WO2013087821A1 - Surproduction de jasmonates dans des plantes transgéniques - Google Patents

Surproduction de jasmonates dans des plantes transgéniques Download PDF

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WO2013087821A1
WO2013087821A1 PCT/EP2012/075504 EP2012075504W WO2013087821A1 WO 2013087821 A1 WO2013087821 A1 WO 2013087821A1 EP 2012075504 W EP2012075504 W EP 2012075504W WO 2013087821 A1 WO2013087821 A1 WO 2013087821A1
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plant
gherf
genus
sequence
seq
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Antony Champion
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Institut De Recherche Pour Le Développement (Ird)
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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
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • 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

Definitions

  • Jasmonic acid and its precursors and derivatives constitute a family of compounds that are synthesized from membrane linoleic acid via the octadecanoids metabolic pathway (Turner et al, Plant Cell, 2002, 14 (suppl.): S153-S164; Atallah et al, In Encyclopedia of Plant and Crop Science, 2004, R.M. Goodman (Ed), New York: Marcel Dekker Inc, pp. 1006-1009).
  • the development, in Arabidopsis, of mutants that exhibit deficiencies in some jasmonate-dependent mechanisms, has revealed the complexity of the signalling pathway of jasmonic acid.
  • the present invention generally relates to the improvement of plants and in particular to the improvement of plant natural defense against bioagressors, via an increase in the production of jasmonates in the plants.
  • the overexpression of GhERF-IIc in cotton plants and tobacco plants, was found to result in production of jasmonic acid that is higher than the production of OPDA.
  • the overexpression of GhERF-IIa or GhERF-IIb was found to result in a production of OPDA that is higher than the production of jasmonic acid.
  • the overexpression of one (or more) of these transcription factors can be used to increase the content in jasmonic acid and/or OPDA in plants and thereby generate plants that exhibit an improved resistance to pathogenic agents.
  • This approach has several advantages compared to existing methods: (1) it is universal, (2) it induces an accumulation of jasmonates within the plant (or in planta), and (3) it exploits natural defense mechanisms of plants with the aim of increasing resistance and therefore is a strategy that is more respectful of the environment than the chemical methods used for fighting plant diseases.
  • the present invention provides a transgenic plant exhibiting an overproduction or accumulation of jasmonic acid, and optionally of OPDA, comprising an exogenous nucleic acid sequence, wherein the exogenous nucleic acid sequence is integrated in the genome of the transgenic plant and comprises the sequence of a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana and wherein expression of the plant functional ortholog in the plant results in overproduction or accumulation of jasmonic acid, and optionally of OPDA, in said transgenic plant.
  • the gene ORA47 of Arabidopsis thaliana has the sequence set forth in SEQ ID
  • the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana may be from any plant.
  • the plant from which the functional ortholog originates may or may not be of the same species as the transgenic plant.
  • the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana is from a plant that belongs to the Gossypium or Cotoneaster genera (cotton), to the Nicotiana genus (tobacco), to the Oryza genus (rice), to the Solanum genus (tomato), to the Cojfea genus (coffee), or to the Vitis genus (vine).
  • the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana is from Gossypium hirsutum and is selected from GhERF-IIa, GhERF-IIb and GhERF-IIc, which encode the transcription factors GhERF-IIa, GhERF- Ilb and GhERF-IIc, respectively.
  • the present invention also provides a transgenic plant exhibiting an overproduction or accumulation of jasmonic acid, and optionally of OPDA, comprising an exogenous nucleic acid sequence encoding a transcription factor selected from the group consisting of GhERF-IIa, GhERF-IIb, GhERF-IIc and any combination thereof, wherein the exogenous nucleic acid sequence is integrated in the genome of the transgenic plant.
  • the exogenous nucleic acid sequence encoding a transcription factor comprises, or consists of, a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 4 ⁇ GhERF-IIa), the sequence set forth in SEQ ID NO: 5 ⁇ GhERF-IIb), the sequence set forth in SEQ ID NO: 6 ⁇ GhERF-IIc), any homologous sequence thereof, and any combination thereof.
  • the exogenous nucleic acid sequence encodes a transcription factor having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 1 (GhERF-IIa), the sequence set forth in SEQ ID NO: 2 (GhERF-IIb), the sequence set forth in SEQ ID NO: 3 (GhERF-IIc), any homologous sequence thereof, and any combination thereof.
  • a transgenic plant according to the invention is characterized in that it exhibits an improved resistance to at least one pathogenic agent selected from the group consisting of bacteria, viruses, fungi, insects and oomycetes, wherein said at least one pathogenic agent is capable of inducing a disease in the plant.
  • a transgenic plant according to the invention belongs to the Malvaceae family, to the Solanaceae family, to the Rubiaceae family, to the Poaceae or Gramineae family or to the Vitaceae family. In certain embodiments, a transgenic plant according to the invention belongs to the
  • Gossypium or Cotoneaster genera (cotton), to the Nicotiana genus (tobacco), to the Oryza genus (rice), to the Solanum genus (tomato), to the Cojfea genus (coffee), or to the Vitis genus (vine).
  • the present invention provides a vegetal material obtained or extracted from a transgenic plant of the invention, wherein said vegetal material is selected from the group consisting of plant cells, plant cell cultures, protoplasts, plant organs, plant calluses, plant seeds, leaves, stems, roots, flowers, fruits, tubers, pollen, and plant cuttings.
  • the vegetal material is capable of regenerating a whole plant.
  • the present invention provides an expression construct or expression vector comprising the nucleic acid sequence of a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana, wherein the nucleic acid sequence is operably linked to at least one element allowing the expression, and optionally the regulation, of the nucleic acid sequence in a plant.
  • the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in an expression construct or expression vector according to the invention may be from any plant.
  • the plant from which the functional ortholog originates may, or may not be, of the same species as the plant to which the expression construction or expression vector is intended.
  • the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in an expression construct or expression vector according to the invention is from a plant that belongs to the Gossypium or Cotoneaster genera (cotton), to the Nicotiana genus (tobacco), to the Oryza genus (rice), to the Solanum genus (tomato), to the Cojfea genus (coffee), or to the Vitis genus (vine).
  • the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in an expression construct or expression vector according to the invention is from Gossypium hirsutum and is selected from GhERF-IIa, GhERF-IIb and GhERF-IIc, which encode the transcription factors GhERF-IIa, GhERF-IIb and GhERF-IIc, respectively.
  • the nucleic acid sequence of a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in an expression construct or expression vector according to the invention comprises, or consists of, a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 4, the sequence set forth in SEQ ID NO: 5, the sequence set forth in SEQ ID NO: 6, any homologous sequence thereof, and any combination thereof.
  • the nucleic acid sequence of a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in an expression construct or expression vector according to the invention encodes a transcription factor having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 1, the sequence set forth in SEQ ID NO: 2, the sequence set forth in SEQ ID NO: 3, any homologous sequence thereof, and any combination thereof.
  • the present invention provides for the use of an expression construct or expression vector according to the invention for transforming a plant and obtaining a transgenic plant exhibiting an overproduction or accumulation of jasmonic acid, and optionally of OPDA.
  • the present invention also provides for the use of an expression construct or expression vector according to the invention for transforming a plant and obtaining a transgenic plant exhibiting an improved resistance to at least one pathogenic agent selected from the group consisting of bacteria, viruses, fungi, insects and oomycetes, wherein said at least one pathogenic agent is capable of inducing a disease in the plant.
  • the present invention also provides for the use of an expression construct or expression vector according to the invention for transforming a plant and obtaining a transgenic plant producing a secondary metabolite whose synthesis is induced by jasmonates, in particular by jasmonic acid and/or OPDA.
  • the secondary metabolite may be, for example, nicotine.
  • the present invention provides a method for obtaining a transgenic plant exhibiting an overproduction or accumulation of jasmonic acid and/or for obtaining a transgenic plant exhibiting an improved resistance to at least one pathogenic agent and/or for obtaining a transgenic plant producing a secondary metabolite whose synthesis is induced by jasmonic acid and/or by OPDA, said method comprising transforming a plant using an expression construct or expression vector of the invention.
  • transforming a plant using an expression construct or expression vector of the invention comprises steps of:
  • transforming a plant using an expression construct or expression vector of the invention comprises steps of:
  • Step (b) may comprise: infecting several plants or several plant cells with recombinant Agwbacterium host cells; optionally culturing infected plant cells in order to regenerate several plants; and selecting, among the infected plants or among the regenerated plants, those plants that comprise, integrating in their genome, the sequence of the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana.
  • the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in the expression construct or expression vector may be from any plant, as already mentioned.
  • the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in the expression construct or expression vector used to transformed a plant is from Gossypium hirsutum and is selected from GhERF-IIa, GhERF-IIb and GhERF-IIc, which encode the transcription factors GhERF-IIa, GhERF- lib and GhERF-IIc, respectively.
