EP1224308A1

EP1224308A1 - Novel class of proteins and uses thereof for plant resistance to various pathogenic agents

Info

Publication number: EP1224308A1
Application number: EP00964380A
Authority: EP
Inventors: Jocelyne Olivier; Laurent Deslandes; Yves Marco
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Recherche Agronomique INRA
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Recherche Agronomique INRA
Priority date: 1999-10-01
Filing date: 2000-09-27
Publication date: 2002-07-24
Also published as: FR2799204A1; US20030196215A1; WO2001025458A1; FR2799204B1

Abstract

The invention concerns a novel class of proteins having an N-terminal part comprising motifs characteristic of a plant protein resistant to pathogenic agents and a C-terminal part comprising an DNA-binding domain. The invention also concerns nucleic acids coding for such proteins involved in plant resistance to various pathogenic agents and means for detecting said proteins and said nucleic acids, such as antibodies or probes and nucleotide primers.

Description

New class of proteins and their applications to plant resistance to various pathogens

The present invention relates to a new class of proteins having an N-terminal part comprising motifs characteristic of a plant protein for resistance to pathogens and a C-terminal part comprising a DNA-binding domain. The invention also relates to the nucleic acids coding for such proteins involved in the resistance of plants to various pathogens as well as to means for detecting these proteins and these nucleic acids, such as antibodies or probes and primers. nucleotide. The invention also relates to recombinant vectors comprising a nucleic acid coding for a polypeptide belonging to this new class of proteins, to host cells transformed by a nucleic acid or a recombinant vector according to the invention, as well as to plants transgenics of which a part or all of the cells are transformed with a nucleic acid or a recombinant vector according to the invention.

The invention also relates to means intended to increase or on the contrary to inhibit the expression of a nucleic acid coding for a protein according to the invention in plants, with the aim of increasing the resistance of said plants to various pathogens. .

The invention also relates to methods of screening for candidate substances which bind to a polypeptide according to the invention, as well as to the candidate substances obtained according to such methods.

Improving the resistance of plants, and particularly plants of agronomic interest, to various pathogens is the subject of intense research with a view to meeting the technical needs of an agricultural industrial sector which is resorting more and more systematically intensive cultivation practices.

The identification of new genes or new proteins capable of conferring on plants an increased resistance to various bacteria, fungi or viruses is of significant economic interest in agriculture today, by making it possible to develop plants of large crop capable of naturally resisting these pathogens and therefore not requiring a massive supply of products phytosanitary, in particular pesticides, which have many disadvantages for the environment.

Bacterial wilt is one of the most widespread pathologies affecting plants of agronomic interest. It is one of the most important phytobacteriosis in the world which is caused in particular by the bacteria Ralstonia solanacearum.

This vascular pathogen, of telluric origin, affects nearly 200 plant species such as tomatoes, tobacco, potatoes and bananas, mainly in tropical and subtropical zones.

Recently, in Europe, different strains of this pathogen have been detected. In tomatoes, the study of resistance to Ralstonia solanacearum is complicated by its polygenic nature. Several genes involved in establishing resistance have been identified.

Recently, a study by DESLANDES et al. (1998) identified a chromosomal locus in the Arabidopsis thaliana plant carrying a genetic determinant involved in resistance to Ralstonia solanacearum. Thus, lines of Arabidopsis thaliana, constituting the Fg generation, obtained by crossing lines of Arabidopsis thaliana respectively sensitive and resistant to Ralstonia solanacearum, were used for the study of the co-segregation of markers of RLFP type with l expression of the resistance phenotype to this pathogen responsible for wilting. These authors have thus shown that a genetic determinant of resistance to Ralstonia solanacearum was localized, on chromosome V of Arabidopsis thaliana, between the RFLP markers mi83 and mi61, that is to say in a region of approximately 6 , 8 cM (approximately 1.7 to 3.4 Mb) of this chromosome. According to these authors, the results of the study suggest that the locus of interest located on chromosome V carries one or more genes, these genes being able either to confer the phenotype of resistance, or on the contrary to confer the phenotype of sensitivity to Ralstonia solanacearum, although the genetic basis for resistance and / or susceptibility to this pathogen has not been identified in Arabidopsis thaliana. In addition, DESLANDES et al. (1998) also observe that the chromosomal region of interest of Arabidopsis thaliana was known to contain many other genetic determinants involved in recognition of pathogens. The applicant has now identified a genetic determinant which is capable of conferring resistance to Ralstonia solanacearum in Arabidopsis thaliana.

It is a single gene encoding a polypeptide belonging to a new structural class of proteins. This new class of proteins identified according to the invention is characterized in that it has an N-terminal part having structural motifs in common with several known resistance genes and a C-terminal part having structural motifs in common with proteins transactivators having a DNA binding domain, in particular designated under the name of WRKY proteins or else WRKY transcription factors.

It has thus been shown according to the invention, that the transformation of an Arabidopsis thaliana plant sensitive to Ralstonia solanacearum with a nucleic acid coding for a protein as defined above was capable of conferring on the transformed plant resistance to different strains of this pathogen.

The subject of the invention is therefore a nucleic acid comprising at least 15 consecutive nucleotides of a nucleotide sequence coding for a protein for resistance of a plant to a pathogen, said protein comprising: a) an N-terminal part comprising at least one amino acid sequence rich in leucine and at least one nucleotide binding site; and b) a C-terminal part comprising a DNA binding domain, said binding domain comprising the amino acid sequence "WRKYGQK".

Preferably, the N-terminal part of this protein also comprises at least one TIR domain (TOLL / IL-1 R), such a TIR domain being defined in particular by its sequence homology with the cytoplasmic domains of the TOLL protein from Drosophila as well that with the mammalian interleukin-1 receptor protein, the consensus sequence of which is described by HAMMOND-KOSACK et al. (1997).

Preferably, the N-terminal part of this protein will also comprise a P loop (P-LOOP), which is a peptide segment forming a loop and capable of binding a phosphate, which is found in protein kinases.

Such a TIR domain will preferably have the amino acid sequence

"DEEFVCISCVEEVRYSFVSHLSEALRRKGINNVWDVDIDDLLFKESQAKI EKAGVSVMVLPGNCDPSEVWLDKFAKVLECQRNNKDQAWSVLYGDSLL RDQWLSELDFRGLSRIHQSRKECDDSILVE

Such a P loop will preferably have the amino acid sequence "CVGIWGMPGIGKTTLAKAV".

Preferably, the N-terminal part also comprises a domain of the NB-ARC type, which is a conserved domain and characteristic of proteins for resistance to pathogens as well as to proteins involved in the mechanisms of apoptosis. Preferably, this NB-ARC domain has the amino acid sequence represented in bold and underlined in FIGS. 1 and 2.

In addition, the N-terminal part preferably comprises a nuclear signaling site of the NLS type.

Preferably, such an NLS domain has the amino acid sequence "KKKLSEMETAFLKLKRRPP".

As already indicated, the C-terminal part of a protein encoded by a nucleic acid according to the invention comprises a WRKY domain characteristic of certain transactivating proteins, in particular certain transcription factors and characterized in that it comprises the acid sequence "WRKYGQK" amines.

Preferably, this WRKY domain comprises the following amino acid sequence:

"DXXXWRKYGQKXIXGXXXPRXYYXCXXXXXXXCXXXKXXXXXEXXXXXXX XXYXSXHXH", wherein X represents any of the 20 natural amino acids.

In addition, the C-terminal part preferably comprises a domain rich in leucine. The C-terminal part preferably also comprises a domain characteristic of a nuclear signaling site (NLS), said nuclear signaling domain preferably having the amino acid sequence "NFHCWAPGKWPKVRKD".

Preferably, the C-terminal part also comprises a motif of the “leucine zipper” type, characteristic of a protein binding site, which preferably has the amino acid sequence “LRVSYDDLQEMDKVLFLYIASL”.

Most preferably, a nucleic acid according to the invention codes for a polypeptide comprising, from the N-terminal to the C-terminal end, (i) a TIR domain, (ii) a P-LOOP motif , (iii) a NB-ARC motif, (iv) an NLS domain, (v) a first region rich in leucine, (vi) a second region rich in leucine,

(vii) an NLS domain, (viii) a leucine zipper type motif, (ix) a WRKY domain, the domains (i) to (ix) being as defined above.

Also part of the invention is a nucleic acid of complementary sequence.

Preferably, a nucleic acid according to the invention is in an isolated and / or purified form. The term "isolated" within the meaning of the present invention designates a biological material (nucleic acid or protein) which has been removed from its original environment (the environment in which it is naturally located).

For example, a polynucleotide naturally occurring in a plant or animal is not isolated. The same polynucleotide separated from Adjacent nucleic acids within which it is naturally inserted into the genome of the plant or animal is considered to be "isolated".

Such a polynucleotide may be included in a vector and / or such a polynucleotide may be included in a composition and nevertheless remain in an isolated state since the vector or the composition does not constitute its natural environment.

The term "purified" does not require that the material be present in a form of absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition. A polynucleotide is in the “purified” state after purification of the starting material or of the natural material of at least one order of magnitude, preferably 2 or 3 and preferably 4 or 5 orders of magnitude.

For the purposes of the present invention, the expression “nucleotide sequence” can be used to denote either a polynucleotide or a nucleic acid. The term "nucleotide sequence" encompasses the genetic material itself and is therefore not limited to information regarding its sequence.

The terms "nucleic acid", "polynucleotide",

"Oligonucleotide" or "nucleotide sequence" include RNA, DNA, cDNA or hybrid sequences

RNA / DNA of more than one nucleotide, either in the single chain form or in the form of duplex.

The term “nucleotide” designates both natural nucleotides (A, T, G, C) as well as modified nucleotides which comprise at least one modification such as (1). An analog of a purine, (2) an analog of a pyrimidine, or (3) an analogous sugar, examples of such modified nucleotides being described for example in PCT application No. WO 95/04 064.

For the purposes of the present invention, a first polynucleotide is considered to be “complementary” to a second polynucleotide when each base of the first nucleotide is paired with the base complementary to the second polynucleotide whose orientation is reversed.

The complementary bases are A and T (or A and U), or C and G.

Without wishing to be bound by any theory, the inventors believe that the different patterns characteristic of the class of proteins according to the invention are such as to confer on it its biological function which results in the observation of a phenotype of resistance towards plant pathogens, in particular towards the bacteria R. solanacearum.

Thus, the N-terminal part, which includes the TIR, NB-ARC domains and one or more regions rich in leucine, are characteristic of many pathogen resistance proteins in plants (R proteins), these proteins being supposed to activate metabolic pathways. cascade signaling systems that coordinate the plant's initial defense responses preventing pathogens from progressing. The WRKY domain of the C-terminal part of a protein according to the invention could have a binding function to regulatory sequences located near or inside promoter regions of plant genes, the binding of a protein according to the invention. he invention of DNA is thus capable of modulating the activity of other genes potentially involved in the resistance of plants to pathogens, and more particularly to R. solanacearum.

The applicant has isolated and characterized a nucleic acid coding for a polypeptide belonging to the class of proteins for resistance to plant pathogens defined above, the protein RRS1-R. More specifically, the applicant isolated, from the genome of a plant of Arabidopsis thaliana resistant to R. solanacearum, the gene RRS1 -R which is capable of conferring on a plant a phenotype of resistance to R.solanacearum, such a nucleic acid constituting the sequence SEQ ID No. 1. The invention relates to a nucleic acid comprising at least 15 consecutive nucleotides of a nucleotide sequence having at least 40% nucleotide identity with a polynucleotide of sequence SEQ ID No. 1, as well as a nucleic acid of complementary sequence.

According to the invention, a first nucleic acid having at least 40% identity with a second reference nucleic acid will have at least 60%, preferably at least 80%, 85%, 90%, 95%, 98%, 99% or 99.5% of nucleotide identity with this second reference polynucleotide, the percentage of identity between two sequences being determined as described below. The “percentage of identity” between two nucleotide or amino acid sequences, within the meaning of the present invention, can be determined by comparing two optimally aligned sequences, through a comparison window. The part of the nucleotide or polypeptide sequence in the comparison window can thus include additions or deletions (for example “gaps”) with respect to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which an identical nucleic base or amino acid residue is observed for the two sequences (nucleic or peptide) compared, then by dividing the number of positions at which there is identity between the two bases or amino acid residues by the total number of positions in the comparison window, then multiplying the result by one hundred to obtain the percentage of sequence identity.

The optimal alignment of the sequences for the comparison can be achieved by computer using known algorithms contained in the software tool of the company WISCONSIN GENETICS SOFTWARE PACKAGE, GENETICS COMPUTER GROUP (GCG), 575 Science Doctor, Madison, Wisconsin.

Most preferably, the percentage of identity between two sequences is carried out using BLAST software (BLAST version 2.06 of September 1998), using exclusively the default parameters (SF ALTSCHUL et al., (1990) ; SF ALTSCHUL et al., 1997).

The genomic sequence of the RRS1 -R gene is referenced as the sequence SEQ ID No. 1.

Another subject of the invention is a nucleic acid which comprises or consists of the sequence SEQ ID No. 1. The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of the polynucleotide of sequence SEQ ID No. 1.

Each of the genomic nucleic acids according to the present invention can be easily obtained by a person skilled in the art who knows its nucleotide sequence disclosed in the present description. Those skilled in the art can also reproduce any of the genomic nucleic acids according to the invention by constructing, on the basis of the sequences disclosed in the present description, oligonucleotide primers capable of amplifying all or part of these nucleic acids from a plant genome, preferably a genome of Arabidopsis thaliana, or even from a vector bank (YACS, BACS, cosmids) containing genomic inserts of a plant, and preferably Arabidopsis thaliana.

After amplification using the appropriate primers, the different amplified nucleic acids are then subjected to a ligation step in a plasmid vector in order to obtain the desired genomic nucleic acid, according to techniques well known to those skilled in the art. .

Furthermore, the isolation of the DNA fragment containing the desired genomic insert can be obtained by subcloning from a library (YACS, BACS, cosmids) containing genomic inserts from a plant, and preferably from Arabidopsis thaliana.

The RRS1-R gene sequence includes seven exons and six introns, as well as potentially regulatory regions located respectively on the 5 'side of the first exon and the 3' side of the last exon, the structural characteristics of these exons and introns being detailed in the Tables 1 and 2 below.

TABLE 1: Sequence of exons of the RRSI-R gene

The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of an exonic polynucleotide of the RRSI-R gene, such as the polynucleotides 1 to 7 described in table 1 above, which are all included in the nucleic acid with sequence SEQ ID No. 1.

In general, a nucleic acid having at least 15 consecutive nucleotides of a sequence according to the invention advantageously has at least 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500 , 1000, 2000, 3000, 4000 consecutive nucleotides of the reference sequence, the length of consecutive nucleotides being naturally limited by the length of the reference sequence.

