FR2807756A1 - New plant polypeptide useful for improving plant resistance to pathogen attack and for identifying specific inducers comprises a polypeptide with phospholipase A2 activity - Google Patents

Abstract

Plant polypeptide (I) with phospholipase A2 (PLA2) activity that catalyzes a stage in the defensive reaction of plants, is new. Independent claims are also included for the following: (1) isolated polynucleotides (II) that encode (I); (2) chimeric gene (CG) comprising, a promoter, (II) and a terminator; (3) expression or transformation vector containing CG; (4) organism, preferably plant, transformed with the vector; (5) producing plants resistant to attack by pathogenic organisms; (6) transgenic plants transformed with the vector, its parts or seeds; (7) mono- or poly-clonal antibodies (Ab) directed against (I); (8) screening for compounds that stimulate synthesis of (I); (9) identified compounds; and (10) increasing plant resistance to pathogens by increasing the expression level of (I).

Description

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PLA2 polypeptides from plants involved in the defense response of plants, polynucleotides encoding these polypeptides and transformed plants containing them.

 The present invention relates to plant polypeptides having phospholipase A2 activity (hereinafter designated PLA2 polypeptides) and involved in the defense reaction of plants against attacks by pathogenic organisms. In particular, the PLA2 polypeptides of the present invention do not show sequence similarity with known PLA2 polypeptides, but with the major potato tuber protein, Patatin. The invention also relates to polynucleotides encoding the PLA2 polypeptides of the invention, as well as chimeric genes comprising said polynucleotides, vectors comprising these chimeric genes, and host organisms transformed with these vectors. Among the transformed host organisms, the invention relates more particularly to plants transformed with a vector of the invention. These plants contain a chimeric gene of the present invention which allows them to overexpress, in a constitutive or induced manner, a PLA2 polypeptide of the invention, which increases their resistance to attack by pathogenic organisms.

 Cultivated plants are attacked by many pathogens such as viruses, bacteria, and fungi, but also by pests such as insects and nematodes. These attacks weaken the plants and decrease the yields of their crop. There is therefore an important need to limit these attacks. Among the solutions implemented to date, pesticides occupy a major place. However, the development of techniques for the isolation of genes and the transformation of plants makes it possible to envisage other pathways. One of these pathways is to isolate a gene encoding a protein toxic to the pathogen or pest and introduce it into the genome of a plant so that it expresses this toxic protein in its tissues. Another way is to increase the natural defenses of plants. The latter route constitutes the technical problem of the present invention. The solution to this technical problem consists in isolating a gene coding for a protein involved in the natural defense system of plants, and in transforming a plant with this gene so that, by overexpressing said protein in its tissues, it acquires a amplified defense system giving it an increased level of resistance to attack by pathogens.

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 The higher plants have developed over the course of evolutionary systems of defense against pathogens. These are reactions induced following the aggression of the plant by a pathogenic agent. One of the most effective induced defense reactions is the hypersensitivity reaction (RH, In The hypersensitive response in plants to pathogens: arésistance phenomenon, Goodman RN and Novacky AJ, 1994, APS Press, St-Paul). During this reaction, the aggressive pathogen (virus, bacteria, fungus, nematode) remains confined in a limited area of the plant surrounding its initial point of penetration where very active defense mechanisms are induced (Hammond-Kozack and Jones , 1996, Plant Cell 8,1773-1791). Plants that have developed RH also generate a second phenomenon of partial and lasting resistance, acquired systemic resistance, which manifests itself in the uninfected parts of the plant. The early molecular events of RH (ion fluxes through the plasma membrane, formation of reactive oxygen species, protein phosphorylations ...) initiated after recognition of the aggressor by the plant, lead to the production of signals and various chemical messengers which have the property of inducing a vast repertoire of defense genes, resulting in a very effective state of resistance (Yang et al., 1997, Genes Dev. 11, 1621-1639; Fritig et al., 1998, Curr, Opin.

Immunol. 10.16-22). Among these signals produced by plants following the assault of a pathogenic agent, two are well known, salicylic acid and ethylene (Dong, 1998, Curr. Opin.

Plant Biol. 1, 316-323). Oxylipins constitute a third class of signals more recently demonstrated and controlling the induction of specific defense genes (Penninckx et al., 1996, Plant Cell 8,2309-2323; Thomma et al., 1998, Proc. Nat. Acad. Sci. USA 95, 15107-15111; Vijayan et al., 1998, Proc. Nat. Acad. Sci. USA 95,7209-7214). Oxylipins are synthesized from unsaturated fatty acids from cell membranes, and some of them (from the jasmonic acid family) have a structure similar to lipid mediators of inflammation in animals (eicosanoids) . However, although several enzymes of the plant oxylipin biosynthesis pathway have been studied (Mueller, 1997, Physiol. Plant 100,653-663; Blée, 1998, Prog. Lipid Res. 37,33-72), the initial enzymatic step which releases precursor fatty acids from cell membranes and controls the synthesis of oxylipins is not known in plants.

 The technical problem which the present invention proposes to solve consists in producing plants having increased capacities of resistance towards pathogens by overexpression in said plants of an enzyme involved in the defense reaction process. This technical problem involves isolating said enzyme and the gene encoding it, then transforming a plant with this gene. The best solution to this

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 technical problem consists in isolating more particularly an enzyme implied in an early and amplifying stage of the defense reaction of plants. The step that best meets these criteria is the step initiating the synthesis of lipid signals of the oxylipin type. Indeed, this step is triggered following the recognition of the pathogenic agent by the plant, and triggers in return, through the lipid signals synthesized, the activation of a large number of genes participating in the defense reaction it -even. This step of mobilizing membrane fatty acids is supposed to be catalyzed by a particular enzyme, phospholipase A2. Indeed, linolenic acid, a major precursor of most oxylipins, is present predominantly in the sn-2 position of plant phospholipids, which sn-2 position is specifically hydrolysed by phospholipases A2 (Figure 1A). On the other hand, in animals, it has been shown that the synthesis of lipids mediating the inflammatory reaction is correlated with the activity of certain forms of phospholipases A2 (Balboa et al., 1996, J. Biol. Chem. 271 , 32381-32384; Bingham and Austen, 1999, Proc. Assoc. Amer. Physic. 111 516-524). In plants, indirect evidence tends to show the involvement of phospholipases (A, C and D) in signaling during a defense reaction. In addition, activation of a phospholipase A2 has been shown in response to auxin (Paul et al., 1998, Plant J. 16,601-611) or injury (Narvâez-Vâsquez et al., 1999, Plant cell 11, 2249-2260), and soybean cells showed increased phospholipase A2 activity following treatment with bacterial and fungal elicitors (Chandra et al., 1996, Plant Physiol. 110,979-986). The state of the art, through these documents, therefore directs a person skilled in the art towards the involvement of a phospholipase A2 in a stage of the defense reaction of plants, but does not describe the isolation or the characterization of such an enzyme, or the isolation of the gene encoding that enzyme.

 It therefore appears, in the light of the state of the art, and in particular the possible involvement of an enzyme having a phospholipase A2 activity in the defense mechanisms of plants without, however, presupposing the level of involvement or the controlled step. by this enzyme, that the solution to the technical problem of the present invention lies in the isolation and characterization of a phospholipase A2 enzyme as well as of the gene encoding it, after demonstration of the effective involvement of this enzyme in the step initial synthesis of oxylipins. No plant phospholipase A2 enzyme involved in defense reactions had been isolated and characterized before this invention. The major difficulty in isolating the PLA2 polypeptides of the present invention resided in the fact that, surprisingly, their structure is different from that of the known phospholipase A2 enzymes (Dennis, 1997, Trends Biochem. Sci. 2,1- 2). Their particularity is to present

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 a strong similarity with the major protein of the potato tuber, Patatine. Therefore, a search for PLA2 polypeptides induced during RH or mRNA encoding such polypeptides on the basis of known sequences of phospholipase A2 enzymes or polynucleotide sequences encoding such enzymes proved unsuccessful. Consequently, the search and the isolation of the PLA2 polypeptides of the invention had to be carried out on the basis of a purification work.

Description of the figures: Figure 1: Examples of glycerolipid structures. In plants, the fatty acid in position sn-2 is mainly linolenic acid (CI 8: 3). B: Structure of 12-oxo-phytodienoic acid (APD). C: jasmonic acid (AJ).

Figure 2: phospholipase A2 activities (round symbols) and quantities of phytodienoic (triangles) and jasmonic acids (squares) in the tobacco leaves. Empty symbols: plants treated with water. Solid symbols: plants inoculated with tobacco mosaic virus. The vertical arrow indicates the time of onset of necrotic lesions.

Figure 3: induction of phospholipase A2 activity and 3 families of PR proteins (Pathogenesis-Related) in leaves defending against the tobacco mosaic virus. PI: protease inhibitor. Chit: chitinase.Gluc: B-l, 3-glucanase.

Figure 4: Isolation of a PLA2 Polypeptide from a soluble protein extract from leaves infected for 6 days with VMT. Solid line: absorbance at 280 nm. Triangles: PLA2 activity. The fractions that have been collected for the next step are topped with a hook.

(a): cation exchange chromatography on SP-Sepharose (b): cation exchange chromatography on CM-TSK under FPLC conditions (c): gel filtration on Superdex HR 75 under FPLC conditions (d): Analysis by polyacrylamide gel denaturing fractions from step (c) and containing PLA2 activity. The arrow indicates the 28 kDa protein that has been sequenced.