  • the nucleic acid sequence of a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in an expression construct or expression vector used to transformed a plant comprises, or consists of, a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 4, the sequence set forth in SEQ ID NO: 5, the sequence set forth in SEQ ID NO: 6, any homologous sequence thereof, and any combination thereof.
  • the nucleic acid sequence of a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana present in an expression construct or expression vector used to transformed a plant encodes a transcription factor having a sequence selected from the group consisting of the sequence set forth in SEQ ID NO: 1, the sequence set forth in SEQ ID NO: 2, the sequence set forth in SEQ ID NO: 3, any homologous sequence thereof, and any combination thereof.
  • the plants that can be transformed using an expression construct or expression vector of the invention, for example using a method according to the invention, may belong to any plant family.
  • the plant belongs to the Malvaceae family, to the Solanaceae family, to the Rubiaceae family, to the Poaceae or Gramineae family or to the Vitaceae family.
  • the plant belongs to the Gossypium or Cotoneaster genera (cotton), to the Nicotiana genus (tobacco), to the Oryza genus (rice), to the Solanum genus (tomato), to the Cojfea genus (coffee), or to the Vitis genus (vine).
  • the plant belongs to the Nicotiana genus and the secondary metabolite is nicotine.
  • Figure 1 shows the amino acid alignment of AtORA47 from Arabidopsis thaliana and the three sequences GhERF-IIa, GhERF-IIb and GhERF-IIc identified in Gossypium hirsutum cv. Reba B50 (see Example 1 for experimental details).
  • Figure 2 is a set of two graphs showing the expression patterns of GhERF-IIa and GhERF-IIc in cotyledons inoculated with virulent (Xcm20), avirulent (Xcml8) Xcm races and water (c) using the Q-RT-PCR method (see Example 1 for experimental details).
  • Figure 3 shows the constructs used in trans-activation experiments (A) and the relative transcription factors activity (B) in cotton cotyledon protoplasts (see Example 2 for experimental details).
  • Figure 4 presents microscopic images showing the subcellular localization of the GhERF-II transcription factor fused to a reporter gene encoding GFP in leaves protoplasts from Arabidopsis. These protoplasts were transformed simultaneously by the nuclear marker NtKISla-DsRFP (see Example 2 for experimental details).
  • Figure 5 is a set of six graphs showing the expression of five genes (GhAOS,
  • the sixth graph shows the quantity of jasmonic acid (J A) and of OPDA accumulated in cotton cotyledons 48 hours after transformation with GFP (control) or with GhERF-IIa, GhERF- IIb and GhERF-IIc (see Example 3 for experimental details).
  • Figure 6 is is a graph showing the XcmlO population growth in GFP-transformed cotton and in G/iER -Z/c-transformed cotton measured 1 day, 6 days and 12 days post inoculation with the bacterium Xanthomonas campestris pv. malvacearum race 20 (see Example 4 for experimental details).
  • Figure 7 shows the intracellular localisation of GhERF-IIc (A) and the quantity of jasmonic acid (JA) and of OPDA (B) accumulated in tobacco leaves transformed with GFP (control) or with CgERF-IIc measured 0, 24 hours and 48 hours after transformation (see Example 5 for experimental details).
  • Figure 8 is a set of four graphs showing the expression of the genes NbAOX, NbODX, NbPMT and NbMPO in tobacco transformed with GFP (control) or with GhERF-IIc measured 0, 24 hours and 48 hours after transformation.
  • the genes NbAOX, NbODX, NbPMT and NbMPO encode enzymes involved in the biosynthesis pathway of nicotine.
  • the present invention relates to the transformation of a plant in order to induce, in said plant, an overproduction or accumulation of jasmonic acid and/or OPDA, which leads to an improvement of the plant resistance against bioagressors.
  • the transformation may also be used to obtain plants producing secondary metabolites whose synthesis is induced by jasmonates.
  • An expression construct (or expression vector) according to the present invention comprises a nucleic acid sequence of a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana.
  • the term "gene ORA47 of Arabidopsis thaliana” refers to the gene ORA47, also called Atlg74930, (GenBank Accession Number: NM_106151 - SEQ ID NO. 8), which encodes ORA47 (GenBank Accession Number: NP_177631 - SEQ ID NO.
  • a transcription factor i.e., a protein that modulates the expression of genes
  • a transcription factor i.e., a protein that modulates the expression of genes
  • plant functional ortholog of the gene ORA47 of Arabidopsis thaliana refers to a gene from a plant species different from Arabidopsis thaliana, wherein said gene and the gene ORA47 of Arabidopsis thaliana originate by vertical descent from a single gene of the last common ancestor.
  • a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana according to the invention may be from any plant that has a common ancestor with the species Arabidopsis thaliana. Using sequence homology and the known sequence of the ORA47 gene, one skilled in the art knows how to identify a functional ortholog of the gene ORA47 of Arabidopsis thaliana in the genome of any plant.
  • a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana may be identified in the genome of a plant that belongs to the Gossypium or Cotoneaster genera (cotton), to the Nicotiana genus (tobacco), to the Oryza genus (rice), to the Solanum genus (tomato), to the Cojfea genus (coffee), or to the Vitis genus (vine).
  • the present Applicants have identified 3 functional orthologs of the gene ORA47 of Arabidopsis thaliana in the Cotton genome. Consequently, in certain preferred embodiments, the plant functional ortholog of the gene ORA47 of Arabidopsis thaliana is from Gossypium hirsutum and is selected from GhERF-IIa, GhERF-IIb and GhERF-IIc, which encode the transcription factors GhERF- Ila, GhERF-IIb and GhERF-IIc, respectively.
  • the invention is hereafter described and illustrated using the Cotton functional orthologs of the gene ORA47 of Arabidopsis thaliana identified by the present Applicants.
  • the invention encompasses any expression construct or vector comprising a plant functional ortholog of the gene ORA47 of Arabidopsis thaliana, any use thereof, any method for preparing transgenic plants using such an expression construct or vector, and any transgenic plant or plant part thus obtained.
  • the nucleic acid sequence encoding GhERF- IIa, GhERF-IIb or GhERF-IIc may be any nucleic acid sequence whose transcription results in the formation of GhERF-IIa, GhERF-IIb or GhERF-IIc (SEQ ID NO: l, SEQ ID NO: 2 and SEQ ID NO: 3, respectively) or a homologous polypeptide thereof.
  • the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc comprises, or consists of, the sequence SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, respectively, or a homologous sequence thereof resulting from the genetic code degeneracy.
  • the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc comprises, or consists of, a sequence which is homologous to the sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, respectively and which encodes a polypeptide that is homologous to GhERF-IIa, GhERF-IIb or GhERF-IIc.
  • the nucleic acid encoding GhERF-IIa, GhERF-IIb or GhERF-IIc comprises, or consists of, a nucleic acid sequence that is complementary to the sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, respectively or a homologous sequence thereof, a nucleic acid sequence that is modified compared to the sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6 or a homologous sequence thereof, or a representative fragment of any one of the preceding sequences (for example an open reading frame).
  • nucleic acid sequence refers to a given sequence of nucleotides, modified or not, which defines a region of a nucleic acid molecule and which may be either under the form a single strain or double strain DNAs or under the form of transcription products thereof.
  • nucleic acid sequence homologous to a particular sequence (for example homologous to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6) refers to any nucleic acid sequence that differs from the particular sequence by substitution, deletion and/or insertion of one nucleotide or of a limited number of nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) at positions such that the homologous nucleic acid sequence encodes a polypeptide that is homologous to the particular sequence (for example homologous to GhERF-IIa, GhERF-IIb or GhERF-IIc and in particular to SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3).
  • such a homologous nucleic acid sequence has a percentage of identity of at least 75% of the sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, preferably at least 85%, more preferably at least 95% or more.
  • homologous (or “homology”), as used herein, is synonymous with the term “identity” and refers to the sequence similarity between two polypeptide molecules or between two nucleic acid molecules. When a position in both compared sequences is occupied by the same base or same amino acid residue, the respective molecules are then homologous at that position.
  • the percentage of homology between two sequences corresponds to the number of matching or homologous positions shared by the two sequences divided by the number of positions compared and multiplied by 100. Generally, a comparison is made when the two sequences are aligned to give maximum homology. This percentage is purely statistical and the differences between the two compared sequences are spread at random and over the whole length of the sequence.
  • optimal alignment and “best alignment”, which are used herein interchangeably, refer to the alignment for which the percentage of identity is determined as described herein to be the highest.
  • the optimal alignment of sequences, that is necessary to the comparison may be performed manually or using softwares (GAP, BESTFIT, BLASTP, BLASTN, FASTA, and TFASTA, which are available either on the NCBI website, or in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI).
  • Homologous amino acid sequences share identical or similar amino acid sequences. Similar residues are conservative substitutions for, or "allowed point mutations" of, corresponding amino acid residues in a reference sequence.