Such a nucleic acid codes for at least part of the RRS1-R polypeptide and can in particular be inserted into a recombinant vector intended for the expression of the corresponding translation product in a host cell or in a plant transformed with this recombinant vector.

Such a nucleic acid can also be used for the synthesis of probes and nucleotide primers intended for the detection or the amplification of nucleotide sequences included in the RRS1-R gene in a sample, if necessary of sequences of the RRS1- genes. R carrying one or more mutations, preferably one or more mutations such as to modify the phenotype of a plant carrying such a mutated RRS1-R gene, for example by modifying the regulation of the mechanisms of resistance to pathogens, and preferably the mechanisms of resistance to R. solanacearum.

TABLE 2

Regulatory and intronic sequences of the RRS-IR gene

The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of an intronic polynucleotide of the gene

RRS1-R, such as polynucleotides 1 to 6 described in Table 2 above, which are all included in the nucleic acid of sequence SEQ ID

# 1.

Such a nucleic acid can be used as a probe or oligonucleotide primer to detect the presence of at least one copy of the gene

RRS1-R in a sample, or to amplify a target sequence determined within the RRS1-R gene.

In addition, the nucleic acid of the RRS1 -R gene of sequence SEQ ID

No. 1 includes an ATG codon starting at the nucleotide at position 260 (1 st

Exon) and a polyadenylation signal between the nucleotides at positions 6642 and 6647 included. The nucleic acid of the RRS1-R gene of sequence SEQ ID No. 1 comprises regulatory sequences located respectively on the 5 'side of exon 1 and on the 3' side of exon 7.

The 5 'regulatory region of the RRS1-R gene comprises the nucleotide sequence SEQ ID No. 2. The polynucleotide of sequence SEQ ID No. 2 is located between the nucleotide in position 1 and the nucleotide in position 259 of the sequence SEQ

ID # 1. The 3 ′ regulatory sequence of the RRSI-R gene comprises the nucleotide sequence SEQ ID No. 3.

The polynucleotide of sequence SEQ ID No. 3 is located between the nucleotide at position 6533 and the nucleotide at position 7936 of the nucleotide sequence SEQ ID No. 1.

The polynucleotide derived from the regulatory regions of the above RRS1-R gene is useful for detecting the presence of at least one copy of this gene in a sample.

The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of a polynucleotide of sequence SEQ ID No. 2 or SEQ ID No. 3.

According to another aspect, the invention also relates to a nucleic acid comprising a polynucleotide having at least 80% identity in nucleotides with a polynucleotide of sequence SEQ ID No 2 or SEQ ID No 3, advantageously at least 90% , 95%, 98%, 99%, 99.5% and very preferably 99.8% of nucleotide identity with a polynucleotide chosen from the sequences SEQ ID No. 2 and SEQ ID No. 3, or a biologically active fragment of the latter.

Preferred fragments of the nucleotide sequences SEQ ID No. 2 or SEQ ID No. 3 advantageously have a length of between 500, 100, 150, 200 to 300, 400, 600, 1000 or 2000 bases.

The term “biologically active” fragment of a polynucleotide of sequence SEQ ID No. 2 or SEQ ID No. 3 according to the invention means a polynucleotide comprising or consisting of a polynucleotide capable of regulating the expression of a nucleic acid placed near the latter, in a recombinant host cell.

For the purposes of the present invention, a nucleic acid constitutes a “functional” regulatory region for expressing a nucleic acid placed close to the latter, for example a nucleic acid coding for a polypeptide or a polynucleotide of interest, if this regulatory polynucleotide contains the nucleotide sequences containing signals for regulating transcription and / or translation, and if the sequences are localized so as to effectively induce or increase the transcription or translation of said polypeptide or polynucleotide of interest. In order to identify the “biologically active” fragments of the polynucleotides of sequence SEQ ID No. 2 or SEQ ID No. 3, a person skilled in the art can advantageously refer to the work by SAMBROOK et al. (1989) which describes the use of a recombinant vector carrying a marker gene (for example β-galactosidase, chloranphenicol acetyltransferase, etc.) the expression of which is detected by placing the sequence of the marker gene under the control of polynucleotide fragments biologically active sequences SEQ ID N ° 2 or SEQ ID N ° 3.

The genomic sequences located upstream of the first exon of the RRS1-R gene are cloned into an appropriate vector containing a marker gene, such as the GUS gene (Jefferson et al., 1987).

Fragments of polynucleotides of sequence SEQ ID No. 2 or

SEQ ID NO: 3 according to the invention can be prepared from any of the sequences SEQ ID No 1, SEQ ID No 2 and SEQ ID No 3 by cleavage using the appropriate restriction endonucleases, as described for example in the work of SAMBROOK et al. (1989) cited above.

The fragments of the regulatory polynucleotides according to the invention can also be prepared by digestion of any of the sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 with an exonuclease enzyme, such as for example Bal31 as described by WABIKO et al.

(1986), WABIKO H. et al., 1986, DNA, volume 5 (4): 305-314.

Such regulatory polynucleotides can also be prepared by chemical synthesis of nucleic acids according to techniques well known to those skilled in the art, such as the abovementioned phosphoramidite techniques.

The activity level and the tissue specificity of the regulatory sequences, in particular the promoter sequences of the RRS1-R gene according to the invention can be determined by measuring the expression levels of a detectable polynucleotide placed under the control of the latter in different types of plant cells and tissues. The detectable polynucleotide can be either a polynucleotide which specifically hybridizes with an oligonucleotide probe of predetermined sequence, or a polynucleotide encoding a detectable protein, including the RRS1-R polypeptide or a fragment thereof. A test allowing such verification is well known to those skilled in the art and is described in particular in US patents No. 5,502,176 and US No. 5, 266,488, incorporated herein by reference.

The invention therefore also relates to a nucleic acid comprising: a) a nucleic acid comprising a nucleotide regulatory sequence of at least 50 consecutive nucleotides of the sequence SEQ ID No. 3 or of a sequence having at least 80% of nucleotide identity with the sequence SEQ ID No. 2; b) a polynucleotide encoding a polypeptide of interest or a polynucleotide of interest whose transcription and / or translation is placed under the control of the regulatory nucleic acid a); c) where appropriate, a regulatory nucleic acid comprising at least 50 consecutive nucleotides of the sequence SEQ ID No. 3 or of a sequence having at least 80% nucleotide identity with the sequence SEQ ID No. 3.

The polypeptide of interest encoded by the nucleic acid b) above can be of varied nature and origin; it may be a protein of eukaryotic or prokaryotic origin. Among the polypeptides of interest are toxic polypeptides, such as, for example, elicitins, for plant cells, their expression being such as to induce the death of the cell in which they are expressed and consequently a containment of the pathogens having infected the plant. , thereby preventing the spread of the pathogen to all organs of the plant. For example, it is possible to generate transgenic tobacco with a high level of resistance to pathogens by expressing elicitins, small toxic peptides produced by a phytopathogenic fungus according to the technique described by Keller et al. (1999). The nucleic acid of interest mentioned in b) above, generally an RNA molecule, can be complementary to a target polynucleotide, for example a polynucleotide located in the coding region of the RRS1-R gene, and can thus be advantageously used as an antisense polynucleotide. It has been shown according to the invention that the RRS1-R gene is transcribed in the form of a messenger RNA which has been isolated and characterized. This messenger RNA comprises a unique open reading frame coding for the protein RRS1-R with a length of 1378 amino acids. The messenger RNA of the RRS1-R gene and the corresponding cDNA can be easily obtained by a person skilled in the art, for example by screening a cDNA band using an appropriate probe or by rapid amplification cDNA ends (PCR, RACE-PCR). Those skilled in the art can again obtain the cDNA for the RRS1-R gene using the techniques described in Example 6, or also by direct chemical synthesis.

Those skilled in the art can also reproduce a nucleic acid according to the invention by direct chemical synthesis, such as the phosphodiester method described by NARANG et al. (1979), the phosphodiester method described by BROWN et al. (1979), the diethylphosphoramidite method described by BEAUCAGE et al. (1981), as well as the method on solid support described in European patent application EP 0 707 592, the content of these documents being incorporated here by reference.

On either side of the open reading frame, this messenger RNA respectively comprises a 5 'untranslated region (5'-UTR) and a 3' untranslated region (3'-UTR).

The cDNA resulting from the transcription of the RRS1-R gene is referenced as the sequence SEQ ID No. 4.

The 5'-UTR sequence of the messenger RNA transcribed by the RRS1-R gene consists of the sequence going from the nucleotide in position 1 to the nucleotide in position 81 of the sequence SEQ ID No. 4.

The 3'-UTR sequence of the messenger RNA transcribed by the RRS1-R gene consists of the sequence going from the nucleotide at position 4219 to the last nucleotide at the 3 'end of the sequence SEQ ID No. 4.

The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of a polynucleotide chosen from the 5'-UTR and 3'-UTR sequences of the messenger RNA of the RRS1-R gene described above, a polynucleotide having at at least 80% nucleotide identity with such a nucleic acid as well as a nucleic acid of sequence complementary to said nucleic acids or to said polynucleotides. Also part of the invention is a nucleic acid which comprises, or which consists of the nucleotide sequence SEQ ID No. 4.

The 5'-UTR and 3'-UTR regions of the messenger RNA of the RRS1-R gene are capable of containing elements for regulating the transcription and / or translation of this gene, such as one or more sites of binding to the ribosome, one or more polyadenylation sites, the sequences allowing the stability of messenger RNA or even all or part of the promoter region of transcription.

The applicant also isolated and characterized, from an ecotype of Arabidopsis thaliana sensitive to the pathogen R. solanacearum, the gene corresponding to the RRS1-R gene, comprising, relative to the sequence of the RRS1-R gene, several additions and substitutions of nucleotides, the mutated gene found in the sensitive ecotype of Arabidopsis thaliana being designated RRS1-S for the purposes of this description. The RRS1-S gene comprises a first mutation in the part coding for the C-terminal region of the protein, leading to the replacement of the amino acid sequence "SEASKLERL" going from the amino acid in position 704 to the amino acid in position 712 of the polypeptide encoded by the RRS1-R gene of sequence SEQ ID No. 1 by the amino acid sequence "SEELERL" ranging from the amino acid in position 704 to the amino acid in position 710 of the polypeptide encoded by the gene RRS1-S. This mutation thus leads, on the one hand, to the substitution of the alanine residue in position 706 of the protein coded by the RRS1-R gene with a glutamic acid residue in position 706 of the protein coded by the RRS1-S gene and , on the other hand, to the deletion of the amino acids serine and lysine respectively in position 707 and 708 of the amino acid sequence of the protein encoded by the RRS1-R gene of sequence SEQ ID No. 1.

A second mutation consists in the insertion of an early stop codon found in the coding region of the RRS1-S gene, leading to a deletion of the 90 amino acids located at the C-terminal end of the RRS1-R protein, the polypeptide encoded by the RRS1-S gene being accordingly 1288 amino acids.

Without wishing to be bound by any theory, the applicant believes that the various mutations carried by the RRS1-S gene identified in the Col-5 ecotype of Arabidopsise thaliana, which is sensitive to the pathogen R. solanacearum, are responsible for the phenotype of sensitivity to R. solanacearum observed in this ecotype.

It follows that the various modifications observed in the nucleotide sequence of the RRS1-S gene with respect to the nucleotide sequence of the RRS1-R gene are such as to significantly alter the biological activity of the resulting polypeptide.

Consequently, the nucleotide sequence of the RRS1-S gene found in the Colid-5 ecotype of Arabidopsis thaliana is useful in particular for the development of various means of specific detection of the RRS1-S gene, such means of detection allowing those skilled in the art to determine whether a plant of interest contains in its genome the RRS1-S gene and is consequently sensitive to the bacterial pathogen R. solanacearum.

The genomic sequence of the RRS1-S gene is referenced as the sequence SEQ ID No. 5. Another subject of the invention is a nucleic acid which comprises where is made up of the sequence SEQ ID No. 5.

The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of a polynucleotide of sequence SEQ ID No. 5.

The invention also relates to a nucleic acid having at least 40% nucleotide identity with the nucleotide sequence SEQ ID

No. 5 or with a polynucleotide comprising at least 15 consecutive nucleotides of the nucleotide sequence SEQ ID No. 5, as well as a nucleic acid of complementary sequence.

The RRS1-S gene sequence comprises 7 exons and 6 introns, the structural characteristics of which are detailed in Tables 3 and 4 respectively.

TABLE 3 EXONIC SEQUENCES OF THE RRS1-S GENE

The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of an exonic polynucleotide of the RRS1-S gene, such as the polynucleotides 1 to 7 described in table 3 above, which are all included in the nucleic acid with sequence SEQ ID No. 5. Such a nucleic acid codes for at least part of the RRS1-S polypeptide and can in particular be inserted into a recombinant vector intended for the expression of the corresponding translation product in a host cell or in a plant transformed with this recombinant vector. Such a nucleic acid can also be used for the synthesis of nucleotide probes and primers intended for the detection or the amplification of nucleotide sequences included in the RRS1-S gene in a sample.

TABLE 4 1NTRONIC SEQUENCES OF THE RRS1-S GENE

The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of an intronic polynucleotide of the RRS1-S gene, such as the polynucleotides 1 to 7 described in table 4 above, which are all included in the nucleic acid with sequence SEQ ID No. 5.

Such a nucleic acid can be used as a probe or oligonucleotide primer to detect the presence of at least one copy of the RRS1-S gene in a sample, or alternatively to amplify a target sequence determined within the RRS1-S gene. Preferably, such probes are constructed so as to specifically hybridize with the regions of the RRS1-S gene which comprise one or more basic substitutions, additions or deletions with respect to the nucleotide sequence of the RRS1-R gene of sequence SEQ ID N 1. Preferably, such primers make it possible to amplify the regions of the RRS1 -S gene comprising one or more basic substitutions, additions or deletions with respect to the nucleotide sequence of the RRS1-R gene of sequence SEQ ID No. 1.

The applicant has also isolated and characterized the non-transcribed nucleotide sequences located respectively on the 5 'side and on the 3' side of the coding regions of the RRS1-S gene. These non-transcribed 5 'and 3' sequences are capable of carrying signals for regulating the transcription and / or translation of the RRS1-S gene and are also part of the invention, and are respectively referenced as sequences SEQ ID N ° 6 and SEQ ID N ° 7. The invention therefore therefore also relates to a nucleic acid comprising at least 50 consecutive nucleotides of a polynucleotide regulating the RRS1-S gene chosen from the nucleotide sequences SEQ ID No. 6 and SEQ ID No. 7 and the sequences having at least 80% d identity in nucleotides with one of the sequences SEQ ID No. 6 and SEQ ID No. 7, as well as a nucleic acid of sequence complementary to said nucleic acids to said polynucleotides.

