WO2001096363A1 - Gene de desaturase d'acide gras et proteine destines a moduler l'activation des voies de signalisation de defense dans des plantes - Google Patents

Gene de desaturase d'acide gras et proteine destines a moduler l'activation des voies de signalisation de defense dans des plantes Download PDF

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WO2001096363A1
WO2001096363A1 PCT/US2001/016134 US0116134W WO0196363A1 WO 2001096363 A1 WO2001096363 A1 WO 2001096363A1 US 0116134 W US0116134 W US 0116134W WO 0196363 A1 WO0196363 A1 WO 0196363A1
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ssi2
plant
nprl
plants
gene
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PCT/US2001/016134
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Daniel F. Klessig
Pradeep Kachroo
Jyoti Shah
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Boyce Thompson Institute For Plant Research
Rutgers, The State University Of New Jersey
Kansas State University Research Foundation
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Priority to AU2001261764A priority Critical patent/AU2001261764A1/en
Priority to US10/297,813 priority patent/US20040049807A1/en
Publication of WO2001096363A1 publication Critical patent/WO2001096363A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C12N9/0083—Miscellaneous (1.14.99)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8281—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for bacterial resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • This invention relates to the field of plant molecular biology and plant pathology. More specifically, this invention relates to a novel pathway for defense responses in plants, and genes involved in that pathway.
  • the hypersensitive response (HR) and systemic acquired resistance (SAR) are important components of a plants defense arsenal against pathogens.
  • the HR is a rapid defense response characterized by localized programmed host cell death and restriction of pathogen to the site of pathogen entry. Subsequent to the HR, a systemic signal is released that induces SAR in uninfected plant tissues. SAR is long- lasting and confers resistance against a broad spectrum of pathogens. Tightly correlated with the appearance of HR and SAR is an increase in the accumulation of salicylic acid (SA) and expression of a subset of the pathogenesis-related (PR) genes, some of which encode proteins with antimicrobial activities. Hence, the expression of these genes serves as an excellent molecular marker for a resistance response.
  • SA salicylic acid
  • PR pathogenesis-related
  • transgenic tobacco and Arabidopsis plants constitutively expressing the Pseudomonas putida nahG gene, which encodes the SA-degrading enzyme salicylate hydroxylase fail to develop SAR and are hypersusceptible to pathogen infection.
  • preventing SA accumulation by application of SA biosynthesis inhibitors also makes otherwise resistant Arabidopsis plants susceptible to Peronospora parasitica.
  • the elevated levels of S A present in the Arabidopsis acd accelerated cell death; Greenberg et al. 1994; Rate et al. 1999), Isd (lesion simulating disease; Dietrich et al. 1994; Weymann et al.
  • the Arabidopsis NPR1INIM1 gene is an important component of the SA signal transduction pathway(s). nprl/niml loss-of-function mutations render the plant insensitive to SA. SA and its functional analogs 2,6-dichloroisonicotinic acid (INA) and benzothiadiazole (BTH) are unable to induce expression of PR genes and SAR in nprl/niml plants. In contrast, the overexpression of NPR1 in Arabidopsis has been shown to increase resistance against bacterial and fungal pathogens. However, overexpression of NPR1 did not cause constitutive activation of defense responses.
  • INA 2,6-dichloroisonicotinic acid
  • BTH benzothiadiazole
  • NPR1 or possibly another coinducer may require activation by pathogen attack, or alternatively by SA before SAR can be activated.
  • NPRl encodes a novel, 65 kD protein containing ankyrin repeats (Cao et al. 1997; Ryals et al. 1997) which are involved in its specific interaction with some members of the TGA family of bZIP DNA binding proteins. Genetic screens for suppressors of nprl have identified additional components of the SA signaling pathway(s). The ssil and snil mutants restore SA responsiveness and resistance in nprl plants (Li et al. 1999; Shah et al., 1999).
  • Elevated levels of SA are essential for the ssil and snil conferred suppression of nprl mutant phenotypes.
  • the SNI1 protein shows no significant homology to any known protein and has been proposed to be a negative regulator of PR gene expression and SAR.
  • the ability of ssil and snil alleles to restore SA responsiveness in various nprl mutant allele backgrounds argues against SSI1 and SNI1 functioning in defense as NPRl -interacting proteins.
  • the ssil and snil mutants could suppress nprl mutant phenotype either by somehow restoring function to the SA/NPR1 pathway or alternatively by activating an NPRl -independent, S A-response pathway.
  • NPRl is a key component of SA signaling and overexpression of NPRl in Arabidopsis confers enhanced resistance against pathogen
  • several lines of evidence suggest the existence of a NPRl -independent pathway, in addition to the NPRl -dependent pathway for SA signaling in plant defense response.
  • loss-of-function mutations in NPRl do not confer complete loss of S A-mediated resistance.
  • the SA-deficient NahG plants are 10-50 fold more susceptible to pathogens than the nprl mutant.
  • the pad4-l mutant which does not accumulate elevated levels of S A upon infection with virulent pathogens, is more susceptible than nprl to powdery mildew caused by the obligate biotrophic fungus Erysiphe orontii.
  • NPRl -independent pathways also activate certain defense responses.
  • expression of the defensin gene PDF1.2 as well as resistance to the fungal pathogen Botrytis cinerea are mediated by a pathway(s) that requires ethylene and jasnionic acid (JA), but not SA or NPRl(Ryals et al., 1997).
  • an SA-dependent but NPRl -independent pathway(s) appears to regulate pathogen-induced PR gene expression in nprl mutant plants (Yang et al., 1997; Cao et al., 1997; Shah et al.., 1997) and resistance to certain pathogens (Shah et al, 1997; Glazebrook et al., 1996; Kachroo et al. 2000).
  • Mutant screens in Arabidopsis have made significant contributions in identifying various components of the SA signaling pathway. However, very few of these mutants have been shown to affect the NPRl -independent SA-signaling pathway.
  • the paucity of mutants in genes affecting the NPRl -independent SA signaling pathway may in part be due to the NPRl pathway masking the role of the NPRl -independent pathway. It would be an advance in the art to develop a genetic screen capable of identifying components of the NPRl -independent SA signaling pathway. More significantly, the art would be further advanced by the identification, isolation and characterization of such components of novel defense pathways in plants.
  • an isolated nucleic acid molecule which comprises an SSI2 gene isolated from Arabidopsis thaliana chromosome 2 at a location within 0.2 cM from marker AthB102 and 3.7 cM from marker GBF.
  • the loss of function of the product of this gene is associated with altered resistance of a plant to plant pathogens or other disease-causing agents.
  • the nucleic acid molecule may comprise a genomic clone of, or a cDNA corresponding to, the SSI2 gene of the invention.
  • the nucleic acid molecule encodes a polypeptide having greater than 60% (more preferably 70%, yet more preferably 80%, even more preferably 90%) identity to SEQ ID NO:3.
  • the nucleic acid molecule comprises a coding sequence of SEQ ID NO:l or SEQ ID NO:2.
  • an isolated nucleic acid molecule comprising a homolog of the Arabidopsis SSI2 gene, isolated from another plant species and encoding a ⁇ 9 fatty acid desaturase.
  • the coding region of the homolog comprises a sequence which is greater than 60% (preferably 70%, yet more preferably 80%, even more preferably 90%) homologous to the coding region of SEQ ID NO:l or SEQ ID NO:2.
  • an isolated plant enzyme comprising a ⁇ 9 fatty acid desaturase.
  • Loss of function of the enzyme in a plant results in altered resistance of the plant to plant pathogens or other disease- causing agents.
  • loss of function of the enzyme in a plant causes the plant to exhibit one or more features including: (a) NPRl- and SA-independent constitutive expression of PR genes; (b) impairment of jasmonic acid-mediated activation of PDF1.2; and (c) accumulation of 18:0 fatty acids and decrease in 18:1 fatty acids.
  • the enzyme possesses a substrate preference for 18:0 fatty acids, and its enzymatic activity produces at least one product that functions in the plant as a defense response signal molecule or a precursor of a defense response signal molecule.
  • Antibodies immunologically specific for the above-described plant enzyme of the invention are also provided.
  • the invention also features a plant-derived defense response signal molecule, produced directly or indirectly by activity of the aforementioned enzyme. This molecule, or combination of molecules, preferably comprises an 18:1 fatty acid or derivative thereof.
  • the defense response signal molecule inhibits a SA-independent defense response and participates in activation of a jasmonic-acid mediated defense response selected from the group consisting of activation of PDF 1.2, resistance to A. brassicicola and resistance to B. cinerea.