  • Constant substitutions of a residue in a reference sequence are substitutions that are physically or functionally similar to the corresponding reference residue, e.g. that have a similar size, shape, electric charge, chemical properties, including the ability to form covalent or hydrogen bonds, or the like. Particularly preferred conservative substitutions are those fulfilling the criteria defined for an "accepted point mutation" as described by Dayhoff et al. ("Atlas of Protein Sequence and Structure", 1978, Nat. Biomed. Res. Foundation, Washington, DC, Suppl. 3, 22: 354-352).
  • a nucleic acid sequence homologous to SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 specifically hybridizes to the complementary sequence of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 under stringent conditions (Sambrook et al., "Molecular Cloning - A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1989).
  • nucleic acid sequence modified compared to a particular sequence (for example modified compared to the sequence set forth in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6) refers to any nucleic acid sequence that is obtained by mutagenesis using techniques well known in the art, and that comprises modifications compared to the particular sequence, for example, mutations in regulatory and/or promoter sequences of the polypeptide expression, in particular leading to the modification of the expression or activity level of said polypeptide.
  • modified nucleic acid sequence also encompasses any nucleic acid sequence encoding a modified GhERF-IIa, GhERF-IIb or GhERF-IIc polypeptide.
  • representative fragment of a particular nucleic acid sequence refers to any fragment of that particular sequence (or of a homologous sequence thereof or of a modified sequence thereof) that encodes a polypeptide exhibiting an activity that is identical or similar to the activity of GhERF-IIa, GhERF-IIb or GhERF-IIc.
  • a representative fragment of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 is an open reading of SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 (see Examples section below).
  • GhERF-IIa GhERF-IIb or GhERF-IIc
  • GhERF-IIa GhERF-IIa, GhERF-IIb or GhERF-IIc
  • Cloning of a gene, or of a nucleic acid sequence encoding a transcription factor, from genomic DNA may be carried out for example using PCR (polymerase chain reaction) or via screening of expression libraries to detect cloned DNA fragments with identical structural characteristics (Innis et al, "PCR: A Guide to Method and Application", 1990, Academic Press: New York).
  • Other methods of amplification of nucleic acids known to those skilled in the art may be used in the context of the present invention, such as for example, ligase chain reaction (LCR), ligation activated transcription (LAT), and Nucleic Acid Sequence Based Amplification (NASBA).
  • LCR ligase chain reaction
  • LAT ligation activated transcription
  • NASBA Nucleic Acid Sequence Based Amplification
  • an expression construct according to the present invention the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc is inserted in the sense orientation and is preferably linked to one or more elements that allows for its expression and optionally for its regulation in a plant or plant cell.
  • an expression construct according to the present invention comprises 5' and 3' regulatory sequences operably linked to a nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF- lie.
  • the term "operably linked” refers to a functional link between the 5' and 3' regulatory sequences and the nucleic acid sequence that they control.
  • An expression construct according the present invention comprises, in the 5' ⁇ 3' direction of transcription, a transcription initiation sequence, the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc and a transcription termination sequence that are functional in a plant or plant cell.
  • a transcription initiation sequence the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc and a transcription termination sequence that are functional in a plant or plant cell.
  • Such a combination is designated herein interchangeably as "nucleic acid sequence allowing expression of GhERF-IIa, GhERF-IIb or GhERF-IIc in a plant” or "nucleic acid sequence allowing synthesis of GhERF-IIa, GhERF-IIb or GhERF-IIc in a plant”.
  • promoter refers to any polynucleotide capable of regulating the expression, in a cell, of a nucleic acid sequence to which the promoter is operably linked. In the context of the invention, a promoter is capable of exerting its regulating action in a plant cell (i.e., it is a "plant promoter").
  • a promoter type regulatory sequence is a regulatory region that is recognized by a RNA polymerase in a cell and that is able to initiate transcription of the nucleic acid sequence of GhERF-IIa, GhERF-IIb or GhERF-IIc in a plant or plant cell.
  • the promoter may be homologous to a cell of the host plant or, alternatively, may be heterologous to a cell of the host plant.
  • the promoter may be a natural sequence (i.e., a sequence that exists in nature) or a synthetic sequence (i.e., a sequence that does not exist as such in nature).
  • a construct according to the present invention comprises a plant constitutive promoter operably linked to the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc.
  • constitutive promoter refers to a promoter that is able to express nucleic acid sequences operably linked to the promoter, in every, or almost every, tissue of the host organism and during the entire development of this organism.
  • Plant constitutive promoters include, but are not limited to, the Cauliflower Mosaic Virus 35S (CaMV) promoter, the CaMV constitutive promoter double 35S (pd35S), the nopaline synthase promoter, the octopine synthase promoter, the 19S promoter, the rice actin promoter and actin intron (PAR-LAR) contained in plasmid pAct-l-F4, the promoter of the rice actin 1 gene, the promoter of the gene AdH of corn, the promoter of ubiquitin of corn, and the promoter pUbil of the gene encoding ubiquitin 1 of corn.
  • Such promoters may be obtained from genomic DNA using PCR, and may then be cloned in an expression construct according to the invention.
  • tissue-Specific Promoters In other embodiments, the expression of GhERF-IIa, GhERF-IIb or GhERF-IIc is targeted to certain tissues of the transgenic plant.
  • tissue- specific promoter refers to a promoter that is able to express, in a selective manner, nucleic acid sequences to which it is operably linked, in specific tissues of the host organism.
  • tissue-specific expression in tissues may be performed for a preferential expression of GhERF-IIa, GhERF-IIb or GhERF-IIc in the leaves and/or the stems and/or the roots of plants rather than in the seeds or fruits of the plants (in order to reduce concerns and worries associated with human consumption of genetically modified organisms).
  • Tissue- specific expression may also be used when the pathogen to which the plant is naturally sensitive to specifically attacks given tissues of the plant.
  • a construct according to the present invention comprises a plant tissue-specific promoter operably linked to the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc.
  • tissue-specific gene regulators and tissue-specific promoters that can be used in plants are known in the art.
  • genes include, but are not limited to, genes encoding zeine-type storage proteins (such as napin, cruciferin, ⁇ -conglycin and phaseolin), genes involved in the biosynthesis of fatty acids (including the ACP protein - acyl carrier protein, stearoyl ACP-desaturase, and desaturases of fatty acids (fad 2-1)), and other genes that are express during the embryonic development such as Bce4 (Kridl et al, Seed Science Res., 1991, 1: 209).
  • Tissue-specific promoters that have been described include, but are not limited to, lectin (Vodkin, Prog.
  • heat shock protein of corn the pea small sub-unit of ribulose 1,5-biphosphate carboxylase, the mannopine synthase in the Tl plasmid, the nopaline synthase in the Tl plasmid, the chalcone isomerise of petunia (van Tunen et al, EMBO J., 1988, 7: 125), the glycin-rich protein I of bean (Keller et al, Genes Dev., 1989, 3: 1639), the truncated CaMV 25S (Odell et al, Nature, 1985, 313: 810), the potato palatine (Wenzler et al, Plant Mol.
  • the transcription termination region present in the expression construct may be of the same origin as ⁇ i.e., homologous) the transcription initiation region or the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc, or of different origin ⁇ i.e., heterologous).
  • Transcription termination regions are for example available from the Agrobacterium tumefaciens ⁇ plasmid, such as the termination regions of the octopine synthase and nopaline synthase (An et al, Plant Cell, 1989, 1: 115-122; Guerineau et al, Mol. Gen.
  • transcription termination regions include, but are not limited to, the polyA 35S of cauliflower mosaic virus (Franck et al., Cell, 1980, 21: 285-294) and the histone gene terminator (EP 0 633 317).
  • sequences that can be present in an expression construct according to the invention are sequences that increase the genetic expression such as introns, enhancer sequences and leader sequences.
  • Introns that are known to increase genetic expression in plants are, for example, introns of the gene Adhl of corn, introns of the gene bronzel of corn (J. Callis et al., Genes Develop., 1987, 1 : 1183-1200), intron DSV of tobacco yellow mosaic (Morris et al., Virology, 1992, 187: 633) and intron of actin-1 of rice (McElroy et al., Plant Cell, 1990, 2: 163-171).
  • Suitable enhancer sequences include, but are not limited to, transcription activator of tobacco mosaic virus TEV (Carrington et al., J. Virol., 1990, 64: 1590-1597).
  • Non-translated leader sequences that are known to increase gene expression in plants are, for example, leader sequences of tobacco mosaic virus (TMV), of maize chlorotic mottle virus (MCMV), and of alfalfa mosaic virus (A1MV) (Gallie et al., Nucl. Acids Res., 1987, 15: 8693-8711; Skuzeski et al., Plant Mol. Biol., 1990, 15: 65-79).