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 Figure 5: N-terminal sequences of the purified PLA2 polypeptide and potato patatin (Bevan et al., 1986, Nucleic Acids Res. 14, 4625-4638).

Figure 6: protein sequences of PLA2 polypeptides according to the invention (NtPatl, NtPat2 and NtPat3) with those of the specific gene of the tobacco petal NtTP12 (Drews, et al., 1992, Plant Cell 4,1383-1404) and that potato tuber patatin (Bevan et al., 1986, Nucleic Acids Res. 14,4625-4638). Amino acid residues stored in more than 3 sequences are outlined in black. Interruptions created in alignment are represented by a dash. The GXSXG motif typical of serine hydrolases is underlined.

FIG. 7: similarity / identity between the protein sequences NtPatl, NtPat2 NtPat3, NtTP12 (Drews, et al., 1992, Plant Cell 4,1383-1404) of tobacco and the potato-type sequences (Bevan et al ., 1986, Nucleic Acids Res. 14,4625-4638), arabidopsis (accession number AJ002596), cucumber (May et al., 1998, Biochim.

Biophys. Acta 1393,267-276), and hevea (Kostyal et al., 1998, Clin. Exp. Immunol. 112, 355-362). Comparisons were made using the University of Wisconsin's GAP program.

 Figure 8: by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of the expression of the NtPat genes during the hypersensitive reaction to VMT. The amplification products have the following sizes: NtPatl: 445 bp, NtPat2: 380 bp, NtPat3: 660 bp.

Figure 9: Acyl hydrolase activities of the recombinant NtPatl and NtPat3 proteins.

(a): Analysis by polyacrylamide gel denaturing of the recombinant NtPatl and NtPat3 proteins produced in E. coli. M: molecular mass markers (b): Specific enzymatic activity against lipid substrates. The PLA1 and PLA2 activities were measured on phosphatidylcholine. The esterase activity was measured on p-nitro-palmitate. ND: detectable.

Figure 10: phenylalanine ammonia lyase (PAL) activities in extracts of tobacco cells treated for 4 h with different doses of recombinant NtPatl (A) and NtPat3 (B) proteins.

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Description of the invention:
The present invention therefore relates to PLA2 polypeptides from plants involved in the defense reaction of plants. By PLA2 polypeptides is meant polypeptides having an enzymatic activity of the Phospholipase A2 type, which enzymatic activity can be demonstrated by tests known to those skilled in the art, preferably the enzymatic test described in Elsbach and Weiss (1991, in Methods in Enzymol, 197, Dennis EA ed., New York, Academic Press, 24-30) or that described in Example 10 using phosphatidylcholine as a substrate. The peculiarity of the PLA2 polypeptides of the present invention is that they contribute to the implementation of the defense reaction of plants, that is to say that they catalyze one of the stages of this reaction. By plant defense reaction, it is preferably meant the hypersensitivity reaction (RH).

 The PLA2 polypeptides of the present invention catalyze the release of oxylipin precursor fatty acids. The fatty acids released by these PLA2 polypeptides are fixed on a biological support. Among the fatty acids released by said PLA2 polypeptides, there is oleic acid (Cl 8: 1), linoleic acid (C18: 2) or linolenic acid (C18: 3), preferably linolenic acid. In addition, by biological support is meant any constituent element of a cell, preferably a plant cell, in particular all cellular lipid membranes, including those of cellular organelles. Preferably, these supports are the plasma membranes made up of a phospholipid bilayer, the membranes of organelles whose basic structure is a lipid bilayer, including the chloroplast membranes made up of lipid bilayers enriched with galactolipids (Figure 1A). The description of these different cellular constituents is listed in reference works (Trémolières A., Plant lipids, 1998, De Broeck University) and is part of the general knowledge of those skilled in the art. In general, the molecular structure of the biological support is that of a glycerolipid, in particular a phospholipid or a galactolipid. The term “glycerolipid” means a lipid molecule consisting of a glycerol skeleton, two alcohol functions of which are each esterified with an unsaturated fatty acid. The third alcohol function is esterified by a variable polar group (Figure 1A). Preferably, the action of the PLA2 polypeptides is specifically carried on the sn-2 position of said glycerolipids.

 The fatty acids released by the PLA2 polypeptides of the invention are precursors of oxylipins, preferably oxylipins of the jasmonate family, and more preferably these oxylipins are 12-oxophytodienoic acid (Figure 1B) or the acid

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 jasmonic (Figure 1C). The fatty acids released by the PLA2s of the invention can also be precursors of other unidentified oxylipins but which can also be activators of genes involved in the defense of plants.

 The PLA2 polypeptides of the present invention are plant PLA2 polypeptides.

They can come from a monocotyledonous plant, preferably rice, corn or wheat, or from a dicotyledonous plant, preferably Arabidopsis, tobacco, rapeseed, sunflower, soybeans, or cotton. Preferably, the PLA2 polypeptides of the invention come from tobacco.

 According to a particular embodiment of the invention, the PLA2 polypeptides are represented by the amino acid sequences described by the sequence identifiers SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6 or a fragment of these sequences. By fragment is meant essentially a biologically active fragment, that is to say a fragment of the sequence of a PLA2 polypeptide having the same enzymatic activity as a complete PLA2 polypeptide, in particular the phospholipase A2 activity as described. and measured above and in Examples 2 and 10.

 One of the particularities of the PLA2 polypeptides of the present invention resides in their similarity with the major reserve protein of the potato tuber, Patatin.

So far, no phospholipase A2 enzyme sequence present in the leaves has been assimilated to that of a Patatin. In addition, Patatin was only known as a reserve protein of the potato tuber but no form inducible during pathogenic infection has been described in any plant species. Patatin extracted from the potato tuber has been shown to have acyl hydrolase activity (Andrews et al., 1988, Biochem. J. 252,199-206), and even low phospholipase A2 activity (Senda et al, 1996, Plant Cell Physiol. 37, 347-353), but no prior art document suggests that Patatin may be involved in the defense reaction of plants. In addition, the PLA2 polypeptides of the present invention have a phospholipase A2 activity (as measured using the test using phosphatidylcholine described in Example 10) greater than 2.5 moles of fatty acid released / mg of protein / min, preferably greater than 10 μmol of released fatty acid / mg of protein / min, even more preferably greater than 50 mol of released fatty acid / mg of protein / min. Preferably, this activity is between 40 and 60 moles of fatty acid released / mg of protein / min.

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 The present invention also relates to isolated polynucleotides encoding PLA2 polypeptides from plants catalyzing a step in the defense reaction of plants. The term “polynucleotide” means a nucleotide polymer of DNA or RNA type, single or double-stranded, preferably double-stranded, which can in particular be a polynucleotide of complementary DNA type (cDNA) or an oligonucleotide, of natural or synthetic origin. The polynucleotides of the present invention have been isolated from their natural environment by molecular biology techniques known to those skilled in the art, in particular those described in Sambrook et al. (1989, Molecular Cloning: Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press).

 According to a particular embodiment of the invention, the polynucleotides coding for PLA2 polypeptides from plants code for amino acid sequences represented by the sequence identifiers SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO : 6, or a fragment of these sequences. It is well known to those skilled in the art that this definition includes all polynucleotides which, although comprising different nucleotide sequences as a result of the degeneracy of the genetic code, code for the same amino acid sequence, which is represented by sequence identifiers SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, or a fragment of these sequences.

 The present invention also comprises polynucleotides encoding PLA2 polypeptides from plants and capable of selectively hybridizing to one of the polynucleotides previously described. The term “polynucleotide capable of hybridizing selectively” means, according to the invention, the polynucleotides which, by one of the usual methods of the prior art (Sambrook et al., 1989, Molecular Cloning: Laboratory Manual, Nolan C. ed., New York: Spring Harbor Laboratory Press), hybridize with the above polynucleotides at a level significantly above background. The background noise can be linked to the hybridization of other polynucleotides present, in particular other cDNAs present in a cDNA library. The level of the signal generated by the interaction between the polynucleotide capable of selective hybridization and the polynucleotides defined by the above sequences ID according to the invention is generally 10 times, preferably 100 times more intense than that of l interaction of other DNA sequences generating background noise. The level of interaction can be measured, for example, by labeling the probe with radioactive elements, such as 32P. Selective hybridization is generally obtained by using very severe environmental conditions (for example 0.03 M NaCl and 0.03 M sodium citrate at about 50 C-60 C).

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 The invention also includes polynucleotides encoding PLA2 polypeptides from plants and homologs of the polynucleotides previously described. By homologous is meant according to the invention polynucleotides having one or more sequence modifications compared to the nucleotide sequences described above and coding for a PLA2 polypeptide. These modifications can be obtained according to the usual mutation techniques leading in particular to the addition, deletion, or substitution of one or more nucleotides with respect to the sequences of the invention. Advantageously, the degree of homology will be at least 70% relative to the sequences of the invention, preferably at least 80%, more preferably at least 90%. The methods for measuring and identifying the homologies between the nucleic acid sequences are well known to those skilled in the art. One can use for example the PILEUP or BLAST programs (in particular Altschul et al., 1993, J. Mol. Evol. 36: 290-300; Altschul et al., 1990, J. Mol. Biol. 215: 403-10) .

