly Val Ser Ser Thr Arg Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Phe Cys Phe Leu Asn Pro65 70 75 80Asp Pro Ile Ser Phe Leu Glu Asn Asp Val Ser Glu Ala Glu Arg Thr 85 90 95Val Val Leu Pro Asp Gly Ser Val Asn Gly Ala Gly Ser Val Asn Gly 100 105 110Tyr His Ser Asp Val Val Pro Gly Arg Asn Val Ala Glu Val Asn Glu 115 120 125Phe Cys Lys Ala Leu Gly Gly Lys Arg Pro Ile His Ser Ile Leu Val 130 135 140Ala Thr Asn Gly Met Ala Ala Val Lys Phe Ile Arg Ser Val Arg Thr145 150 155 160Trp Ala Tyr Glu Thr Phe Gly Ser Glu Lys Ala Val Lys Leu Val Ala 165 170 175Met Ala Thr Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile 180 185 190Ala Asp Gln Phe Val 19530292PRTArtificial Sequenceconsensus sequenceSITE(20)..(20)Xaa is any one of N,D,Q, E, B, and Z.SITE(20)..(20)Xaa is any one of N, D, Q, E, B, and Z.SITE(22)..(22)Xaa is any one of N,D,Q, E, B, and Z.SITE(39)..(39)Xaa is I or V.SITE(67)..(67)Xaa is I or V. 302Gly Ala Gly Ser Val Asn Gly Asn His Ser Ala Val Gly Pro Gly Arg1 5 10 15Asn Val Ala Xaa Val Xaa Glu Phe Cys Lys Ala Leu Arg Gly Lys Arg 20 25 30Pro Ile His Ser Ile Leu Xaa Ala Asn Asn Gly Met Ala Ala Val Lys 35 40 45Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe Gly Ser Glu 50 55 60Lys Ala Xaa Leu Leu Val Ala Met Ala Thr Pro Glu Asp Met Arg Ile65 70 75 80Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val 85 90303200PRTArabidopsis thaliana 303Met Ala Gly Ser Val Asn Gly Asn His Ser Ala Val Gly Pro Gly Ile1 5 10 15Asn Tyr Glu Thr Val Ser Gln Val Asp Glu Phe Cys Lys Ala Leu Arg 20 25 30Gly Lys Arg Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala 35 40 45Ala Val Lys Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe 50 55 60Gly Thr Glu Lys Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp65 70 75 80Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu 85 90 95Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile 100 105 110Val Glu Met Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp 115 120 125Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Asp Ala Lys 130 135 140Gly Ile Ile Phe Leu Gly Pro Pro Ala Ser Ser Met Ala Ala Leu Gly145 150 155 160Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr 165 170 175Leu Pro Trp Ser Gly Ser His Val Lys Ile Pro Pro Asn Ser Asn Leu 180 185 190Val Thr Ile Pro Glu Glu Ile Tyr 195 200304200PRTArabidopsis lyrata 304Met Ala Gly Ser Val Asn Gly Tyr Gln Ser Ala Ile Gly Pro Gly Ile1 5 10 15Asn Tyr Glu Thr Val Ser Gln Val Asp Glu Phe Cys Lys Ala Leu Gly 20 25 30Gly Lys Arg Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala 35 40 45Ala Val Lys Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe 50 55 60Gly Thr Glu Lys Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp65 70 75 80Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu 85 90 95Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile 100 105 110Val Glu Met Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp 115 120 125Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Asp Ala Lys 130 135 140Gly Ile Ile Phe Leu Gly Pro Pro Ala Ala Ser Met Ala Ala Leu Gly145 150 155 160Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr 165 170 175Leu Pro Trp Ser Gly Ser His Val Lys Met Pro Pro Asn Ser Asn Leu 180 185 190Val Thr Ile Pro Glu Glu Ile Tyr 195 200305200PRTCamelina sativa 305Met Ala Gly Ser Val Asn Gly Tyr Gln Ser Ala Val Gly Pro Gly Ile1 5 10 15Asn Tyr Glu Ser Val Ser Gln Val Asp Glu Phe Cys Lys Ala Leu Gly 20 25 30Gly Lys Arg Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala 35 40 45Ala Val Lys Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe 50 55 60Gly Thr Glu Lys Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp65 70 75 80Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu 85 90 95Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile 100 105 110Val Glu Met Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp 115 120 125Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Asp Ala Lys 130 135 140Gly Ile Ile Phe Leu Gly Pro Pro Ala Ala Ser Met Gly Ala Leu Gly145 150 155 160Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr 165 170 175Leu Pro Trp Ser Gly Ser His Val Lys Ile Pro Pro Asn Ser Asn Leu 180 185 190Val Thr Ile Pro Glu Glu Ile Tyr 195 200306200PRTCamelina sativa 306Met Ala Gly Ser Val Asn Gly Tyr Gln Ser Ala Val Gly Pro Gly Ile1 5 10 15Asn Tyr Glu Thr Val Ser Gln Val Asp Glu Phe Cys Lys Ala Leu Gly 20 25 30Gly Asn Arg Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala 35 40 45Ala Val Lys Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe 50 55 60Gly Thr Glu Lys Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp65 70 75 80Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu 85 90 95Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile 100 105 110Val Glu Met Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp 115 120 125Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Asp Ala Lys 130 135 140Gly Ile Ile Phe Leu Gly Pro Pro Ala Ala Ser Met Gly Ala Leu Gly145 150 155 160Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr 165 170 175Leu Pro Trp Ser Gly Ser His Val Lys Ile Pro Pro Asn Ser Asn Leu 180 185 190Val Thr Ile Pro Glu Glu Ile Tyr 195 200307200PRTCapsella rubella 307Met Ala Gly Ser Val Asn Gly Tyr Gln Ser Ser Val Gly Pro Gly Ile1 5 10 15Asn Tyr Glu Thr Val Ser Gln Val Asp Glu Phe Cys Lys Ser Leu Gly 20 25 30Gly Lys Arg Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala 35 40 45Ala Val Lys Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe 50 55 60Gly Thr Glu Lys Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp65 70 75 80Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu 85 90 95Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile 100 105 110Val Glu Met Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp 115 120 125Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Asp Ala Lys 130 135 140Gly Ile Ile Phe Leu Gly Pro Pro Ala Ala Ser Met Ala Ala Leu Gly145 150 155 160Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr 165 170 175Leu Pro Trp Ser Gly Ser His Val Lys Ile Pro Pro Asn Ser Asn Leu 180 185 190Val Thr Ile Pro Glu Glu Ile Tyr 195 200308197PRTBrassica oleracea 308Met Ala Gly Ser Val Asn Gly Tyr Gln Thr Pro Gly Arg Asn His Val1 5 10 15Ser Val Ser Glu Val Asp Asp Phe Cys Ile Ala Leu Gly Gly Lys Arg 20 25 30Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala Ala Val Lys 35 40 45Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe Gly Thr Glu 50 55 60Arg Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp Met Arg Ile65 70 75 80Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu Val Pro Gly 85 90 95Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile Val Glu Met 100 105 110Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp Gly His Ala 115 120 125Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys Ala Lys Gly Ile Ile 130 135 140Phe Leu Gly Pro Pro Ala Ala Ser Met Ala Ala Leu Gly Asp Lys Ile145 150 155 160Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr Leu Pro Trp 165 170 175Ser Gly Ser His Val Lys Ile Pro Pro Asp Ser Ser Leu Val Thr Ile 180 185 190Pro Glu Glu Ile Tyr 195309197PRTBrassica napus 309Met Ala Gly Ser Val Asn Gly Tyr Gln Thr Pro Gly Arg Asn His Val1 5 10 15Ser Val Ser Glu Val Asp Asp Phe Cys Ile Ala Leu Gly Gly Lys Arg 20 25 30Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala Ala Val Lys 35 40 45Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe Gly Thr Glu 50 55 60Arg Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp Met Arg Ile65 70 75 80Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu Val Pro Gly 85 90 95Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile Val Glu Met 100 105 110Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp Gly His Ala 115 120 125Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys Ala Lys Gly Ile Ile 130 135 140Phe Leu Gly Pro Pro Ala Ala Ser Met Ala Ala Leu Gly Asp Lys Ile145 150 155 160Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr Leu Pro Trp 165 170 175Ser Gly Ser His Val Lys Ile Pro Pro Asp Ser Ser Leu Val Thr Ile 180 185