  • leader sequences include, but are not limited to, the EMCV leader (Encephalomyocarditis 5'noncoding region; Elroy-Stein et al., PNAS USA, 1989, 86: 6126-3130), the leader of human BiP-protein (Macejack et al., Nature, 1991, 353: 90-94), and the leader AMV RNA 4 from the Alfalfa mosaic virus protein (Jobling et al., Nature, 1987, 325: 622-625).
  • the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc may be modified to include codons that are optimized for expression in a transformed plant (Campbell et al., Plant Physiol., 1990, 92: 1-11; Muray et al., Nucleic Acids Res., 1989, 17: 477-498; Wada et al., Nucl. Acids Res., 1990, 19: 2367; and U.S. Pat. Nos. 5,096,825; 5,380,831; 5,436,391; 5,625,136, 5,670,356 and 5,874,304).
  • the sequences of such modified codons are generally synthetic sequences.
  • an expression construct according to the present invention further comprises one or more marker genes.
  • Marker genes are genes that confer a distinct phenotype to cells expressing said marker gene, which distinguishes cells that have been transformed from cells that have not been transformed. These marker genes encode a selection marker.
  • a distinct phenotype may be used to identify plant cells, group of plant cells, plant tissues, plant organs, parts of plants or whole plants that contain in their genome an expression construct. Numerous examples of marker genes are known in the art. Some markers confer an additional advantage to the transgenic plant, such as for example resistance to a herbicide, to diseases, to bioagressors or to environmental stress.
  • markers that confer a resistance to herbicides and that can be used in the practice of the present invention include, but are not limited to, the gene bar of Streptomyces hygroscopicus which encodes phosphinothricin acetylase (PAT) providing a resistance to glufosinate, mutant genes that confer a resistance to imidazalinone or to sulfonylurea such as the genes encoding the mutant form of the ALS and AHAS enzymes (Lee et al, EMBO J., 1988, 7: 1241; Miki et al, Theor. Appl. Genet., 1990, 80: 449; and U.S. Pat. No.
  • PAT phosphinothricin acetylase
  • genes that confer a resistance to glycophosphate such as the mutant forms of EPSP synthase and aroA, a resistance to L-phophinothricine such as the genes of glytamine synthase, a resistance to kanamycin such as the nptl and nptll genes of omycin phosphotransferase, or a resistance to phenoxypropionic acids and to cyclohexones such as the genes encoding the ACCAse inhibitor (Marshall et al, Theor. Appl. Genet., 1992, 83: 435).
  • Marker genes that confer a resistance to diseases or to bioagressors and which may be used in the practice of the present invention include, but are not limited to, genes encoding a protein of Bacillus thuringiensis such as delta-endotoxin (U.S. Pat. No. 6,100,456); genes encoding proteins that bind to vitamins such as avidin and homologs thereof that are used as larvicides against insects; genes encoding protease inhibitors or amylase inhibitors such as rice cystein proteinase (Abe et al., J. Biol. Chem., 1987, 262: 16793) and tobacco proteinase inhibitor I (Hubb et al, Plant Mol.
  • genes encoding hormones specific of insects or of pheromones such as ecdysteroid hormone or juvenile hormone and equivalents thereof; genes encoding peptides or neuropeptides that are specific of insects and whose expression disturb said insects' physiology; genes encoding venom specific of insects, genes encoding enzymes responsible for the accumulation of monoterpenes, sesquiterpenes, hydroxamic acid, phenylpropanoid derivative or other non-proteinic molecules that exhibit an insecticidal activity, genes encoding enzymes involved in the modification of the biological activity of a molecule (U.S. Pat. No.
  • genes encoding hydrophobic peptides such as Tachyplesin derivatives that inhibit fungal pathogens; genes encoding a viral invasive protein or a toxin derivative (Beachy et al, Ann. Rev. Phytopathol., 1990, 28: 451); and genes encoding an antibody or antitoxin specific of insects or an antibody specific of a virus (Tavladoraki et al, Nature, 1993, 366: 469).
  • Marker genes that confer a resistance to environmental stress and that may be used in the practice of the present invention include, but are not limited to, mtld and HVAI ; rd29A et rdl9B, which are genes of Arabidopsis thaliana encoding hydrophilic proteins that are induced in response to dehydration, low temperatures, stress due to salinity, or exposure to abscisic acid (Yamaguchi-Shinozaki et al, Plant Cell, 1994, 6: 251-26). Other examples of such genes are described in U.S. Pat. Nos. 5,296,462 and 6,356,816.
  • a marker gene may cause, in plant cells transformed or in plants transformed, a visible response ⁇ e.g., a distinctive appearance, such as a different color or different growth compared to plant cells or plants that do not express the marker gene).
  • These marker genes encode a reporter. It is known in the art that transcription activators of the biosynthesis of anthocyanine operably linked to a suitable promoter in an expression construct is of great utility as non-phyto toxic marker for the transformation of plant cells.
  • the location of a protein may be altered by modifying the nucleic acid sequence encoding the protein by addition of a region encoding a signal peptide.
  • Methods for adding such regions have been described (Dai et al, Trans. Res., 2005, 14: 627; Keegstra et al, Physiol. Plant., 1995, 93: 157-162; Zoubenko et al, Nucleic Acids Res., 1994, 22: 3819-3824; Jones et al, Plant. Physiol., 1993, 101: 595-606; Nhakamura et al, Plant.
  • An expression construct according to the present invention may also further comprise any other nucleic acid sequence which, following transcription, confers an additional desirable property to the transformed plant obtained.
  • Such desirable properties include, but are not limited to, the ability to grow under different climate conditions and/or in different soils; incorporation of bio-confinement characteristics such as for example sterile flowers (for males only or for both males and females); incorporation of phytoremediation characteristics, and increased biomass.
  • an expression construct of the present invention is inserted into a suitable vector.
  • vector refers to a circular or linear, DNA or RNA molecule that is indifferently under a single strain or double strain form.
  • a recombinant vector according to the present invention is preferably an expression vector or more specifically an insertion vector, a transformation vector or an integration vector.
  • a vector may be of bacterial or viral origin.
  • the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc is placed under the control of one or more sequences comprising regulatory signals that regulate the expression of the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc in a given plant, as mentioned above.
  • these regulatory signals are contained in the expression construct that is inserted in the vector.
  • one or more regulatory signals are contained in the expression construct and one or more other regulatory signals are contained in the vector.
  • all the regulatory signals are contained in the vector.
  • a recombinant vector according to the present invention may preferably comprise suitable transcription initiation and termination sequences. Furthermore, a recombinant vector according to the present invention may comprise one or more origins of replication sequences that are functional in plants in which their expression is desired, as well as optionally selection marker sequence(s). Recombinant vectors according to the present invention may include one or more regulatory signals as defined above. In certain embodiments, a recombinant vector according to the present invention is an integration vector that allows the insertion of multiple functional copies of the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc in the plant genome.
  • a vector according to the present invention is selected among those vectors specifically suitable for the expression of sequences of interest in plant cells, such as for example the cambia 1302 vector (Hajdukiiewicz et al., Plant Mol. Biol., 1994, 25: 989-994) and the vectors commercialized by Clontech; the pBIN19 vector (Bevan et al., Nucleic Acids Res., 1984, 12: 8711-8721), the pBI 101 vector (Jefferson, Plant Mol. Biol. Report., 1987, 5: 387-405), the pBI 121 vector (Jefferson, Plant Mol. Biol.
  • plasmid refers to an autonomous circular DNA molecule that is capable of replication in a cell. If a microorganism or a recombinant cell culture is described as host of an expression plasmid, said plasmid comprises both extrachromosomic circular DNA and DNA having integrated host chromosome(s). If the plasmid is maintained in a host cell, the plasmid is either stably replicated during mitosis as an autonomous structure, or is integrated into the host's genome.
  • Plasmids that may be used in the practice of the present invention include, but are not limited to, the Ti plasmids of Agwbacterium tumefaciens (Darnell, Lodish, Baltimore, "Molecular Cell Biology", 2 nd Ed., 1990, Scientific American Books: New York), a plasmid comprising a ⁇ -glucuronidase gene and a Cauliflower mosaic virus (CaMV) promoter with a leader sequence from the Alfalfa Mosaic virus (Sanford et al., Plant Mol.
  • CaMV Cauliflower mosaic virus
  • the plasmid may comprise an origin of replication that allows replication in Agwbacterium and a high number of origins of replication that are functional in E. Coli. This allows for the easy production and testing of transgenes in E. Coli before transfer to Agwbacterium for subsequent introduction into plants.
  • an expression construct or expression vector according to the invention may comprise a nucleic acid sequence encoding only one of the transcription factors GhERF-IIa, GhERF-IIb and GhERF-IIc, or may comprise nucleic acid sequence(s) encoding two of these factors or all of the these factors;
  • this construct or vector may be prepared using any of a variety of suitable methods, the method used for preparing the expression construct or vector being a non-critical or limiting element of the invention.