 The present invention also relates to fragments of the polynucleotides described above. The term "fragment" denotes in particular a fragment of at least 20 nucleotides, in particular of at least 100 nucleotides, advantageously of at least 500 nucleotides and preferably of at least 1000 nucleotides.

 In a particular embodiment, the subject of the present invention is polynucleotides comprising a nucleotide sequence represented by the sequence identifiers SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, polynucleotides capable of selectively hybridizing to one of these polynucleotides, polynucleotides homologous to these polynucleotides, and a portion of these polynucleotides.

 The present invention also relates to a chimeric gene comprising at least, operatively linked, a promoter functional in a host organism, a polynucleotide encoding a PLA2 polypeptide as defined in the present invention, and a functional terminator in the same. host organism. The different elements that a chimeric gene can contain are, on the one hand, regulatory elements for the transcription, translation and maturation of proteins, such as a promoter, a sequence coding for a signal peptide or a peptide transit, or a terminator constituting a polyadenylation signal, and on the other hand a polynucleotide coding for a protein. The expression "operably linked" means that said elements of the chimeric gene are linked together so that the functioning of one of these elements is affected by that of another. For example, a promoter is operationally linked

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 to a coding sequence when it is capable of affecting the expression of said coding sequence.

The construction of the chimeric gene according to the invention and the assembly of its different elements is achievable by the use of techniques well known to those skilled in the art, in particular those described in Sambrook et al. (1989, Molecular Cloning: Laboratory Manual, Nolan C. ed., New York: Spring Harbor Laboratory Press). The choice of regulatory elements constituting the chimeric gene is essentially a function of the host species in which they must function, and those skilled in the art are capable of selecting functional regulatory elements in a given host organism. By "functional" is meant capable of functioning in a given host organism.

 According to a particular embodiment of the invention, the chimeric gene contains a promoter called "constitutive". A constitutive promoter according to the present invention is a promoter which induces the expression of a coding sequence in all the tissues of an organism host and permanently, that is to say throughout the life cycle of said host organism.

Some of these promoters can be tissue-specific, that is to say express the coding sequence permanently, but only in a particular tissue of the host organism. Constituent promoters can come from any type of organism. Among the constitutive promoters which can be used in the chimeric gene of the present invention, we can cite by way of example, bacterial promoters, such as that of the octopine synthase gene or that of the nopaline synthase gene, promoters Viral, such as that of the gene controlling the transcription of RNA19S or 35S of the cauliflower mosaic virus (Odell et al., 1985, Nature, 313,810-812), or the promoters of the cassava rib mosaic virus (as described in patent application WO 97/48819). Among the promoters of plant origin, mention will be made of the promoter of the gene for the small ribulose-biscarboxylase / oxygenase subunit (RuBisCO), the promoter of a histone gene as described in application EP 0 507 698, or the promoter of a rice actin gene (US 5,641,876).

 According to another particular embodiment of the invention, the chimeric gene contains an inducible promoter. An inducible promoter is a promoter which functions, that is to say which induces the expression of a coding sequence, only when it is itself induced by an inducing agent. This inducing agent is generally a substance which can be synthesized in the host organism following a stimulus external to said organism, this external stimulus possibly being for example a pathogenic agent. The inducing agent can also be a substance external to this host organism capable of penetrating inside of it. Advantageously, the promoter used in the present invention is inducible following the aggression of the host organism by a pathogenic agent. Such promoters are known,

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 as for example the promoter of the plant O-methyltransferase class II (COMT II) gene described in patent application FR 99 03700, the PR-1 promoter from Arabidopsis (Lebel et al., 1998, Plant J. 16 (2): 223-233), EAS4 promoter of the sesquiterpene synthase gene from Tobacco (Yin et al., 1997, Plant Physiol. 115 (2), 437-451), or the promoter of the gene encoding 3- hydroxy-3-methylglutaryl coenzyme A reductase (Nelson et al., 1994, Plant Mol. Biol.

25 (3): 401-412).

 Among the terminating elements which can be used in the chimeric gene of the present invention, we can cite by way of example the terminating element nos of the gene coding for nopaline synthase from Agrobacterium tumefaciens (Bevan et al., 1983, Nucleic Acids Res 11 (2), or the terminator of a histone gene as described in application EP 0 633 317.

 According to a particular embodiment of the invention, the promoter and the terminator element of the chimeric gene according to the invention are both functional in plants.

 The present invention also relates to a vector containing a chimeric gene according to the invention. The vector according to the invention is useful for transforming a host organism and expressing therein a PLA2 polypeptide. This vector can be a plasmid, a cosmid, a bacteriophage or a virus. In general, the main qualities of this vector must be an ability to maintain and self-replicate in the cells of the host organism, in particular thanks to the presence of an origin of replication, and to express therein a PLA2 polypeptide. . The choice of such a vector as well as the techniques for inserting into it the chimeric gene according to the invention are widely described in Sambrook et al. (1989, Molecular Cloning: Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press) and are part of the general knowledge of a person skilled in the art. Advantageously, the vector used in the present invention also contains, in addition to the chimeric gene of the invention, a chimeric gene containing a selection marker. This selection marker makes it possible to select the host organisms actually transformed, that is to say those which have incorporated the vector. Preferably, the host organism to be transformed is a plant. Among the selection markers that can be used in plants, mention may be made of markers containing antibiotic resistance genes such as that of the hygromycin phosphotransferase gene (Gritz et al., 1983, Gene 25: 179-188), but also markers containing herbicide tolerance genes such as the bar gene (White et al., NAR 18: 1062, 1990) for bialaphos tolerance, the EPSPS gene (US 5,188,642) for glyphosate tolerance or the HPPD gene (WO 96/38567) for tolerance to isoxazoles. Mention may also be made of genes coding for easily identifiable enzymes such as the enzyme GUS, genes coding

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 for pigments or enzymes regulating the production of pigments in transformed cells. Such selection marker genes are described in particular in patent applications WO 91/02071 and WO 95/06128.

 The present invention also relates to transformed host organisms, containing a vector as described above. Preferably, the transformed host organisms are plants. The term "transformed host organism" means a host organism which has incorporated the chimeric gene of the invention into its genome, and consequently produces a PLA2 polypeptide in its tissues. To obtain the host organisms according to the invention, those skilled in the art can use one of the many known transformation methods.

One of these methods consists in placing the cells to be transformed in the presence of polyethylene glycol (PEG) and of the vectors of the invention (Chang and Cohen, 1979, Mol. Gen. Genet. 168 (1), 111-115; Mercenier and Chassy, 1988, Biochemistry 70 (4), Electroporation is another method which consists in subjecting the cells or tissues to be transformed and the vectors of the invention to an electric field (Andreason and Evans, 1988, Biotechniques 6 (7) , 650-660; Shigekawa and Dower, 1989, Aust. J. Biotechnol. 3 (1), 56-62). Another method is to directly inject the vectors into host cells or tissues by microinjection (Gordon and Ruddle, 1985, Gene 33 (2), 121-136). Advantageously, the so-called "biolistic" method can be used, which consists in bombarding cells or tissues with particles on which the vectors of l are adsorbed. invention (Bruce et al., 1989, Proc. Natl. Acad. Sci. USA 86 (24), Klein et al., 1992, Biote chnology
10 (3), 286-291; US Patent No. 4,945,050). Preferably, the transformation of plants will be carried out using bacteria of the genus Agrobacterium, preferably by infection of the cells or tissues of said plants with A. tumefaciens (Knopf, 1979, Subcell. Biochem. 6,143-
173; Shaw et al., 1983, Gene 23 (3): 315-330) or A. rhizogenes (Bevan and Chilton, 1982, Annu.

Rev. Broom. 16: 357-384; Tepfer and Casse-Delbart, 1987, Microbiol. Sci. 4 (1), 24-28). Preferably, the transformation of plant cells by Agrobacterium tumefaciens is carried out according to the protocol described by Ishida et al. (1996, Nat. Biotechnol. 14 (6), These different techniques are described in particular in the following patents and patent applications: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267,159, EP 604 662, EP 672,752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,254 615, EP 442 174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, WO 91/02071 and WO

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The present invention also relates to a process for the production of plants resistant to attack by pathogenic organisms. This method consists in transforming said plants with a chimeric gene according to the invention, that is to say in incorporating said chimeric gene into their genome, so that they express in their tissues a PLA2 polypeptide of the present invention.

 According to a particular embodiment of the invention, the plants obtained by said process have incorporated into their genome a chimeric gene containing a constitutive promoter, which allows the expression of at least one PLA2 polypeptide constitutively in the tissues of the plant. Plants produced by this process permanently express a PLA2 polypeptide, which gives said plant continuous resistance to attack by pathogenic organisms throughout its development, in all or part of its tissues.

 According to another embodiment of the invention, the plants obtained by said process have incorporated into their genome a chimeric gene containing an inducible promoter. Preferably, this promoter is induced following the aggression of the plant by a pathogenic organism, and the expression of a PLA2 polypeptide in the tissues of the plant is only achieved during such aggressions. Plants produced by this process only develop a state of resistance to pathogenic organisms when they are attacked by a pathogenic organism.

 The present invention also includes the plants obtained by this process, parts of these plants, and the offspring of these plants. "Part of these plants" means any organ of these plants, whether aerial or underground. The aerial organs are stems, leaves, flowers. The underground organs are mainly the roots, but they can also be tubers. By "offspring" is meant mainly the seeds containing the embryos resulting from the reproduction of these plants among themselves. By extension, the term "offspring" applies to all the seeds formed in each new generation resulting from crosses between a plant and the plant transformed by the process according to the invention.