The invention also relates to a nucleic acid comprising: a) a 5 'regulatory polynucleotide of the RRS1-S gene as defined above; b) a polynucleotide encoding a polypeptide or a polynucleotide of interest; c) where appropriate, a 3 ′ regulatory polynucleotide of the RRS1-S gene as defined above,

the nucleic acid coding for the polypeptide or polynucleotide of interest being placed under the control of the regulatory polynucleotide 5 'of the RRS1-S gene and, if appropriate, also under the control of the polynucleotide 3' of the RRS1-S gene.

The applicant has shown that the RRS1-S gene is transcribed in the form of a messenger RNA which has been isolated and characterized. This messenger RNA comprises a unique open reading frame coding for the protein RRS1-S with a length of 1288 amino acids.

On either side of the open reading frame, this messenger RNA respectively comprises a 5 'untranslated region (5'-UTR) and a 3' untranslated region (3'UTR).

The cDNA of the RRS1-S gene is referenced as the sequence SEQ ID No. 8.

The 5'-UTR sequence of the messenger RNA transcribed by the RRS1-S gene consists of the sequence going from the nucleotide in position 1 to the nucleotide in position 81 of the sequence SEQ ID No. 8. The 3'-UTR sequence of the messenger RNA transcribed by the RRS1-S gene consists of the sequence going from the nucleotide at position 3949 to the last nucleotide at the 3 'end of the sequence SEQ ID No. 8.

The invention also relates to a nucleic acid comprising at least 15 consecutive nucleotides of a polynucleotide of sequence SEQ ID

No. 8 or 15 consecutive nucleotides of a sequence having at least 40% nucleotide identity with the sequence SEQ ID No. 8, as well as a sequence complementary to said nucleic acid.

The nucleic acids according to the invention, and in particular the nucleotide sequences SEQ ID No. 1 to SEQ ID No. 8, their fragments of at least 15 nucleotides, the sequences having at least 40% identity in nucleotides with at least part of the sequences SEQ ID No. 1 to SEQ ID No. 8, as well as the nucleic acids of complementary sequences, are useful for detecting the presence of at least one copy of a nucleotide sequence of the RRS1-R gene or RRS1-S or a fragment or an allelic variant of the latter in a sample.

Also included in the invention are the nucleotide probes and primers hybridizing, under high stringency hybridization conditions, with a nucleic acid chosen from the sequences SEQ ID No. 1 to SEQ ID No. 8.

By high stringency hybridization conditions within the meaning of the invention means the following hybridization conditions:

- the DNA to be tested is immobilized on Hybond-N ⁺ type membranes (Amersham, Buckingamshire, United Kingdom) according to the manufacturer's instructions, in the presence of 0.4 M NaOH overnight;

- the membranes are washed with a 5 x SSC buffer (1 x SSC and corresponds to 0.15 M NaCl + 0.015 M sodium citrate), then the membranes are treated with UV at 312 nm for 3 minutes, then prehybridized for at least 4 hours at 65 ° C in a hybridization buffer (6 x SSC, 5 x Denhardt's, 100 μg of single-stranded DNA of calf thymus / ml and 0.5% SDS [weight / volume]).

The probes are added to the membranes and incubated at 65 ° C overnight. After the hybridization step, the membranes are washed in 500 ml of a 2 × SSC, 1% SDS (weight / volume) buffer at laboratory temperature for 30 minutes;

- a second washing is carried out in 500 ml of a 0.1 x SSC, 0.1% SDS (weight / volume) buffer for 15 minutes at 42 ° C.

The hybridization conditions described above are suitable for hybridization under conditions of high stringency, a nucleic acid molecule with a length of 300 to 400 nucleotides. It goes without saying that the hybridization conditions described above can be adapted as a function of the length of the nucleic acid for which hybridization is sought or of the type of labeling chosen, according to techniques known to those skilled in the art. .

The suitable hybridization conditions can for example be adapted according to the teaching contained in the work of HAMES and HIGGINS (1985) or even in the work of AUSUBEL et al; (1989).

The nucleotide probes or primers according to the invention comprise at least 15 consecutive nucleotides of a nucleic acid according to the invention, in particular of a nucleic acid of sequence SEQ ID No. 1 to SEQ ID No. 8 or of its sequence complementary, of a nucleic acid having at least 40% nucleotide identity with a sequence chosen from the sequences SEQ ID N ° 1 to SEQ ID N ° 8 or of its complementary sequence or of a hybridizing nucleic acid, in high stringency hybridization conditions, with a sequence chosen from the sequences SEQ ID No. 1 to SEQ ID No. 8 or its complementary sequence.

Preferably, nucleotide probes or primers according to the invention have a length of at least 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400 or 500 consecutive nucleotides of a nucleic acid according to the invention, in particular a nucleic acid of nucleotide sequence chosen from the sequences SEQ ID No. 1 to SEQ ID No. 8, or of a nucleic acid of complementary sequence.

According to another aspect a probe or a primer of the nucleotide according to the invention will consist and / or include fragments with a length of 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300 , 400 or 500 nucleotides consecutive of a nucleic acid according to the invention, more particularly of a nucleic acid chosen from the sequences SEQ ID No. 1 to SEQ ID No. 8, or of a nucleic acid of complementary sequences.

Examples of primers and of pairs of primers making it possible to amplify different regions of the RRS1-R gene are, for example, the sequences SEQ ID No. 11 to SEQ ID No. 61 shown in Table 6.

A primer or a nucleotide probe according to the invention can be prepared by any suitable method well known to those skilled in the art, including by cloning and action of restriction enzymes or also by direct chemical synthesis according to techniques such as methods of phosphodiester from Narang et al. (1979) or Brown et al. (1979) cited above.

Each of the nucleic acids according to the invention, including oligonucleotide probes and primers described above, can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical or even chemical means.

For example, such markers can consist of radioactive isotopes ( ³² P, ³ H, ³⁵ S), fluorescent molecules (5-bromodeoxyuridine, fluorescein, acetylaminofluorene, digoxigenin) or also ligands such as biotin.

The labeling of a nucleic acid is preferably carried out by incorporating labeled molecules within these nucleotides by extension of primers, or else by adding to the 5 ′ or 3 ′ ends.

Examples of non-radioactive labeling of nucleic acid fragments are described in particular in patent FR 78 19 175 or also in the articles of URDEA et al. (1988), or SANCHEZ-PESCADOR et al. (1988).

Advantageously, the probes according to the invention can have structural characteristics such as to allow amplification of the signal, such as the probes described by URDEA et al; (1991) or in European patent No. EP 0 225 807 (Chiron).

The oligonucleotide probes according to the invention can be used in particular in Southern type hybridizations with genomic DNA, or in hybridizations with gene messenger RNA RRS1-R and RRS1-S, when the expression of the corresponding transcript is sought in a sample.

The probes according to the invention can also be used for the detection of PCR amplification products or even for the detection of mismatches.

Nucleotide probes or primers according to the invention can be immobilized on a solid support. Such solid supports are well known to those skilled in the art and include surfaces of the microtiter plate wells, polystyrene beads, magnetic beads, nitrocellulose strips or even microparticles such as latex particles.

The present invention also relates to a method for detecting the presence of a nucleic acid of the RRS1-R or RRS1-S gene in a sample, said method comprising the steps of:

- contacting a probe or a plurality of nucleotide probes according to the invention with the sample to be tested;

- detecting the complex possibly formed between the probe (s) and the nucleic acid present in the sample.

According to a particular embodiment of the detection method according to the invention, the oligonucleotide probe (s) are immobilized on a support. In another aspect, the oligonucleotide probes include a detectable marker.

The invention further relates to a kit or kit for detecting the presence of a nucleic acid of the RRS1-R or RRS1-S gene in a sample, said kit comprising:

a) one or more nucleotide probes, as described above;

b) where appropriate, the reagents necessary for the hybridization reaction. According to a first aspect, the detection kit or kit is characterized in that the probe or probes are immobilized on a support.

According to a second aspect, the detection kit or kit is characterized in that the oligonucleotide probes comprise a detectable marker.

According to a particular embodiment of the detection kit described above, such a kit will comprise a plurality of oligonucleotide probes in accordance with the invention which can be used to detect target sequences of interest of the RRS1-R or RRS1-S gene or else to detect mutations in the coding regions or the non-coding regions of the RRS1 -R gene, more particularly of the nucleic acids of the sequences SEQ ID No. 1 to SEQ ID No. 8 where the nucleic acids of complementary sequences.

The nucleotide primers according to the invention can be used to amplify any nucleotide fragment (gDNA, cDNA, mRNA) of RRS1-R or RRS1-S, and more particularly all or part of a nucleic acid of sequence SEQ ID No. 1 to SEQ ID N ° 8.

Another subject of the invention relates to a method for the amplification of a nucleic acid of the RRS1 -R or RRS1-S gene, and more particularly a nucleic acid of sequence SEQ ID No. 1 to SEQ ID No. 8 or a fragment or a nucleic acid of sequence complementary to the latter, contained in a sample, said method comprising the steps of:

a) contacting the sample in which the presence of the target nucleic acid is suspected with a pair of nucleotide primers whose hybridization position is located respectively on the 5 ′ side and on the 3 ′ side of the region of l the target nucleic acid of RRS1-R or RRS1 -S whose amplification is sought, in the presence of the reagents necessary for the amplification reaction and;

b) detection of the possibly amplified nucleic acid.

According to the above amplification method, at least one amplification cycle of the nucleic acid contained in the sample is carried out. prior to the detection of the optionally amplified nucleic acid, preferably at least 10, and very preferably at least 20, amplification cycles.

To implement the above amplification method, advantage will be had to any of the nucleotide primers previously described.

The subject of the invention is also a kit or kit for the amplification of a nucleic acid of the RRS1-R or RRS1-S gene according to the invention, and more particularly all or part of a nucleic acid of sequence SEQ ID N ° 1 to SEQ ID N ° 8, said kit or kit comprising:

a) a pair of nucleotide primers in accordance with the invention, the hybridization position of which is located respectively on the 5 ′ side and on the 3 ′ side of the target nucleic acid of the RRS1-R or RRS1-S gene, the amplification is sought;

b) where appropriate, the reagents necessary for the amplification reaction.

Such an amplification kit or kit will advantageously comprise at least one pair of nucleotide primers as previously described.

According to a preferred embodiment, primers according to the invention comprise all or part of a polynucleotide chosen from nucleotide sequences SEQ ID No. 11 to SEQ ID No. 61.

The applicant has shown that plants carrying the RRS1 -R gene have a resistance to R. solanacearum phenotype. In addition, the applicant has also shown that the insertion of the RRS1-R gene or even the cDNA of the RRS1-R gene into the genome of a plant initially sensitive to R. solanacearum gives this plant a phenotype of resistance to this pathogen.

The invention also relates to methods and means intended to inhibit or block the expression of the RRS1-S gene found in plants sensitive to R. solanacearum, in particular with a view to increasing their resistance to different pathogens and this by any technique known to those skilled in the art.

In order to inhibit or block the expression of the RRS1-S gene in a plant, a person skilled in the art may use the use of antisense polynucleotides.

Thus, the invention also relates to an antisense polynucleotide, capable of specifically hybridizing to a determined region of the gene

RRS1-S and capable of inhibiting or blocking its transcription and / or its translation. Such a polynucleotide has the general structure which has been defined above for the probes and primers according to the invention.

Preferably, an antisense polynucleotide according to the invention hybridized with a sequence corresponding to a sequence localized in the region of the 5 ′ end of the messenger RNA RRS1-S, and very preferably close to the codon of initiation of translation (ATG) of the RRS1-S gene.

According to a second preferred embodiment, an antisense polynucleotide according to the invention comprises a sequence corresponding to one of the sequences located at the exon / intron junctions of the RRS1-S gene and preferably sequences corresponding to a splicing site, which can be determined according to techniques well known to those skilled in the art, on the basis of the description of the sequences of the RRS1-S gene of the present description.

To synthesize antisense polynucleotides as defined above, a person skilled in the art can refer to Tables 3 and 4 in which are detailed the positions of the various exons and introns of the RRS1-S gene in the sequence SEQ ID No. 5.

In general, the antisense polynucleotides must have a sufficient length and melting temperature to allow the formation of an intracellular duplex hybrid having sufficient stability to inhibit the expression of RRS1-S mRNA.

Strategies for constructing antisense polynucleotides are notably described by GREEN et al. (1986) and IZANT and WEINTRAIB (1984), the content of these two articles being incorporated here by reference.

Methods of constructing antisense polynucleotides are also described by ROSSI et al. (1991) as well as in the applications PCT No. WO 94/23 026, WO 95/04141, WO 92/18522 and in European patent application No. EP 0 572 287, the content of these documents being incorporated by reference.

Advantageously, an antisense polynucleotide according to the invention has a length of 15 to 4000 nucleotides. An antisense polynucleotide of the invention preferably has a length ranging from 15, 20, 25, 30, 35, 40, 45 or 50 to 75, 100, 200, 500, 1000, 2000, 3000 or 4000 nucleotides.

Among the antisense polynucleotides according to the invention, those having a length of approximately 300 nucleotides or a length of approximately 4000 nucleotides are preferred respectively.

In order to inhibit or block the expression of the RRS1-S gene, it is also possible to use simultaneously a plurality of antisense polynucleotides as defined above, each of the antisense polynucleotides hybridizing with a distinct region of the RRS1-S gene or of its messenger RNA.

In an entirely preferred manner, the antisense polynucleotides according to the invention are defined in such a way that they hybridize with a region of the RRS1-S gene having, with respect to the corresponding sequence of the RRS1-R gene, one or more substitutions, deletions, additions of at least one base.

Other methods of implementing the antisense polynucleotides are for example those described by SCZAKIEL et al. (1995).

Another strategy for inhibiting or blocking the expression of the RRS1-S gene consists in using polynucleotides capable of forming a triple DNA helix with the genomic region of double stranded DNA carrying the RRS1-S gene.

In general, homopurine sequences are considered to be most useful in this type of strategy, although homopyrimidine sequences may also be used. Those skilled in the art can advantageously refer to the genomic sequence SEQ ID No. 5 of RRS1-S in order to select from this sequence sequences of homopurine or homopyrimidine 10 to 20 nucleotides long, which will be capable of inhibit expression of the RRS1-S gene. Methods of inhibiting the expression of a gene by the triple helix technique are for example described by GRIFFIN et al. (1989)

The invention therefore also relates to the use of an antisense polynucleotide or a homopyrimidine polynucleotide, as defined above, or of a recombinant vector comprising such a polynucleotide, for inhibiting or blocking the expression of the RRS1 gene -S in a plant cell or in a whole plant.

As already mentioned, experiments in complementing Arabidopsis thaliana plants belonging to an ecotype sensitive to R. solanacearum by a recombinant vector containing the genomic sequence or the cDNA sequence of the RRS1-R gene made it possible to observe, in the plant thus transformed, the appearance of a phenotype of resistance to R. solanacearum.