  • transgenic plant refers to a plant that has been obtained using techniques involving genetic manipulations. More specifically, a transgenic plant is a plant with ⁇ i.e. containing) at least one cell comprising heterologous nucleic acid sequences that were introduced by the hand of man. Typically, transgenic plants express DNA sequences which confer to these plants one or more characters that are different from those of non-transgenic plants of the same species.
  • the present invention generally provides a method for obtaining a transgenic plant that overproduces or accumulates jasmonic acid and/or OPDA, said method comprising transforming a plant using an expression construct comprising a nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc or a combination thereof, or an expression vector comprising said expression construct.
  • the terms "overproduction of jasmonic acid”, “accumulation of jasmonic acid” and related terms are used herein interchangeably. They refer to a production or accumulation of jasmonic acid in the transformed plant that is higher than the production or accumulation of jasmonic acid in a non-transformed plant of the same species and at the same development stage.
  • the production of jasmonic acid in the transformed plant is at least 2 times higher than that in the non-transformed plant, preferably at least 5 times higher, at least 10 times higher, at least 25 times higher, at least 50 times higher, at least 75 times higher, at least 100 times higher, or more than 100 times higher than the production or accumulation of jasmonic acid in the non-transformed plant.
  • overproduction of OPDA “accumulation of OPDA” and related terms are used herein interchangeably. They refer to a production or accumulation of OPDA in the transformed plant that is higher than the production or accumulation of OPDA in a non-transformed plant of the same species and at the same development stage.
  • the production of OPDA is at least 2 times higher than that in the non-transformed plant, preferably at least 5 times higher, at least 10 times higher, at least 15 times higher, at least 25 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 75 times higher, at least 100 times higher, or more than 100 times higher than the production or accumulation of OPDA in the non-transformed plant.
  • Transformation of a plant using an expression construct or expression vector according to the invention may be performed using any suitable method, since the transformation method used is not critical to the present invention.
  • suitable methods include, but are not limited to, non-biological methods (e.g., micro-injection, microprojectile bombardment, electroporation, infiltration under vacuum, or direct precipitation) and biological methods (e.g., infection with a transformed bacterial strain such as an Agwbacterium strain).
  • non-biological methods e.g., micro-injection, microprojectile bombardment, electroporation, infiltration under vacuum, or direct precipitation
  • biological methods e.g., infection with a transformed bacterial strain such as an Agwbacterium strain.
  • any combination of these methods that allows for an efficient transformation of plant cells or of plants may be used in the practice of the present invention.
  • a method for obtaining a transgenic plant that overproduces or accumulates jasmonic acid and/or OPDA comprises steps of: (a) transforming a plant cell with an expression construct or expression vector comprising a nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc (or a combination thereof) in order to obtain a plant cell stably transformed; and (b) culturing the plant stably transformed in order to regenerate a whole plant comprising, integrated in its genome, a nucleic acid sequence allowing the expression of GhERF-IIa, GhERF-IIb or GhERF-IIc (or a combination thereof) in the plant.
  • the culturing step may comprise regenerating several plants and selecting, among the regenerated plants, those plants that comprise, integrated in their genome, a nucleic acid allowing the expression of GhERF- Ila, GhERF-IIb or GhERF-IIc (or a combination thereof) in the plant.
  • a method for obtaining a transgenic plant that overproduces or accumulates jasmonic acid and/or OPDA comprises: (a) transforming an Agrobacterium tumefaciens or Agrobacterium rhizogenes host cell in order to obtain a recombinant host cell; and (b) transforming a plant or plant cell via infection with the recombinant host cell in order to obtain a whole plant comprising, integrated in its genome, a nucleic acid sequence allowing the expression of GhERF-IIa, GhERF-IIb or GhERF-IIc (or a combination thereof) in the plant.
  • the first transformation step may comprise: infecting several plants or several plant cells with recombinant host cells; optionally culturing several infected plant cells in order to regenerate several plants; and selecting, among the infected plants or among the regenerated plants, those plants that comprise, integrated in their genome, a nucleic acid allowing the expression of GhERF- IIa, GhERF-IIb or GhERF-IIc (or a combination thereof) in the plant.
  • these methods may further comprise the following additional steps: (c) crossing two transformed plants in order to obtain crossed plants; and (d) selecting, among the crossed plants obtained, those plants that are homozygous for the transgene.
  • these methods may further comprise the following additional steps: (c) crossing a transformed plant with a plant of the same species in order to obtain hybrid plants; and (d) selecting, among the hybrid plants obtained, those plants that have conserved the transgene. Transformation of Plant Cells and Host Cells
  • plant cell include protoplasts (plant cells without walls), plant germ cells or somatic cells, and more generally any cell or cell group capable of regenerating a whole plant.
  • a seed which comprises multiple plant cells and which can regenerate a whole plant is encompassed within the term "plant cell”.
  • a cell plant used in a method of the present invention may be isolated from the plant from which it originates ⁇ e.g., cell line) or from the culture of a plant tissue or organ. Plant cells used in a method of the present invention may originate from any plant of any species (see below).
  • Plant cells may be obtained from a large number of different sources such as the American Type Culture Collection (Rockland, MD) or from other commercial sources of seeds such as for example A. Atlee Burpee Seed Co. (Warminster, PA), Park Seed Co. (Greenwood, SC), Johnny Seed Co. (Albion, ME), or Northrup King Seeds (Hartsville, SC), Vilmorin, France, Thompson & Morgan, Graines Baumaux: Clause vegetable seeds.
  • A. Atlee Burpee Seed Co. Warminster, PA
  • Park Seed Co. Greenwood, SC
  • Johnny Seed Co. Albion, ME
  • Northrup King Seeds Hardrup King Seeds
  • Vilmorin France
  • Thompson & Morgan, Graines Baumaux Clause vegetable seeds.
  • Transformation of plant cells may be performed using any method known to those skilled in the art. Methods for introducing expression constructs in plant cells have been described. See, for example, “Methods for Plant Molecular Biology”, Weissbach and Weissbach (Eds.), 1989, Academic Press, Inc; “Plant Cell, Tissue and Organ Culture: Fundamental Methods", 1995, Springer- Verlag: Berlin, Germany; and U.S. Pat. Nos.
  • electroporation has often been used to transform plant cells (U.S. Pat. No. 5,384,253).
  • This method is generally carried out on friable tissues (such as, for example, a suspension of cells or an embryogenic callus) or embryo cells or other organized tissues that have been rendered more susceptible to electroporation by exposition to enzymes that degrade pectin or via mechanical treatment.
  • friable tissues such as, for example, a suspension of cells or an embryogenic callus
  • embryo cells or other organized tissues that have been rendered more susceptible to electroporation by exposition to enzymes that degrade pectin or via mechanical treatment.
  • intact cells of corn, wheat, tomato, soybean and tobacco have been transformed by electroporation (D'Halluin et al., Plant cell, 1992, 4: 1495-1505; Rhodes et al., Methods Mol. Biol. 1995, 55: 121-131; and U.S. Pat. No. 5,384,253).
  • Electroporation can also be used to transform protoplasts
  • Particle bombardment techniques may be used to transform almost any species of monocotyledon or dicotyledon plant (U.S. Pat. Nos. 5,036,006; 5,302,523; 5,322,783 and 5,563,055, WO 95/06128; Ritala et al., Plant Mol. Biol. 1994, 24: 317-325; Hengens et al., Plant Mol. Biol. 1993, 23: 643-669; Hengens et al., Plant Mol. Biol. 1993, 22: 1101- 1127; Buising et al., Mol. Gen. Genet. 1994, 243: 71-81; Singsit et al., Transgenic Res. 1997, 6: 169-176).
  • Transformation of plant protoplasts may be carried out using methods such as precipitation by calcium phosphate, treatment with polyethylene glycol, electroporation or any combination thereof (Potrykus et al., Mol. Gen. Genet. 1985, 199: 169-177; Fromm et al., Nature, 1986, 31: 791-793; Callis et al., Genes Dev. 1987, 1: 1183-1200; OmiruUeh et al., Plant Mol. Biol. 1993, 21: 415-428).
  • Other methods for transforming plant cells that are known or have been described in the art (for example Rakoczy-Trojanowska, Cell Mol. Biol. Lett. 2002, 7: 849-858) may alternatively or additionally be used in the practice of the present invention.
  • Coli then the plasmid comprising nucleic sequences of interest is transferred by conjugation or electroporation in a bacterial strain of Agwbacterium (generally Agwbacterium tumefaciens or Agwbacterium rhizogenes), and the recombinant Agwbacterium cells obtained are used to infect plants or plant cells.
  • a bacterial strain of Agwbacterium generally Agwbacterium tumefaciens or Agwbacterium rhizogenes
  • plant cells that can be transformed using this method are typically callus cells, embryo cells, meristematic cells, or cell cultures in suspension.
  • plant cells are stably transformed.
  • stably transformed refers to a cell, a callus or a protoplast in which an exogenous nucleic acid molecule that has been introduced by a method of transformation is capable of replication.