 The plants obtained by the present process can be monocots or dicots. Preferably, these plants are plants of agronomic interest. Advantageously, the monocotyledonous plants are wheat, corn, rice. Advantageously, the dicotyledonous plants are rapeseed, soybeans, tobacco, cotton.

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 The pathogenic organisms against which the plants obtained by the present process have acquired resistance may be viruses, bacteria, fungi, or nematodes. Among the viruses, there may advantageously be mentioned the mosaic viruses, in particular the tobacco mosaic virus.

 According to a particular embodiment of the invention, the plants obtained by the method according to the invention contain, in addition to a chimeric gene according to the invention, at least one other chimeric gene containing a polynucleotide encoding a protein of interest. Among the polynucleotides encoding a protein of interest, mention may be made of polynucleotides encoding an enzyme for resistance to a herbicide, for example the polynucleotide encoding the enzyme bar (White et al., NAR 18: 1062, for tolerance to bialaphos, the polynucleotide coding for the EPSPS enzyme (US 5,188,642; 97/04103) for tolerance to glyphosate or the polynucleotide coding for the HPPD enzyme (WO 96/38567). polynucleotide coding for an insecticidal toxin, for example a polynucleotide coding for a toxin of the bacterium Bacillus thuringiensis (for example, see International Patent Application WO 98/40490). Other disease resistance polynucleotides may also be contained in these plants, for example a polynucleotide coding for the oxalate oxidase enzyme as described in patent application EP 0 531 498 or US patent 5,866,778, or a polynucleotide coda nt for an antibacterial and / or antifungal peptide such as those described in patent applications WO 97/30082, WO 99/24594, WO 99/02717, WO 99/53053, and WO99 / 91089. Mention may also be made of polynucleotides coding for agronomic characteristics of the plant, in particular a polynucleotide coding for a delta-6 desaturase enzyme as described in patents US 5,552,306, US 5,614,313, and patent applications WO 98/46763 and WO 98 / 46764.

 The present invention also relates to monoclonal or polyclonal antibodies directed against a PLA2 polypeptide according to the invention, or a fragment thereof.

Antibody production techniques are widely described in the general literature and in reference works such as Immunological Techniques Made Easy (1998, O. Cochet, J.-L. Teillaud, C. Sautès eds, John Wiley & Sons, Chichester).

 The present invention also relates to a process for searching for molecules stimulating the synthesis of at least one PLA2 polypeptide according to the invention in plants. This

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 process is characterized in that two groups of plants are used, one of which is treated with a molecule. The level of expression (ie, the quantity) of at least one of the PLA2 polypeptides according to the invention is then measured in the tissues of the two groups of plants. If the expression level of at least one PLA2 polypeptide is significantly higher in the group of treated plants compared to the group of untreated plants, the applied molecule is retained as being a molecule stimulating the synthesis of PLA2 polypeptides according to the invention in plants. Among the techniques for measuring the level of expression of the PLA2 polypeptide, a person skilled in the art can use the well-known techniques of amplification after reverse transcription (RT-PCR) and Northern blot for the measurement of messenger RNA (mRNA ) coding for PLA2 polypeptides, or Western blot for the measurement of PLA2 polypeptides. The protocols for these techniques are described in particular in Sambrook et al. (1989, Molecular Cloning: Laboratory Manual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press). The technique of amplification by RTPCR consists in extracting the mRNAs from plants following the application of the molecule, in subjecting them to reverse transcription, in subjecting the products of this reverse transcription to selective amplification by PCR with pairs of primers consisting of fragments of polynucleotides according to the invention, to migrate the amplification products on an electrophoresis gel, then to measure the intensity of the fragments in the gel.

The Northern blot technique consists in extracting the mRNAs from plants following the application of the molecule, in separating these mRNAs on a gel by electrophoresis, in transferring these mRNAs to a membrane, then in hybridizing these mRNAs with a probe constituted by a polynucleotide according to the invention or a fragment thereof on which a marker molecule is grafted make it possible to detect it after hybridization to measure the amount of mRNA hybridized. The Western blot technique consists in extracting proteins from plants following application of the molecule, in separating these proteins on a gel by electrophoresis, in transferring these proteins to a membrane, then in hybridizing these proteins with antibodies according to the invention on which is grafted a marker molecule making it possible to detect them after hybridization to measure the quantity of PLA2 polypeptide hybridized.

 The present invention therefore also includes molecules obtained by the process described above. The main characteristic of these molecules is an ability to stimulate the natural synthesis in plants of at least one PLA2 polypeptide according to the invention.

 The present invention also relates to a process for improving the defense of plants against attack by pathogenic organisms. This process consists of

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 increasing the level of expression of at least one PLA2 polypeptide of the invention in said plants. This increase can be carried out in a constitutive or induced manner in the transformed plants of the present invention, but also by application to transformed plants or not, of a molecule stimulating the synthesis of at least one PLA2 polypeptide in said plants. By attacks of pathogenic organisms is meant according to the present invention, attacks on plants by viruses, bacteria, fungi or nematodes.

Examples: Example 1. Plant material and virus inoculation technique
The plants (Nicotiana tabacum cv. Samsun NN) are grown in a greenhouse and then in an air-conditioned cubicle at 22 C 4 C under a 16-hour photoperiod.

 The leaves of tobacco plants aged 6 to 8 weeks are rubbed with cotton wool impregnated with a suspension of purified tobacco mosaic virus (VMT, common strain U1) mixed with an abrasive, celite ( 10 mg / ml). After a few minutes of contact, the leaves are rinsed with lukewarm water to remove excess celite and viral particles. The leaves are harvested and denervated after different infection times before being stored at -80 C until use.

Example 2. Method for Measuring the Phospholipase A2 Activity
2.1. Preparation of enzyme extracts
One gram of tobacco leaves is ground in liquid nitrogen. The grinding is continued in ice in the presence of quartz and 2 ml of cold extraction buffer (100 mM sodium acetate pH 5.2, 5 mM CaCl 2, 15 mM B-mercaptoethanol). The whole is centrifuged for 15 min at 13000 g at 4 C. The supernatant (1.5 ml) is deposited on a Sephadex G-25 column (Hi Trap Desalting, Pharmacia) balanced with a 20 mM sodium acetate solution, CaCl2 5 mM. The elution is carried out with 2 ml of equilibration buffer and the excluded fraction containing the proteins is collected. This step eliminates pigments and low molecular weight compounds.

 2. 2. Determination of the phospholipase A2 activity (Elsbach and Weiss, 1991, in Methods in Enzymol. 197, Dennis EA ed., New York, Academic Press, 24-30) - preparation of the substrate: the substrate used consists of cells of strain NM 522 of Escherichia coli cultured in LB medium in the presence of oleic acid labeled with

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 [14C] (C18: 1). The radioactive fatty acid being added to the culture medium, its incorporation takes place mainly during the exponential growth phase of the bacteria and over several generations, allowing uniform labeling of the membrane phospholipids.

The labeled bacteria are washed with 1% bovine serum albumin (BSA), then resuspended in water. In order to inactivate the bacterial phospholipases and to allow the exposure of the phospholipids to phospholipases A contained in the test sample, the bacteria are autoclaved for 15 min at 121 C. The labeled cells are diluted with unlabeled cells to have a final concentration of 1.5 x 1010 cells / ml containing 7 x 105 cpm / ml then the substrate is stored at -80 C.

 - assay of the activity: the reaction medium is composed of 50 (it of enzymatic extract, 50 l of 100 mM Tris-HCl buffer pH 7.5, 5 mM CaCl 2 and 50 l of labeled substrate. The reaction is carried out 30 min at 37 C. A control (substrate without enzyme) is carried out in parallel.The reaction is stopped by adding 0.5% of cold BSA then centrifugation at 13000 g for 3 min at room temperature The sedimented bacteria contain the phospholipids not degraded and the hydrolysis products complexed with albumin are in the supernatant In order to quantify the rate of hydrolysis, 100 l of the supernatant are removed and counted by scintillation. The amount of radioactivity released is a measure of the activity phospholipase.

Example 3. Phospholipase A2 Activity During the Hypersensitive Reaction (RH)
The enzymatic test developed by Elsbach and Weiss (1991, in Methods in Enzymol.

197, Dennis E.A. ed., New York, Academic Press, 24-30) using membranes of radiolabelled E. coli bacteria as a substrate was used to measure phospholipase A2 activity. This test has the advantage of presenting the phospholipids in a membrane environment, which brings it closer to physiological conditions than the use of solubilized synthetic substrates. The culture of the bacteria is carried out in the presence of oleic acid- [14C] (Cl 8: 1) which is incorporated almost exclusively in the phospholipids of the bacterial membranes in position sn-2, which makes this test specific for phospholipases A2 .

 The phospholipase A2 activity was measured in soluble protein extracts from tobacco leaves developing an RH to the tobacco mosaic virus (VMT). The enzymatic activity, very weak in healthy leaves, increases rapidly between 2 and 4 days after inoculation to reach a maximum from 4 days post-inoculation and stabilize until late RH times (Figure 2). The appearance of phospholipase A2 activity coincides with the formation of necrotic lesions, typical of RH (36-48 hours). Such stimulation

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 in infected plants was not known and suggests that the metabolism of fatty acid mobilization is activated during this defense response.