190Pro Glu Glu Ile Tyr 195310197PRTBrassica napus 310Met Ala Gly Ser Val Asn Gly Tyr Gln Thr Pro Gly Arg Asn His Val1 5 10 15Ser Val Ser Glu Val Asp Asp Phe Cys Ile Ala Leu Gly Gly Lys Arg 20 25 30Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala Ala Val Lys 35 40 45Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe Gly Thr Glu 50 55 60Arg Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp Met Arg Ile65 70 75 80Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu Val Pro Gly 85 90 95Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile Val Glu Met 100 105 110Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp Gly His Ala 115 120 125Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys Ala Lys Gly Ile Ile 130 135 140Phe Leu Gly Pro Pro Ala Ala Ser Met Ala Ala Leu Gly Asp Lys Ile145 150 155 160Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr Leu Pro Trp 165 170 175Ser Gly Ser His Val Lys Ile Pro Pro Asp Ser Ser Leu Val Thr Ile 180 185 190Pro Glu Glu Ile Tyr 195311197PRTBrassica rapa 311Met Ala Gly Ser Val Asn Gly Tyr Gln Thr Pro Gly Arg Asn His Val1 5 10 15Ser Val Ser Glu Val Asp Asp Phe Cys Ile Ala Leu Gly Gly Lys Arg 20 25 30Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala Ala Val Lys 35 40 45Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe Gly Thr Glu 50 55 60Arg Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp Met Arg Ile65 70 75 80Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu Val Pro Gly 85 90 95Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile Val Glu Met 100 105 110Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp Gly His Ala 115 120 125Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys Ala Lys Gly Ile Ile 130 135 140Phe Leu Gly Pro Pro Ala Ala Ser Met Ala Ala Leu Gly Asp Lys Ile145 150 155 160Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr Leu Pro Trp 165 170 175Ser Gly Ser His Val Lys Ile Pro Pro Asp Ser Ser Leu Val Thr Ile 180 185 190Pro Glu Glu Ile Tyr 195312197PRTArtificial Sequenceconsensus sequenceSITE(20)..(20)Xaa is any one of N, D, Q, E, B, and Z.SITE(23)..(23)Xaa is any one of N, D, Q, E, B, and Z.SITE(186)..(186)Xaa is any one of N, D, Q, E, B, and Z. 312Met Ala Gly Ser Val Asn Gly Tyr Gln Thr Pro Gly Arg Asn His Val1 5 10 15Ser Val Ser Xaa Val Asp Xaa Phe Cys Ile Ala Leu Gly Gly Lys Arg 20 25 30Pro Ile His Ser Ile Leu Ile Ala Asn Asn Gly Met Ala Ala Val Lys 35 40 45Phe Ile Arg Ser Val Arg Thr Trp Ala Tyr Glu Thr Phe Gly Thr Glu 50 55 60Arg Ala Ile Leu Leu Val Gly Met Ala Thr Pro Glu Asp Met Arg Ile65 70 75 80Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Glu Val Pro Gly 85 90 95Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile Val Glu Met 100 105 110Ala Glu Val Thr Arg Val Asp Ala Val Trp Pro Gly Trp Gly His Ala 115 120 125Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys Ala Lys Gly Ile Ile 130 135 140Phe Leu Gly Pro Pro Ala Ala Ser Met Ala Ala Leu Gly Asp Lys Ile145 150 155 160Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr Leu Pro Trp 165 170 175Ser Gly Ser His Val Lys Ile Pro Pro Xaa Ser Ser Leu Val Thr Ile 180 185 190Pro Glu Glu Ile Tyr 195313279PRTArabidopsis thaliana 313Met Glu Met Arg Ala Leu Gly Ser Ser Cys Ser Thr Gly Asn Gly Gly1 5 10 15Ser Ala Pro Ile Thr Leu Thr Asn Ile Ser Pro Trp Ile Thr Thr Val 20 25 30Phe Pro Ser Thr Val Lys Leu Arg Ser Ser Leu Arg Thr Phe Lys Gly 35 40 45Val Ser Ser Arg Val Arg Thr Phe Lys Gly Val Ser Ser Thr Arg Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Phe Cys Phe Leu Asn Pro65 70 75 80Asp Pro Ile Ser Phe Leu Glu Asn Asp Val Ser Glu Ala Glu Arg Thr 85 90 95Val Val Leu Pro Asp Gly Ser Val Asn Gly Ala Gly Ser Val Asn Gly 100 105 110Tyr His Ser Asp Val Val Pro Gly Arg Asn Val Ala Glu Val Asn Glu 115 120 125Phe Cys Lys Ala Leu Gly Gly Lys Arg Pro Ile His Ser Ile Leu Val 130 135 140Ala Thr Asn Gly Met Ala Ala Val Lys Phe Ile Arg Ser Val Arg Thr145 150 155 160Trp Ala Tyr Glu Thr Phe Gly Ser Glu Lys Ala Val Lys Leu Val Ala 165 170 