  • a ssi2 mutant plant which displays a phenotype characterized by one or more features including: (a) NPRl- and SA-independent constitutive expression of PR genes; (b) impairment of jasmonic acid-mediated activation of PDF1.2; and (c) accumulation of 18:0 fatty acids and decrease in 18:1 fatty acids.
  • the phenotype is conferred by a loss-of-function mutation in the SSI2 gene.
  • the invention also features a method to enhance resistance of a plant to plant pathogens or other disease causing agents, comprising reducing or preventing function of a SSI2 gene product in the plant. Specifically, this method results in a plant having features that include (a) NPRl- and SA-independent constitutive expression of PR genes; (b) impairment of jasmonic acid-mediated activation of PDF 1.2; and (c) accumulation of 18:0 fatty acids and decrease in 18:1 fatty acids.
  • Another method provided in accordance with the present invention is a method to enhance resistance of a plant to plant pathogens or other disease causing agents, comprising increasing production or activity of a SSI2 gene product in the plant.
  • the enhanced resistance results from increased activity of jasmonic acid- mediated defense responses.
  • FIG. 1A Expression of the PR-1 and BGL2 genes in water-treated (W) and SA- treated (S) wild-type (SSI2 NPRl), nprl-5 (SSI2 nprl-5) and ssi2-l (ssi2-l NPRl and ssi2-l nprl-5) plants.
  • RNA was extracted from leaves of 4 week old soil grown plants 24 h after treatment.
  • FIG. 1 Growth of P. syringae in ssi2 (marked in figure as ssi2-l).
  • SSI2 NPRl wild-type
  • nprl-5 nprl-5
  • ssi2-l ssi2-l NPRl and ssi2-l nprl-5
  • SA and SAG Levels in ssi2 (marked in figure as ssi2-l). Leaves from 4-week-old soil-grown wild-type (SSI2 NPRl), nprl-5 (SSI2 nprl-5) and ssi2-l (ssi2-l NPRl and ssi2-l nprl-5) plants were harvested, extracted, and analyzed by HPLC. The SA and SAG values ⁇ SD, presented as micrograms of SA per gram fresh weight (FW) of tissue, are averages of three to six sets of samples per line.
  • FIG. 4A Microscopy of trypan blue-stained leaf containing lesions from an ssi2-l nprl-5 and ssi2-l nprl-5 nahG plant showing intensely stained dead cells.
  • FIG. 4B Comparison of constitutive PR-1 and BGL2 expression in SSI2 and ssi2-l plants with or without the nahG transgene. All lines were either homozygous for the wild-type NPRl or the nprl-5 mutant alleles.
  • the wild-type (wt) or mutant (m) genotype at NPRl and SSI2 loci is indicated on the top of the blot. Presence of the nahG transgene is indicated by the presence of a strong signal for the nahG (NahG) transcript in these lines. Gel loading was monitored by photographing the ethidium bromide stained gel before transfer of RNA to Nytran Plus membrane. The blots were sequentially probed for the indicated genes. All plants were grown in soil and sampled when 4 weeks old.
  • FIG. 6B The morphological phenotype of T 2 transgenic plants complemented by the SSI2 gene in comparison with that of the ssi2 mutant.
  • FIG. 6C Northern blot analysis showing PR-1 gene expression in the ssi2 mutant, SSI2 (No) and ⁇ and T 2 progeny of the F23 complemented transgenic ssi2 plant.
  • FIG. 6D dCAPS analysis of same set of plants shown in Fig. 6C.
  • FIG. 6E Approximately 50-60 plants of wt, ssi2 and T 2 progeny of F23 transformed ssi2 plants were spray inoculated with P. p ⁇ r ⁇ sitic ⁇ spores.
  • Plants were sampled at 7 days post inoculation and scored as susceptible if they developed 10 or more sporangiophores per cotyledon.
  • Cotyledons of SSI2 or ssi2lssi2::SSI2 plants showed an average of 30-40 sporagiophores per cotyledon and 95% of these plants were susceptible.
  • only 5% of ssi2 plants were susceptible and they developed 2-4 fold fewer sporangiophores per cotyledon.
  • Fungal structures and HR-like cell death were visualized by trypan blue staining.
  • SSI2 encodes a stearoyl-ACP desaturase (S-ACP DES) with reduced activity.
  • S-ACP DES stearoyl-ACP desaturase
  • thaliana ecotype Nossen also referred to herein as wild-type (wt) or SS12
  • SS12 a portion of SEQ ID NO:3
  • ssi2 is polypeptide encoded by A. thaliana ssi2 mutant (SEQ ID NO:4)
  • polypeptides from other organisms are as follows: Brassica napus (SEQ ID NO:5); Brassica juncea (SEQ ID NO:6); Ricinis (SEQ ID NOJ); Sesamum (SEQ ID NO:8); Glycine (SEQ ID NO:9); Cucumis (SEQ ID NO:10); Carthamus (SEQ ID NO:ll); Arachis (SEQ ID NO:12); Solanum (SEQ ID NO:13); Oryza (SEQ ID NO:14); and Mycobacterium (SEQ ID NO:15).
  • Variable aa are boxed and the mutated aa in ssi2 is marked by an asterisk.
  • Fig. 7B Enzymatic studies were carried out with a nearly homogeneous preparation of bacterial-expressed SSI2 (No) and mutant proteins. Desaturase activity was determined using either 18:0 or 16:0 as a substrate.
  • Fig. 7C GC-MS analysis of the double bond position in the 18:1 FA methyl ester product generated by wt (I) and mutant (II) S-ACP DES.
  • FIG. 8 A Histochemical staining of GUS activity in the leaves and inflorescence of transgenic plants expressing an SSI2::GUS reporter gene. A 1631 bp fragment containing the SSI2 promoter was transcriptionally fused upstream of GUS in pBI121 and three independent transgenic lines were analyzed in both T x and T 2 generations. The control is a stained leaf from a wt plant.
  • FIG. 8B Northern blot analysis of SSI2 (No), nprl- 5,j ⁇ rl-l and ssi2 plants treated with water or 50 ⁇ M JA.
  • Fig. 8C Northern blot analysis of plants inoculated with spores of Altern ⁇ ri ⁇ br ⁇ ssicicol ⁇ . Mock (M) or fungal (A) inoculations were carried out as described previously (12). RNA was extracted at 72 h post inoculation and PDF1.2 gene expression was monitored.
  • Figure 9 Analysis of disease resistance to B. cinerea. Infections with B. cinerea were carried out by wounding the leaves by needle pricks and subsequently spot inoculating spores at the wounded site. The number of pricks made per leaf were based on the leaf size and ranged from three per leaf for SSI2 (No) to one per leaf for the ssi2 mutant. Plants were treated with either water or 50 ⁇ M JA for 48 h prior to and throughout the infection and the inoculated leaves were photographed at 10 dpi. Figure 10. Complementation of JA-dependent PDF1.2 expression in
  • FIG. 11 Schematic diagram showing role of SSI2 in defense signaling in plants.
  • Stearoyl-ACP desaturase encoded by SSI2 catalyzes the first step in the pathway from stearic acid (18:0) to linolenic acid (18:3), which is a precursor for signaling molecule JA.
  • a mutation in SSI2 leads to increased levels of 18:0 and a reduction in the levels of 18 : 1.
  • JA or pathogen-induced expression of defense gene PDF 1.2 and resistance to B. cinerea is compromised in the ssi2 mutant. Activation of some of these JA-dependent responses may require a second signal that is generated by SSI2.
  • SSI2 is used to designate the naturally-occurring or wild- type genotype. This genotype has the phenotype of the naturally-occurring spectrum of disease resistance and susceptibility.
  • ssi2 refers to a genotype having recessive mutation(s) in the wild-type SSI2 gene. The phenotype of ssi2 individuals is enhanced resistance to selected plant pathogens by a novel defense pathway, as described in greater detail below.
  • SSI2 refers to the protein product of the SSI2. gene.
  • the Arabidopsis SSI2 gene is exemplified herein, as is a ssi2 mutant of Arabidopsis.
  • the mutant Arabidopsis is referred to herein either as ssi2 or ssi2-l.
  • the wild-type Arabidopsis is referred to herein in one of three ways: (1) as wild-type (wt), (2) as SSI2, and (3) as N ⁇ ssen (No).
  • mutant'Or “loss-of-function mutant” may be used to designate an organism or genomic DNA sequence with a mutation that causes the product of the SSI2 gene to be non- functional or largely absent. Such mutations may occur in the coding and/or regulatory regions of the SSI2 gene, and may be changes of individual residues, or insertions or deletions of regions of nucleic acids. These mutations may also occur in the coding and or regulatory regions of other genes which may regulate or control the SSI2 gene and/or the product of the SSI2 gene so as to cause the gene product to be non-functional or largely absent. Though not exemplified herein, it should also be understood that a mutation in SSI2 can result in an increase in gene expression or in production of a protein with increased activity.