Example 4. Relation between the phospholipase A2 activity and the defense signals derived from fatty acids during RH
In order to determine whether the appearance of phospholipase A2 activity during RH is followed by the accumulation of fatty acid derivatives, the oxylipin contents were determined in the infected plants. The quantities of 12-oxophytodienoic acid (APD), precursor of jasmonic acid (AJ), and those of jasmonic acid, were measured, these two compounds being signals inducing defense responses.

4.1. Determination of jasmonic acid (AJ).

 Extraction. The method is that developed by Albrecht et al., (1993, Planta 191.86-94), slightly modified. The tobacco leaves (1 g) are finely ground in liquid nitrogen. The grinding is continued for 5 min at 4 ° C. in a Waring mixer containing 50 ml of 80% methanol and 0.1 Ci of () [3H] -dihydrojasmonic acid, which will make it possible to calculate the yield of the extraction of the AJ endogenous. One gram of Polyclar AT is then added to trap the soluble phenolic compounds. After filtration through Büchner, the methanol is evaporated on a rotary evaporator. The aqueous phase recovered (- 10 ml) is adjusted to 12.5 ml with water and then acidified with acetic acid (0.2% final). After centrifugation for 30 min at 4 ° C. and at 12,000 g, the clarified supernatant is deposited on a column C18 (Lichroprep RP 18, Merck) previously rinsed with twice 5 ml of methanol and then equilibrated with twice 5 ml of acetic acid 0, 2%. The elution is carried out with 5 ml of a 70% methanol / 0.2% acetic acid solution. After evaporation of the methanol under a stream of nitrogen, the aqueous phase is acidified with 100 l of glacial acetic acid and then extracted twice with 2 ml of ethyl acetate. The organic phases are combined and then evaporated to dryness under a stream of nitrogen and the dry residue is taken up in 300 l of methanol. The samples are methylated with 300 l of cold diazomethane (in ether). After 15 sec, the reaction is stopped by evaporation of the ether under a stream of nitrogen to avoid overmethylation. The dry extracts are then taken up in 300 l of 20% methanol / TBS. 50 l are taken for radioactivity counting to calculate the yield of the extraction of the AJ.

 Quantitative analysis by ELISA test. The quantitative analysis is carried out by competitive ELISA (Enzyme Linked Immuno-Sorbant Assay). The plates are "coated" with a

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 purified antibody, RAMIG (Rabbit Anti-Mouse Immunoglobulin G, Pierce), then with an anti-methyl-jasmonate monoclonal antibody (Prof. Weiler, Bochum, Germany). RAMIG recognizes the Fc region of the specific anti-MJ antibody. This configuration ensures that the anti-MJ antibody is immobilized in the solid phase by its Fc part and therefore that the binding sites of the antigen are accessible. The sample is then added. The number of specific MJ binding sites that remain free (because they are not occupied by the MJ contained in the sample) is measured using a tracer (AJ coupled to alkaline phosphatase). The tracer is detected by an enzymatic test: alkaline phosphatase cleaves its substrate, pNPP (sodium p-nitrophenyl phosphate) into p-nitrophenol, which results in the appearance of a yellow coloration whose intensity is measured by colorimetry (absorbance = 405 nm).

The quantity of tracer fixed is inversely proportional to the quantity of MJ present in the samples and makes it possible to calculate the quantity of AJ contained in the extracts.

 A standard range is carried out in parallel with MJ (0.1 to 100 pmol) as well as two types of controls (reaction without antigen or without tracer).

4.2. Determination of 12-oxo-phytodienoic acid (APD).

 Extraction. The leaves (2 g) are ground in liquid nitrogen and then transferred to an Erlenmeyer flask. The extraction is carried out at room temperature for 4 h with three times 20 ml of ether, stirring from time to time. During the first extraction, 30 l of [2H5] -APD (Prof. Weiler, Bochum, Germany) are added (to calculate the yield of the extraction of endogenous APD). The three supernatants are collected and then filtered on Büchner. The filtrate is then loaded on an aminopropyl column in reverse phase (Aminopropyl 500 mg, Macherey-Nagel). After washing the column with 8 ml of CHCl3 / isopropanol (2: 1), the samples are eluted with 13 ml of ether / acetic acid (98: 2). The eluate is evaporated under nitrogen flow. The dry residues are taken up in 200 l of hexane / isopropanol / acetic acid solution (100: 1.61: 0.11) (v / v / v) and then centrifuged for 2 min at 13000 g.

 Quantitative analysis by HPLC and mass spectrometry. (Prof. E. Weiler, Bochum, Germany). 100 l of the samples are injected onto an HPLC system (Nucleosil column 25 x 4 mm, 10 m). The elution, programmed in isocratic mode, is carried out with a hexane / isopropanol / acetic acid solution (100: 1.61: 0.11) (v / v / v). A fraction is collected every 2 min from 6.5 min to 14.5 min. The column is then washed for 5.5 min before a new injection. The fraction containing the APD is identified by comparison of the retention time with that of the reference APD and then evaporated in a rotary evaporator. Drying is complete under nitrogen flow. The dry residues are taken up in 100 l of methanol and

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 methylated with 200 l of diazomethane in the same way as the AJ. After stopping the reaction under nitrogen flow, the samples are taken up in 50 l of chloroform and then analyzed immediately by mass spectrometry (Laudert et al., 1997, Plant J. 15,675- 684). The amount of APD contained in the sample is calculated by comparison with the area of the standard peak [2H5] -APD.

4.3. Assay results
The results of these assays are shown in Figure 2. The contents of APD and AJ are extremely low in plants not treated or rubbed with water. In response to a VMT infection, the APD accumulates from 48 hours and reaches a maximum around 6-7 days. YI increases more slowly and later. The most interesting point is that the phospholipase A2 activity clearly precedes the accumulation of lipid derivatives.

This is a strong argument in favor of an involvement of this enzymatic activity in the initiation of the synthesis of oxylipins regulating defense responses.

 To situate the chronology of this lipolytic activity in relation to defense responses well described in this pathosystem, the kinetics of appearance of phospholipase A2 activity was compared to the induction of proteins "Pathogenesis-Related" (PR). PR proteins have antimicrobial properties which contribute to resistance against infections and several families of PR proteins are induced in a coordinated manner during RH. Three families of PR proteins (chitinase, 13-1,3-glucanase and a protease inhibitor) have been measured by their enzymatic activities, and it appears that these reach their maximum around 7 days post-inoculation, while the maximum phospholipase A2 activity is reached between 3 and 4 days (Figure 3). This result shows that phospholipase A2 activity is an early event in RH and is involved in cell signaling phenomena which determine the establishment of the resistance state.

Example 5. Identification of PLA2 Polypeptides
The early inducibility characteristics of phospholipase A2 activity stimulated a search for the protein (s) of this activity. The soluble protein fraction is extracted from 180 g of tobacco leaves (Nicotiana tabacum cv. Samsun NN) infected for 6 days by VMT. The grinding is carried out at 4 ° C. in 270 ml of 0.1 M sodium acetate buffer pH 5.2,13-15 mM mercaptoethanol, 2 mM CaCl 2. The ground material is filtered through gauze and is then centrifuged at 10,000 g for 30 min at 4 C. The supernatant is loaded onto

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 a column of Sephadex G25 filtration gel (3.5 x 70 cm, Pharmacia) equilibrated in 20 mM sodium acetate solution pH 5.2, 2 mM CaCl2 (solution A). This step eliminates molecules of small molecular mass. The eluted protein fraction is stored overnight at 4 ° C., which results in the precipitation of a large part of the cellular proteins. The usual technique for preparing the total protein extract at a pH of 5.2 makes it possible to preserve the phospholipase A2 activity and to eliminate "non-PR" proteins by selective precipitation. A research without a priori consisted in a fractionation of this soluble protein extract on different chromatographic supports, the different eluted fractions being tested for their phospholipase A2 activity using the test on bacterial substrate described above (Elsbach and Weiss, 1991 , in Methods in Enzymol. 197, Dennis EA ed., New York, Academic Press, 24-30). The clarified supernatant constitutes the starting protein extract for the purification of the phospholipase. The extract is loaded onto a cation exchange column (2.1 × 8 cm) (S-Sepharose Fast Flow, Pharmacia) balanced in solution A.

The cationic proteins retained on this support are eluted with a NaCl gradient of 0 to 0.3 M in solution A. No significant activity is detected in the "acid" (anionic) proteins, all of the enzymatic activity. being retained on a cation exchange chromatography support. The elution of this 1st column with a salt gradient makes it possible to separate three peaks of activity (Figure 4a).

 The fractions containing the major peak of phospholipase A2 activity (PLA2) were combined, dialyzed against solution A and then loaded onto a cation exchange column (CM-TSK 3 SW, Altex) mounted on an FPLC system and balanced in the solution. AT.

Elution is carried out with a gradient of 0.1 to 0.2 M NaCl in solution A (Figure 4b). The fractions containing a high PLA2 activity are combined, concentrated on Centricon 10 filters (Amicon), then loaded onto a molecular sieving column (Superdex 75, Pharmacia) equilibrated in solution A containing 0.2 M NaCl (Figure 4c). One g of cytochrome C is added to the collection tubes before harvesting to stabilize the PLA2 activity.