175Met Ala Thr Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile 180 185 190Ala Asp Gln Phe Val Glu Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr 195 200 205Ala Asn Val Gln Leu Ile Val Glu Met Ala Glu Val Thr Arg Val Asp 210 215 220Ala Val Trp Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro225 230 235 240Asp Ala Leu Lys Glu Lys Gly Ile Ile Phe Leu Gly Pro Pro Ala Asp 245 250 255Ser Met Ile Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln 260 265 270Ala Ala Asp Val Pro Thr Leu 275314282PRTArabidopsis lyrata 314Met Glu Met Arg Ala Leu Val Ser Ser Cys Ala Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Phe Ser Phe Thr Lys Val Ser Pro Trp Ile Thr Thr Val 20 25 30Gly Gly Lys Asp Arg Asp Phe Pro Thr Thr Val Lys Leu Arg Thr Ser 35 40 45Met Arg Thr Phe Lys Gly Val Ser Ile Arg Gly Arg Thr Phe Lys Gly 50 55 60Val Ser Thr Arg Val Leu Ser Arg Asn Lys Gln Gln Phe Pro Leu Phe65 70 75 80Cys Phe Leu Asn Pro Asp Pro Thr Ser Phe Arg Asp Asn Asp Ile Ser 85 90 95Glu Ala Gln Arg Thr Val Val Leu Pro Gly Gly Ser Val Asn Gly Tyr 100 105 110His Gln Ser Glu Val Val Pro Gly Arg Asn Asp Gly Thr Val Ala Glu 115 120 125Val Asp Glu Phe Cys Lys Ala Leu Gly Gly Lys Arg Pro Ile His Ser 130 135 140Ile Leu Val Ala Thr Asn Gly Met Ala Ala Val Lys Phe Ile Arg Ser145 150 155 160Ile Arg Thr Trp Ala Tyr Glu Thr Phe Gly Thr Glu Lys Ala Val Lys 165 170 175Leu Val Ala Met Ala Thr Pro Glu Asp Met Arg Ile Asn Ala Glu His 180 185 190Ile Arg Ile Ala Asp Gln Phe Val Glu Val Pro Gly Gly Thr Asn Asn 195 200 205Asn Asn Tyr Ala Asn Val Gln Leu Ile Val Glu Met Ala Glu Val Thr 210 215 220Arg Val Asp Ala Val Trp Pro Gly Trp Gly His Ala Ser Glu Asn Pro225 230 235 240Glu Leu Pro Asp Ala Leu Lys Ala Lys Gly Ile Ile Phe Leu Gly Pro 245 250 255Pro Ala Ala Ser Met Ile Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu 260 265 270Ile Ala Gln Ala Ala Asp Val Pro Thr Leu 275 280315280PRTCamelina sativa 315Met Glu Met Arg Ala Leu Val Ser Ser Tyr Ser Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Ile Ser Leu Thr Asn Gly Ser Pro Trp Ile Thr Thr Val 20 25 30Gly Gly Gly Ala Ser Thr Met Asp Arg Glu Phe Pro Leu Thr Val Lys 35 40 45Leu Gly Ser Ser Met Arg Ala Phe Lys Gly Val Ser Thr Thr Thr Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Val Cys Leu Ala Arg Asn65 70 75 80Asn Ala Asn Ser Thr Asp Pro Thr Ser Phe Trp Glu Asn Asp Ile Ser 85 90 95Glu Val Gln Arg Thr Val Leu Pro Ala Glu Ser Ile Asn Gly Asp Lys 100 105 110Ser Ala Val Glu Pro Gly Arg Asn Asp Val Thr Val Ser Glu Val Asp 115 120 125Glu Phe Cys Lys Ala Leu Gly Gly Lys Arg Pro Ile His Ser Ile Leu 130 135 140Val Ala Thr Asn Gly Met Ala Ser Val Lys Phe Ile Arg Ser Ile Arg145 150 155 160Thr Trp Ala Tyr Gln Thr Phe Gly Ser Glu Lys Ala Ile Ser Leu Val 165 170 175Ala Met Ala Thr Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg 180 185 190Ile Ala Asp Gln Phe Val Glu Val Pro Gly Gly Thr Asn Asn Asn Asn 195 200 205Tyr Ala Asn Val Gln Leu Ile Val Glu Met Ala Glu Ala Thr Arg Val 210 215 220Asp Ala Val Trp Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu225 230 235 240Pro Asp Ala Leu Lys Ala Lys Gly Ile Ile Phe Leu Gly Pro Pro Ala 245 250 255Thr Ser Met Val Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala 260 265 270Gln Ala Ala Asp Val Pro Thr Leu 275 280316280PRTCamelina sativa 316Met Glu Met Arg Ala Leu Val Ser Ser Cys Ser Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Ile Ser Leu Thr Asn Gly Ser Pro Trp Ile Thr Thr Val 20 25 30Gly Gly Gly Ala Ser Thr Met Asp Arg Glu Phe Pro Ala Thr Val Lys 35 40 45Leu Gly Ser Ser Met Arg Ala Phe Lys Gly Val Ser Thr Ile Thr Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Val Cys Leu Ala Arg Asn65 70 75 80Asn Gly Asn Ser Thr Asp Pro Thr Ser Phe Trp Glu Asn Asp Ile Ser 85 90 95Glu Thr Gln Arg Thr Val Leu Pro Ala Glu Ser Ile Asn Gly Asp Lys 100 105 110Ser Ala Val Glu Pro Gly Arg Asn Asp Val Thr Val Ser Glu Val Asp 115 120 125Glu Phe Cys Lys Ala Leu Gly Gly Lys Arg Pro Ile His Arg Ile Met 130 135 140Val Ala Thr Asn Gly Met