In order to stimulate the resistance of a plant to different pathogens, and more particularly to R. solanacearum, it will therefore be advantageous either to introduce or increase the number of copies of the RRS1-R gene in this plant or, or to promote the expression of the RRS1-R gene, these two methods being capable of preventing the development of such a pathogen. High expression of the RRS1-R gene in a plant can be achieved either by overexpression of the RRS1-R gene, or by the insertion of multiple copies of a polynucleotide encoding the RRS1-R protein in the plant, or by a combination of these two strategies. For the insertion of multiple copies of a polynucleotide encoding the RRS1-R protein into the genome of a plant, use will be advantageously made of a recombinant vector according to the invention.

The invention also relates to a recombinant vector comprising a nucleic acid according to the invention.

Advantageously, such a recombinant vector comprises a nucleic acid chosen from the following nucleic acids:

A nucleic acid comprising at least 15 consecutive nucleotides of a nucleotide sequence coding for a protein for resistance of a plant to a pathogen, said protein comprising: (i) an N-terminal part comprising at least one amino acid sequence rich in leucine and at least one nucleotide binding site; and

(ii) a C-terminal part comprising a DNA binding domain comprising the amino acid sequence "WRKYGQK", as well as a nucleic acid of complementary sequence;

b) a nucleic acid comprising a sequence having at least 40% nucleotide identity with the nucleotide sequence SEQ ID No. 1, as well as a nucleic acid of complementary sequence;

c) a nucleic acid comprising a sequence having at least 40% nucleotide identity with the nucleotide sequence SEQ ID No. 5;

d) a nucleic acid comprising at least 15 consecutive nucleotides of a polynucleotide consisting of one of exons 1 to 7 of the RRS1-R gene or of the RRS1-S gene, as defined above;

e) a nucleic acid comprising a polynucleotide of at least 15 consecutive nucleotides of a polynucleotide consisting of one of introns 1 to 7 of the RRS1-R or RRS1-S gene, as defined above;

f) a nucleic acid having at least 15 consecutive nucleotides of a polynucleotide regulating the RRS1-R or RRS1-S gene as defined above;

g) an antisense polynucleotide or a homopurine or homopyrimidine polynucleotide, as defined above, useful for inhibiting the expression of the RRS1-S gene;

By “vector” in the sense of the present invention, is meant a circular or linear DNA or RNA molecule which is either in the form of single strand or double strand. A recombinant vector according to the invention is either a cloning vector, an expression vector, or more specifically an insertion vector, a transformation vector or an integration vector.

It can be a vector of bacterial or viral origin. According to a first embodiment, a recombinant vector according to the invention is used for the purpose of amplifying the nucleic acid which is inserted therein after transfection transformation of the desired cellular host.

According to a second embodiment, it is an expression vector comprising, in addition to a nucleic acid coding for a polypeptide in accordance with the invention, in particular the polypeptides coded by the RRS1-R and RRS1-S genes, regulatory sequences for directing transcription and / or translation.

According to an advantageous embodiment, a recombinant vector according to the invention will comprise in particular the following elements: 1) elements for regulating the expression of the nucleic acid of the RRS1-R gene or of the RRS1-S gene to be inserted, such as promoters and activator sequences ("enhancers");

2) the sequence included in the nucleic acid of the RRS1-R or RRS1-S gene according to the invention to be inserted into such a vector, said sequence being placed under the control of the regulatory signals described in (1); and

3) sequences for initiating and stopping the appropriate transcription.

In addition, the recombinant vectors according to the invention may include one or more origins of replication in cellular hosts in which their amplification or expression is sought as well as selection markers.

In a particular embodiment, a recombinant vector according to the invention comprises an antisense polynucleotide or a homopurine or homopyridine polynucleotide, as defined above, if necessary placed under the control of appropriate regulatory sequences making it possible to ensure expression thereof. in a selected host cell or plant. Such a recombinant vector is preferably used to inhibit the expression of the RRS1-S gene in the cell or in the plant. According to another particular embodiment, a recombinant vector according to the invention comprises a polynucleotide encoding the RRS1-R polypeptide or a polypeptide having at least 40% amino acid identity with the latter and retaining the biological activity of RRS1 -R, placed under the control of regulatory sequence (s) allowing high level expression of RRS1-R or its homolog in a host cell or in a chosen plant. Such a recombinant vector is useful for allowing a high level of expression of RRS1-R in a plant.

According to an advantageous aspect, such a recombinant vector is an integrative vector allowing the insertion of multiple copies of the coding sequence of RRS1-R into the genome of a plant.

By way of example, the bacterial promoters could be the Lacl, LacZ promoters, the RNA polymerase promoters of bacteriophage T3 or T7, the PR or PL promoters of phage lambda. Promoters for the expression of a nucleic acid of RRS1-R or RRS1-S according to the invention in plants are the CaMV 35 S promoter of the cauliflower mosaic virus Odell et al., (1985) or still the promoter of the lactin 1 gene of rice McEIroy et al. (1990).

Other promoters useful for the expression of a polynucleotide of interest in plants are described in patents No. US 5,750,866 and US No. 5,633,363, incorporated herein by reference.

In general, for choosing a suitable promoter, those skilled in the art may advantageously refer to the work by Sambrook et al. (1989) cited above or to the techniques described by Fuller et al. (1996), and Ausubel et al. (1989).

The preferred bacterial vectors according to the invention are for example the vectors pBR 322 (ATCC No. 37017) or also vectors such as pAA223-3 (Pharmacia Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, WU, USA) and pUC19 ( marketed by Boehringer Mannheim, Germany). Mention may also be made of other commercial vectors such as the vectors pQE70, pQE60, pQE9 (Qiagen, psuX 174, pBluescript SA, pNH8A, pMH16A, pMH18A, pMH46A, pWLNEO, pSG2CAT, pOG44, pXTI, pSG (Stratagene). They can also be baculovirus type vectors such as the vector pVL1392 / 1393 (Pharmingen) used to transfect cells of the Sf9 line (ATC No. CRL 1711) derived from Spodoptera frugidera.

Preferably, use will be made of vectors specially adapted for the expression of sequence of interest in plant cells, such as the following vectors:

• vector pBIN19 (Bevan et al., Nucleic Acids Research, Vol.12: 8711-8721, sold by the Clotench Company, Palo Alto, California, USA);

• vector pB101 (Jefferson 1987, Plant Molecular Biology Reporter, vol. 5: 387-405, sold by the Clotench company);

• vector pBI121 (Jefferson et al. 1987, Plant Molecular Biology

Reporter, vol. 5: 387: 405, sold by the Clotench Company);

• vector pEGFP (Cormack BP et al., 1996, sold by the Clotench company);

• vectors SLJ75515 and SLJ75516 (donation from Dr. J. Jones, The Sainsbury Laboratory, Norwich, UK);

• vectors pDHB321 (donation from Dr. Bouchez, INRA Versailles, France).

• vectors pAOV, pOV2, pSOV, pSOV2, pkMB and pSMB (Mylne et al., 1996).

To allow the expression of the polynucleotides according to the invention, these vectors must be introduced into a host cell. The introduction of the polynucleotides according to the invention into a host cell can be carried out in vitro, according to techniques well known to those skilled in the art for transforming or transfecting cells, either in primary culture or in the form of cell lines. The invention further relates to a host cell transformed with a nucleic acid or with a recombinant vector according to the invention.

Such a transformed host cell is preferably of prokaryotic or eukaryotic origin, in particular bacterial, fungal or vegetable origin. Thus, bacteria cells of different from E. coli or from Agrobacterium tumefaciens can in particular be used.

Preferably, the transformed host cell is a plant cell or also a plant protoplast.

Most preferably, it is a cell or a protoplast of rapeseed, tobacco, corn, tomato, potato or Arabidopsis thaliana.

The invention also relates to a transformed multicellular plant organism, characterized in that it comprises a transformed host cell or a plurality of host cells transformed with a nucleic acid of the RRS1-R or RRS1-S gene or with a recombinant vector according to the invention.

According to a first aspect, the plant multicellular organism is transformed with one or more antisense nucleotides and / or one or more homopurine or homopyrimidine polynucleotides in order to inhibit or block the expression of the RRS1-S gene in this organism. According to a second aspect, the plant multicellular organism is transformed with one or more copies of a polynucleotides encoding the RRS1-R protein or for a polypeptide having at least 40% amino acid identity with the RRS1-R polypeptide and retaining the biological activity making it possible to confer a resistance phenotype on R. solanacearum on the transformed organism.

The subject of the invention is also a transgenic plant, that is to say a transformed plant comprising, preferably in a form integrated into its genome, a nucleic acid of the RRS1-R or RRS1 -S gene and preferably an antisense polynucleotide or a homopurine or homopyrimidine polynucleotide or a nucleic acid coding for the RRS1-S polypeptide or a homologous polypeptide, said nucleic acid having been inserted into the genome of the plant by transformation with a nucleic aid of RRS1-R or RRS1-S or a recombinant vector according to the invention. Preferably, a plant transformed according to the invention is rapeseed, tobacco, corn, tomato, potato or Arabidopsis thaliana.

According to a first aspect, the transgenic plants as defined above have a reduced expression, an undetectable expression or an absence of expression of the RRS1-S gene and are therefore capable of exhibiting increased resistance to pathogens such as

R.solanacearum.

According to a second aspect, the transgenic plants as defined above have the property of strongly expressing the RRS1- polypeptide

R and consequently possess a phenotype of resistance to various pathogens, and in particular to bacterial pathogens such as

R. solanacearum.

The subject of the invention is also a process for obtaining a transgenic plant transformed with a nucleic acid according to the invention, characterized in that it comprises the following steps:

a) obtaining a recombinant plant host cell as defined above;

b) regeneration of an entire plant from the transformed plant host cell obtained in step a);

c) selection of the plants obtained in step b) having integrated the nucleic acid of the RRS1-R or RRS1-S gene of interest.

The invention also relates to a process for obtaining a transgenic plant, transformed with a nucleic acid according to the invention, characterized in that it comprises the following steps:

a) transforming a plant cell with a nucleic acid of the RRS1-R or RRS1-S gene or with a recombinant vector according to the invention;

b) regenerating an entire plant from transformed plant cells obtained in step a); c) select the plants having integrated the nucleic acid of the RRS1-R or RRS1-S gene of interest.

The invention also relates to a process for obtaining a transformed plant, characterized in that it comprises the following steps:

a) obtaining an Agrobacte um tumefaciens host cell transformed with a nucleic acid or a recombinant vector according to the invention;

b) transformation of the chosen plant by infection with Agrobactehum tumefaciens cells obtained in step a);

c) selection of plants having integrated the nucleic acid according to the invention.

Any of the methods for obtaining a transgenic plant described above can also comprise the following additional steps:

d) crossing between them of the two transformed plants as obtained in step c);

e) selection of plants homozygous for the transgene.

According to another aspect, any of the methods described above may further comprise the following steps:

f) crossing a transformed plant obtained in step c) with a plant of the same species;

g) selection of the plants resulting from the crossing of step d) having conserved the transgene. A person skilled in the art is capable of implementing numerous methods of the state of the art in order to obtain plants transformed with a nucleic acid of the RRS1-R or RRS1-S gene according to the invention.

Those skilled in the art may advantageously refer to the technique described by BECHTOLD et al. (1993) in order to transform a plant using the bacterium Agrobacterium tumefaciens.

The techniques used in other types of vectors can also be used such as the techniques described by BOUCHEZ et al. (1993) or by HORSCH et al. (1984). The invention further relates to a transformed plant as obtained according to any one of the production methods described above.

The invention also relates to a plant seed, the constituent cells of which comprise a nucleic acid of the RRS1-R gene or of the RRS1-S gene according to the invention which has been artificially inserted into their genome.

The invention also relates to a seed of a transgenic plant as defined above.

Another subject of the invention consists in the use of a nucleic acid of the RRS1 -R gene or of the RRS1-S gene according to the invention for the expression in vitro or in vivo, preferably in planta of the protein RRS1 -R or RRS1-S or a peptide fragment thereof.

The invention also relates to the use of an antisense nucleic acid, or of a homopurine or homopyrimidine nucleic acid according to the invention for inhibiting or blocking the expression of the gene coding for the protein RRS1-R or RRS1 -S.

Preferably, the above uses are characterized in that it is an expression in vivo in a plant transformed with such a nucleic acid. As already mentioned previously, the RRS1-R gene codes for a polypeptide of 1378 amino acids in length.

In addition, the RRS1-R polypeptide has the structural characteristics defined previously in the description, namely the characteristic presence of functional domains found in combination for the first time in the amino acid sequence of a polypeptide, which gives the protein RRS1-R the quality of first known member of a new class of proteins defined most generally as proteins comprising:

a) an N-terminal part comprising at least one amino acid sequence rich in leucine and at least one nucleotide binding site; and

b) a C-terminal part comprising a DNA binding domain comprising the amino acid sequence "WRKYGQK".

The RRS1-R polypeptide is referenced as the sequence SEQ ID No. 9.

As already mentioned, the applicant has shown that mutations in the amino acid sequence of the RRS1-R polypeptide could lead to a protein lacking the biological activity of the wild protein RRS1-R, the expression of the mutated protein in a plant no longer making it possible to observe a phenotype of resistance to certain pathogens, such as R. solanacearum. A polypeptide representative of the mutated RRS1-R polypeptides altered in their biological function is the RRS1-S polypeptide, the amino acid sequence of which is referenced as the sequence SEQ ID No. 10.

According to another aspect, the invention also relates to a polypeptide encoded by a nucleic acid of the RRS1-R gene or of the RRS1-S gene, and preferably a polypeptide comprising at least 5 consecutive amino acids of a protein chosen from RRS1-R and RRS1-S.

Preferably, such a polypeptide comprises at least 10, 15, 20,

25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,

600, 700, 1200 consecutive amino acids of the RRS1-R polypeptide of amino acid sequence SEQ ID No. 9 or of the RRS1-S polypeptide of amino acid sequence SEQ ID No. 10.

. The invention also relates to a polypeptide comprising the amino acid sequences having at least 40% amino acid identity with the sequence of the RRS1-R SEQ ID No. 9 polypeptide, with the sequence of the RRS1-S SEQ ID No. 10 polypeptide, or to a peptide fragment thereof.

Advantageously, part of the invention is a polypeptide having at least 60%, 80%, 85%, 90%, 95% or 99% of amino acid identity with the sequence of the RRS1-R polypeptide of SEQ ID No. 9 , with the sequence of the RRS1-S SEQ ID NO: 10 polypeptide, or a peptide fragment thereof.