The active fractions are analyzed by electrophoresis on 12.5% polyacrylamide gel under denaturing conditions (Laemmli, 1970, Nature 227,680-685) stained with silver nitrate (Geoffroy et al., 1990, Mol. Plant-Microbe Interact. 3 , 327-333). The fractions containing a 28 kDa protein, the abundance of which is correlated with the PLA2 activity, are combined and loaded onto a preparative gel (FIG. 4d). The separated proteins are transferred to a PVDF membrane, stained with Coomassie blue according to the manufacturer's instructions (Problott, Applied Biosystems). The area containing the 28 kDa protein was excised from the membrane for N-terminal sequencing by degradation of Edman (Applied sequencer

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 Biosystems model 473A). The 50 amino acid sequence obtained is compared with the databases using the BLAST program (National Center for Biotechnology Information, USA, http://www.ncbi.nlm.nih.gov/blast) which made it possible to identify the similarity between the sequence encoding the isolated PLA2 polypeptide and patatin, the major reserve protein in the potato tuber (Figure 5).

 The identification of a tobacco leaf phospholipase as a "patatin-like" protein is new. If acyl hydrolase activity (on different substrates comprising at least one fatty acid chain) has been reported for patatin (Andrews et al., 1988, Biochem.

J. 252,199-206), the role of proteins of this type in defense responses has not been studied, in particular in relation to lipid signaling and the production of oxylipins. A soluble extract of potato tubers (2 varieties) in which the patatin has been identified immunologically as representing approximately 30% of the proteins was then prepared. When the phospholipase A2 activity is measured in these extracts with the bacterial substrate described above, the phospholipase A2 activity specific for potato patatin is up to 500 times lower than that measured for the PLA2 polypeptide isolated from leaves of infected tobacco.

Example 6 Cloning of cDNA Coding for PLA2 Polypeptides
Only one patatin type sequence is known in tobacco (Drews et al., 1992, Plant Cell 4,1383-1404). It is a cDNA specifically expressed during the development of tobacco petals, but the corresponding protein has not been studied. A strategy of cloning by PCR of new isoforms was undertaken using on the one hand a primer derived from the N-terminal sequence of the PLA2 polypeptide isolated from infected leaves, and on the other hand primers derived from conserved regions identified in 5 sequences. patatine type nucleotides (tobacco, potato, cucumber, hevea and arabidopsis) available in databases. The usual molecular biology techniques used in this invention are described in Sambrook et al. (1989, Molecular Cloning: Laboratory Manual, Nolan C. ed., New York: Spring Harbor Laboratory Press).

 RNA extraction. Total RNAs are isolated from one gram of plant material ground in a buffer based on sodium tetraborate (Heitz et al., 1993, J. Biol.

Chem. 268.16987-16992). Two extractions with phenol / chloroform / isoamyl alcohol (25: 24: 1) are carried out in order to deproteinize the samples. The RNAs are then selectively precipitated with a LiCl solution, then washed before being taken up in water.

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 The RNAs are quantified by measuring the absorbance at 260 nm (one absorbance unit corresponds to 40 μg / ml of RNA) and then stored at -80 C. The ratio of the absorbances recorded at 260 nm and 280 nm gives an indication the purity of the RNAs (1.8 <R <2).

 Back transcription. Five g of total RNA to which 500 ng of oligodT are added in a final volume of 13.5 l are denatured at 65 ° C. and then rapidly cooled in ice. The reaction buffer (50 mM Tris-HCl pH 8.3, 6 mM MgCl2, 40 mM KC1, 1 mM DTT), the 4 dNTP at 1 mM and 20 units of RNAsin (Biotech), an RNAses inhibitor are added.

Incubation takes place in a total volume of 20 l for 1 h at 37 ° C. in the presence of 200 units of reverse transcriptase from the Moloney murine leukemia virus (Promega).

 Amplification by PCR. The reverse transcription reaction mixture is precipitated by adding one volume of 5 M ammonium acetate pH 5.0 and two volumes of absolute ethanol, then washed and taken up in 20 l of water. One liter of reverse transcript is used as a template for PCR. The reaction is carried out in a final volume of 50 l containing the 20 mM Tris-HCl mixture pH 8.4, 50 mM KC1, 1.5 mM MgCl2, 0.01% gelatin (w / v), each dNTP at 200 M and 500 ng of a mixture of primers defined as follows: three degenerate primers were used to amplify cDNA fragments similar to patatin: Primer 1: 5'-GCIACIAAA / GGGIAAA / GATT / C / AGTIACIGT-3 ' (sense) Primer 2: 5'-TT / AGCA / GGATTAC / TTTT / CGAT / CG / ATG / AATT / AGC / G-3 '(sense) Primer 3: 5'-TA / TG / TC / TA / TAIC / TAACTACCA / GCC / GICAAC / TG / AA / TC / TG / AG-3 '(antisense) Primer 1 is derived from amino acids 1 to 9 of the N-terminal sequence of the purified PLA2 polypeptide from tobacco leaves. Primers 2 and 3 are derived from two consensus regions identified by alignment of 5 tobacco patatin (NtTP12, Genbank U68484), potato (Z27221), cucumber (Y12793), rubber tree (U80598) and arabidopsis (AJ002596) genes. The PCR experiments are carried out under the following conditions on RNAs transcribed from healthy leaves or infected for 3 days: denaturation: 94 C, hybridization: 47 C, elongation: 72 C (1 min each step) for 35 cycles with combinations of primers 1 and 3 or 2 and 3.

 The pair of primers 2 + 3 made it possible to amplify a DNA fragment of expected size (0.5 kb) from infected leaves. This fragment is re-amplified under the same conditions, purified on agarose gel, then cloned in the vector pGEM-T (Promega) and multiplied in the strain E. coli DH5 [alpha]. This fragment is identified by sequencing as a sequence of the type

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 "patatine" and is used as a probe to screen a cDNA library constructed in vector 1 ZAP II from RNA of plants infected for 5 days by VMT (Heitz, et al., 1993, J. Biol. Chem . 268.16987-16992). The isolated clones all have a sequence identical to that of the probe and the longest of these clones (1505 nt) is called NtPatl.

The use of the pair of primers 1 + 3 leads to the amplification of a DNA fragment of 0.65 kb from RNA of infected leaves. After cloning into the vector pGEM-T and sequencing, this fragment is identified as a second “patatin” type sequence, and the predicted protein sequence corresponds to the N-terminal sequence of the purified PLA2 polypeptide. This partial fragment of 0.65 kb is called NtPat2 and is used as a probe to screen the cDNA library. The isolated complete clones represent a third sequence, 80% similar to NtPat2 and define a third gene called NtPat3. Surprisingly, no NtPat2 clone could be isolated from the library. We therefore have full-length cDNAs NtPatl (1506 nt) (SEQ ID NO: 1) and NtPat3 (1505 nt) (SEQ ID NO: 5) and a partial sequence NtPat2 (620 nt) (SEQ ID NO: 3 ). These genes are described for the first time in the context of this invention.

Example 7. Analysis of the protein sequences deduced from the cDNAs
The cDNAs NtPatl and NtPat3 code for proteins of predicted masses of 46.7 and 45.1 kDa, respectively. An alignment of the NtPat-1, -2 and -3 protein sequences with that of the tobacco NtTP12 gene expressed in the petals, and that of the potato tuber patatin is shown in Figure 6. This alignment presents new information on the variability existing in the primary structure of this class of proteins which is poorly understood. The N-terminal half of the proteins is the best preserved while more divergence exists in the C-terminal half. The similarities / identities between NtPatl and NtPat2 or NtPat3 are 75/55%, and these tobacco proteins are similar to 64-82% and identical to 46-61% to protein sequences deduced from cucumber, hevea and arabidopsis (Figure 7). The GXSXG motif, typical of serine hydrolases, is present in all of these sequences.

 The protein sequence of potato patatin has a hydrophobic Nterminal extension (Figure 6), in agreement with the vacuolar localization which has been demonstrated for this protein (Sonnewald et al., 1990, Plant Cell 2, 345-355). Extensions of this type are also identifiable in the sequences NtTP12 and NtPat3, but not in the sequence NtPatl, which indicates that the latter protein is characterized by a

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 non vacuolar localization. These data show that the two new genes isolated from infected tobacco leaves encode proteins located in separate cell compartments. These enzymes are therefore in contact with several plant membrane systems which are potential sources of fatty acids precursors of oxylipins.

EXAMPLE 8 Induction of the NtPat Genes in the Hypersensitivity Reaction
The total RNAs were extracted from plants inoculated with water or with VMT and then harvested at increasing times after treatment. The signals detected by Northern blot using the NtPatl and NtPat3 probes being very weak, the expression of the genes was studied by semi-quantitative RT-PCR using specific primers chosen in regions divergent from the NtPatl, NtPat2 and NtPat3. Three pairs of specific primers derived from divergent regions of the NtPatl, NtPat2 and NtPat3 genes were prepared.

These primers are: NtPatl - (sense): 5'-AGCTGGAACGAGCACTGGTGGTCTT-3 '- (antisense): 5'-GAGATGATGTTCTTTTACATTGCCTTTGG-3' NtPat2 - (sense): 5'-ACATAGATGGACCGAATGCAAGens-3 ' CGTGAGTTTTGGCATCAGCAGTTG-3 'NtPat3 - (sense): 5'-AGCTTCGTGAGGAGGGTCACA-3' - (antisense): 5'-CCTCTCCAGTCAAATTGTCGTC-3 '
The total RNA is extracted at various times after treatment of the plants and reverse transcribed as described above. RT-PCR procedure control reactions are performed with primers derived from consensus regions of the tobacco actin genes Tob25, Tob53 and Tob66 (Moniz de Sa and Drouin, 1996, Mol. Biol. Evol. 13, 1198 -1212).