  • the stability of the transformation is demonstrated by the ability of the transformed cell to establish cell lines or clones comprising a population of daughter cells that also comprise the exogenous nucleic acid molecule.
  • the success of the transformation of a plant cell may preliminarily be evaluated visually when the expression construct or expression vector used comprises a marker gene (as described herein).
  • plant cells which comprise the nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc (or any combination thereof) and which express GhERF-IIa, GhERF-IIb or GhERF-IIc (or any combination thereof) may be identified and selected using any of a variety of suitable procedures such as via DNA- DNA or DNA-RNA hybridizations, protein or immunologic assays known to detect and quantify nucleic acids and proteins.
  • Plant cells including protoplasts, calluses, etc ..
  • stably transformed by a method of the present invention are also encompassed within the scope of the invention. Regeneration of Plants
  • plant cells stably transformed may be cultured to obtain transgenic plants using any standard method known in the art (see, for example, McCormick et ah, Plant Cell Reports, 1986, 5: 81-84). Regeneration of plants from protoplasts has also been described, for example by Evans et al., "Handbook of Plant Cell Cultures", Vol. 1, 1983, MacMilan Publishing Co: New York; and I.R.
  • the term “regeneration” refers to a process whereby a plant is grown from a plant cell.
  • Means of regeneration may vary from one plant species to another. However, generally, a suspension of transformed plant cells or of transformed explants contained in a Petri dish is used. A plant callus is formed, from which appear sprouts and then roots. Alternatively, a technique of somatic embryogenesis may be used. Using this technique, it is possible from a single seed or a single callus to obtain an unlimited number of copies of this seed or callus, wherein each of the copies is morphologically and genetically identical to the starting seed or callus.
  • Primary transgenic plants may be cultivated using conventional methods. Numerous techniques for plant cultivation are known in the art. Thus, the plants of the present invention may be cultivated in the soil, or alternatively may be grown via hydroponic cultivation (i.e., in the absence of soil - see, for example, U.S. Pat. Nos. 5,364,451; 5,393,426; and 5,785,735).
  • Selection of plants that have been transformed may be carried out using any suitable method, for example Northern Blot, Southern Blot, detection of resistance to a herbicide or to an antibiotic agent, or any combination of these methods or other methods known to those skilled in the art.
  • the techniques of Southern Blot and Northern Blot which respectively test the presence, (in a plant tissue), of a nucleic acid sequence of interest (e.g., sequence encoding GhERF-IIa, GhERF-IIb, GhERF-IIc or any combination thereof) and of the corresponding RNA, are standard methods known in the art (see, for example, Sambrook & Russell, "Molecular Cloning", 2001, Cold Spring Harbor Laboratory Press: Cold Spring Harbor).
  • the selection may be carried out based on the detection of an overproduction of jasmonic acid and/or OPDA (see Examples).
  • Primary transformed (and optionally selected) transgenic plants may be crossed among themselves, or crossed with plants of the same species.
  • the plants that exhibit desired phenotypic characteristics may be selected among the crossed plants or hybrid plants obtained.
  • Several generations of plants may be generated in order to ensure that the desired phenotypic characteristics are indeed inherited and stably maintained, and seeds of these plants may then be harvested.
  • a plant transformed according to the present invention may be crossed with a plant of the same species but which is considered to be of high agronomic value.
  • Hybrid plants obtained that have conserved the transgene may then be submitted to another crossing procedure with the plant of high agronomic value in order to obtain plants that have conserved the transgene and that possess a genetic background that is close or identical to the genetic background of the plant of high agronomic value.
  • the present invention also provides transgenic plants obtained by a method described herein, i.e. transgenic plants comprising, integrated in their genome, an exogenous nucleic acid sequence allowing the expression of GhERF-IIa, GhERF-IIb or GhERF-IIc (or any combination thereof).
  • transgenic plants of the present invention are characterized in that the expression of GhERF-IIa, GhERF-IIb or GhERF- IIc (or any combination thereof) induces an overexpression or accumulation of jasmonic acid and/or OPDA in the plants.
  • an overproduction or accumulation of jasmonic acid in a transgenic plant corresponds to a production or accumulation of jasmonic acid in the transformed plant which is higher than the production or accumulation of jasmonic acid observed in a plant of the same species and at the same development stage but that has not been transformed.
  • the overproduction of jasmonic acid in the transformed plant is at least 2 times higher, at least 5 times higher, at least 10 times higher, at least 25 times higher, at least 50 times higher, at least 75 times higher, at least 100 times higher or more than 100 times higher than the production or accumulation of jasmonic acid in the non-transformed plant.
  • an overproduction or accumulation of OPDA in a transgenic plant corresponds to a production or accumulation of OPDA in the transformed plant which is higher than the production or accumulation of OPDA observed in a plant of the same species and at the same development stage but that has not been transformed.
  • the overproduction or accumulation of OPDA in the transformed plant is at least 2 times higher, at least 5 times higher, at least 10 times higher, at least 20 times higher, at least 25 times higher, at least 30 times higher, at least 40 times higher, at least 50 times higher, at least 75 times higher, at least 100 times higher, or more than 100 times higher than the production or accumulation of OPDA in the non-transformed plant.
  • the overexpression of GhERF-IIc in a transgenic plant according to the invention results in an overproduction of jasmonic acid and of OPDA, such that the jasmonic acid content in the plant is higher than the OPDA content in said plant.
  • the overexpression of GhERF-IIa or GhERF-IIb in a transgenic plant according to the invention results in an overproduction of jasmonic acid and of OPDA, such that the OPDA content in the transgenic plant is higher that the jasmonic acid content in said plant.
  • the term "higher” refers to a content in one jasmonate that is at least 1.1 times higher than the content in the other jasmonate, preferably at least 1.25 times higher, more preferably at least 1.5 times higher and even more preferably at least 1.75 times higher, for example 2 times, 2.25 times, 2.5 times, 2.75 times, 3 times or more than 3 times higher.
  • the transgenic plants of the present invention may belong to any plant genus or plant species for which a transformation via introduction of an expression construct or expression vector comprising a nucleic acid sequence encoding GhERF-IIa, GhERF-IIb or GhERF-IIc (or any combination thereof) results in overproduction or accumulation of jasmonic acid and/or OPDA.
  • transgenic plants of the present invention may be plants of large cultures, vegetables, flowers or trees.
  • Transgenic plants of the present invention may be dicotyledons, such as Malvaceae (e.g. , Cotton, etc .), Solanaceae (e.g., tobacco, tomato, potato, eggplant, etc .), Cucurbitaceae (e.g., melon, cucumber, watermelon, squaches, etc .), Brassicaceae (e.g. , colza, mustard, etc .), Asteraceae (e.g., cichorium, etc .), Apiaceae (e.g.
  • Rosaceae in particular trees and arbusts whose fruits are economically valuable
  • monocotyledons such as for example in particular cereals (e.g. , wheat, barley, oat, rice, corn, etc .) or liliaceae (e.g., onion, garlic, etc .).
  • a transgenic plant of the present invention belongs to the Malvaceae family (e.g. , cotton, cocoa, okra, etc .), to the Solanaceae family (e.g., tobacco, tomato, potato, eggplant, etc .), to the Rubiaceae family (e.g., coffee, etc .), to the Poaceae or Gramineae family (e.g. , rice, corn, wheat, barley, oat, rye, mil, sugarcane, etc...) or to the Vitaceae family (e.g., vine, etc.).
  • Malvaceae family e.g. , cotton, cocoa, okra, etc .
  • Solanaceae family e.g., tobacco, tomato, potato, eggplant, etc .
  • Rubiaceae family e.g., coffee, etc .
  • the Poaceae or Gramineae family e.g. , rice, corn, wheat, barley, oat, rye
  • a transgenic plant of the present invention belongs to the Gossypium or Cotoneaster genera (cotton), to the Nicotiana genus (tobacco), to the Oryza genus (rice), to the Solanum genus (tomato), to the Cojfea genus (coffee), or to the Vitis genus (vine).
  • the invention encompasses whole transgenic plants, their progeny (or descendants) including cross-progeny, as well as any vegetal material obtained or extracted from these plants.
  • vegetal material includes plant cells, plant organs, protoplasts, plant calluses, cultures of plant cells or other plant cells organized as functional and/or structural units, plant seeds, leaves, stems, roots, flowers, fruits, tubers, pollen, plant cuttings and the like.
  • the methods according to the present invention may be used to generate plants exhibiting an improved resistance to pathogenic agents.
  • improved resistance to pathogenic agent refers to the resistance of a transformed plant ⁇ i.e. the ability to defend itself) against at least one pathogenic agent, wherein the resistance is higher than the resistance exhibited by a non-transformed plant of the same species.