In general, the polypeptides according to the invention are in an isolated or purified form. The invention also relates to a process for the production of the RRS1-R polypeptide of sequence SEQ ID No. 9, the RRS1-S polypeptide of sequence SEQ ID No. 10 or a peptide fragment thereof, the method comprising Steps :

a) inserting a nucleic acid coding for the RRS1-R polypeptide,

RRS1-S or a peptide fragment thereof, in an appropriate vector;

b) cultivating, in an appropriate culture medium, a host cell previously transformed or transfected with the recombinant vector of step a);

c) recovering the conditioned culture medium or lysing the transformed host cell, for example by sonication or by cosmetic shock;

d) separating and purifying from the culture medium or from the cell lysates obtained in step c), said polypeptide;

e) where appropriate, characterize the recombinant polypeptide produced.

The peptides according to the invention can be characterized by attachment to an immunoaffinity chromatography column on which the antibodies directed against these polypeptides in which a fragment or a variant of the latter have been immobilized beforehand. According to another aspect, a recombinant polypeptide according to the invention can be purified by passage through a chromatography column according to the methods known to those skilled in the art and described for example by AUSUBEL F. et al. (1989) cited above. A polypeptide according to the invention can also be prepared by conventional techniques of chemical synthesis either in homogeneous solution or in solid phase.

By way of illustration, a polypeptide according to the invention may be prepared by the technique in homogeneous solution described by HOUBEN WEYL (1974) or also the solid phase synthesis technique described by MERRIFIELD (1965a, 1965b).

Also part of the invention are so-called "homologous" polypeptides to the RRS1-R or RRS1-S polypeptides, or their fragments. Such homologous polypeptides have amino acid sequences having one or more substitutions of an amino acid with an equivalent amino acid, relative to the reference polypeptide.

The equivalent amino acids according to the present invention will be understood, for example the replacement of a residue in the L form with a residue in the D form or else the replacement of a glutamic acid (E) by a pyro-glutamic acid according to techniques well known to those skilled in the art.

By way of illustration, the synthesis of peptides containing at least one residue in the D form is described by KOCH et al. (1977). According to another aspect, two amino acids belonging to the same class are also considered to be equivalent amino acids, that is to say two amino acids, basic, non-polar or even uncharged polar.

Also part of the invention is a polypeptide comprising amino acid modifications of 1, 2, 3, 4, 5, 10 to 20 substitutions, additions or deletions of an amino acid with respect to the amino acid sequence of the RRS1-R polypeptide or RRS1-S polypeptide according to the invention.

Preferably, the polypeptides according to the invention comprising one or more additions, deletions, substitutions of at least one amino acid retain their ability to induce resistance to various pathogens, especially R. solanacearum

According to another preferred embodiment, the polypeptides according to the invention comprising one or more additions, deletions, substitutions of at least one amino acid retain their ability to be recognized by antibodies directed against the RRS1-R polypeptides or

RRS1-S not modified.

A polypeptide derived from the RRS1-R protein or from the RRS1-S protein is useful in particular for the preparation of antibodies intended for the detection of the presence of one or other of these polypeptides or of a peptide fragment of these in a sample.

In addition to detecting the presence of the RRS1-R or RRS1-S polypeptide or of a peptide fragment of the latter in a sample, antibodies directed against these polypeptides are used to quantify the synthesis of RRS1-R or RRS1-S, for example in the cells of a plant, and thus determine the capacity of this plant to resist certain pathogens, and more particularly to R. solanacearum.

Preferred antibodies according to the invention are the antibodies specifically recognizing the amino acid sequence ranging from the amino acid in position 704 to the amino acid in position 712 of the sequence of the RRS1-R polypeptide of SEQ ID No. 9.

A second class of preferred antibodies according to the invention are the antibodies specifically recognizing the amino acid sequence ranging from the amino acid at position 704 to the amino acid at position 710 of the sequence of the RRS1-S polypeptide of SEQ ID # 10.

A third class of preferred antibodies according to the invention are the antibodies specifically recognizing the amino acid sequence ranging from the amino acid at position 1291 to the amino acid at position 1378 of the RRS1-R polypeptide of sequence SEQ ID No. 9. By “antibody” within the meaning of the present invention, is meant in particular polyclonal or monoclonal antibodies or fragments (for example fragments F (ab) ′ ₂₁ F (ab) or also any polypeptide comprising a domain of the initial antibody recognizing the target polypeptide or fragment of the polypeptide according to the invention. Monoclonal antibodies can be prepared from hybridomas using the technique described by KOHLER and MILSTEIN (1975).

The present invention also relates to antibodies directed against a polypeptide as described above or a fragment or a variant thereof, as produced in the triome technique or also the hybridoma technique described by KOZBOR et al. (1983).

The invention also relates to fragments of single chain Fv antibody (ScFv) as described in US Patent No. 4,946,768 or by MARTINEAU et al. (1998). The antibodies according to the invention also include fragments of antibodies obtained using phage libraries (RIDDER et al., 1995), REINMANN K.A. et al., 1997).

The antibody preparations according to the invention are useful in immunological detection tests intended for the identification of the presence and / or the amount of the protein RRS1-R or RRS1-S or also of a peptide fragment of l one of these proteins, present in a sample.

An antibody according to the invention may also comprise an isotopic or non-isotopic detectable marker, for example fluorescent, or else be coupled to a molecule such as biotin, according to techniques well known to those skilled in the art.

Thus, the subject of the invention is also a method for detecting the presence of an RRS1-R polypeptide, an RRS1-S polypeptide or also a peptide fragment of one of these polypeptides in accordance with the invention , in a sample, said method comprising the steps of:

a) bringing the test sample into contact with an antibody as defined above;

b) detecting the antigen / antibody complex formed.

The invention also relates to a diagnostic kit or kit for detecting the presence of a polypeptide according to the invention in a sample, said kit comprising:

a) an antibody as defined above; b) where appropriate, a reagent allowing the detection of the antigen / antibody complexes formed.

The invention also relates to fusion proteins comprising an amino acid sequence of one of the two C-terminal or N-terminal functional domains of a polypeptide according to the invention, on the one hand, and, on the other hand, all or part of the amino acid sequence of a heterologous polypeptide. By “heterologous polypeptide” is meant for the purposes of this description an amino acid sequence which is not naturally present in the amino acid sequence of the N-terminal or C-terminal polypeptide fragment or fragment according to the invention with which this "heterologous" amino acid sequence is covalently linked, preferably by a peptide bond.

According to a first aspect, a fusion protein, or chimera according to the invention comprises (1) the N-terminal domain of a first polypeptide belonging to the new class of proteins of resistance to plant pathogens according to the invention, linked to covalently, preferably by a normal peptide bond, to a second polypeptide comprising the C-terminal domain of a second protein also belonging to the class of proteins for resistance to plant pathogens according to the invention.

The N-terminal domain of the first polypeptide and the C-terminal domain of the second polypeptide can be linked directly to each other by a peptide bond.

According to a second aspect, the N-terminal domain of the first polypeptide and the C-terminal domain of the second polypeptide can be separated, within the fusion protein, by a spacer amino acid sequence which can be of any kind.

Such a chimeric protein therefore comprises an N-terminal domain comprising the motifs characteristic of a protein for resistance to pathogens, covalently linked to a C-terminal domain comprising a nucleotide binding site capable of having a specificity different from that of the C-terminal domain which is found naturally present within the amino acid sequence of the natural polypeptide from which the N-terminal domain of the chimeric protein originates.

Such chimeric proteins constitute new means making it possible, by choosing the N-terminal domain, to confer in a plant resistance to a determined pathogen.

In addition, the choice of the C-terminal nucleotide binding domain allows those skilled in the art to specifically target the regulatory sequences which are activated by such a chimeric protein and thus to control both the type and the level of resistance. desired. According to a second embodiment, a fusion protein according to the invention consists of (1) the amino acid sequence of all or part of a protein whose mechanisms of induction of synthesis in a plant are known and ( 2) (i) either the complete amino acid sequence of a pathogen resistance polypeptide according to the invention, or (i.) The amino acid sequence of the C-terminal domain comprising a nucleotide binding site of a polypeptide according to the invention.

Most preferably, the synthesis of such a chimeric protein is inducible by stress signals from the plant, such as stress due to a sudden change in the temperature of the environment. The invention also relates to a nucleic acid coding for a chimeric protein as defined above.

According to a preferred embodiment, such a nucleic acid comprises, near and advantageously upstream of the coding region, a constitutive promoter or an inducible promoter. By way of illustration, such a nucleic acid comprises, from the 5 ′ to the 3 ′ end a plant heat shock protein promoter, (2) a nucleotide sequence coding for the N-terminal part of the heat shock protein the synthesis of which is naturally regulated by the promoter (1), (3) the nucleotide sequence coding for a polypeptide of resistance to plant pathogens having retained its function of transcription factor (functional WRKY domain) according to the invention.

By way of example, preferred promoters are those which are inducible by: - Heat (Athsp 17.6; Prandl et al., 1995);

- H ₂ 0 ₂ , auxin, salicylic acid (GST6; Chen et al., 1996);

- salicylic acid (PR1; Lebel et al., 1998).

A chimeric protein according to the invention can be obtained according to the technique described in Example 7.

As described above, a polypeptide for resistance to plant pathogens according to the invention, has, both in the N-terminal domain and in the C-terminal domain, characteristic patterns of binding to proteins.

Proteins interacting with a polypeptide according to the invention therefore also represent means involved in the resistance of plants to infection by certain pathogens, in particular by R.solanacearum. The invention also relates to methods and means intended for screening for candidate substances or molecules capable of binding with a polypeptide according to the invention.

The invention therefore relates to a method for screening a candidate substance which binds to a polypeptide according to the invention, and very particularly an RRS1-R or RRS1-S polypeptide, characterized in that it comprises the steps of:

a) preparing a polypeptide according to the invention;

b) obtaining a candidate substance to be tested;

c) bringing the polypeptide of step a) into contact with the candidate substance of step b);

d) detecting the complex possibly formed between the polypeptide of the invention and the candidate substance.

By way of illustration, of a screening method as defined above, a polypeptide according to the invention is immobilized by adsorption or by covalent bonding on a suitable surface, the surface of a well of a plate of microtiter. The immobilized polypeptide of the invention is then brought into contact with the candidate substance or molecule to be tested.

The detection of the binding of the candidate molecule to the immobilized polypeptide of the invention can then be carried out for example with an antibody specifically recognizing the candidate molecule to be tested, said antibody optionally comprising a detectable marker.

According to another embodiment of a screening method as defined above, the candidate substance or molecule is immobilized beforehand on the surface of a support before being brought into contact with a polypeptide according to the invention.

The detection of the complex possibly formed between the polypeptide according to the invention and the candidate molecule immobilized on the support can then be carried out using an antibody specifically recognizing a polypeptide according to the invention, said antibody being optionally labeled.

To carry out such a screening method, a person skilled in the art can advantageously refer to the use of ELISA type immunodetection techniques.

The invention also relates to a kit or kit for screening a candidate substance which binds to a polypeptide of the invention, such a kit or kit comprising:

a) a polypeptide according to the invention;

b) where appropriate, the reagents necessary for the detection of the complexes formed between the polypeptide of the invention and the candidate substance to be tested.

A method of screening for molecules interacting with a polypeptide according to the invention can also be carried out using double hybrid techniques. Double-hybrid type screening techniques are used to study protein-protein interactions in vivo and are based on the fusion of a “bait” protein to the DNA binding domain of the yeast protein Gal4. The double hybrid technique is described in particular in US Pat. Nos. 5,667,973 and US Pat. No. 5,283,173, the technical teaching of these two patents here being incorporated by reference.

The screening of cDNA bands containing inserts encoding translation products potentially interacting with a polypeptide according to the invention or a peptide fragment containing a protein binding domain of a polypeptide according to the invention can be carried out as described by HARPER et al. (1993), by CHO et al. (1998), or by FROMONT-RACINE et al. (1997). The invention also relates to a method for screening a candidate substance which binds to a polypeptide according to the invention, and very particularly to the RRS1-R or RRS1-S polypeptide, said method comprising the steps of:

a) obtaining a first nucleic acid coding for a fusion protein comprising a part of the polypeptide of interest fused to the DNA binding domain of a transactivating protein such as Gal4;

b) obtaining a second nucleic acid coding for a fusion protein comprising the candidate substance fused to the transcription domain of a transactivating protein such as Gal4;

c) providing a nucleic acid comprising a nucleotide sequence coding for a detectable marker, placed under the control of a regulatory sequence recognized by the transactivating protein such as Gal4.

The nucleic acids a), b) and c) being inserted into suitable vectors; and d) co-transfected yeast cells simultaneously with said vectors; e) detecting the expression of the nucleotide sequence coding for the detectable marker.

The above screening method may also comprise an additional step f) during which the nucleotide sequence and / or the amino acid sequence of the “prey” nucleic acid inducing the expression of the detectable marker is characterized.

The invention also relates to a kit or kit for screening a candidate substance which binds to a polypeptide according to the invention, and very particularly the RRS1-R or RRS1-S polypeptide, said kit or kit comprising:

a) a first nucleic acid coding for a fusion protein comprising a part of the polypeptide of interest fused to the DNA binding domain of a transactivating protein such as Gal4;

b) where appropriate, a second nucleic acid comprising a nucleotide sequence coding for a detectable marker, placed under the control of a regulatory sequence recognized by the transactivating protein such as Gal4;

c) a third nucleic acid coding for a fusion protein comprising the candidate substance fused to the transcription domain of a transactivating protein such as Gal4.

The invention also relates to a substance capable of binding to a polypeptide of the invention, preferably to the RRS1 -R or RRS1 -S polypeptide, this substance being characterized in that it is capable of being obtained by a method of screening as defined above. A screening technique using a double hybrid type system can be easily performed by a person skilled in the art, in particular in accordance with the teaching of Example 8.

As already described above, a polypeptide according to the invention comprises several nucleotide binding domains both in its N-terminal part and in its C-terminal part. The nucleotide sequences to which these different nucleotide binding domains bind are therefore of decisive importance in the mechanisms of resistance of plants to various pathogens, and more particularly to R. solanacearum. The isolation and characterization of the polypeptides of resistance to plant pathogens according to the invention now allows a person skilled in the art to determine these nucleotide sequences involved in the induction of a phenotype of resistance to pathogens in a plant. The invention also relates to a method of screening for a nucleic acid interacting with a polypeptide according to the invention, said screening method comprising the steps of:

a) obtaining a statistical population of nucleic acids 20 to 50 nucleotides in length;

b) bringing the population of nucleic acids of step a) into contact with a polypeptide according to the invention;

c) characterizing the nucleic acid or the plurality of nucleic acids interacting with said polypeptide.

According to such a method, it is first prepared, for example by direct chemical synthesis, nucleic acids 20 to 50 nucleotides in length, and preferably 20 to 30 nucleotides in length, the nucleotide sequence of which is random, each of the acids synthesized nucleic acids being further covalently linked, at their 5 ′ or 3 ′ end, to a single and known nucleotide sequence.

The nucleic acids forming a statistical population of nucleic acids described above are then brought into contact with a polypeptide according to the invention, said polypeptide possibly having been immobilized beforehand on a support.