  • isolated nucleic acid may be 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 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived.
  • the "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 procaryote or eucaryote.
  • An “isolated nucleic acid molecule” may also comprise a cDNA molecule.
  • isolated nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form (the term “substantially pure” is defined below).
  • isolated protein or peptide
  • isolated and purified protein or peptide
  • This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.
  • immunologically specific refers to antibodies that bind to one or more epitopes of a protein of interest, but which do not substantially recognize and bind other molecules in a sample containing a mixed population of antigenic biological molecules.
  • substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). More preferably, the preparation comprises at least 75%) by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis, HPLC analysis, and the like).
  • Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids thus define the differences.
  • the BLAST programs (NCBI) and parameters used therein are employed, and the DNAstar system
  • nucleic acid or amino acid sequences having sequence variation that do not materially affect the nature of the protein (i.e. the structure, stability characteristics, substrate specificity and/or biological activity of the protein).
  • nucleic acid sequences the term “substantially the same” is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide.
  • amino acid sequences refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.
  • percent identical refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program.
  • Percent similar refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein. Conservative substitutions are defined in Taylor (1986, J. Theor. Biol. 119:205).
  • percent identical refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program.
  • the term “specifically hybridizing” refers to the association between two single-stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under pre-determined conditions generally used in the art (sometimes termed “substantially complementary”).
  • the term refers to hybridization of an oligonucleotide with a substantially complementary sequence contained within a single-stranded DNA or RNA molecule, to the substantial exclusion of hybridization of the oligonucleotide with single-stranded nucleic acids of non-complementary sequence.
  • a “coding sequence” or “coding region” refers to a nucleic acid molecule having sequence information necessary to produce a gene product, when the sequence is expressed.
  • operably linked means that the regulatory sequences necessary for expression of the coding sequence are placed in a nucleic acid molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence.
  • This same definition is sometimes applied to the arrangement other transcription control elements (e.g. enhancers) in an expression vector.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • promoter refers generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the coding region, or within the coding region, or within introns.
  • a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease S 1), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a “vector” is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
  • the term "nucleic acid construct” or “DNA construct” is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term "transforming DNA”.
  • Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.
  • selectable marker gene refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.
  • reporter gene refers to a gene that encodes a product which is easily detectable by standard methods, either directly or indirectly.
  • a “heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • the term "DNA construct", as defined above, is also used to refer to a heterologous region, particularly one constructed for use in transformation of a cell.
  • a cell has been "transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • NPRl gene In Arabidopsis thaliana, the NPRl gene is required for salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance.
  • SA salicylic acid
  • PR pathogenesis-related
  • loss-of-function mutations in NPRl do not confer complete loss of SA-dependent resistance.
  • both resistance and PR genes expression can also be induced via an NPRl -independent pathway that heretofore had not been elucidated.
  • SSI2 a novel gene, which is involved with a SA- and NPRl -independent pathway for expression of PR genes and resistance.
  • the gene has been cloned and characterized.
  • a loss-of-function mutation of SSI2 in Arabidopsis has been characterized.
  • the features of the gene, its encoded protein and the ssi2 mutant are summarized below and are described in detail in the examples.
  • the recessive ssi2 mutant identified in a genetic screen for suppressors of nprl-5, defines a new component of the NPRl -independent defense pathway.
  • the ssi2 nprl-5 double mutant and the ssi2 NPRl single mutant constitutively express their PR (PR-1, BGL2 [PR-2], and PR-5) genes, accumulate elevated levels of S A, spontaneously develop lesions, and possess enhanced resistance to a virulent strain of P. p ⁇ r ⁇ sitic ⁇ .
  • SSI2 might function as a negative regulator of an SA-dependent, NPRl -independent defense pathway or alternatively an SA- and NPRl -independent defense pathway.
  • the SSI2 gene is located on chromosome 2, approximately 0.2 cM from the AthB102 marker on the centromeric side and 3.7 cM from GBF on the telomeric side. Recombination analysis with these markers placed ssi2 within a 41 kb region encompassed by the bacterial artificial chromosome (BAC) F 18019 (Genbank Accession No. AC002333). Four open reading frames were identified within a corresponding 11.7 kb sub-region in clone F23 of a transformation- competent artificial chromosome (TAC) library (Pieterse et al., 1999). Open reading frame (ORF) 2 was determined to be SSI2. Further details are set forth in Example 2.
  • the genomic nucleotide sequence of Arabidopsis SSI2 is set forth as SEQ ID NO: 1 or 2.
  • the nucleotide sequence of the corresponding cDNA is set forth as SEQ ID NO:2.
  • the predicted amino acid sequence of the encoded SSI2 protein is set forth as SEQ ID NO:3. Sequence analysis predicts that SSI2 encodes a fatty acid desaturase, of which the archetype is the stearoyl-ACP desaturase (S-ACP-DES).
  • This enzyme is a ⁇ 9 fatty acid desaturase that preferentially desaturates stearoyl-ACP (18:0 ACP).
  • the wt SSI2 was expressed in E. coli and its gene product assayed in vitro.
  • the encoded SSI2 enzyme had a specific activity and substrate preference (88:1 for 18- versus 16-carbon chain length FA) that are characteristic of S-ACP-DES.
  • the ssi2 mutant gene was analyzed by comparative sequence analysis, and was found to possess a C to T point mutation that changed the leucine (L) at position 146 of SEQ ID NO:3 to a phenylalanine (F).
  • plants carrying the recessive ssi2 mutation exhibit the following characteristics: (1) a significant decrease in activity of the encoded S-ACP- DES, resulting in elevated 18:0 FA levels at the expense of 18:1 FA; (2) constitutive activation of an NPRl -independent pathway leading to PR gene expression and resistance to P. parasitica; and (3) impairment of some JA-dependent defense responses.
  • the fact that a defect in the FA desaturation pathway leads to activation of certain defense responses and inhibition of others indicates that one or more FA- derived signals modulates cross-talk between different defense pathways. Since the 18:1 FA pool is decreased when SSI2 activity is impaired, the FA-derived signal molecule(s) may be derived from 18:1 fatty acids.
  • the FA-derived signal that co- activates certain JA-mediated defense responses also inhibits the NPRl -independent pathway. Loss of this signal in ssi2 plants would result in constitutive activation of the NPRl -independent responses. Alternatively, an increase in 18:0 content might lead to activation of lipid signaling, which could then induce the PR signal transduction pathway (23 - NEED REF).
  • the present invention features a novel gene, SSI2, that encodes a S-ACP-DES in plants and plays a key role in modulating plant defense responses.
  • the invention further features a FA-derived signaling molecule(s) that can be manipulated through the up- or down-regulation of the SSI2 FA desaturase, resulting in specific modifications of plant defense responses.
  • This FA-derived signaling molecule(s) comprises at least an 18:1 FA or a derivative thereof.
  • SSI2 genomic clone and cDNA from Arabidopsis thaliana are described and exemplified herein, this invention is intended to encompass nucleic acid sequences and proteins from other plants that are sufficiently similar to be used instead of the Arabidopsis SSI2 nucleic acid and proteins for the purposes described below. These include, but are not limited to, allelic variants and natural mutants of SEQ ID NO: 1 or 2, which are likely to be found in different species of plants or varieties of Arabidopsis.
  • this invention provides an isolated SSI2 nucleic acid molecule having at least about 50% (preferably 60%, more preferably 70% and even more preferably over 80%) sequence identity in the coding regions with the nucleotide sequence set forth as SEQ ID NO: 1 or 2 (and, most preferably, specifically comprising the coding region of SEQ ID NO: 1 or 2 or the ssi2 mutant of SEQ ID NO: 1 or 2 described herein).
  • This invention also provides isolated polypeptide products of SEQ ID NO: 1 or 2, having at least about 50% (preferably 60%, 70%, 80% or greater) sequence identity with the amino acid sequences of SEQ ID NO:3.
  • the ssi2 mutant from Arabidopsis is also part of the present invention. It exhibits an altered defense response with characteristics of regulation that have not been observed previously.
  • This mutant is novel in its ability to constitutively express PR genes, which are known to provide defense against a wide variety of pathogens, and suppresses activation of the jasmonate-inducedP Pi.2 gene.
  • the present invention encompasses not only other plant homologs of the SSI2 gene, but also using these homologs to engineer enhanced disease resistance or to customize a defense response in other plant species.
  • the ssi2 mutant establishes that mutations in this gene result in plants with enhanced resistance to some pathogens.