Ala Ala Val Lys Phe Ile Arg Ser Ile Arg145 150 155 160Thr Trp Ala Tyr Gln Thr Phe Gly Ser Glu Lys Ala Ile Ser Leu Val 165 170 175Ala Met Ala Thr Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg 180 185 190Ile Ala Asp Gln Phe Val Glu Val Pro Gly Gly Thr Asn Asn Asn Asn 195 200 205Tyr Ala Asn Val Gln Leu Ile Val Glu Met Ala Glu Ala Thr Arg Val 210 215 220Asp Ala Val Trp Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu225 230 235 240Pro Asp Ala Leu Lys Ala Lys Gly Ile Ile Phe Leu Gly Pro Pro Ala 245 250 255Thr Ser Met Val Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala 260 265 270Gln Ala Ala Asp Val Pro Thr Leu 275 280317277PRTCapsella rubella 317Met Glu Met Arg Ala Leu Val Ser Ser Cys Ser Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Ile Ser Leu Thr Asn Val Ser Pro Trp Ile Thr Thr Val 20 25 30Gly Gly Gly Ala Ser Ser Ile Asp Arg Glu Phe Pro Thr Thr Val Lys 35 40 45Leu Gly Ser Ser Leu Arg Thr Phe Lys Gly Val Ser Ser Thr Thr Val 50 55 60Leu Ser Arg Thr Lys Gln Gln Phe Pro Leu Val Cys Leu Ala Arg Asn65 70 75 80Asn Ala Asn Ser Thr Asp Pro Thr Leu Phe Trp Glu Asn Asp Ile Ser 85 90 95Glu Ala Gln Ser Thr Val Leu Pro Ser Gly Ser Asp Glu Ser Ala Val 100 105 110Val Pro Ser Gly Asn Asp Val Lys Val Ser Glu Val Asp Glu Phe Cys 115 120 125Lys Ala Leu Gly Gly Lys Arg Pro Ile His Ser Ile Leu Val Ala Thr 130 135 140Asn Gly Met Ala Ala Val Lys Phe Ile Arg Ser Ile Arg Thr Trp Ala145 150 155 160Tyr Gln Thr Phe Gly Thr Glu Lys Ala Ile Leu Leu Val Ala Met Ala 165 170 175Thr Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp 180 185 190Gln Phe Val Glu Val Pro Gly Gly Thr Asn Asn His Asn Tyr Ala Asn 195 200 205Val Gln Leu Ile Val Glu Met Ala Glu Ala Ala Ser Val Asp Ala Val 210 215 220Trp Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala225 230 235 240Leu Lys Ala Lys Gly Ile Ile Phe Leu Gly Pro Ser Ala Ala Ser Met 245 250 255Val Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala 260 265 270Asp Val Pro Thr Leu 275318276PRTBrassica oleracea 318Met Glu Met Arg Ala Leu Val Ser Cys Ser Ala Ala Gly Asn Gly Ala1 5 10 15Ser Asp Arg Phe Arg Leu Ser Asn Val Ser Pro Trp Ile Thr Ser Ala 20 25 30Arg Gly Ala Ser Gly Ser Asp Ser Pro Ala Thr Val Lys Leu Arg Ser 35 40 45Ser Ser Met Ile Arg Ala Phe Lys Gly Val Ser Ile Tyr Lys Asn Lys 50 55 60Thr Arg Arg Asn Val Leu Ser Gln Arg Asn Lys Gln Phe Arg Pro Met65 70 75 80Ala Tyr Leu Gly Arg Lys Asp Leu Ser Ser Pro Asp Pro Thr Ser Phe 85 90 95Cys Asp Asn Asp Ile Ser Glu Pro Gln Gly Thr Gly Ser Ile Asn Gly 100 105 110Asn Asp His Ser Ala Val Arg Val Ser Gln Val Asp Glu Phe Cys Lys 115 120 125Ala His Gly Gly Lys Arg Pro Ile His Ser Ile Leu Val Ala Thr Asn 130 135 140Gly Met Ala Ala Val Lys Leu Ile Arg Ser Val Arg Ala Trp Ser Cys145 150 155 160Gln Thr Phe Gly Ser Glu Lys Ser Ile Ser Leu Val Ala Met Ala Thr 165 170 175Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln 180 185 190Phe Met Gln Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val 195 200 205His Leu Ile Val Glu Met Ala Gln Ala Thr Gly Val Asp Ala Val Trp 210 215 220Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu225 230 235 240Lys Ala Lys Gly Val Ile Phe Leu Gly Pro Thr Ala Ala Ser Met Leu 245 250 255Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp 260 265 270Val Pro Thr Leu 275319276PRTBrassica napus 319Met Glu Met Arg Ala Leu Val Ser Cys Ser Ala Ala Gly Asn Gly Ala1 5 10 15Ser Asp Arg Phe Arg Leu Ser Asn Val Ser Pro Trp Ile Thr Ser Ala 20 25 30Arg Gly Ala Ser Gly Ser Asp Ser Pro Ala Thr Val Lys Leu Gly Ser 35 40 45Ser Ser Met Ile Arg Ala Phe Lys Gly Val Ser Ile Tyr Lys Asn Lys 50 55 60Thr Arg Arg Asn Val Leu Ser Gln Arg Asn Lys Gln Phe Arg Pro Met65 70 75 80Ala Tyr Leu Gly Arg Lys Asp Leu Ser Ser Pro Asp Pro Thr Ser Phe 85 90 95Cys Asp Asn Asp Ile Ser Glu Pro Gln Gly Thr Gly Ser Ile Asn Gly 100 105 110Asn Asp His Ser Ala Val Arg Val Ser Gln Val Asp Glu Phe Cys Lys 115 120 125Ala His Gly Gly Lys Arg Pro Ile His Ser Ile Leu Val Ala Thr Asn 130 135 140Gly Met Ala Ala Val Lys Leu Ile