After a step making it possible to eliminate the nucleic acids which have not interacted with the polypeptide according to the invention, the polypeptide / nucleic acid complexes are brought into contact with an oligonucleotide primer which hybridizes specifically, under determined hybridization conditions, with the unique and known nucleotide sequence common to all of the nucleic acids of the above statistical population, then at least one amplification cycle is carried out using the primer hybridizing the unique and known nucleotide sequence of l 'acid nucleic acid retained by the polypeptide of the invention, for example an amplification by PCR.

According to a particular embodiment of this screening method, the nucleic acids thus amplified and isolated are again brought into contact with a polypeptide according to the invention, then the nucleic acid or the plurality of nucleic acids having formed a complex with said polypeptide are again hybridized with the nucleotide primer hybridizing with the single and known nucleotide sequence and a new amplification cycle (s) is carried out. Preferably, the screening method as defined above comprises from 1 to 10, and very preferably from 1 to 5 cycles of bringing into contact / amplification of the nucleic acids attached to the polypeptide of the invention.

The nucleic acids selected for their specific binding to a polypeptide according to the invention are then characterized, preferably by sequencing.

The invention further relates to a kit or kit for the screening of a nucleic acid interacting with a polypeptide according to the invention, comprising:

a) a polypeptide according to the invention;

b) where appropriate, a statistical population of nucleic acids 20 to 50 nucleotides in length.

The invention is further illustrated, without being limited, by the figures and examples.

Figure 1 illustrates the amino acid sequence of the RRS1-R polypeptide. The different characteristic areas are represented.

Figure 2 illustrates the amino acid sequence of the RRS1-S polypeptide. The different characteristic areas are represented. FIG. 3 illustrates the representation and the localization of the YACS, BACS and cosmid clones covering the genomic region of interest of A.thaliana.

Material and methods

at. Southern blot analysis

Genomic DNA was isolated from the leaves of Arabidopsis plants or seedlings according to the technique described by Deslandes et al. (1998). Southern type experiments are carried out as described by Sambrook et al. (1989) using 2 μg of genomic DNA.

b. DNA extraction from BAC, YAC, TAC, cosmidigues, and A. clones. tumefaciens

The extraction of DNA from BAC and TAC clones was carried out according to a modification of the protocol provided by the CARL. 25 mL of a 12 hour culture of a BAC clone in the appropriate antibiotic are centrifuged for 5 min at 6000 rpm. The pellet is resuspended in 4 ml of a buffer containing: 50 mM Glucose, 10 mM EDTA, 25 mM Tris, pH 8.0, 5 mg / ml of lyzozyme. After 5 min of incubation at room temperature, the cells are lysed by addition of 8 ml of 0.2 N NaOH, 1% SDS. A further 5 min incubation at room temperature is followed by the addition of 6 ml of 3M potassium acetate, pH 4.8. The mixture is incubated at 0 ° C for 10 min. then centrifuged for 15 min at 13,000 rpm. The supernatant is recovered, precipitated by adding 0.7 volume of isopropanol, then centrifuged for 15 min at 13,000 rpm. The pellet obtained is washed with 70% ethanol, dried for 15 min at room temperature and then resuspended in 0.6 ml of TE buffer (10 mM tris, pH 8, 1 mM EDTA).

The purification of cosmid DNA was carried out according to the protocol of Sambrook et al (1989).

DNA extraction from A. tumefaciens was performed according to a protocol developed in the laboratory. 2 mL of a 12 hour culture in medium L supplemented with the appropriate antibiotics are centrifuged for 4 min at 13000 rpm. The pellet is resuspended in 700 μl of CTAB buffer (100 mM Tris HCI, pH 8, 1.4M NaCI, 20 mM EDTA, pH 8, 2% w / v CTAB (hexadecyltrimethylammonium bromide) to which 0.07N of B-mercaptoethanol is added immediately The mixture incubated for 10 min at 65 ° C. is then extracted twice with an equal volume of phenol / chloroform (1V / 1V) followed by an extraction with chloroform. The aqueous phase is then precipitated with 0.7 volume of isopropanol, the mixture centrifuged for 10 min at 13,000 rpm and the pellet obtained is resuspended in 50 μL of distilled water.

vs. Plant material and Arabidopsis inoculation protocol by R. solanacearum

The growth of Arabidopsis plants and the conditions of inoculation of these plants with the different strains of R. solanacearum have been described by Deslandes et al. (1998).

d. DNA sequence analysis

The BLAST research program (Altschul et al. 1990, 1997) was used for sequence analysis and comparison in Genbank, EMBL and Swissprot databases as well as in Arabidopsis databases (AatDB Database Arabidopsis ).

Example 1) Cloning positions! from RRS1

This cloning was carried out on the sensitive Col-5 ecotype which contains the RRS1 allele. Resistance to the GMI1000 strain of Ralstonia solanacearum is conferred by the rrsl allele of the resistant Nd-1 ecotype.

A series of overlapping YACs (Yeast Artificial

Chromosome) built by the Japanese consortium in charge of the V chromosome sequencing program of Arabidopsis thaliana

(http://www.kazusa.or.jp/arabi/chr5/pmap/P1_map_16.html) was obtained from ABRC (Arabidopsis Biological Resource Center, Ohio State University, USA). RFLP markers showing a polymorphism between the parental ecotypes Col-5 and Nd-1 were generated from the left and right ends of the inserts of the YACs clones according to the protocol of Schmidt et al (1996). These different markers, EW7G12LE (LE, Left End: generated from the left end of the YAC insert), 11 H2RE (RE, Right End: generated from the right end of the YAC insert) , 11 H2LE, 4H11 RE as well as a marker T43968 (cDNA clone positioned on the YAC CIC4E12 and obtained from the CARL) were used as RFLP markers in order to more precisely locate the RRS1 locus. Two of these markers (T43968 and EW7G12LE) were found to be polymorphic on the parental ecotypes and made it possible to restrict the region containing the RRS1 locus to a region of approximately 600 kb. Of the 18 remaining recombinant plants, only 11 lines still exhibited a recombination event between these 2 markers and the RRS1 locus. Furthermore, the markers generated from the ends of the YACs clones were used to screen a TAMU bank (Texas A & M University) of BACs (Bacterial Artificial Chromosome), a bank obtained through the CARL. This screening carried out according to the conditions provided by the CARL led to the isolation of a large number of BAC clones. Markers were then generated from the ends of some of these BACs by the techniques of "plasmid rescue" and reverse PCR (Woo et al., 1994). These markers, 1 H19LE, 2G14LE, 21 F10LE, 9N23LE, 27P17LE and 29D23LE were used to position the different BACs selected with respect to each other (by digestion of the DNA of the different BACs and hybridization with each of the isolated ends). These data made it possible to construct a series of overlapping BAC clones. Later, we also took advantage of the existence of a series of overlapping TACs (Transformation Artificial Chromosome) clones generated by the Japanese consortium. The corresponding TACs clones (K9E15, K18C1, K15I22, K2N11) and the clone MFC19 (bank P1, Liu et al., 1995) were obtained by CARL.

In parallel with these studies, the number of lines presenting a recombination event being relatively low, 650 F2 plants resulting from a cross between the 2 parental ecotypes were tested in order to characterize other lines presenting such an event between the markers T43968 and EW7G12LE and the RRS1 locus. For this purpose, the DNA of a few leaves of each F2 plant was extracted (Deslandes et al., 1998), digested with the restriction enzyme Bglll which makes it possible to observe a polymorphism with the markers T43968 and EW7G12LE on the 2 parental genotypes. The F2 plants thus selected were then self-fertilized and the seeds obtained (F3) sown. The phenotype of these third generation plants was then determined by inoculation of the GMI1000 strain. This approach made it possible to obtain 15 recombinant F3 families between the markers T43968 and EW7G12LE.

The markers corresponding to the ends of BACs were then used as RFLP markers on the 11 RIL lines as well as on the 15 F3 families. The subcloning of BACs and cosmids by different restriction enzymes has also made it possible to generate other RFLP markers (Table 5).

Table 5: RFLP markers used to map the RRS1 locus on 15 F3 families from the Col-5 and Nd-1 crossing.

For each F3 family, the genotype revealed by the use of the different RFLP markers is indicated in this table (A: Col-5 genotype, B: Nd-1 genotype, AB: heterozygous genotype).

• T43968: marker obtained from the CARL (Ohio, USA).

• 1 H19 n ° 1,, 9N23 N ° 2, 9N23 N ° 18. fragments of the BACs T1 H19 and T9N23 clones (BamHI-Xhol digestion) cloned in the vector pKS (Stratagene, USA).

• # 419, B1: cosmids derived from clones K18C1 and T29K4 respectively. • MFC5, MFC61 and MFC14: cosmids resulting from partial digestion

BamHI of the clone MFC19.

• # 6.1: phagemid from a phage bank Col-0 (zap, Stratagene, gift from Pr Lescure).

• B1 n ° 4: subclone of cosmid B1 • EW7G12LE: left end of the insert of the YAC clone EW7G12 (gift from W. Gassman).

These experiments made it possible to localize the RRS1 gene on 3 overlapping BAC clones (T29K4, T25P9 and MFC19), covering a region of approximately 170 kb (Figure 3).

Cosmic contigs covering all of the 2 BACs were obtained by partial digestion with the BamHI enzyme and subcloning of the DNA of the 2 BACs into the cosmid vector SLJ75515 (gift from Dr. J. Jones, The Sainsbury Laboratory ). The use of these cosmids and subclones of these cosmids obtained by digestion with the enzyme HindIII and insertion into the vector pBlueScript made it possible to locate RRS1 on a cosmid of 18 Kb, the cosmid B1 derived from the clone BAC T29K4. A cDNA library of the Col-0 ecotype prepared in the IZAP vector (gift from B. Lescure in our institute) was then screened with the DNA of the insert of cosmid B1 and made it possible to isolate several clones cDNA whose nucleotide sequence has been determined. At the same time, the nucleotide sequence of a TAC clone, K9E15 which covers the cosmid B1, was delivered to databases by the Japanese consortium. Certain cDNA clones whose nucleotide sequence was known were thus able to be located on cosmid B1. One of them, noted # 6.1, used as RFLP markers as well that a CAPS marker, B69.1, made it possible to delimit a region of approximately 10 kb from the cosmid B1 containing the RRS1 locus. Knowing the sequence of cosmid B1, it was possible to generate several primer pairs in order to identify PCR markers of the CAPS type. Two recombinant F3 plants, 324 and 566, presenting a recombination event within the cosmid B1 made it possible to identify the RRS1 gene.

Example 2) Isolation and characterization of RRS1

A cosmid bank prepared from DNA of the Nd- ecotype

1 was constructed in the vector SLJ75515 in the group of J. Beynon (Horticultural Research International, Wellesbourne, GB). This library was graciously given to us and its screening was carried out (according to the Southern hybridization conditions described by Deslandes et al., 1998) using the DNA of the clone T1 H19 which contains the RRS1 locus. Several cosmid clones have been isolated and characterized. One of them, clone H, was selected because it has a restriction profile identical to that of B1, a cosmid on which RRS1-S has been located by positional cloning. The sequence of the RRS1-S gene being known, it was possible to generate oligonucleotide primers which made it possible to determine the nucleotide sequence of the region corresponding to RRS1 of cosmid H. The determination of the nucleotide sequence was carried out on a Perkin sequencer -Elmer 373 (Dye Terminator kit) and the different sequences were assembled using the "Staden" sequence assembly programs, available on UNIX stations (Roger Staden, MRC, Cambridge, UK). The different primers used are the primers of the "K" series, ie all the primers whose name begins with the letter "K" (see Table 6).

Example 3) Complementing sensitive and resistant ecotypes with RRS1-R and RRS1-S a. constructions

The cosmid vector SLJ 75515 of clones H and B1 has the advantage of being directly transferable to Agrobacterium tumefaciens. This transfer of cosmids from E. coli (strain XLI Blue for cosmid B1 and strain DH12S for cosmid H) in A. tumefaciens (strain GV3101 containing the helper plasmid pMP90) is carried out by triparental conjugation using a third strain, pRK2013 (E. coli ) according to the Tolmasky et al. (1984). The colonies of A. tumefaciens containing the cosmids H or B1 are selected on an L agar medium supplemented with tetracycline (10 μg / ml) and gentamycin (25 μg / ml). The dishes are then incubated at 28 ° C for 3 days.

The presence of cosmid DNA is checked by PCR amplification using primers specific to the inserts of clones H and B1 (pair

RT1 / RT2 for example). Furthermore, Southern type experiments make it possible to verify the stability of the cosmid DNAs in A. tumefaciens

Furthermore, the insert of cosmid B1 was cloned using the enzyme BamHI which generates two fragments: 9.68 and 8.03 kb. For cosmid H, 2 BamHI fragments of size comparable to those of cosmid B1 were obtained. The BamHI site is present only once in the genomic sequences of RRS1-R and RRS1-S. Each of the 2 subfragments from cosmids B1 and H was introduced into the binary vector pDHB321.1 provided by Dr. Bouchez (INRA Versailles). After transformation into a strain of E. coli (XLI BlueMR, Stratagene, USA), the plasmid DNA is extracted (Maniatis 1978). The strain of A. tumefaciens GV3101 is then transformed by the thermal shock method (Holsters et al., 1978). The colonies of A. tumefaciens containing the subclones of cosmids B1 and H are then selected on medium L agar containing gentamycin (25 μg / ml) and Kanamycin (50 μg / ml). The dishes are then incubated at 28 ° C for 48 h.

The presence of the plasmid DNA is checked by PCR amplification using primers specific for the 2 subfragments of clones H and B1. Furthermore, Southern type experiments make it possible to verify the stability of the plasmid DNAs in A. tumefaciens. These constructs are then introduced into the genome of the Col-5 and Nd-1 plants. b. Transformation of the Nd-1 and Col-5 ecotypes by Agrobacterium tumefaciens

In order to demonstrate the function of the candidate genes, experiments to complement sensitive and resistant phenotypes with cosmids H and B1 were carried out. The classical transformation techniques of Arabidopsis by A. tumefaciens (Clough and Bent 1999) were used to generate the transgenic plants described in this work. The seeds of plants transformed by A. tumefaciens (generation T1) are deposited in terrines containing a layer of perlite (about 2cm) covered with a layer of sand of the same thickness soaked in a solution of the herbicide gluphosinate (trade name : BASTA) at a concentration of 15 mg / L. The terrines are then placed for 4 days in the dark at 4 ° C, then transferred in the condition of long days (16h of day / 8h of night). After about 10 days, the resistant plants are selected and transplanted individually. The T2 progeny of each T1 transgenic plant obtained by self-fertilization is used for inoculation experiments with the pathogenic agent.

vs. Results obtained

c.1 The RRS1-R gene confers resistance to the GMI1000 strain of Ralstonia solanacearum.