  • A. Isolation of SSI2 Genetic Mutants Populations of plant mutants are available from which ssi2 mutants in other plant species can be isolated. Many of these populations are very likely to contain plants with mutations in the SSI2 gene. Such populations can be made by chemical mutagenesis, radiation mutagenesis, and transposon or T-DNA insertions. The methods to make mutant populations are well known in the art.
  • the nucleic acids of the invention can be used to isolate ssi2 mutants in other species. In species such as maize where transposon insertion lines are available, oligonucleotide primers can be designed to screen lines for insertions in the SSI2 gene.
  • Plants with transposon or T-DNA insertions in the SSZ2 gene are very likely to have lost the function of the gene product.
  • a plant line may then be developed that is homozygous for the non-functional copy of the SSI2 gene.
  • the PCR primers for this purpose are designed so that a large portion of the coding sequence the SSI2 gene are specifically amplified using the sequence of the SSI2 gene from the species to be probed (see Baumann et al., 1998, Theor. Appl. Genet. 97:729-734).
  • ssi2-like mutants can easily be isolated from mutant populations using the distinctive phenotype characterized in accordance with the present invention. This approach is particularly appropriate in, but not limited to, plants with low ploidy numbers where the phenotype of a recessive mutation is more easily detected. That the phenotype is caused by an ssi2 mutation is then established by molecular means well known in the art.
  • Species contemplated to be screened with this approach include but are not limited to: alfalfa, aster, barley, begonia, beet, canola, cantaloupe, carrot, chrysanthemum, clover, cucumber, delphinium, grape, lawn and turf grasses, lettuce, pea, peppermint, rice, rutabaga, sorghum, sugar beet, sunflower, tobacco, tomatillo, tomato, turnip, and zinnia.
  • a gene can be defined by its mapped position in the plant genome.
  • SSI2 gene on BAC clones or to map the chromosomal location of the SSI2 gene using recombination frequencies.
  • Nucleic acid molecules encoding the SSI2 protein may be isolated from Arabidopsis or any other plant of interest using methods well known in the art. Nucleic acid molecules from Arabidopsis may be isolated by screening Arabidopsis cDNA or genomic libraries with oligonucleotides designed to match the Arabidopsis nucleic acid sequence of SSI2 gene (SEQ ID NO: 1 or 2). In order to isolate SSI2- encoding nucleic acids from plants other than Arabidopsis, oligonucleotides designed to match the nucleic acids encoding the Arabidopsis SSI2 protein may be likewise used with cDNA or genomic libraries from the desired species.
  • the genomic library is screened.
  • the protein coding sequence is of particular interest, the cDNA library is screened.
  • all the appropriate nucleic acids residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used.
  • the strategy of oligonucleotide design is well known in the art (see also Sambrook et al.).
  • PCR polymerase chain reaction
  • primers may be designed by the above method to encode a portion of the Arabidopsis SSI2 protein, and these primers used to amplify nucleic acids from isolated cDNA or genomic DNA.
  • nucleic acids having the appropriate sequence homology with an Arabidopsis SSI2 nucleic acid molecule may be identified by using hybridization and washing conditions of appropriate stringency.
  • hybridizations may be performed, according to the method of Sambrook et al. (1989, supra), using a hybridization solution comprising: 5X SSC, 5X
  • Denhardt's reagent 1.0% SDS, 100 ⁇ g/ml denatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate and up to 50% formamide.
  • Hybridization is carried out at 37-42°C for at least six hours. Following hybridization, filters are washed as follows: (1) 5 minutes at room temperature in 2X SSC and 1% SDS; (2) 15 minutes at room temperature in 2X SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37°C in IX SSC and 1% SDS; (4) 2 hours at 42-65° in IX SSC and 1% SDS, changing the solution every 30 minutes.
  • T m 81.5°C + 16.6Log [Na+] + 0.41 (% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • the T m is 57°C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42°C.
  • the hybridization is at 37°C and the final wash is at 42°C, in a more preferred embodiment the hybridization is at 42°C and the final wash is at 50°C, and in a most preferred embodiment the hybridization is at 42°C and final wash is at 65°C, with the above hybridization and wash solutions.
  • Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector.
  • clones are maintained in plasmid cloning/expression vector, such as pBfuescript (Stratagene, La Jolla, CA), which is propagated in a suitable E. coli host cell.
  • Arabidopsis SSI2 nucleic acid molecules of the invention include DNA, RNA, and fragments thereof which may be single- or double-stranded.
  • this invention provides oligonucleotides (sense or antisense strands of DNA or RNA) having sequences capable of hybridizing with at least one sequence of a nucleic acid molecule encoding the protein of the present invention. Such oligonucleotides are useful as probes for detecting Arabidopsis SSI2 genes or transcripts.
  • any plant may be transgenically engineered to display a similar phenotype. While the natural ssi2 mutant has lost the functional product of the SSI2 gene due to a single point mutation, a transgenic plant can be made that also has a similar loss of the SSI2 product. This approach is particularly appropriate to plants with high ploidy numbers, including but not limited to wheat, corn and cotton.
  • a synthetic mutant can be created by a expressing a mutant form of the SSI2 protein to create a "dominant negative effect". While not limiting the invention to any one mechanism, this mutant SSI2 protein will compete with wild-type SSI2 protein for interacting proteins in a transgenic plant. By over-producing the mutant form of the protein, the signaling pathway used by the wild-type SSI2 protein can be effectively blocked. Examples of this type of "dominant negative" effect are well known for both insect and vertebrate systems (Radke et al, 1997, Genetics 145:163- 171; Kolch et al., 1991, Nature 349:426-428).
  • a second kind of synthetic mutant can be created by inhibiting the translation of the SSI2 mRNA by "post-transcriptional gene silencing".
  • the SSI2 gene from the species targeted for down-regulation, or a fragment thereof, may be utilized to control the production of the encoded protein.
  • Full-length antisense molecules can be used for this purpose.
  • antisense oligonucleotides targeted to specific regions of the SSI2-encoded RNA that are critical for translation may be utilized.
  • the use of antisense molecules to decrease expression levels of a pre-determined gene is known in the art.
  • Antisense molecules may be provided in situ by transforming plant cells with a DNA construct which, upon transcription, produces the antisense RNA sequences.
  • Such constructs can be designed to produce full-length or partial antisense sequences. This gene silencing effect can be enhanced by transgenically over-producing both sense and antisense RNA of the gene coding sequence so that a high amount of dsRNA is produced (for example see Waterhouse et al., 1998, PNAS 95:13959-13964).
  • part or all of the SSI2 coding sequence antisense strand is expressed by a fransgene.
  • hybridizing sense and antisense strands of part or all of the SSI2 coding sequence are transgenically expressed.
  • a third type of synthetic mutant can also be created by the technique of "co-suppression". Plant cells are transformed with a copy of the endogenous gene targeted for repression. In many cases, this results in the complete repression of the native gene as well as the fransgene.
  • the SSI2 gene from the plant species of interest is isolated and used to transform cells of that same species.
  • Transgenic plants displaying enhanced SSI2 activity can also be created. This is accomplished by transforming plant cells with a transgene that expresses part of all of an SSI2 coding sequence, or a sequence that encodes the either the SSI2 protein or a protein functionally similar to it. In a preferred embodiment, the complete SSI2 coding sequence is transgenically over-expressed.
  • Transgenic plants with one of the transgenes mentioned above can be generated using standard plant transformation methods known to those skilled in the art. These include, but are not limited to, Agrobacterium vectors, polyethylene glycol treatment of protoplasts, biolistic DNA delivery, UN laser microbeam, gemim virus vectors, calcium phosphate treatment of protoplasts, electroporation of isolated protoplasts, agitation of cell suspensions in solution with microbeads coated with the transforming D ⁇ A, agitation of cell suspension in solution with silicon fibers coated with transforming D ⁇ A, direct D ⁇ A uptake, liposome-mediated D ⁇ A uptake, and the like. Such methods have been published in the art.
  • Agrobacterium vectors are often used to transform dicot species.
  • Agrobacterium binary vectors include, but are not limited to, BI ⁇ 19 (Bevan, 1984) and derivatives thereof, the pBI vector series (Jefferson et al., 1987), and binary vectors pGA482 and pGA492 (An, 1986)
  • biolistic bombardment with particles coated with transforming DNA and silicon fibers coated with transforming DNA are often useful for nuclear transformation.
  • DNA constructs for transforming a selected plant comprise a coding sequence of interest operably linked to appropriate 5' (e.g., promoters and franslational regulatory sequences) and 3' regulatory sequences (e.g., terminators).
  • the coding region is placed under a powerful constitutive promoter, such as the Cauliflower Mosaic Virus (CaMV) 35S promoter or the figwort mosaic virus 35S promoter.