Arg Ser Val Arg Ala Trp Ser Tyr145 150 155 160Gln Thr Phe Gly Ser Glu Lys Ser Ile Ser Leu Val Ala Met Ala Thr 165 170 175Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln 180 185 190Phe Met Gln Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val 195 200 205His Leu Ile Val Glu Met Ala Gln Ala Thr Gly Val Asp Ala Val Trp 210 215 220Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu225 230 235 240Lys Ala Lys Gly Val Ile Phe Leu Gly Pro Thr Ala Ala Ser Met Leu 245 250 255Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp 260 265 270Val Pro Thr Leu 275320276PRTBrassica napus 320Met Glu Met Arg Ala Leu Val Ser Cys Ser Ala Ala Gly Asn Gly Ala1 5 10 15Ser Asp Arg Phe Arg Leu Ser Asn Val Ser Pro Trp Ile Thr Ser Ala 20 25 30Arg Gly Ala Ser Gly Ser Asp Ser Pro Ala Thr Val Lys Leu Gly Ser 35 40 45Ser Ser Met Ile Arg Ala Phe Lys Gly Val Ser Ile Tyr Lys Asn Lys 50 55 60Thr Arg Arg Asn Val Leu Ser Gln Arg Asn Lys Gln Phe Arg Pro Met65 70 75 80Ala Tyr Leu Gly Arg Lys Asp Leu Ser Ser Pro Asp Pro Thr Ser Phe 85 90 95Cys Asp Asn Asp Ile Ser Glu Pro Gln Gly Thr Gly Ser Ile Asn Gly 100 105 110Asn Asp His Ser Ala Val Arg Val Ser Gln Val Asp Glu Phe Cys Lys 115 120 125Ala His Gly Gly Lys Arg Pro Ile His Arg Ile Leu Val Ala Thr Asn 130 135 140Gly Met Ala Ala Val Lys Phe Ile Arg Ser Val Arg Ala Trp Ser Tyr145 150 155 160Gln Thr Phe Gly Ser Glu Lys Ser Ile Ser Leu Val Ala Met Ala Thr 165 170 175Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln 180 185 190Phe Met Gln Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val 195 200 205His Leu Ile Val Glu Met Ala Glu Ala Thr Gly Val Asp Ala Val Trp 210 215 220Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu225 230 235 240Lys Ala Lys Gly Val Ile Phe Leu Gly Pro Thr Ala Ala Ser Met Leu 245 250 255Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp 260 265 270Val Pro Thr Leu 275321276PRTBrassica rapa 321Met Glu Met Arg Ala Leu Val Ser Cys Ser Ala Ala Gly Asn Gly Ala1 5 10 15Ser Asp Arg Phe Arg Leu Ser Asn Val Ser Pro Trp Ile Thr Ser Ala 20 25 30Arg Gly Ala Ser Gly Ser Asp Ser Pro Ala Thr Val Lys Leu Gly Ser 35 40 45Ser Ser Met Ile Arg Ala Phe Lys Gly Val Ser Ile Tyr Lys Asn Lys 50 55 60Ser Arg Arg Asn Val Leu Ser Gln Arg Asn Lys Gln Phe Arg Pro Met65 70 75 80Ala Tyr Leu Gly Arg Lys Asp Leu Ser Ser Pro Asp Pro Thr Ser Phe 85 90 95Cys Asp Asn Asp Ile Ser Glu Pro Gln Gly Thr Gly Ser Ile Asn Gly 100 105 110Asn Asp His Ser Ala Val Arg Val Ser Gln Val Asp Glu Phe Cys Lys 115 120 125Ala His Gly Gly Lys Arg Pro Ile His Arg Ile Leu Val Ala Thr Asn 130 135 140Gly Met Ala Ala Val Lys Phe Ile Arg Ser Val Arg Ala Trp Ser Tyr145 150 155 160Gln Thr Phe Gly Ser Glu Lys Ser Ile Ser Leu Val Ala Met Ala Thr 165 170 175Pro Glu Asp Met Arg Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln 180 185 190Phe Met Gln Val Pro Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val 195 200 205His Leu Ile Val Glu Met Ala Glu Ala Thr Gly Val Asp Ala Val Trp 210 215 220Pro Gly Trp Gly His Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu225 230 235 240Lys Ala Lys Gly Val Ile Phe Leu Gly Pro Thr Ala Ala Ser Met Leu 245 250 255Ala Leu Gly Asp Lys Ile Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp 260 265 270Val Pro Thr Leu 275322254PRTArtificial Sequenceconsensus sequenceSITE(62)..(62)Xaa is L or M.SITE(73)..(73)Xaa is any one of N, D, Q, E, B, and Z.SITE(76)..(76)Xaa is I or V.SITE(79)..(79)Xaa is any one of N, D, Q, E, B, and Z.SITE(101)..(101)Xaa is any one of N, D, Q, E, B, and Z.SITE(103)..