Complementation experiments carried out using cosmid H containing the RRS1-R allele introduced into the Col-5 ecotype were carried out. After selection with the herbicide BASTA, 12 transgenic plants could be isolated (lines noted CH1.1 to CH1.12). These plants were self-fertilized in order to obtain the T2 progeny of each of them. and 24 individuals of each transgenic line obtained were inoculated with the GMI1000 strain of R. solanacearum. While control plants (Col-5) withered 7 days after inoculation with the pathogen, the transgenic plants containing the RRS1-R allele of Nd-1 did not show any symptoms of wilting. These results therefore indicate that the RRS1-R gene can confer resistance to the GMI1000 strain of R. solanacearum in plants of genetic background Col-5. The expression of RRS1-R can cause a decrease in bacterial multiplication in the infected plant, which could explain the absence of symptoms.

c.2 The RRS1-R gene confers resistance to different strains of Ralstonia solanacearum.

A certain number of R. solanacearum strains provoke responses (disease or absence of symptoms) identical to that of the strain GMI1000 on the Nd-1 and Col-5 ecotypes. These strains isolated from very diverse host plants (see table) come from different regions of the globe. The ability of the RRS1-R gene to confer resistance to these different strains was therefore tested. The Col-5 transgenic plants containing the RRS1-R gene were therefore infected with these different strains. While control plants (Col-5) withered 7 days after inoculation with the pathogen, the transgenic plants containing the RRS1-R allele Nd-1 showed no symptoms of wilting. This result indicates that the RRS1-R gene is capable of conferring resistance to different strains of R. solanacearum.

c.3 Does the RRS1-S gene confer sensitivity to the GMI1000 strain of

R. solanacearum.

The response of Nd-1 plants transformed with the RRS1-S gene of the sensitive Col-5 ecotype was tested. 104 plants were selected for their resistance to BASTA (B1.1 to B1 .104) and self-fertilized. These transgenic plants were inoculated with the GMI1000 strain. Only one transgenic line (B1.3) developed symptoms of wilting while the other lines showed no symptoms under conditions causing total wilting of control plants Col-5.

Example 4) Complementation experiments with genomic clones RRS1-S and RRS1-R.

A Sphl digestion of the cosmid B1 made it possible to obtain a fragment of 9393 bp (fragment going from base 6557 to base 15950, according to the numbering of the sequence of the clone BAC K9E15 published by the consortium Japanese) covering the entire RRS1-S gene (promoter, introns / exons, polyadenylation signal sequence). This Sphl fragment was ligated into a plasmid pUC19 digested with the enzyme Sphl and dephosphorylated. This construction was then introduced into the DH5α strain of E. coli. This clone is defined as the genomic clone RRS1 and is called B1puc3.

The same type of experiment was carried out using cosmid H. The genomic clone obtained is called Hpuc2.

In parallel, the binary vector pDHB321.1 was digested with the enzyme BamHI (single cloning site). The ligation of the inserts (Sphl / Sphl) of the clones B1 puc3 and Hpuc2 (from a Sphl digestion) in the binary vector (BamHI / BamHI) therefore required the use of an adapter in order to ligate the BamHI and Sphl ends between them. Two oligonucleotide primers are involved in the synthesis of this adapter: it consists of an equimolar mixture (5 μM) of the primers noted "BamSph" (5 '-GAT CGC GGC CGC CAT G- 3') and "Not" (5 ' -GCG GCC GC -3 '). The mixture is subjected to a temperature of 95 ° C for 3 minutes. The return to ambient temperature then takes place naturally.

To 100 ng of vector pDHB321.1 digested with the BamHI enzyme, 1 μl of the 5 μM adapter solution is added. The linearized vector and the adapter are ligated in a final volume of 10 μl for 12 hours at 4 ° C. The reaction is then stopped by dilution (addition of 20 μl of water). The excess adapter is subsequently removed by passing the ligation mixture through a microspin S400 column (Pharmacia, ref).

The DNA of clones B1puc3 and Hpuc2 is digested with the enzyme Sphl. After inactivation of the enzyme at 65 ° C for 20 minutes, an aliquot of each digestion (approximately 100 ng) is used to carry out a ligation with the vector pDHB321.1 + adapter. These constructions are then introduced into the strain DH5α of E. coli and selected on an L agar medium containing 50 μg / ml of kanamycin (resistance conferred by the binary vector): the clones which contain the insert originating from clones B1 and H (approximately 9400 bp) are called B1 MT9 and HMTB respectively. The plasmid DNA extracted from these clones is then used to transform the GV3101 strain of A. tumefaciens by thermal shock. The colonies are selected on an agar medium L containing 25 μg / ml of gentamycin (resistance conferred by the helper plasmid pMP90) and 50 μg / ml of kanamycin. The colonies of A. tumefaciens transformed by the DNA of clones B1 MT9 and HMTB are called B1 T9.1 and HMTB2 respectively.

EXAMPLE 5 Expression of the RRS1-S and RRS1-R Genes by Northern Experiments

The expression of the RRS1-S and RRS1-R genes was studied in plants not infected by Northern type experiments. After extraction of the total RNA by the technique described by Lummerzheim (1993), the polyadenylated RNAs were purified by using the Dynabeads Kit (Dynal, USA). After dosing at 260 nm, the mRNAs were deposited on a gel in a denaturing condition (Marco et al. 1990), then transferred to a nitrocellulose membrane (Hybond N +, Amersham Pharmacia Biotech). The hybridization was carried out according to the protocol given by the supplier.

Example 6) Obtaining full-length cDNA clones by RACE PCR

Obtaining full-length cDNA clones required the use of a Rapid Amplification of cDNA Ends (RACE) PCR kit. The details of the experimental protocols presented below are valid for the cDNA clones derived from the Col-5 and Nd-1 ecotype.

at. RACE PCR (rapid amplification of cDNA ends)

The 5 'and 3' ends of the cDNA clones were generated by RACE PCR using the "Smart Race cDNA Amplification" kit (Clontech, USA). The total RNAs were extracted from seedlings of the Col-5 and Nd-1 ecotypes two weeks old according to the protocol of Lummerzheim et al. (1993). The reverse transcriptase reactions were carried out on 1 μg of total RNA according to the conditions provided by the manufacturer. The first strands of the cDNAs were synthesized using the following primers:

- 5'CDS and SMART II oligo (Clontech) to obtain the 5 'ends. - 3'CDS (Clontech) to obtain the 3 'ends.

The oligonucleotide primers used for the amplification of the 5 ′ end of the RRS1 (Col-5) and rrsl (Nd-1) cDNAs are:

- the universal primer UPM (Universal Primer Mix, Smart Race kit) - the RT4 antisense primer, specific for RRS1-S / RRS1-R cDNAs (see Table 2).

The oligonucleotide primers used for the amplification of the 3 'end of the RRS1 (Col-5) and rrsl (Nd-1) cDNAs are:

- the universal primer UPM (Universal Primer Mix, Smart Race kit) - the RT6 primer, specific for RRS1-S / RRS1-R cDNAs (see Table 2). Primers RT4 and RT6 were chosen such that the 5 'and 3' end amplification products have in common a 500 bp region containing a unique BamHI restriction site. The amplification conditions for the 5 ′ and 3 ′ ends are as follows:

-5 cycles (94 ° C for 30 seconds, 72 ° C for 5 minutes) -5 cycles (94 ° C for 30 seconds, 70 ° C for 30 seconds, 72 ° C for 5 minutes) -30 cycles (94 ° C for 30 seconds, 68 ° C for 30 seconds, 72 ° C for 5 minutes)

The size of the amplification products for the 5 ′ and 3 ′ ends is: 3708 bp and 1230 bp for Col-5; 3714 and 1231 bp for Nd-1 respectively. These PCR products were cloned into the vector pGemT-easy (Promega). The sequencing of these clones made it possible to precisely determine the start (5 ') and the end (3') of the untranslated transcribed region of the cDNA clones originating from the ecotypes Col-5 and Nd-1. The size of the full-length cDNA clones from the Col-5 and Nd-1 plants is 4336 and 4343 bp, respectively.

b. Obtaining full-size cDNA clones

Full-size cDNA clones were generated (i.e. 4336 and 4343 bp) by carrying out a double digestion BamHI (single site) and NotI (site present at the level of the multiple cloning site of the vector pGemT-easy) on a mixture of 2 plasmids in order to excise the corresponding inserts at the 5 'and 3' ends. The 5 'and 3' inserts were then ligated together in the plasmid pGemT-easy digested with NotI and dephosphorylated. After transformation of DH5α bacteria with the ligation mixture and plating on selective medium (medium L + ampicillin 50 μg / ml + 0.5 mM IPTG + 80 μg / ml X-Gal), the positive clones containing the full-size cDNA clone were selected by carrying out PCR screening on colonies using primers K3.5 and RT2 (see Table 6) which are specific for each of the 5 'and 3' ends respectively.

A second RACE PCR experiment was carried out on the first strands of cDNAs from the reverse transcriptase reaction described above with the aim of introducing two SalI restriction sites, one being located just upstream from the ATG initiator codon. (translation initiation point), the other being located after the polyadenylation signal. The two pairs of primers used to amplify the 5 'and 3' ends are 5'RRSall / RT8 and 3'RRSall / RT7 respectively.

The amplification conditions, the cloning of the PCR products as well as the method for obtaining the full length cDNA clones are the same as those described above except that the single restriction site common to the 5 'and 3 ends 'is an Aflll site. The purpose of introducing SalI sites on either side of the sequence of the cDNA clone is to facilitate its cloning in different types of vectors.

Example 7) Domain Exchange Experiment by Obtaining Chimeric RRS1 -S / RRS1-R Clones The plasmid DNA used to carry out the domain exchange experiments comes from the genomic clones B1 puc3 and Hpuc2 described above. These two plasmids were digested with the enzyme BamHI whose restriction site is located in the region with similarities to a resistance gene (exon 5 ^ιeme). This digest generates two fragments, one of 4881 bp (clone B1 puc3 RRS1 containing the S-gene) containing the 5 'portion of the genomic clone (promoter and coding portion of the gene RRS1-S to the level of 5 ^ιeme exon) and a fragment of 7180 bp (clone B1 puc3) containing the 3 'part of the genomic clone (4512 bp containing the end of the region showing similarities with the resistance gene, the region similar to the gene encoding a WRKY-type transcription factor and the 3 'region transcribed untranslated; 2668 bp of plasmid vector pUC19).

The size of the fragments generated by the BamHI digestion of the plasmid Hpuc2 is of the same order of magnitude as that of the fragments obtained by BamHI digestion of the plasmid B1 puc3.

After digestion with the BamHI enzyme, an aliquot of each of the DNA fragments generated is dephosphorylated. The rest is deposited on gel in order to purify the fragments corresponding to the 5 ′ part of each genomic clone. The DNA fragment corresponding to the 5 'part of the RRS1-S gene is then ligated with the 3' part of the RRS1-R gene. Likewise, the DNA fragment corresponding to the 5 'part of the RRS1-R gene is ligated with the 3' part of the RRS1-S gene. These constructs are then introduced into E. coli (strain DH5α) and were respectively called 3BH (5'RRS1-S / 3'RRS1-R) and 6HB (5'RRS1-R 3'RRS1-S). Primers specific to the 5 'and 3' regions of the RRS1-S and RRS1-R genes made it possible to verify the constructs. The DNA of each plasmid was purified, digested with Sphl, an enzyme allowing the excision of genes in their entirety. The inserts were then ligated into the plasmid vector pDHB321.1 digested with the enzyme BamHI and the ends of which were made compatible with Sphl ends by virtue of the presence of an adapter (cf. above). These constructions were successively introduced into the DH5α strain (the clones are called 3BHMT7 (5'RRS1-S / 3'RRS1-R) and 6HBMT2 (5'RRS1-R / 3'RRS1-S)) then in the strain GV3101 A.. tumefaciens (clones called 3BHMT7.1 and 6HBMT2.1).

Example 8) Search for proteins interacting with the rrsl and RRS1 proteins by the double hybrid technique in yeast

In order to identify proteins interacting with the RRS1-S and RRS1-R proteins, the double hybrid system in yeast will be used (Clontech kit, Matchmaker Gal4 Two-Hybrid System 3). The whole cDNA clones corresponding to the RRS1-S and RRS1-R genes as well as subfragments corresponding to certain particular domains of these genes (TIR, NBS, LRR, WRKY domains) will be used to screen cDNA libraries in the purpose of isolating genes whose products interact with the products of these DNA fragments. EXAMPLE 9 Modulation of the Expression of the RRS1-S and RRS1-R Genes by Sense and Antisense Approaches

With regard to antisense approaches, two different constructions have been made.

The first consists of a 299 bp fragment (position 3642 to 3941 of the sequence SEQ ID No. 8) fragment corresponding to the WRKY domain of the protein RRS1-1. PCR primers (B1AS5 'and B1AS3' which allow the introduction of an Ncol and SalI site respectively) were generated to amplify this DNA fragment. The amplification was carried out on single-stranded cDNAs resulting from the "reverse transcription" of total RNA from leaves of 2-week-old Col-5 plants. The polymerase used is Deep Vent (Biolabs, New England) and the amplification conditions are those described by the manufacturer. The amplification product B1AS57B1AS3 is digested by the Ncol and Sali enzymes and the digestion product deposited on gel and purified (Kit Jetsorb, Quantum Appligene). This DNA fragment is then ligated into the vector PA1 proterm 2 (gift from Pr. Lescure, our institute) digested with the enzymes Ncol and Sali. This construction was introduced into the DH5α strain of E. coli. The corresponding plasmid DNA has been purified and digested with the BamHI enzyme, which allows excision of an insert comprising the promoter of the EF1α gene, the 299 bp of the RRS1-S gene in antisense orientation as well as the gene terminator. EF1a (Axelos et al., 1989). This BamHI fragment was then inserted by ligation into the binary vector pDHB321.1 digested with BamHI. This new construction was introduced into the strain DH5α of E. coli. Purified plasmid DNA was used to transform strain GV3101 from A. tumefaciens.

The second antisense construct was carried out by excising an EcoRI fragment from the insert of the full-length cDNA clone RRS1-R (sequence SEQ ID No. 4). The DNA fragment thus generated (size of approximately 3930 bp) was introduced into the vector pKMB (Mylne and Botella, 1998) digested with EcoRI. This vector contains the promoter 35S of the cauliflower mosaic virus (CaMV 35S) as well as the corresponding terminator. This construction was successively introduced into the DH5α strains of E coli and GV3101 of A. tumefaciens by triparental conjugation. The strains of A. tumefaciens obtained (29B1ASMT2: fragment of 299 bp and J1: fragment of 3930 bp) allowed the transformation of the ecotypes Nd-1 and Col-5.

Table 6:

TABLE 6 continued:

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Claims

1. Nucleic acid comprising at least 15 consecutive nucleotides of a nucleotide sequence coding for a protein for resistance of a plant to a pathogen, said protein comprising:

b) a C-terminal part comprising a DNA binding domain comprising the amino acid sequence "WRKYGQK", as well as a nucleic acid of complementary sequence.