  • a pathogenic agent may be any of a variety of microorganisms (bacteria, fungi, mycoplasma, viruses), insects and other bioagressor capable of causing a disease in a plant. Production of Secondary Metabolites
  • a lot of plant secondary metabolites have an economic value as pharmaceutical products ⁇ e.g., taxol, digoxine, colchicine, codeine, morphine, quinine, quinidine, shikonine, ajmaline, ajmalicine, vinblastine, vincristine, reserpin, rescinnamine, camptothecine, ellipticine, nicotine, etc .), colorants or food flavors ⁇ e.g., anthocyanins, vanillin, etc .), and fragrances.
  • the industrial use of these secondary metabolites is limited due to the fact that the plants only produce low quantities of these metabolites.
  • jasmonates The biosynthesis of numerous classes of secondary metabolites is stimulated by jasmonates and some of their precursors (Memelink, Curr. Opin. Plant Biol., 2005, 8: 230-235).
  • jasmonic acid induces the expression of genes involved in the biosynthesis of secondary metabolites (Menke et ah, EMBO J., 1999, 18: 4455-4463).
  • a non-exhaustive list of classes of secondary metabolites whose biosynthesis is induced by jasmonates includes taxoids, phenylpropanoids, flavanoids, anthocyanins, guaianolides, anthraquinones, sesquiterpenoids, and several types of alkaloids such as terpenoid indole alkaloids. Therefore, the transgenic plants according to the present invention, which overproduce jasmonic acid and/or OPDA, can be used for the production of secondary metabolites. Similarly, the methods according to the present invention can be used to produce plants that overproduce at least one secondary metabolite whose biosynthesis is induced by jasmonates.
  • Any plant known to produce a secondary metabolite whose synthesis is induced by jasmonates, and in particular by jasmonic acid and/or OPDA, can be transformed using a method of the present invention with the goal of obtaining a transgenic plant which overproduces said secondary metabolite.
  • plants known to produce such secondary metabolites include, but are not limited to, Madagascar Periwinkle ⁇ Catharanthus roseus) whose leaves synthesize vinblastine and vincristine - compounds that are used in the treatment of cancer - and whose roots synthesize ajmalicine, which is used to improve cerebral blood flow; opium poppy (Papaver somniferum), which produces latex comprising narcotic alkaloids such as morphine and codeine; Rauwolfia serpentine, which produces several bioactive compounds such as reserpine which is used as a hypotensive agent; plants from the Cinchona genus which produce quinine used in the treatment of malaria and quinidine, an antiarrythmic agent; henbanes such as the White Henbane ⁇ Hyoscyamus albus L.) and the Black Henbane ⁇ Hyoscyamus niger L.) or plants of the Datura genus which comprise several alkaloids such as atrop
  • Example 1 Induction of Transcription Factors GhERF-IIa and GhERF-IIc
  • Cotton cultivar Gossypium hirsutum cv. Reba B50 which carries the B2B3 blight- resistance genes, was used in the present study. This cultivar is resistant to race 18 of the bacterium Xanthomonas campestris pv. Malvacearum (Xcml8), but develops disease symptoms in response to race 20 of the same bacterium (Xcm20) (Innes et ah, Biol. Rev., 1983, 58: 157el76; Hillocks et al, Cotton Diseases, 1992, Redwood Press CAB, International Melksham).
  • Bacteria Xcml8 and XcmlO were maintained at 28°C on LPG agar (0.5%w/v yeast extract, 0.5% w/v bacteriological peptone, 0.5% w/v glucose as a carbon source, solidified with 1.5% w/v agar; Difco, Detroit, MI) in distilled water. Prior to inoculation, the bacteria were grown in 20 ml LPG medium in a shaking incubator at 150 rpm/minute. After about 18 hours under these conditions, the cultures were centrifuged at 4 000 min "1 for 20 minutes at 4°C and washed twice with tap water by centrifugation at 4 000 min "1 for 10 minutes at 4°C to remove nutrients and exopolysaccharides. Then, the bacterial pellets thus obtained were resuspended in tap water and adjusted to 10 8 cfu ml - " 1 , (corresponding to an absorbance of 0.2 at 600 nm).
  • the plants were grown in a greenhouse with a natural light/dark cycle at 29°C/24°C and a relative humidity averaging 80% - conditions that are optimal for a cotton/Xcm interaction.
  • the bacterial suspensions (10 8 cfu ml - " 1 ), or sterile water used as control, were injected in intercellular domains of 10-day old half cotton cotyledons using a needle-less syringe.
  • the three sequences were cloned from cDNA of Gossypium hirsutum cv. Reba B50, and called GhERF-IIa (SEQ ID NO: 4), GhERF-IIb (SEQ ID NO: 5) and GhERF-IIc (SEQ ID NO: 6).
  • the protein sequences deduced from these genomic sequences (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively) share with the sequence of ORA47 (SEQ ID NO: 7) a common DNA-binding domain, called AP2-domain, and two domains: CMII-1 and CMII-3, that are characteristic of the Group II of the ERF family (see Figure 1).
  • the control treatment water does not modify, or only slightly modifies, the expression of the GhERF-IIa gene.
  • GhERF-IIa GhERF-IIc was found to be specifically induced in response to the race Xcml8, but much more so than GhERF-IIa compared to TO.
  • Cotton protoplasts were transformed with polyethylene glycol using a method known in the art (Schirawski et al., J. Virol. Methods, 2000, 86: 85-94) and 3 constructs in a ratio of 2:2:6
  • the protoplasts of cotton cotyledons were produced and transformed using a protocol adapted from the method described by Sheen et al. (Sheen J, 2002, A transient expression assay using Arabidopsis mesophyll protoplasts. http//genetics.mgh.harvard.edu/sheeweb/).
  • the protoplasts were collected 18 hours after transformation and frozen in liquid nitrogen. GUS and LUC activity tests were performed as previously described (Zarei et al., Plant Mol.
  • Protoplasts of leaves of Arabidopsis were prepared and transformed as previously described (Sheen et al., 2002 A transient expression assay using Arabidopsis mesophyll protoplasts. http//genetics.mgh.harvard.edu/sheeweb/). The protoplasts were co- transformed with 10 ⁇ g of GFP-fusion and the NtKISla-DsRFP DNA plasmid (Jasinski et al., Plant Physiol., 2002, 130: 1871-1882) and incubated in the absence of light for at least 16 hours.
  • the nuclear localization was studied via expression, in the protoplasts, of GhERF-IIa, GhERF-IIb and GhERF-IIc fused to the C-terminal portion of the fluorescent green protein 6 (GFP6) and by fluorescence microscopy.
  • the biosynthesis of jasmonic acid occurs via a metabolic route of octadecanoids (Turner et ah, Plant Cell, 2002, 14 (suppl.): S153-S164), which comprises several well- characterized enzymatic steps.
  • One of these steps includes the action of a plastid enzyme, the allene oxide cyclase (AOC).
  • Zarei et al. Plant Mol. Biol., 2011, 75: 321-331 have shown that AtOR47 binds to the promoter OAC2 in vitro.
  • GhERF-IIa, GhERF-IIb and GhERF-IIc are capable of activating the expression of the AOC2 gene by binding to the gene promoter.
  • the ability of these transcription factors to transactivate the promoter OAC2 was tested in a transitory assay.
  • the constructs used in experiments on protoplasts of cotton cotyledons are presented in Figure 3(A).
  • the 'effector' constructs consist of an expression vector comprising the promoter CAMV 35S (arrow) with or without the cDNA of GhERF-IIa, GhERF-IIb, GhERF-IIc or GhERF-IXa5.
  • the gene LUC (Firefly luciferase) was fused to the promoter CAMV 35S and was used as reference or control gene to correct for the differences in the efficacy of transformation and of protein extraction between samples.
  • GhERF-IIa, GhERF-IIb and GhERF-IIc activate the gene AOC2 2.5, 6.5 and 5 times, respectively.
  • GhERF-IXa5 and the empty vector do not have any effects on AOC2.
  • Figure 4 shows images obtained by microscopy of protoplasts of Arabidopsis transformed simultaneously by the nuclear marker NtKISla-DsRFP and an expression plasmid of GhERF-IIa-GFP6 of, GhERF-IIb-GFP6 or of GhERF-IIc-GFP6. A total overlap of the localizations was observed, indicating that the transcription factors, GhERF-IIa, GhERF-IIb and GhERF-IIc , are expressed in the nucleus of protoplasts.
  • the Agrobacterium tumefaciens strain LBA1119 has been used to transitorily transform a cotton plant.
  • the transformation system used is called "ternary". For numerous plant species, this system causes an increase in the frequency of T-DNA transfer (Van der fits et ah, Plant Mol. Biol., 2000, 43: 495-502). Briefly, after contact between the agrobacterium and wounded plant cells, phenolic compounds, oses and an acid pH created an environment that favored the induction of the genes vir.
  • the transcription factor VirG activated by phosphorylation can induce the expression of the vir genes which is necessary to the transfer of T-DNA.
  • the ternary system used a mutated form of the virG protein (virGN54D) which mimics the active form.