(103)Xaa is any one of N, D, Q, E, B, and Z.SITE(134)..(134)Xaa is I or V.SITE(140)..(140)Xaa is any one of N, D, Q, E, B, and Z.SITE(148)..(148)Xaa is I or V.SITE(174)..(174)Xaa is any one of N, D, Q, E, B, and Z.SITE(195)..(195)Xaa is any one of N, D, Q, E, B, and Z.SITE(224)..(224)Xaa is I or V. 322Met Glu Met Arg Ala Leu Val Ser Ser Cys Ala Thr Gly Asn Gly Gly1 5 10 15Ser Asp Pro Phe Leu Thr Asn Val Ser Pro Trp Ile Thr Thr Val Gly 20 25 30Asp Phe Pro Thr Val Lys Leu Gly Ser Ser Arg Ala Phe Lys Gly Val 35 40 45Ser Ile Lys Thr Val Leu Ser Arg Lys Gln Gln Phe Pro Xaa Cys Leu 50 55 60Arg Ser Pro Asp Pro Thr Ser Phe Xaa Asn Asp Xaa Ser Glu Xaa Arg65 70 75 80Thr Val Leu Pro Gly Ser Ile Asn Gly Asp His Ser Ala Val Pro Gly 85 90 95Arg Asn Val Ser Xaa Val Xaa Glu Phe Cys Lys Ala Leu Gly Gly Lys 100 105 110Arg Pro Ile His Ser Ile Leu Val Ala Thr Asn Gly Met Ala Ala Val 115 120 125Lys Phe Ile Arg Ser Xaa Arg Thr Trp Ala Tyr Xaa Thr Phe Gly Ser 130 135 140Glu Lys Ala Xaa Ser Leu Val Ala Met Ala Thr Pro Glu Asp Met Arg145 150 155 160Ile Asn Ala Glu His Ile Arg Ile Ala Asp Gln Phe Val Xaa Val Pro 165 170 175Gly Gly Thr Asn Asn Asn Asn Tyr Ala Asn Val Gln Leu Ile Val Glu 180 185 190Met Ala Xaa Ala Thr Arg Val Asp Ala Val Trp Pro Gly Trp Gly His 195 200 205Ala Ser Glu Asn Pro Glu Leu Pro Asp Ala Leu Lys Ala Lys Gly Xaa 210 215 220Ile Phe Leu Gly Pro Pro Ala Ala Ser Met Ala Leu Gly Asp Lys Ile225 230 235 240Gly Ser Ser Leu Ile Ala Gln Ala Ala Asp Val Pro Thr Leu 245 25032324DNAArtificial Sequenceoligonucleotide sequence 323gcctcacgaa tatatctcca gttt 24324104DNAArtificial Sequencethe parental Ws/RLD sequence 324atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatctcc atggatcaca acagtttttc cgtc 104325105DNAArtificial SequenceWs-6-2 325atggagatga aagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc catggatcac aacagttttt ccgtc 105326101DNAArtificial SequenceRLD-6-2 326atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc catggatcac aacagttttt c 101327105DNAArtificial SequenceWs-10-35 327atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc catggatcac aacagttttt ccgtc 10532896DNAArtificial SequenceWs-11-5 328atggagatga aagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatggatca caacagtttt tccgtc 96329105DNAArtificial SequenceWs-11-28 329atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc cagggatcac aacatttttt ccgcc 105330105DNAArtificial SequenceWs-6-23 330atggagatga aagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatccctgc attggaacac attttctttc ccctc 105331105DNAArtificial SequenceWs-6-11 331atggagatga aagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60tccctcacca atggatctcc cctgtttttc caccgttttt ccatc 105332103DNAArtificial SequenceWs-6-9 332atggagatga aagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatccca ggaaccaaaa aatttttccc tcc 10333394DNAArtificial SequenceWs-11-96 333atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcaca acagtttttc cgtc 94334105DNAArtificial SequenceWs-6-19misc_feature(67)..