2. Nucleic acid according to claim 1, characterized in that it has at least 40%, advantageously 60%, preferably 80% and quite preferably 90%, of identity in nucleotides with the nucleotide sequence SEQ ID N ° 1, as well as a nucleic acid of complementary sequence.

3. Nucleic acid according to claim 2, characterized in that it has at least 40%, advantageously 60%, preferably 80% and very preferably 90%, of identity in nucleotides with a nucleotide sequence chosen from following sequences:

a) the nucleotide sequence going from the nucleotide in position 260 to the nucleotide in position 636 of the nucleic acid SEQ ID N ° 1.

b) the nucleotide sequence going from the nucleotide in position 746 to the nucleotide in position 1856 of the nucleic acid SEQ ID N ° 1.

c) the nucleotide sequence going from the nucleotide in position 1937 to the nucleotide in position 2236 of the nucleic acid SEQ ID No. 1. d) the nucleotide sequence going from the nucleotide in position 2326 to the nucleotide in position 3249 of the nucleic acid SEQ ID No. 1.

e) the nucleotide sequence going from the nucleotide in position 3438 to the nucleotide in position 4291 of the nucleic acid SEQ ID N ° 1.

f) the nucleotide sequence going from the nucleotide in position 5377 to the nucleotide in position 5499 of the nucleic acid SEQ ID N ° 1.

g) the nucleotide sequence going from the nucleotide in position 6085 to the nucleotide in position 6532 of the nucleic acid SEQ ID No. 1.

as well as a nucleic acid of complementary sequence.

4. Nucleic acid according to claim 2, characterized in that it has at least 40%, advantageously 60%, preferably 80% and quite preferably 90%, of identity in nucleotides with a nucleotide sequence chosen from following sequences:

a) the nucleotide sequence going from the nucleotide in position 637 to the nucleotide in position 745 of the nucleic acid SEQ ID N ° 1.

b) the nucleotide sequence going from the nucleotide in position 1857 to the nucleotide in position 1936 of the nucleic acid SEQ ID N ° 1;

c) the nucleotide sequence going from the nucleotide in position 2237 to the nucleotide in position 2325 of the nucleic acid SEQ ID N ° 1;

d) the nucleotide sequence extending from the nucleotide at position 3250 to the nucleotide at position 3437 of the nucleic acid SEQ ID No. 1;

e) the nucleotide sequence extending from the nucleotide at position 4292 to the nucleotide at position 5376 of the nucleic acid SEQ ID No. 1; f) the nucleotide sequence going from the nucleotide in position 5500 to the nucleotide in position 6084 of the nucleic acid SEQ ID N ° 1.

as well as a nucleic acid of complementary sequence.

5. Nucleic acid according to claim 1, characterized in that it comprises at least 15 consecutive nucleotides of the nucleotide sequence SEQ ID No. 4, as well as a nucleic acid of complementary sequence.

6. Nucleic acid according to claim 1, characterized in that it has at least 40%, advantageously 60%, preferably 80% and very preferably 90%, of identity in nucleotides with the nucleotide sequence SEQ ID N ° 5, as well as a nucleic acid of complementary sequence.

7. Nucleic acid according to claim 6, characterized in that it has at least 40%, advantageously 60%, preferably 80% and quite preferably 90%, of identity in nucleotides with a nucleotide sequence chosen from following sequences:

a) the nucleotide sequence going from the nucleotide in position 1184 to the nucleotide in position 1560 of the nucleic acid SEQ ID N ° 5.

b) the nucleotide sequence extending from the nucleotide at position 1670 to the nucleotide at position 278 of the nucleic acid SEQ ID No. 5 .;

c) the nucleotide sequence going from the nucleotide in position 2861 to the nucleotide in position 3160 of the nucleic acid SEQ ID N ° 5;

d) the nucleotide sequence going from the nucleotide in position 3254 to the nucleotide in position 4171 of the nucleic acid SEQ ID N ° 5;

e) the nucleotide sequence going from the nucleotide in position 4360 to the nucleotide in position 5213 of the nucleic acid SEQ ID N ° 5: f) the nucleotide sequence going from the nucleotide in position 6302 to the nucleotide in position 6424 of the nucleic acid SEQ ID N ^c 5;

g) the nucleotide sequence extending from the nucleotide at position 6953 to the nucleotide at position 7136 of the nucleic acid SEQ ID No. 5.

as well as a nucleic acid of complementary sequence.

8. Nucleic acid according to claim 6, characterized in that it has at least 40%, advantageously 60%, preferably 80% and very preferably 90%, of identity in nucleotides with a nucleotide sequence chosen from following sequences:

a) the nucleotide sequence going from the nucleotide in position 1561 to the nucleotide in position 1669 the nucleic acid SEQ ID N ° 5;

b) the nucleotide sequence ranging from the nucleotide at position 2781 nucleotide at position 2860 the nucleic acid SEQ ID No. 5. ;

c) the nucleotide sequence ranging from the nucleotide at position 3161 nucleotide at position 3253 'nucleic acid SEQ ID No. 5;

d) the nucleotide sequence ranging from the nucleotide at position 4172 nucleotide at position 4359 the nucleic acid SEQ ID No. 5;

e) the nucleotide sequence ranging from the nucleotide at position 5214 nucleotide at position 6301 the nucleic acid SEQ ID No. 5;

f) the nucleotide sequence going from the nucleotide in position 6425 nucleotide in position 6592 the nucleic acid SEQ ID N ° 5.

as well as a nucleic acid of complementary sequence.

9. Nucleic acid according to claim 1, characterized in that it comprises at least 15 consecutive nucleotides of the nucleotide sequence SEQ ID No. 8, as well as a nucleic acid of complementary sequence.

10. Nucleic acid hybridizing, under high stringency hybridization conditions, with a nucleic acid according to any one of claims 1 to 9.

11. Nucleotide probe hybridizing, under conditions of high stringency hybridization with a nucleic acid according to any one of claims 1 to 10.

12. A nucleotide primer hybridizing, under conditions of high stringency hybridization with a nucleic acid according to any one of claims 1 to 10.

13. Nucleic acid according to claim 10, characterized in that it is chosen from polynucleotides of sequences SEQ ID No. 1 1 to 61.

14. Antisense nucleotide sequence comprising at least 15 consecutive nucleotides of a nucleic acid according to any one of claims 1 to 10.

15. Recombinant vector, characterized in that it comprises a nucleic acid according to one of claims 1 to 10.

16. Recombinant vector according to claim 15, characterized in that it is a functional expression vector in a plant host cell.

17. Recombinant vector according to one of claims 15 and 16, characterized in that it is a vector of fungal, bacterial or viral origin.

18. Host cell transformed with a nucleic acid according to one of claims 1 to 10 or with a recombinant vector according to one of claims 15 to 17

19. A transformed host cell according to claim 18, characterized in that it is a cell of prokaryotic or eukaryotic origin.

20. Host cell transformed according to claim 19, characterized in that it is an Agrobactehum tumefaciens cell.

21. A transformed host cell according to claim 19, characterized in that it is a plant cell of Arabidopsis thaliana, rapeseed, corn or tobacco.

22. Recombinant plant multicellular organism, characterized in that it comprises at least one transformed host cell according to one of claims 18 to 21.

23. Plant transformed with a nucleic acid according to one of claims 1 to 10, a nucleotide sequence according to claim 13 or a recombinant vector according to one of claims 15 to 17.

24. A transformed plant comprising, in a form integrated into its genome, a nucleic acid according to one of claims 1 to 10, a nucleotide sequence according to claim 13 or a recombinant vector according to one of claims 15 to 17

25. Process for obtaining a transformed plant, characterized in that it comprises the following stages:

a) Obtaining a transformed plant host cell according to one of claims 19 or 21;

b) Regeneration of an entire plant from the recombinant host cell obtained in step a); c) Selection of the plants obtained in step b) having integrated a polynucleotide of interest chosen from the nucleic acids according to one of claims 1 to 10.

26. Process for obtaining a transformed plant, characterized in that it comprises the following stages:

a) obtaining a host cell of Agrobactehum tumefaciens according to claim 20;

b) transformation of the plant by infection with Agrobactehum tumefaciens cells obtained in step a);

c) selection of the plants having integrated a polynucleotide of interest chosen from the nucleic acids according to one of claims 1 to 10.

27. Process for obtaining a transformed plant, characterized in that it comprises the following stages:

a) transfecting a plant cell with a nucleic acid according to one of claims 1 to 10 or a recombinant vector according to one of claims 15 to 17;

b) regenerating an entire plant from the recombinant plant cells obtained in step a);

c) selecting the plants having integrated the nucleic acid according to one of claims 1 to 10 or the recombinant vector according to one of claims 15 to 17.

28. Method for obtaining a transformed plant according to one of claims 25 to 27, characterized in that it also comprises the steps of: d) crossing between them of two transformed plants as obtained in step c);

e) selection of the homozygous plants for the nucleic acid of interest.

29. Method for obtaining a transformed plant according to one of claims 25 to 27, characterized in that it further comprises the steps of:

d) crossing a transformed plant obtained in step c) with a plant of the same species;

e) selection of the plants resulting from the crossing of step d) having conserved the nucleic acid of interest.

30. Processed plant as obtained according to the method according to any one of claims 25 to 29.

31. Seed of a transformed plant according to any one of claims 23, 24 and 29.

32. Plant seed whose constituent cells comprise in their genome a nucleic acid according to one of claims 1 to 10.

33. Use of a nucleic acid according to one of claims 1 to 10 for the expression in vitro or in vivo of a protein chosen from the RRS1-S protein RRS1-R of a peptide fragment thereof.

34. Use according to claim 32, characterized in that it is an expression in vivo in a plant transformed with such a nucleic acid.

35. Use of a nucleotide sequence according to claim 14, or of a recombinant vector comprising a nucleotide sequence according to claim 14, for inhibiting or blocking the expression of the gene coding for the RRS1-S protein or for the RRS1-R protein.

36. Method for detecting a nucleic acid constituting the RRS1-S or RRS1-R gene in a sample, comprising the steps of:

a) bringing a probe or a plurality of probes according to claim 11 into contact with the nucleic acid capable of being contained in the sample;

b) detecting the hybrid possibly formed between the nucleic acid of the sample and the probe (s).

37. Kit or kit for detecting a nucleic acid constituting the RRS1-S or RRS1-R gene in a sample, comprising:

a) a probe or a plurality of probes according to claim 11;

b) where appropriate, the reagents necessary for the hybridization reaction.

38. A method for amplifying an acid constituting the RRS1-S or RRS1-R gene in a sample, comprising the steps of:

a) bringing a pair of primers according to one of claims 12 and 13 into contact with the nucleic acid capable of being contained in the sample;

b) performing at least one amplification cycle of the nucleic acid contained in the sample;

c) detecting the possibly amplified nucleic acid.

39. Kit or kit for the amplification of a nucleic acid in a sample, comprising: a) a pair of primers according to one of claims 12 and 13;

b) where appropriate, the reagents necessary for carrying out the amplification reaction.

40. A polypeptide encoded by a nucleic acid according to any one of claims 1 to 10.

41. Polypeptide according to claim 40, characterized in that it comprises an amino acid sequence SEQ ID No 9 or a polypeptide having at least 40% amino acid identity with the sequence SEQ ID No 9.

42. Polypeptide according to claim 40, characterized in that it comprises an amino acid sequence SEQ ID No 10 or a polypeptide having at least 40% amino acid identity with the sequence SEQ ID No 10.

43. Polypeptide comprising modifications of amino acids of

1, 2, 3, 4, 5, 10 to 20 substitutions, additions or deletions of an amino acid with respect to the amino acid sequence of a polypeptide according to one of claims 41 and 42.

44. A polypeptide comprising at least 5 consecutive amino acids of a polypeptide according to one of claims 40 to 43.

45. Fusion polypeptide, characterized in that it comprises the N-terminal part of the RRS1-S polypeptide fused with the C-terminal part of the RRS1-R polypeptide.

46. Fusion polypeptide, characterized in that it comprises the N-terminal part of the RRS1-R polypeptide fused with the C-terminal part of the RRS1 -S polypeptide.

47. Nucleic acid coding for a polypeptide according to one of claims 45 and 46.

48. Antibody directed against a polypeptide according to one of claims 40 to 46.

49. Method for detecting the presence of a polypeptide according to one of claims 40 to 44 in a sample, comprising the steps of:

a) bringing the sample into contact with an antibody according to claim 45;

b) detecting the antigen / antibody complex possibly formed.

50. Kit or diagnostic kit for detecting the presence of a polypeptide according to one of claims 40 to 44 in a sample, characterized in that it comprises:

a) an antibody according to claim 48;

b) where appropriate, the reagents necessary for the detection of the antigen / antibody complexes formed.

51. Method for screening a candidate substance which binds to an RRS1-S or RRS1-R polypeptide, characterized in that it comprises the steps of:

a) preparing a polypeptide according to one of claims 40 to 44;

b) obtain a candidate substance to be tested

c) bringing the polypeptide of step a) into contact with the candidate substance of step b); d) detecting the complex possibly formed between the polypeptide and the candidate substance.

52. Kit or kit for screening a candidate substance which binds to an RRS1-S or RRS1-R polypeptide, comprising:

a) a polypeptide according to one of claims 40 to 44.

b) where appropriate, the reagents necessary for the detection of the complexes formed between the polypeptide and a candidate substance to be tested.

53. Method for screening a candidate substance which binds to the RRS1-S or RRS1-R polypeptide, comprising the steps of:

c) providing a nucleic acid comprising a nucleotide sequence coding for a detectable marker, placed under the control of a regulatory sequence recognized by the transactivating protein such as Gal4;

the nucleic acids a), b) and c) being inserted into suitable vectors; and

d) co-transfect yeast cells simultaneously with said vectors:

e) detecting the expression of the nucleotide sequence coding for the detectable marker.

54. Kit or kit for the screening of a candidate substance which binds to the RRS1-S or RRS1-R polypeptide, comprising:

c) a third nucleic acid coding for a fusion protein comprising the candidate substance fused to the transcription domain of a transactivating protein such as Gal4;

55. Substance capable of binding to the RRS1-S or RRS1-R polypeptide, characterized in that it is capable of being obtained by a process according to one of claims 48 and 50.

56. A method of screening for a nucleic acid interacting with a polypeptide according to one of claims 40 to 44, comprising the steps of: a) obtaining a statistical population of nucleic acids 20 to 50 nucleotides in length;

b) bringing the population of nucleic acids of step a) into contact with a polypeptide according to one of claims 40 to 44;

57. Kit or kit for the screening of a nucleic acid interacting with a polypeptide according to one of claims 40 to 44, comprising: a) a polypeptide according to one of claims 40 to 44;