  • CaMV Cauliflower Mosaic Virus
  • Other constitutive promoters contemplated for use in the present invention include, but are not limited to: T-DNA mannopine synthetase, nopaline synthase (NOS) and octopine synthase (OCS) promoters.
  • Transgenic plants expressing a sense or antisense SSI2 coding sequence under an inducible promoter are also contemplated to be within the scope of the present invention.
  • Inducible plant promoters include the tetracycline repressor/operator controlled promoter, the heat shock gene promoters, stress (e.g., wounding)-induced promoters, defense responsive gene promoters (e.g. phenylalanine ammonia lyase genes), wound induced gene promoters (e.g.
  • hydroxyprohne rich cell wall protein genes hydroxyprohne rich cell wall protein genes
  • chemically-inducible gene promoters e.g., nitrate reductase genes, glucanase genes, chitinase genes, etc.
  • dark-inducible gene promoters e.g., asparagine synthetase gene
  • Tissue specific and development-specific promoters are also contemplated for use in the present invention. Examples of these included, but are not limited to: the ribulose bisphosphate carboxylase (RuBisCo) small subunit gene promoters or chlorophyll a/b binding protein (CAB) gene promoters for expression in photosynthetic tissue; the various seed storage protein gene promoters for expression in seeds; and the root-specific glutamine synthetase gene promoters where expression in roots is desired.
  • RuBisCo ribulose bisphosphate carboxylase
  • CAB chlorophyll a/b binding protein
  • the coding region is also operably linked to an appropriate 3' regulatory sequence.
  • the nopaline synthetase polyadenylation region NOS
  • Other useful 3' regulatory regions include, but are not limited to the octopine (OCS) polyadenylation region.
  • selectable marker systems include, but are not limited to: other genes that confer antibiotic resistances (e.g., resistance to hygromycin or bialaphos) or herbicide resistance (e.g., resistance to sulfonylurea, phosphinothricin, or glyphosate). Plants are transformed and thereafter screened for one or more properties, including the lack of SSI2 protein, SSI2 mRNA, constitutive HR-like lesions or expression of PR genes, altered FA metabolism or enhanced resistance to a selected plant pathogen, such asP. parasitica. It should be recognized that the amount of expression, as well as the tissue-specific pattern of expression of the transgenes in transformed plants can vary depending on the position of their insertion into the nuclear genome.
  • amino acid sequence information such as the full length sequence in SEQ ID NO: 2
  • sequence encoding Arabidopsis SSI2 from isolated native nucleic acid molecules can be utilized.
  • an isolated nucleic acid that encodes the amino acid sequences of the invention can be prepared by oligonucleotide synthesis.
  • Codon usage tables can be used to design a synthetic sequence that encodes the protein of the invention.
  • the codon usage table has been derived from the organism in which the synthetic nucleic acid will be expressed. For example, the codon usage for pea
  • the SSI2 protein of the present invention may also be prepared by in vitro transcription and translation of either native or synthetic nucleic acid sequences that encode the proteins of the present invention.
  • the SSI2 proteins of the present invention may be prepared by various synthetic methods of peptide synthesis via condensation of one or more amino acid residues, in accordance with conventional peptide synthesis methods.
  • the SSI2 produced by native cells or by gene expression in a recombinant procaryotic or eukaryotic system may be purified according to methods known in the art.
  • the present invention also provides antibodies that are immunologically specific to the Arabidopsis SSI2 of the invention.
  • Polyclonal antibodies may be prepared according to standard methods. In a preferred embodiment, monoclonal antibodies are prepared, which are specific to various epitopes of the protein. Monoclonal antibodies may be prepared according to general methods of K ⁇ hler and Milstein, following standard protocols.
  • Polyclonal or monoclonal antibodies that are immunologically specific for the Arabidopsis SSI2 can be utilized for identifying and purifying SSI2 from Arabidopsis and other species. For example, antibodies may be utilized for affinity separation of proteins for which they are specific or to quantify the protein.
  • Antibodies may also be used to immunoprecipitate proteins from a sample containing a mixture of proteins and other biological molecules. IV. Use of SSI2 nucleic acids, SSI2 proteins and antibodies, ssi2 mutants and transgenic plants.
  • SSI2 nucleic acids may be used for a variety of purposes in accordance with the present invention.
  • DNA, RNA, or fragments thereof may be used as probes to detect the presence and/or expression of SSI2 genes.
  • Methods in which SSI2 nucleic acids may be utilized as probes for such assays include, but are not limited to: (1) in situ hybridization; (2) Southern hybridization (3) Northern hybridization; and (4) assorted amplification reactions such as polymerase chain reactions (PCR).
  • the SSI2 nucleic acids of the invention may also be utilized as probes to identify related genes from other plant species.
  • hybridization stringencies may be adjusted to allow hybridization of nucleic acid probes with complementary sequences of varying degrees of homology.
  • SSI2 nucleic acids may be used to advantage to produce large quantities of substantially pure SSI2, or selected portions thereof.
  • the SSI2 nucleic acids can be used to identify and isolate further members of this novel disease resistance signal transduction pathway in vivo.
  • a yeast two hybrid system can be used to identify proteins that physically interact with the SSI2 protein, as well as isolate their nucleic acids.
  • the sequence encoding the protein of interest is operably linked to the sequence encoding half of a activator protein.
  • This construct is used to transform a yeast cell library which has been transformed with DNA constructs that contain the coding sequence for the other half of the activator protein operably linked to a random coding sequence from the organism of interest.
  • the two halves of the activator protein are physically associated and form a functional unit that activates the reporter gene.
  • all or part of the Arabidopsis SSI2 coding sequence may be operably linked to the coding sequence of the first half of the activator, and the library of random coding sequences may be constructed with cDNA from Arabidopsis and operably linked to the coding sequence of the second half of the activator protein.
  • activator protein/reporter genes are customarily used in the yeast two hybrid system.
  • the bacterial repressor LexA DNA-binding domain and the Gal4 transcription activation domain fusion proteins associate to activate the LacZ reporter gene (see Clark et al., 1998, PNAS 95:5401- 5406). Kits for the two hybrid system are also commercially available from Clontech, Palo Alto CA, among others.
  • the SSI2 proteins of the present invention can be used to identify molecules with binding affinity for SSI2, which are likely to be novel participants in this resistance pathway.
  • the known protein is allowed to form a physical interaction with the unknown binding molecule(s), often in a heterogenous solution of proteins.
  • the known protein in complex with associated molecules is then isolated, and the nature of the associated protein(s) and/or other molecules is determined.
  • Antibodies that are immunologically specific for SSI2 may be utilized in affinity chromatography to isolate the SSI2 protein, to quantify the SSI2 protein utilizing techniques such as western blotting and ELISA, or to immuno-precipitate SSI2 from a sample containing a mixture of proteins and other biological materials.
  • the immuno-precipitation of SSI2 is particularly advantageous when utilized to isolate affinity binding complexes of SSI2, as described above.
  • the ssi2 mutants of the invention display a unique combination of defense responses that include constitutive HR and expression of PR genes and enhanced disease resistance to certain plant pathogens, and therefore can be used to improve crop and horticultural plant species by customizing the defense response.
  • Plants species contemplated in regard to this invention include, but are not limited to: alfalfa, aster, barley, begonia, beet, canola, cantaloupe, carrot, chrysanthemum, clover, corn, cotton, cucumber, delphinium, grape, lawn and turf grasses, lettuce, pea, peppermint, rice, rutabaga, sorghum, sugar beet, sunflower, tobacco, tomatillo, tomato, turnip, wheat, and zinnia.
  • the ssi2 mutant of Arabidopsis exhibits constitutive activation of an
  • the ssi2 mutants will exhibit broad-spectrum resistance against a wide range of fungal, bacterial and viral pathogens.
  • pathogens include, but are not limited to: P. syringae and P. parasitica.
  • the ssi2 mutants of the invention can be used to identify and isolate additional members of this disease resistance pathway. Mutations that, when combined with ssi2, suppress the ssi2 phenotype, are likely to interact directly with SSI2, or to compensate in some other way for the loss of SSI2 function.
  • transgenic plants of the invention are particularly useful in conferring the SSI2 phenotype to many different plant species.
  • a host of plant species with enhanced or modified defense responses can be made, to be used as breeding lines or directly in commercial operations.
  • Such plants can have uses as crop species, or for ornamental use.
  • a plant that has had functional SSI2 transgenically depleted should exhibit a defense response profile similar to that of the ssi2 Arabidopsis mutant described above.
  • a transgenic approach is advantageous because it allows ssi2- phenotype plants to be created quickly, without time-consuming mutant generation, selection, and back-crossing.