(67)n is a, c, g, or t 334atggagatga aagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcncga atatatcttc catggatcac aacagttttt ccgtc 105335101DNAArtificial SequenceRLD-10-25 335atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc catggatcac aacagttttt c 101336101DNAArtificial SequenceRLD-6-10 336atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc catggatcac aacagttttt c 10133799DNAArtificial SequenceRLD-10-2 337atggagatga aagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatctaa tggatcacaa cagtttttc 99338101DNAArtificial SequenceRLD-6-13 338atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc catggatcac aacagttttt c 101339101DNAArtificial SequenceRLD-6-15 339atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc catggatcac aacagttttt c 10134098DNAArtificial SequenceRLD-10-10 340atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatccat ggatcacaac agtttttc 98341101DNAArtificial SequenceRLD-6-6 341atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcttc catggatcac aacagttttt c 10134298DNAArtificial SequenceRLD-10-29 342atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatccat ggatcacaac agtttttc 98343101DNAArtificial SequenceRLD-11-14misc_feature(94)..(94)n is a, c, g, or t 343atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacca agaatccctc catgttttcc aacnactttt c 101344101DNAArtificial SequenceRLD-10-8 344atggagatga gagctttggg ttcttcgtgt tctactggta atggaggttc tgctccaatt 60accctcacga atatatcctc ggaggatcac aatttttttt c 101345399DNAArtificial SequenceN-terminal nucleotides of Brassica napus cv Darmor-bzh ACC2-Br BnaA06g04070D 345atggagatga gagctttggt ttcgtgttct gctgccggaa atggagcttc tgatcggttt 60agactctcca atgtttcacc atggatcaca tcagctcgtg gtgcaagtgg cagtgactcc 120ccagccacag tgaagctggg aagcagctct atgatcagag ccttcaaagg agtttcgatt 180tacaaaaaca agaccagaag aaatgttttg tctcaaagga acaaacagtt tcgtcctatg 240gcctacttag gaaggaagga cttgagcagc cctgatccga cctccttctg cgataatgat 300atatctgaac ctcaagggac tggatccatt aatgggaatg atcatagtgc tgtaagagtg 360tctcaagtcg atgagttctg taaggctcac ggtggaaaa 399346399DNAArtificial SequenceN-terminal nucleotides of Brassica napus cv Darmor-bzh ACC2-Bo BnaC06g01580D 346atggagatga gagctttagt ttcgtgttct gctgccggaa atggagcttc tgatcggttt 60agactctcca atgtttcacc atggatcaca tcagctcgtg gtgcaagtgg cagtgactcc 120ccagccacag tgaagctggg aagcagctct atgattagag ctttcaaagg agtttcgatt 180tacaaaaaca agaccagaag gaatgttctg tctcaaagga acaaacagtt ccgtcctatg 240gcctacttag gaaggaagga cttgagcagc cctgatccga cctccttctg cgataatgat 300atatctgaac ctcaagggac cggatccatt aatgggaatg atcatagtgc tgtaagagtg 360tctcaagtcg atgagttctg taaggctcat ggtggaaaa 39934793DNAArtificial SequenceN-terminal nucleotides of Brassica napus cv Darmor-bzh ACC1-Br BnaA08g06180D 347atggctggct ctgttaacgg gtatcaaact cccggtagaa atcatgtttc ggtgtctgaa 60gtggatgact tttgcattgc acttggaggg aaa 9334893DNAArtificial SequenceN-terminal nucleotides of Brassica napus cv Darmor-bzh ACC1-Bo BnaC08g06560D 348atggctggct ctgttaacgg gtatcaaact cccggtagaa atcatgtttc ggtgtctgaa 60gtggatgact tttgcattgc acttggaggg aaa 9334922DNAArtificial SequencesgRNA-target 349ggtttagact ctccaatgtt tc 2235022DNAArtificial SequencesgRNA-target 350ggaaggaagg acttgagcag cc 2235122DNAArtificial SequencesgRNA-target 351ttagactctc caatgtttca cc 22352298DNABrassica napus 352atggagatga gagctttagt ttcgtgttct gctgccggaa atggagcttc tgatcggttt 60agactctcca atgtttcacc atggatcaca tcagctcgtg gtgcaagtgg cagtgactcc 120ccagccacag tgaagctggg aagcagctct atgattagag ctttcaaagg cgtttcgatt 180tacaaaaaca agaccagaag gaatgttctg tctcaaagga acaaacagtt ccgtcctatg 240gcctacttag gaaggaagga cttgagcagc cctgatccga cctccttctg cgataatg 298353298DNABrassica napus 353atggagatga gagctttggt ttcgtgttct gctgccggaa atggagcttc tgatcggttt 60agactctcca atgtttcacc atggatcaca tcagctcgtg gtgcaagtgg cagtgactcc 120ccagccacag tgaagctggg aagcagctct atgatcagag ccttcaaagg agtttcgatt 180tacaaaaaca agaccagaag aaatgttttg tctcaaagga acaaacagtt tcgtcctatg 240gcctacttag gaaggaagga cttgagcagc cctgatccga cctccttctg cgataatg 298