NtAct - (sense): 5'-GATATGGAGAAG / AATC / ATGGCATCAT / CAC-3 'NtAct - (antisense): 5'-GTTTCA / GTGAATT / ACCT / AGCT-3'
These primers make it possible to amplify specific fragments which can be separated by their sizes: NtPatl: 445 base pairs (bp), NtPat2: 380 bp, NtPat3: 660 bp.

We have verified that the same signal intensities are obtained when the three pairs of primers are mixed in the same reaction. Primers derived from constitutively expressed actin genes were used to standardize the RT-PCR procedure. The amounts of 1st strand cDNA used for the amplification are 100 ng to visualize the NtPat transcripts and 20 ng to visualize the actin transcripts. The PCR conditions are

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 the following: denaturation 94 C, hybridization 50 C, elongation 72 C (1 min each step) for 25 cycles. Under these conditions, the amplification is linearly related to the quantity of starting RNA. To verify that the amplification does not result from the presence of genomic DNA in the RNA preparations, PCR experiments are carried out with 10 ng of purified genomic DNA and the profiles obtained with the different pairs of primers are compared with those resulting from amplification with retro-transcribed RNA. As illustrated in FIG. 8, no NtPat transcript can be detected in the control plants (time 0) or in the plants rubbed with water. NtPat transcripts begin to be detectable around 36 h after inoculation with VMT and maximum and constant levels are reached and then maintained between 2 and 7 days. The accumulation of the 3 mRNAs is remarkably coordinated and the NtPat3 transcripts appear more abundant than the NtPatl and NtPat2 transcripts during the hypersensitive reaction.

 These data show that the expression of the NtPat genes precedes by 12-24 h the appearance of the phospholipase A2 activity with a superimposable profile (FIG. 2). This means that the expression of the NtPat genes is responsible for the phospholipase A2 activity soluble in the extracts of infected tobacco.

 The invention described above identifies the first genes encoding PLA2 polypeptides from plants rapidly induced in response to microbial infection. On the contrary, the tobacco gene NtTP12 previously identified as specific for petals (Drews et al., 1992, Plant Cell 4,1383-1404) is not expressed in healthy or infected leaves and therefore does not play a crucial role in defense reactions.

 The early inducibility of NtPat genes during the hypersensitive reaction is a strong argument in favor of their involvement in the production of precursors of lipid signals leading to hypersensitive resistance. These genes could also be used as markers in the search for inducers of lipid signaling and / or of resistance.

EXAMPLE 9 Expression of the NtPatl and NtPat3 Genes in E. coli: Production of Heterologous Proteins
9.1. Cloning of the coding regions of the cDNAs NtPatl and NtPat3 in the vector pGEXKG

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The bacterial vector pGEX-KG (Pharmacia Biotech) is chosen as an expression vector for its high level of expression and because it allows the production of a fusion protein between the protein of interest and the glutathione S-transferase. (GST). This fusion protein is easily purifiable by affinity for immobilized glutathione. The coding regions of the cDNAs NtPatl and NtPat3 are amplified by PCR using the DNA polymerase Pfu (Stratagene) allowing a very low error rate, and primers comprising the restriction sites XbaI and HindIII. These sites are also present in the plasmid pGEX-KG and are used for the cloning of cDNAs downstream of the GST. The primers are: NtPatlpGEX (sense): 5'-GACTCTAGAGATGACTAAATTACCACAAATGGAGAG-3 'NtPatlpGEX (antisense): 5'-CTCAAGCTTCTAGGATTTTCTTGATAGACTGTATTTC-3' NtPat3pGEX (sense): 5'-GACTCTAGAGGTTACCAAAGGAAAGATGGTAACAG-3 'NtPat3pGEX (antisense): 5'-CTCAAGCTTCTAGTTGTTTAATCTGAGTTTCCTTTC- 3 'The PCR amplification products and the plasmid pGEX-KG are digested independently with the enzymes XbaI and HindIII. The DNA fragments are purified after separation on an agarose gel and each cDNA is inserted by ligation into the vector to create the expression vectors pGEX-PATI and pGEX-PAT3. E. coli DH5a bacteria are transformed with the ligation products. The bacteria which have integrated the plasmid pGEX-PAT1 or pGEXPAT3 are identified by PCR screening on the colonies using the primers specific for the NtPat cDNAs. The plasmid DNA is extracted and the GST-NtPat junction as well as all the coding part of the NtPat cDNAs are sequenced in order to ensure that no error has been introduced during the cloning. The plasmids are then introduced into the E. coli BL21 strain (Stratagene) suitable for the expression of heterologous proteins.

9.2. Purification of fusion proteins
A preculture of BL21 bacteria containing the plasmids pGEX-PAT1 or pGEXPAT3 in LB medium is prepared in the presence of ampicillin. One liter of medium is then inoculated with this preculture. The culture develops for approximately 2 h at 37 ° C. with vigorous stirring until an absorbance of 0.6-0.8 is obtained. IPTG (0.1 mM) is then added and the induction of the expression of the fusion proteins takes place at 18 ° C. for 3 hours. After centrifugation at 4000 g for 20 min, the cell pellet is taken up in PBS buffer (150 mM NaCl, 16 mM Na2HP04, 4 mM NaH2PO4) supplemented with 1% Triton X-100, 2 mM EDTA, B-mercaptoethanol 0.1 %, PMSF 0.2 mM benzamidine. The bacteria are lysed in the French press. The lysate is centrifuged at 10,000 g for 30 min at 4 C then placed in the presence of 1 ml of glutathione-agarose beads (50% v / v) and incubated with gentle shaking at 4 C for 1

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 hour. After centrifugation at 500 g, the beads are recovered and the supernatant is returned to the presence of fresh beads and reincubated under the same conditions. The beads are combined and washed twice with PBS-Triton, then washed twice with 50 mM Tris-HCl buffer pH 7.5 and taken up in a final volume of 1 ml. Fifty units of thrombin are added and the cleavage is continued overnight at 10 C. After centrifugation at 500 g, the supernatant is recovered. The purified protein is analyzed by electrophoresis on denaturing polyacrylamide gel and quantified by comparison with known amounts of SAB.

Example 10. Enzymatic Activities of the Recombinant NtPatl and NtPat3 Polypeptides
To study the enzymatic activity, in particular phospholipase A2, polypeptides coded by the NtPat genes, the cDNAs NtPatl and NtPat3 were expressed in the E. coli bacteria in translational fusion with glutathione-S-transferase (GST) in the vector pGEXKG . About 50% of the fusion proteins were found in the soluble protein fraction of the bacterial lysate and were purified by affinity and then cleaved with thrombin (Figure 9a). In the test using the bacterial membranes as substrate, the proteins NtPatl and NtPat3 exhibit specific phospholipase A2 activities comparable to that of the native PLA2 polypeptide isolated from infected tobacco leaves.

10.1. Esterase activity measurement
The substrate (p-nitrophenyl-palmitate) is dissolved in the Triton X-100 1% SDS 0.01% solution. The reaction medium is composed of 750 l of 0.1 M Tris-HCl supplemented with 150 l (130 nmol) of substrate and this mixture is preincubated at 37 C. The enzyme is brought into a volume of 100 l then the mixture is incubated at 37 C. The absorbance of the released p-nitrophenol is measured at 400 nm.

Like potato patatin, the proteins NtPatl and NtPat3 have a low esterase activity, tested with p-nitrophenyl palmitate (Figure 9b).

10.2. Measurement of phospholipase A2 activity
To demonstrate their phospholipase A2 activity on a pure substrate, the proteins NtPatl and NtPat3 were incubated with phosphatidylcholine radiolabelled at the level of linoleic acid in position sn-2. bacteria test

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The phospholipase A2 activity was tested on the bacterial membranes labeled with 14C oleic acid prepared as described above. Amounts of 1 to 20 ng of enzyme protein are used in the test. phosphatidylcholine test
The reaction medium (90 l) contains 22.5 nmol of phosphatidylcholine (1-palmitoyl- 2- [14C] -linoleoyl-phosphatidylcholine, PC) in 50 mM Tris-HCl buffer, 3 mM CaCl2, Triton X-100 0, 05%. The enzyme extract (10 l) is added and the mixture is incubated at 37 C for 15 min. The reaction is stopped with 300 l of cold chloroform / methanol (2/1). The two phases are separated by rapid centrifugation. The organic phase containing the lipids is evaporated under nitrogen flow and then resuspended in 50 l of chloroform / methanol (2/1). The mixture is deposited on a thin layer silica chromatography plate (0.25 mm).

Migration is carried out in the chloroform / methanol / water solvent system (65/25/4).

Radioactive products are visualized by autoradiography. A bee venom phospholipase A2 is used as a reference.

 The phospholipase A2 activities measured are high and of the same level as that of the reference phospholipase A2 originating from insect venom (FIG. 9b), and much higher than that described for potato patatin (Andrews, et al., 1988, Biochem. J. 252,199-206; Senda et al., 1996, Plant Cell physiol. 37,347-353; Strickland et al., 1995, Plant Physiol.