  • Transgenically created ssi2-pheno ype plants have special utility in polyploid plants, such as wheat, where recessive mutations are difficult to detect.
  • a plant with increased functional SSI2, produced either by over- expression of an endogenous gene, expression of a fransgene or other means of modifying the activity of SSI2, are expected to have additional defense response properties consistent with increased production of the SSI2-associated FA-derived signal molecule(s) discovered in accordance with the present invention.
  • transgenic plants expressing a yeast ⁇ 9 FA desaturase exhibit increased resistance to a wide variety of plant pathogens, including fungi such as Erisyphe, Phytophthora, Verticillium and Fusarium, bacteria such as Pseudomonas, and viruses such as tobacco mosaic virus (U.S. Patent No.
  • RESULTS Growth conditions for plant and bacteria.
  • Arabidopsis plants were grown in soil at 22°C in growth chambers programmed for a 16-h light (8000 to 10,000 lux) and 8-h dark cycle unless otherwise stated.
  • P. syringae pv. tomato DC3000 carrying a plasmid-borne avrRpt2 gene was propagated at 30°C on King's B medium containing rifampicin (100 mg/ml) and kanamycin (25 mg/ml).
  • P. parasitica isolate Emco5 was cultivated on the susceptible ecotype N ⁇ ssen.
  • Plants for infection with P. syringae pv. tomato DC3000 were grown in soil at 22°C in a growth chamber programmed for 12- h light and 12-h dark cycle. Three days before infection the plants were transferred to a 16-h light and 8-h dark cycle.
  • Infection with P. syringae pv. tomato DC3000 carrying a plasmid-borne avrRpt2 gene were performed as described earlier (Shah et al. 1997).
  • Four leaves per plant were infiltrated with a suspension (OD 600 of 0.001) in 10 mM MgCl 2 .
  • Inoculated plants were kept covered with a clear plastic dome to maintain high humidity throughout the course of the experiment, and fungal growth was evaluated under a dissecting microscope eight days post inoculation by counting the number of sporangiophores per leaf. Plants with leaves containing 5 or more sporangiophores were scored as infected.
  • RNA extraction northern and dot blot analyses. Large-scale preparation of RNA from Arabidopsis was carried out. Small-scale extraction of RNA from one or two leaves was performed in the TRIzol reagent (GIBCO-BRL,
  • Leaf samples for trypan blue staining and epifluorescence microscopy were obtained from three-week-old soil grown plants. Trypan blue staining on P. parasitica infected leaves was carried out on samples harvested eight days post inoculation. Samples were processed and analyzed.
  • SA and SAG estimations were extracted and estimated from 0.25 to 0.5 g of fresh weight leaf tissue. Mutagenesis and selection of ssi2 mutant. M 2 seeds derived from ethyl methyl sulfonate mutagenized nprl-5 seeds (ecotype No) were screened for constitutive PR gene expression as previously described (Shah et al. 1999).
  • CAPS analysis was performed as previously described (Shah et al., 1999) on DNA from these phenotypically ssi2 plants to identify plants homozygous for the wild-type NPRl or the niml-1 mutant allele.
  • pollen from a ssi2 nprl-5 double mutant (ecotype No) was used to pollinate flowers from a wild-type plant of the ecotype Columbia.
  • F 2 progeny plants from the above cross were monitored for spontaneous lesion and constitutive PR-1 expression phenotype by dot blot analysis.
  • DNA for PCR was isolated from leaf tissue and used for CAPS or SSLP marker analysis.
  • ssi2 mutant lines containing the n ⁇ hG fransgene were generated by fertilizing flowers from a ssi2 nprl-5 plant with pollen from a transgenic NahG plant (ecotype No). Success of the cross was confirmed by analyzing expression of the n ⁇ hG gene in the Fj plants. A quarter of the F 2 plants had the A5 ⁇ ' 2-conferred lesion "1" phenotype suggesting that the nahG gene did not suppress the lesion + phenotype of ssi2 plants. Northern blot analysis showed constitutive expression of PR genes in these plants, reaffirming that these were truly ssi2 plants. Northern blot analysis also identified expression of the nahG gene in roughly three quarter of these plants. Analysis of the F 3 progeny of some of these F 2 lines identified F 2 plants that were homozygous for ssi2, NPRl and nahG.
  • Loss-of-function mutations in the SSI2 gene confer constitutive R gene expression and spontaneous development of HR-like lesions in nprl-5 plants.
  • the ssi2 mutant was isolated in a screen for suppressors of the SA-insensitive nprl-5 mutant; the details of this screen have been previously described (Shah et al., 1999). Briefly, three-to four- week old M 2 progeny of an ethyl methyl sulfonate mutagenized population of nprl-5 were screened by RNA blot analysis for mutants that constitutively accumulated elevated levels of the PR (PR-1, BGL2, and PR-5) gene transcripts. As shown in Figs.
  • the ssi2 nprl-5 double mutant constitutively expressed the PR-1, BGL2 and PR-5 gene transcripts at elevated levels.
  • Exogenous application of SA did not further increase accumulation of the PR gene transcripts in the ssi2 nprl-5 plant.
  • the ssi2 nprl-5 double mutant plants were smaller, had curled leaves with more prominent indentations of the leaf margin, and spontaneously developed necrotic lesions (data not shown).
  • ssi2 mutant phenotypes do not require the NPRl protein.
  • ssi2 confers enhanced resistance to P. parasitica and P. syringae .
  • PR gene expression is an useful marker for resistance response. Since the ssi2 mutant constitutively expresses PR genes we tested whether it also shows enhanced resistance against pathogens.
  • the ssi2 mutant is in the Arabidopsis ecotype N ⁇ ssen.
  • the fungal pathogen isolate Emco5 is virulent on Arabidopsis ecotype No, causing extensive growth and sporulation.
  • the ssil mutant which restored SA responsiveness in nprl-5 plants, also restored resistance against avirulent P. syringae pv tomato in nprl-5 plants suggesting that the mutation in ssil somehow activates signaling downstream of NPRl in the SA signaling pathway.
  • the mutation in ssi2 may likewise activate signaling downstream of NPRl and restore resistance against P. syringae pv tomato. We therefore compared the growth of P.
  • syringae pv tomato carrying the AvrRpt2 avirulence gene in ssi2 nprl-5, SSI2 nprl-5 and ssi2 NPRl plants, and as control in wild-type SSI2 NPRl plant.
  • Fig. 2 while the mutation in ssi2 enhanced resistance approximately five-fold in plants containing the wild-type NPRl allele, it did not enhance resistance in the plants containing the nprl-5 allele. This result suggests that, unlike ssil, NPRl is required for the ssi2- conferred enhanced resistance against P. syringae pv tomato.
  • the ssi2 mutant constitutively accumulates high levels of SA and SAG.
  • the high levels of endogenous SA are required for the phenotypes of these mutants.
  • the cpr 6 mutant accumulates elevated levels of S A, expresses PR genes and confers enhanced resistance against P. syringae and P. parasitica.
  • PR gene expression in cpr ⁇ is NPRl independent, resistance to P. syringae requires NPRl.
  • the cpr ⁇ phenotypes are dependent on its ability to accumulate elevated levels of S A. It is likely that, like cpr ⁇ , the enhanced resistance against P.
  • S A and its glucoside (SAG) in SSI2 and ssi2 plants were tested. As shown in Fig. 3, in plants homozygous for the ssi2 mutant allele, ssi2 NPRl and ssi2 nprl-5, SA levels were 7- and 15-fold higher than in the SSI2 NPRl and SSI2 nprl-5 plants, respectively. Likewise, SAG levels were >100- fold higher in the ssi2 plants as compared to SSI2 plants.
  • the ssi2 small plant phenotype and constitutive PR expression cosegregated with the lesion 4" phenotype. Approximately three quarters of these ssi2-like plants also expressed the nahG transcript suggesting high levels of S A and SAG are not required for the 55t2-conferred phenotypes. These results were confirmed in the F 3 generation.
  • the ssi2-conferred spontaneous lesions were present in plants homozygous for ssi2 and the nahG fransgene (Fig. 4A).
  • PR genes were also constitutively expressed at elevated levels in ssi2 NPRl nahG and ssi2 nprl-5 n ⁇ hG plants (Fig. 4B).
  • BTH treatment of the ssi2 NPRl n ⁇ hG plants increased the accumulation of PR-1 transcript to a level similar to those seen in the untreated ssi2 NPRl, and BTH- freated wild-type (SSI2 NPRl) plants (data not shown), confirming the role of SA in enhancing the ssi2-c r_ferred constitutive PR-1 phenotype.