  • This system was introduced in the agrobacterium strain LBA1119.
  • This strain also possesses one of the four following binary vectors: pMDC32-GhERF-IIa, pMDC32- GhERF-IIb, pMDC32-GhERF-IIc and pMDC32-GFP.
  • the strains were spread and cultured for 48 hours at 29°C in LB medium comprising three antibiotics: Gentamicin (Gen), Rifampicin (Rif) and Kanamycin (Kan). Some colonies were taken and cultured in liquid (medium comprising 20 mL of LB, 20 of Gen, 20 of Rif and 20 of Kan). Then, the culture was agitated at 200 rpm for 18 hours at 29°C.
  • the level of agrobacteria was then measured via spectrophotometry at 600 nm.
  • optical density (OD) measured was found to be between 0.6 and 1 (without dilution), then a centrifugation was performed at 3000 g at 4°C for 20 minutes.
  • An infiltration medium (comprising MgS0 4 (10 mM), Acetosyringone (200 ⁇ ) and MES pH 5.5 (20mM)) was added to the pellet obtained by centrifugation in order to get an OD equal to 0.5.
  • Agroinfiltration was carried out on 10 days old cotyledons (cotton plants). Each cotyledon was inoculated using a needle-less syringe on the lower face. Infiltration was performed from bottom to top and between 2 main veins in order for the inoculum to spread over the whole surface of the demi-cotyledon. For each construct, 6 demi- cotyledons were agroinfiltrated. Two days (48 hours) later, the demi-cotyledons were collected and rapidly placed in liquid nitrogen. Storage was at -80°C. The experiment was repeated three times. No visible effects were observed on the cotyledons that were inoculated for 48 hours following transformation with the two constructions.
  • the methanol was then eliminated by evaporation under nitrogen (40°C for 1 to 1.5 hours).
  • the dried residues obtained were taken up with 5 mL of phosphate buffer (phosphate sodium 100 mM, pH 7.8, NaCl 5%).
  • An extraction was then performed on the aqueous extract using 2.5 mL of hexane, and the hexane-phase (upper phase) was then eliminated. This extraction was repeated three times.
  • the purified aqueous phase was then acidified at pH 1.4 using a solution of HC1 5N.
  • a second purification by extraction was performed using 2.5 mL of chloroform and the chloroform-phase (lower phase) was recovered using a Pasteur pipette in a different series of glass tubes. This purification step by extraction was repeated three times and the chloroform phases were combined. Then, the chloroform was eliminated by evaporation under nitrogen (40°C for about 10 to 30 minutes) and the tubes were stored at -80°C.
  • the vegetal extract was taken up with 100 of methanol, and 20 of the resulting solution were injected in the column.
  • the separation was performed using a gradient of formic acid 15 mM : methanol (0-2 min, 40/60 % (v/v); 2-14 min, de 40/60 % a 60/40%; with a flow of 0.25 ⁇ 7 ⁇ ).
  • the retention time of jasmonic acid and of its deuterated form was 9.3 minutes
  • the retention of OPDA and of its deuterated form was 16.50 minutes.
  • Jasmonic acid and OPDA were detected and quantified in negative ESI (Electron Spray Ionisation) mode.
  • the analysis conditions for mass spectrometry were as follows: vaporization temperature of 120°C, source temperature of 450°C, Capillary voltage 2.5 KV, Cone voltage 20 V.
  • the quantitation was carried out using the mode "selected ion monitoring" with ion 209 for jasmonic acid, ion 215 for deuterated jasmonic acid, ion 291 for OPDA, and ion 295 for deuterated OPDA, and using ratio of peak surfaces for jasmonic acid/deuterated jasmonic acid and OPDA/deuterated OPDA, wherein the peak surface for deuterated jasmonic acid represented 100 ng.
  • tRNA Extraction The quantitation was carried out using the mode "selected ion monitoring" with ion 209 for jasmonic acid, ion 215 for deuterated jasmonic acid, ion 291 for OPDA, and ion 295 for deuterated OPDA, and using ratio of peak surfaces for jas
  • qPCR Inverse transcription and quantitative real-time PCR analyses were performed using the samples that were prepared for the dosage of OPDA and jasmonic acid.
  • the qPCR method used was based on the detection and quantification of a fluorescent reporter whose emission was directly proportional to the quantity of amplicons generated during the reaction.
  • the qPCR reactions were controlled using the thermocycler MX 3500P (Stratagene, US).
  • the detection system used SYBR Green, which binds to double stranded DNA.
  • the primers used were designed using the software Beacon Designer (Premier Biosoft International, United States); their efficacy and optimal concentrations were checked.
  • the qPCR reaction was performed using the Mesa Green qPCR Master Mix Plus for SYBER Assay (Eurogentec, Belgium) with a total volume of 20 (4 ⁇ L ⁇ of cDNA diluted to the 10 th , 0.6 of each qPCR sense and antisense primers at 10 ⁇ , 10 ⁇ L ⁇ of Master Mix (Taq polymerase, nucleotides and Syber Green) and 4.8 ⁇ ⁇ of sterile water).
  • the program used during the qPCR reaction was as follows: a cycle at 50°C/2 min followed by a cycle at 95°C/10 min (denaturation phase), then 40 cycles at 95°C/15 s, 58°C/20 s, 72°C/40 s and a dissociation cycle at 95°C/1 min, 60°C/30 s, 95°C/30 s.
  • the quantitation of transcripts was performed using the software MXPro, and the values obtained were normalized to the calibrator (TO, time before infection) and to the normalizator (actin, GhACT2, Champion et ah, Mol. Plant Pathol, 2009, 10: 471-485).
  • GhAOS GhAOC2
  • GhACXla which encode Allene Oxyde Synthase, Allene Oxyde Cyclase 2 and Acyl-CoA Oxydase, respectively.
  • Figure 5(A)-(C) shows that the expression of the 3 genes associated to the synthesis of jasmonic acid are induced in response to the overexpression of each of the genes GhERF-IIa, GhERF-IIb, and GhERF-IIc.
  • the overexpression of the negative control GFP did not modify the expression of any of the genes GhAOC2, GhAOS and GhACXla.
  • GhLOXl The two genes, called GhLOXl and GhERF-Ixa2, are linked to the resistance of cotton plants to Xcm.
  • GhLOXl is inducible, on the one hand by Xcml8 and on the other hand by overexpression of AtORA47 (see international patent application number PCT/EP2011/060026 of the present Applicants).
  • GhERF-IXa2 is a gene whose expression is specifically regulated by Xcml8 in the cotton plant.
  • the accumulation levels of OPDA and jasmonic acid are multiplied by a factor of 4 and of 8, respectively in cotton plants transformed with the gene GhERF-IIa, by a factor of 32 and of 56, respectively in cotton plants transformed by the gene GhERF-IIb, and by a factor of 10 and of 60, respectively in cotton plants transformed by the gene GhERF-IIc.
  • the tobacco plants were cultivated in pots filled with nitrogen-containing potting soil in green houses under climate conditions of 24°C during the day, 22°C during the night, 50% relative humidity and a long-day lighting (16h/24h). Tobacco plants were 25 days old when they were genetically engineered.
  • the process used to transform the tobacco leaves by agro-infiltration was the same as that used for the cotton cotyledons (see Example 3). The only difference was that the concentration of agro-bacterium used for the transformation of tobacco leaves was 2.5 times lower than in the case of the transformation of cotton plants.
  • tARNt The method used is similar to that described in Example 2 with the exception that the genes Nubiquitine, NbAOX, NbODC, NbPMT and NbMPO were tested and amplified using primers published by Todd et al. (Plant J., 2010, 62: 589- 600), and the expression data were normalized to the expression of the gene Nubiquitine.
  • NbAOX, NbODC, NbPMT and NbMPO encode respectively the enzymes aspartate oxidase, ornithine decarboxylase, putrescine N-methyltransferase and methylputrescine oxidase, which are all implicated in nicotine biosynthesis.
  • Figure 7(A) shows the intracellular localization of the fusion protein GhERF-IIc-
  • GFP in tobacco This protein is localized in the nuclei of epidermal cells of tobacco leaves (similar to protoplasts produced from leaves of Arabidopsis). In contrast, GFP (alone) is localized in the cells cytoplasm.

Abstract

La présente invention concerne l'utilisation d'une séquence nucléique permettant la synthèse de Gh ERF-IIa, Gh ERF-IIb ou Gh ERF-IIc dans une plante afin d'induire, dans la plante, une surproduction ou une accumulation d'acide jasmonique et/ou OPDA. L'accumulation d'acide jasmonique et/ou OPDA confère, en particulier, à la plante transformée une résistance améliorée à des bio-agresseurs. Les séquences nucléiques, procédés de transformation et plantes transformées selon l'invention peuvent également être utilisés pour la production de métabolites secondaires pharmaceutiquement importants dont la synthèse est induite par des jasmonates.
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