109,667-674). The PLA1 activity, measured from the amount of 2-lysophosphatidic acid generated from the same substrate, is 10 and 100 times lower than the phospholipase A2 activity for NtPatl and NtPat3, respectively.

EXAMPLE 11 Inducing Effect of the NtPatl and NtPat3 Proteins on the Defense Responses
If the PLA2 NtPatl and NtPat3 polypeptides have endogenous substrates in plant cells, incubation of these polypeptides with a tobacco cell suspension should result in the release of fatty acids which will be converted to oxylipins. These lipid signals could then in turn induce defense responses. Phenylalanine ammonia lyase (PAL) is a key enzyme essential for the synthesis of many defense compounds in plants. PAL activity is rapidly induced by exogenous methyljasmonate in tobacco. We measured PAL activity in extracts from tobacco cells incubated for 4 h with different doses of enzymes NtPatl and NtPat3. The results presented in Figure 10 show that the two PLA2 polypeptides induce PAL activity in these cells. Results indicate that PLA2 polypeptides have released

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 fatty acids from endogenous substrates and that these fatty acids have been converted to jasmonate-type oxylipins.

Claims (49)

Claims: 1- Plant polypeptide having phospholipase A2 activity, characterized in that it catalyzes a step in the defense reaction of plants 2- Polypeptide according to claim 1, characterized in that the plant defense reaction is the Hypersensitivity Reaction 3- Polypeptide according to one of claims 1 or 2, characterized in that the step which it catalyzes is the release of fatty acids precursors of oxylipins from biological supports 4- Polypeptide according to claim 3, characterized in that said fatty acids are oleic acid, linoleic acid or linolenic acid 5- Polypeptide according to claim 3, characterized in that the oxylipins are jasmonic acid or 12-oxophytodienoic acid 6- Polypeptide according to one of claims 1 to 5, characterized in that the plant from which it comes is Tobacco 7- Polypeptide according to one of claims 1 to 6, characterized in that it comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6 , or a fragment of these sequences.  8- Isolated polynucleotide coding for a plant polypeptide having a phospholipase A2 activity, characterized in that it catalyzes a stage of the defense reaction of plants 9- Isolated polynucleotide according to claim 8, characterized in that it codes for a polypeptide according to one of claims 1 to 7 <Desc / Clms Page number 32> 10- Isolated polynucleotide according to claim 9, characterized in that it is selected from the group consisting of: (a) an isolated polynucleotide encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6, or a portion of these sequences.  (b) an isolated polynucleotide hybridizing to an isolated polynucleotide according to (a) (c) an isolated polynucleotide homologous to an isolated polynucleotide according to (a) or (b) (d) a fragment of an isolated polynucleotide according to (a ), (goat) 11- Isolated polynucleotide according to one of claims 8 to 10, characterized in that it comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5, or a fragment of these sequences.  12- chimeric gene comprising at least, operatively linked to each other: (a) a functional promoter in a host organism (b) a polynucleotide according to one of claims 8 to 11 (c) a functional terminator in a host organism 13- chimeric gene according to claim 12, characterized in that the promoter is a constitutive promoter 14- chimeric gene according to claim 12, characterized in that the promoter is an inducible promoter 15- chimeric gene according to one of claims 12 to 14, characterized in that the promoter and the terminating element are functional in plants 16- Expression or transformation vector containing a chimeric gene according to one of claims 12 to 15 17- Vector according to claim 16, characterized in that it is a plasmid, a phage or a virus <Desc / Clms Page number 33> 18- Host organism transformed with one of the vectors according to one of claims 16 or 17 19- Host organism according to claim 18, characterized in that it is a plant 20- A method of producing plants resistant to attack by pathogenic organisms characterized in that one introduces into the genome of said plants a chimeric gene according to one of claims 12 to 15 so that they express in their tissues a polypeptide according one of claims 1 to 7.  21- Method according to claim 20, characterized in that the expression of said polypeptide in said plants is constitutive 22- Method according to claim 20, characterized in that the expression of said polypeptide in said plants is induced 23- Method according to claim 22, characterized in that the expression of said polypeptide in said plants is induced during the aggression of said plants by a pathogenic organism 24- The method of claim 23, characterized in that the pathogenic organism is a virus, a bacterium, a fungus, or a nematode 25- The method of claim 24, characterized in that the virus is a mosaic virus 26- Plant resistant to attack by pathogenic organisms characterized in that it is obtained by the method according to one of claims 20 to 25 27- Plant according to claim 26, characterized in that it contains, in addition to a chimeric gene according to one of claims 12 to 15, at least one other chimeric gene containing a polynucleotide encoding a protein of interest <Desc / Clms Page number 34> 28- Plant according to claim 27, characterized in that the polynucleotide coding for a protein of interest is a polynucleotide coding for an enzyme for resistance to a herbicide 29- Plant according to claim 28, characterized in that the polynucleotide coding for an enzyme of resistance to a herbicide codes for the enzyme bar of tolerance to bialaphos, the enzyme EPSPS of tolerance to glyphosate or even the enzyme HPPD of tolerance with isoxazoles 30- Plant according to claim 27, characterized in that the polynucleotide coding for a protein of interest is a polynucleotide coding for an insecticidal toxin 31- Plant according to claim 30, characterized in that the polynucleotide coding for an insecticidal toxin codes for a toxin of the bacterium Bacillus thuringiensis 32- Plant according to claim 27, characterized in that the polynucleotide coding for a protein of interest is a polynucleotide coding for a disease resistance protein 33- Plant according to claim 32, characterized in that the polynucleotide coding for a disease resistance protein codes for the enzyme oxalate oxidase, or for an antibacterial or antifungal peptide 34- Plant according to claim 27, characterized in that the polynucleotide coding for a protein of interest is a polynucleotide coding for an agronomic character of plant 35- Plant according to claim 34, characterized in that the polynucleotide encoding an agronomic plant character codes for the enzyme delta-6 desaturase 36- Part of a plant according to one of claims 26 to 35 37- Seeds of a plant according to one of claims 26 to 35 <Desc / Clms Page number 35> 38- Monoclonal or polyclonal antibodies, characterized in that they are directed against a polypeptide according to one of claims 1 to 7 39- Method for searching for molecules stimulating the synthesis of at least one polypeptide according to one of claims 1 to 7 in plants, characterized in that: (a) two groups of plants are used, one of which is treated with a molecule (b) the quantity of at least one of the polypeptides according to one of claims 1 to 7 is measured in the tissues of each of the groups of plants according to (a) (c) the molecule according to (a) is retained when the quantity according to (b) is significantly higher in the group of treated plants compared to the group of untreated plants 40- Method according to claim 39, characterized in that: (a) two groups of plants are used, one of which is treated with a molecule (b) the mRNAs of these plants are extracted (c) these mRNAs are subjected to reverse transcription (d) subjecting the reverse transcription products to selective amplification by PCR with pairs of primers made up of polynucleotide fragments according to one of claims 8 to 11 (e) migrating the products of the amplification to an electrophoresis gel (f) the intensity of the fragments corresponding to the mRNA of a polypeptide according to one of claims 1 to 7 is measured in the gel (g) the molecule according to (a) is retained when the intensity according to (f) is significantly higher in the group of treated plants compared to the group of untreated plants 41- Method according to claim 40, characterized in that the pair of primers is selected from the group consisting of: (a) SEQ ID NO: 10 and SEQ ID NO: 11 (b) SEQ ID NO: 12 and SEQ ID NO: 13 (c) SEQ ID NO: 14 and SEQ ID NO: 15 42- Method according to claim 39, characterized in that: (a) two groups of plants are used, one of which is treated with a molecule (b) the mRNAs of these plants are extracted (c) these mRNAs are separated by electrophoresis, then we transfer them to a membrane <Desc / Clms Page number 36>  (d) the mRNAs transferred are hybridized with a probe constituted by a polynucleotide according to one of claims 8 to 11, and onto which a marker molecule is grafted (e) the intensity of the hybridization of the probe (f ) the molecule according to (a) is retained when the hybridization intensity of the probe according to (e) is significantly higher in the group of treated plants compared to the group of untreated plants 43- Method according to claim 39, characterized in that: (a) two groups of plants are used, one of which is treated with a molecule (b) the proteins of these plants are extracted (c) these proteins are separated by electrophoresis, then they are transferred to a membrane (d) the proteins transferred are hybridized with antibodies according to claim 38 onto which a marker molecule is grafted (e) the intensity of the hybridization of the antibodies is measured (f) the molecule is retained according to (a) when the antibody hybridization intensity according to (e) is significantly higher in the group of treated plants compared to the group of untreated plants 44- Molecules obtained by a process according to one of claims 39 to 43 45- A method of improving the defense of plants against attack by pathogenic organisms, characterized in that the level of expression of at least one polypeptide according to one of claims 1 is increased 7 in said plants 46- The method of claim 45, characterized in that said pathogenic organisms are viruses, bacteria, fungi or nematodes.  47- Method according to one of claims 45 or 46, characterized in that it applies to plants or parts of plants transformed according to one of claims 26 to 37 48- Method according to one of claims 45 or 46, characterized in that it applies to plants or parts of plants unprocessed <Desc / Clms Page number 37> 49- Method according to one of claims 45 to 48, characterized in that the increase in the level of expression of a polypeptide according to one of claims 1 to 7 is carried out by application to said plants of a molecule obtained by a method according to one of claims 39 to 43