  • the effect of n ⁇ hG on ssi2- conferred BGL2 and PR-5 expression was more variable, with n ⁇ hG having no effect on the levels of BGL2 and PR-5 expression in two out of four experiments.
  • SA also enhances the small plant size phenotype of ssi2.
  • ssi2 NPRl n ⁇ hG and ssi2 nprl-5 n ⁇ hG plants were slightly larger and developed visible lesions later than ssi2 plants lacking the n ⁇ hG fransgene (data not shown).
  • ssi2 confers resistance to P. parasitica and P. syringae in the SA- deficient NahG plants. Since elevated levels of SA and SAG are not essential for the manifestation of &s7 ' 2-conferred constitutive PR gene expression and lesion phenotypes even though they can affect these phenotypes, we tested whether ⁇ /2-conferred resistance was also independent of SA accumulation. Resistance against P. parasitica Emco5 and P.
  • SSI2 NPRl nahG plants are hypersusceptible to P. parasitica Emco5 as compared to the susceptible SSI2 NPRl plant of ecotype N ⁇ ssen. Not only did all the SSI2 NPRl nahG plants show disease symptoms but they also supported higher levels of sporulation. Furthermore, the newly emerging leaves also showed the presence of sporangiophores (data not shown).
  • the ssi2 allele partially restored resistance in the ssi2 NPRl nahG plants. Approximately 50% of ssi2 NPRl nahG plants showed little or no signs of infection. Furthermore, the ssi2 NPRl nahG plants that did show infection had 2-3 fold fewer sporangiophores compared to the SSI2 NPRl nahG plants.
  • the SSI2 NPRl nahG plants were also hypersusceptible to P. syringae pv tomato carrying the avirulence gene avrRpt2, suppporting -250 fold more bacterial growth than the SSI2 NPRl plants.
  • presence of the ssi2 allele in the ssi2 NPRl nahG plants partially restored resistance (20-fold increase).
  • NPRl is not required for the ⁇ t2-conferred constitutive expression of PR genes, which are expressed at elevated levels in ssi2 nprl-5 and ssi2 niml-1 plants (Fig. 1 A and IB). However, in comparison to ssi2 nprl-5, presence of NPRl enhanced accumulation of the PR-1 transcript in ssi2 NPRl plants (Fig. 1 A). This increased expression of PR-1 in ssi2
  • NPRl plants is likely due to elevated levels of SA (Fig. 3) activating signaling through the NPRl pathway.
  • This is supported by the observation that salicylate hydroxylase expression repeatedly reduced PR-1 expression in ssi2 NPRl nahG plants (Fig. 4B).
  • BTH application increased PR-1 expression in ssi2 NPRl nahG plants but not in ssi2 nprl-5 nahG plants (data not shown).
  • ssi2 activates SA signaling through the NPRl -dependent as well as NPRl -independent pathways (Fig. 11). The existence of an NPRl -independent SA signaling pathway has previously been reported.
  • PR genes are expressed at elevated levels in pathogen-infected nprl plants. This is in contrast to the poor expression of PR genes seen in pathogen-infected NahG plants.
  • the pathogen activated accumulation of PAD4 transcript also occurs via a SA-dependent, NPRl -independent pathway besides the SA- and NPRl -dependent pathway.
  • nprl mutation reduced 55t2-conferred constitutive PR-1 expression, it however had little if any effect on constitutive BGL2 and PR-5 expression (Figs. 1A and 4B, and data not shown).
  • the NPRl -dependent pathway is the primary activator(s) of PR-1 expression in ssi2 plants
  • BGL2 and PR-5 expression are induced primarily by the NPRl -independent pathway.
  • Similar differences in the requirement of NPRl for PR-1 expression, as compared to BGL2 and PR-5 expression have previously been noted in pathogen infected nprl plants.
  • ss 2-conferred constitutive PR expression is not dependent on SA accumulation.
  • constitutive expression of PR genes in the ssil, snil, cpr ⁇ and acd ⁇ mutants also occurs independently of NPRl.
  • SA is required for constitutive PR gene expression in these mutants. This is in contrast to the SA- independent expression of PR genes in the ssi2 NPRl nahG andssi2 nprl-5 nahG plants (Fig. 4B).
  • SA and SAG levels in the ssi2 NPRl nahG and ssi2 nprl-5 nahG plants were comparable to those seen in uninfected wild-type plants (data not shown).
  • the NahG transgenic line used in these experiments is hypersusceptible to P. syringae and P.
  • ssil and ssi2 mutants accumulate comparable levels of SA and SAG it is highly unlikely that the residual SA and SAG in ssi2 NPRl nahG andssi2 nprl-5 nahG plants activate PR gene expression.
  • SSI2 negatively regulates the NPR1- independent pathway at a step downstream of SA (Fig. 11).
  • the ssi2 phenotypes might be due to activation of an SA- and NPRl -independent pathway.
  • ssi2 confers enhanced resistance against P. parasitica and P.
  • syringae is primarily dependent on the NPR1-dependent SA signal transduction pathway, while resistance against P. parasitica is conferred primarily by the NPRl -independent S A signal fransduction pathway.
  • SSI2 acts as a negative regulator of the NPRl -independent pathway at a step downstream of SA action (Fig. 11)
  • the lack of SSI2 repressor activity in ssi2 plants will allow significant level of signaling through this pathway even in the absence of elevated SA levels. This could account for the strong resistance against P. parasitica observed in ssi2 nprl-5 plants. Constitutive signaling through this NPRl -independent pathway could also account for the partial resistance against P.
  • S A is not required for the development of HR-like lesions in ssi2.
  • ssi2 plants spontaneously develop HR-like lesions.
  • Spontaneous lesions have been observed in several Arabidopsis mutants exhibiting constitutive SAR. hi all these cases lesion formation was associated with elevated levels of S A and SAG.
  • acd ⁇ Rh et al. 1999
  • cep Silva et al. 1999
  • Isdl Danangl et al. 1996)
  • Isd ⁇ and lsd7 Weymann et al 1995
  • lesion formation is independent of SA in the lsd2, lsd4 (Hunt et al. 1997) and cpr 5 (Bowling et al. 1997) mutants. Similar to lsd2, lsd4 and cpr5 plants, lesion formation in ssi2 plants is not dependent on elevated levels of SA (Fig. 5A). Lesion formation in ssi2 could be a result of the metabolic stress caused by constitutively active defense responses.
  • Spontaneous lesions have been observed in plants exposed to metabolic stress.
  • expression of the bacterial proton pump bacterio-opsin, a subunit of the cholera toxin gene and yeast vacuolar invertase, and inhibition of protoporphyrinogen oxidase in plants causes the development of lesions.
  • lesion formation is associated with the accumulation of elevated levels of SA and SAG.
  • RNA from one or two leaves was perfo ⁇ ned in the TRIzol reagent (GIBCO-BRL, Gaithersburg, MD) following the manufacturer's instructions.
  • Northern blot analysis and synthesis of random primed probes for PR-1, BGL2 and PR-5, PDF 1.2 and THI2.1 were synthesized. Arabidopsis transformation.
  • the putative signal peptide region of SSI2 was predicted by aligning it with the protein sequence from castor bean S-ACP DES.
  • cDNA's from both wt and ssi2 were amplified such that they lacked N-terminal 34 aa of the putative signal peptide and the 35th aa was converted to a methionine.
  • the cDNA's were isolated as a NcoI/EcoRI-linkered PCR products and cloned into p ⁇ T-28a vector. Purification and determination of desaturase activity were performed. Dimethyl disulfide adducts of fatty acid methyl esters were prepared. Methyl esters of unsaturated FA and their dimethyl disulfide derivatives were identified by MS analysis.
  • ssi2 gene was mapped to a 41 kb region of chromosome 2 that is encompassed by the bacterial artificial chromosome (BAC) clone F18O19 (Fig. 6A).
  • BAC bacterial artificial chromosome
  • ssi2 nprl-5 double mutant plants were transformed with subclones of F 18019 that had been inserted into a binary-BAC (BIB AC) vector (Hamilton, 1997).
  • TAC transformation-competent artificial chromosome
  • PCR polymerase chain reaction
  • ORF open reading frame
  • Transformants were screened for restoration of the wt morphology and absence of constitutive PR-1 gene expression. Only TAC clone F23 complemented the ssi2 mutation (Fig. 6B and 6C).
  • the SSI2- containing region of F23 was reduced to 11.7 kb.
  • This region contains 4 ORFs, which were amplified by PCR and sequenced. Comparison with sequences from wt N ⁇ plants revealed only one difference; a C to T transition was detected in ORF2. Since this variation between wt and ssi2 sequences could not be distinguished by restriction enzyme polymo ⁇ hism, a derived-CAPS (dCAPS) marker (Neff et al., 1998) was used to confirm the identity of the ssi2 mutation.