Claims

1. A method for increasing plastid transformation efficiency in plastids of a Brassica ssp. plant, comprising; a) providing a plant comprising a nonfunctional or defective ACC2 nuclear gene; b) introducing one or more plastid transformation vectors into the plastids in cells from said plant, said one or more vectors comprising an aadA spectinomycin resistance marker sequence and a nucleic acid sequence encoding a protein of interest; c) contacting said cells with spectinomycin and selecting plant cells which are resistant to spectinomycin and accumulate said protein of interest in said plastids; and d) culturing said plant cells under conditions suitable to regenerate a transplastomic plant therefrom.

2. (canceled)

3. The method of claim 1, wherein said protein of interest is green fluorescent protein.

4. The method of claim 1, wherein the plant of step a) is a naturally occurring mutant which encodes non-functional or defective ACC2.

5. The method of claim 1, wherein said ACC2 gene is inactivated in said plant using CRISPR/Cas prior to plastid transformation.

6.-7. (canceled)

8. The method of claim 1, further comprising excising said aadA spectinomycin resistance marker sequence from said plant.

9. The method of claim 5, wherein said protein of interest is selected from the group consisting of a protein conferring herbicide resistance, a protein conferring insect resistance, a vaccine, an antibody, regulatory RNA, dsRNA, siRNA, shRNA and insecticidal proteins.

10. A method for seed-specific plastid expression comprising: a) introducing a nuclear expression vector encoding a modified PPR10 binding protein driven by a seed-specific promoter and b) a plastid expression vector encoding a gene of interest linked to an upstream PPR10 binding site, wherein nuclear-expressed PPR10 is imported into plastids and binds said PPR10 binding site to drive expression of the gene of interest in seed plastids.

11. The method of claim 10, wherein said vector comprises a seed specific promoter selected from a napin or a phaseolin gene promoter.

12. The method of claim 10, wherein said modified PPR10 binding protein is PPR10.sup.GG encoded by SEQ ID NO: 265.

13. The method of claim 10, wherein said PPR10 binding site encoded by SEQ ID NO: 261.

14. The method of claim 10, further comprising plastid expression of an aadA spectinomycin resistance gene.

15. The method of claim 10, wherein the plastid expressed gene of interest is linked to an upstream sequence encoding a maize atpH gene and/or tRNA sequence in said plastid vector.

16. A method for increasing plastid transformation efficiency in plastids of a Brassica ssp. plant recalcitrant to plastid transformation, comprising; a) providing a plant comprising a nonfunctional ACC2 nuclear gene; b) introducing one or more plastid transformation vectors into the plastids in cells from said plant, said one or more vectors comprising a nucleic acid sequence conferring resistance to said plastid translation inhibitor, and a nucleic acid sequence encoding a protein of interest; c) contacting said cells with said inhibitor and selecting plant cells which are resistant to said inhibitor and accumulate said protein of interest in said plastids; and d) culturing said plant cells under conditions suitable to regenerate a transplastomic plant therefrom.

17. The method of claim 16, wherein said plastid translation inhibitor is selected from the group consisting of kanamycin, chloramphenicol, tobramycin and gentamycin.

18. The method of claim 17, wherein inhibitor is kanamycin.

19. The method of claim 17, wherein said inhibitor is chloramphenicol and said nucleic acid encodes chloramphenicol acetyl transferase.

20. The method of claim 17, wherein said inhibitor is tobramycin.

21. The method of claim 17, wherein said inhibitor is gentamycin.