  • dCAPS derived-CAPS
  • S-ACP-DES preferentially desaturates stearoyl-ACP (18:0-ACP) between carbons 9 and 10, yielding oleoyl-ACP (18:1 ⁇ 9 -ACP).
  • the wt gene was expressed in Escherichia coli and the activity of the purified enzyme was assessed by in vitro assays. Wt SSI2 had specific activity ( ⁇ 800nm min mg; Fig. 7B) and substrate preference (88:1, for 18 versus 16 carbon chain length FA) characteristic of S-ACP-DES.
  • Gas chromatography-mass spectroscopy (GC-MS) analysis confirmed the regiospecificity as ⁇ 9 (Fig. 7C).
  • the C to T mutation in ssi2 changes the leucine (L) at amino acid (aa) position 146 to a phenylalanine (F).
  • L leucine
  • aa amino acid
  • F phenylalanine
  • the high degree of conservation for L 146 combined with the recessive nature of the ssi2 mutation, suggested that ssi2 might have reduced and/or altered enzymatic activity.
  • the mutant protein was approximately 10- and 20-fold less active on both 18:0 and 16:0 substrates, respectively, but the 18:16 substrate preference ratio and the ⁇ 9 regiospecificity were unaltered (FigJ).
  • the FA composition in ssi2 plants is altered.
  • S-ACP DES activity affects the FA composition in ssi2 plants
  • the levels of various 16 and 18 carbon fatty acids were monitored using GC-MS (Table 2).
  • Leaves of the ssi2 mutant contained considerably elevated levels of 18:0 compared to the wt and decreased levels of 16:3, 18:1 and 18:2 (Table 2).
  • the levels of other FAs including 18:3 were similar to or slightly reduced from those observed in wt plants.
  • the presence of nearly wt levels of these FAs in ssi2 plants is likely due to the activity of other S-ACP DES isoforms, several of which have been identified in Arabidopsis (Maleck & Dietrich, 1999).
  • Table 2 Fatty acid composition of total leaf lipids from wild type and ssi2. All measurements were made on 22°C grown plants and data are described as mol% ⁇ standard error calculated for a sample size of six.
  • JA-inducible defense responses Activation of some JA-inducible defense responses is impaired in ssi2 plants.
  • S-ACP-DES catalyzes the first step in the pathway from stearic acid (18:0) to linolenic acid (18:3), and linolenic acid is a precursor for the defense signaling molecule JA (Farmer & Ryan, 1992). Since JA is required to activate the wounding response and defenses against insect pests and certain microbial pathogens, we monitored SSI2 gene expression after wounding, pathogen infection or treatment with SA, JA or ethylene.
  • JA plus 18:1 induce PDF 1.2 expression in ssi2 nahG plants.
  • a likely explanation for the failure of JA to activate PDF1.2 and resistance to B. cinerea in ssi2 nahG plants is that certain JA-dependent responses require a second signal that is generated by S-ACP DES. ssi2 or ssi2 nahG plants would lack or have reduced levels of this co-activating signal. Consistent with this hypothesis, treatment of ssi2 nahG plants with a combination of JA and 18:1 activated PDFl.2 (Fig. 10). ssi2 plants failed to respond to JA plus 18:1 (which is reduced three fold in ssi2), probably because of antagonistic effects of the high levels of endogenous SA.
  • the recessive ssi2 mutation was identified as a suppressor of the nprl-5 allele.
  • SSI2 encodes S-ACP DES.
  • This enzyme along with other soluble FA desaturases, is a key determinant of the overall level of unsaturated FAs.
  • Analyses of the ssi2 protein revealed that its substrate preference and regiospecificity were unaltered; however, its activity was 10-20 fold lower than that of the wt enzyme.
  • ssi2 plants exhibit constitutive expression of several SA-associated defense responses. Since pathogen infection of wt plants generally induces the expression of either PDF 1.2 or the PR genes, our results suggest that components of the FA desaturation pathway may cross regulate the activation of these defenses. Possibly, the co-activating signal inhibits the NPRl -independent pathway; loss of this signal in ssi2 plants would allow constitutive activation ofthe NPRi-independent responses (Fig. 11). Alternatively, the ratio of saturated versus unsaturated FAs or changes in their subcellular distribution might regulate cross-talk between defense signaling pathways.
  • an increase in 18:0 content might lead to activation of lipid signaling, which could then induce the PR signal transduction pathway (Anderson et al., 1998).
  • Increases in unsaturated FAs also could stimulate (Klumpp et al., 1998) or inhibit (Baudouin et al., 1999) protein phosphatase(s) activity, which might then alter protein kinase- or mitogen activated protein kinase (MAPK)-regulated pathway(s), respectively.
  • MAPK mitogen activated protein kinase
  • an Arabidopsis mutant defective in the MAPK mpk4 exhibits a phenotype similar to that of ssi2, including constitutive PR gene expression and suppressed PDF 1.2 expression (Petersen et al., 2000).

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Abstract

L'invention concerne un nouveau gène de plante SSI2 codant pour une stéaroyl-ACP désaturase dans des plantes et jouant un rôle clé dans la modulation des réponses de défense des plantes. L'invention concerne également une/ou des molécules de signalisation dérivées de FA, pouvant être manipulées par régulation positive ou négative de la désaturase SSI2 FA, ce qui entraîne des modifications spécifiques des réponses de défense des plantes. Cette/ou ces molécules de signalisation dérivées de FA comprennent au moins un FA 18 :1 ou un dérivé de celui-ci. L'invention concerne en outre des plantes mutantes à activité SSI2 sensiblement réduite qui, conjointement avec des plantes transgéniques, sur-expriment ou sous-expriment le gène SSI2.
PCT/US2001/016134 2000-06-12 2001-05-18 Gene de desaturase d'acide gras et proteine destines a moduler l'activation des voies de signalisation de defense dans des plantes WO2001096363A1 (fr)

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US10513717B2 (en) 2006-08-29 2019-12-24 Commonwealth Scientific And Industrial Research Organisation Synthesis of fatty acids
US11976287B2 (en) 2008-11-18 2024-05-07 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing ω-3 fatty acids
US9938486B2 (en) 2008-11-18 2018-04-10 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing omega-3 fatty acids
US9976107B2 (en) 2008-11-18 2018-05-22 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing ω-3 fatty acids
US9994792B2 (en) 2008-11-18 2018-06-12 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing omega-3 fatty acids
US10648046B2 (en) 2008-11-18 2020-05-12 Commonwealth Scientific And Industrial Research Organisation Enzymes and methods for producing omega-3 fatty acids
US10335386B2 (en) 2012-06-15 2019-07-02 Commonwealth Scientific And Industrial Research Organisation Lipid comprising polyunsaturated fatty acids
US8946460B2 (en) 2012-06-15 2015-02-03 Commonwealth Scientific And Industrial Research Organisation Process for producing polyunsaturated fatty acids in an esterified form
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US9556102B2 (en) 2012-06-15 2017-01-31 Commonwealth Scientific And Industrial Research Organisation Process for producing ethyl esters of polyunsaturated fatty acids
US9932289B2 (en) 2012-06-15 2018-04-03 Commonwealth Scientific And Industrial Research Ogranisation Process for producing ethyl esters of polyunsaturated fatty acids
US9725399B2 (en) 2013-12-18 2017-08-08 Commonwealth Scientific And Industrial Research Organisation Lipid comprising long chain polyunsaturated fatty acids
US10190073B2 (en) 2013-12-18 2019-01-29 Commonwealth Scientific And Industrial Research Organisation Lipid comprising long chain polyunsaturated fatty acids
US10125084B2 (en) 2013-12-18 2018-11-13 Commonwealth Scientific And Industrial Research Organisation Lipid comprising docosapentaenoic acid
US10800729B2 (en) 2013-12-18 2020-10-13 Commonwealth Scientific And Industrial Research Organisation Lipid comprising long chain polyunsaturated fatty acids
US11623911B2 (en) 2013-12-18 2023-04-11 Commonwealth Scientific And Industrial Research Organisation Lipid comprising docosapentaenoic acid
US9718759B2 (en) 2013-12-18 2017-08-01 Commonwealth Scientific And Industrial Research Organisation Lipid comprising docosapentaenoic acid
US10005713B2 (en) 2014-06-27 2018-06-26 Commonwealth Scientific And Industrial Research Organisation Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the sn-2 position
US10793507B2 (en) 2014-06-27 2020-10-06 Commonwealth Scientific And Industrial Research Organisation Lipid compositions comprising triacylglycerol with long-chain polyunsaturated fatty acids at the SN-2 position

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