WO2002038780A2

WO2002038780A2 - Use of a nucleic acid to provide a plant with resistance to attack by a pathogen

Info

Publication number: WO2002038780A2
Application number: PCT/FR2001/003457
Authority: WO
Inventors: Dominique Roby; Claudine Balague; François GODARD; Marie Lummerzheim
Original assignee: Genoplante-Valor
Priority date: 2000-11-08
Filing date: 2001-11-07
Publication date: 2002-05-16
Also published as: WO2002038780A3; AU2002223779A1; FR2816320A1

Abstract

The invention concerns the use of a nucleic acid for synthesizing polypeptide AKIN11 in a plant to induce or enhance, in said plant, resistance to attack by a pathogen.

Description

Use of a nucleic acid to give a plant resistance to aggression by a pathogen, and plant transformed with such a nucleic acid

Field of the invention

The present invention relates to the field of inducing or increasing the resistance of plants to aggression by a pathogen.

Technical condition

The resistance of plants to diseases caused by phytopathogenic organisms is often induced by the specific recognition of a given pathogen. This recognition of the pathogen leads to a rapid induction of the defense mechanisms of the plant which limit the multiplication and propagation of the pathogen in the various tissues. Among these mechanisms, the hypersensitive response constitutes a form of programmed cell death induced rapidly in the plant cells which are in direct contact with the pathogen. The hypersensitive response (HR) probably plays an essential role in the containment of the pathogen within the cells initially exposed to the latter, giving this plant resistance to said pathogen.

When a pathogen having a specific avirulence gene (avr) comes into contact with a plant having the corresponding resistance gene, rapid cell death is localized at the site of inoculation is initiated, which constitutes the hypersensitive response which limits progression of the pathogen.

During infection, the pathogen is recognized in the case of bacterial pathogenic microorganisms, most often in the cytoplasm where the avirulence factors are injected via a secretion system coded by the hrp cluster ( "Hypersensibility and pathogenicity").

The resistance proteins have structural similarities with each other and are also capable of intervening in the HR signaling pathway. Mutation analyzes in Arabidopsis have made it possible to identify genes necessary for the resistance associated with the resistance gene (Glazebrook et al. 1997). The EDS1 and NDR1 genes of Arabidopsis code for essential components of breed specific resistance (Century et al., 1995; Faik et al., 1999).

These two genes have recently been shown to define two different signaling pathways dependent on the structural type of the resistance protein (AARTS et al., 1998).

There is a need in the prior art to identify new genes capable of inducing a phenotype of resistance to a pathogen in a plant, in particular in a field crop of agronomic interest, in order to avoid, or at the very least to reduce the treatment of crops with chemical pesticides which have been shown to be harmful to the environment.

SUMMARY OF THE INVENTION

The subject of the invention is the use of a nucleic acid allowing the synthesis of the AKIN 11 polypeptide in a plant to induce or increase, in this plant, resistance to aggression by a pathogen.

Preferably, the nucleic acid allowing the synthesis of the AKIN 11 polypeptide comprises a polynucleotide coding for the AKIN 11 polypeptide chosen from the sequences SEQ ID No. 1 and SEQ ID No. 2 and their homologous sequences resulting from the degeneration of the genetic code.

Advantageously, the nucleic acid allowing the synthesis of the AKIN 11 polypeptide comprises a polynucleotide regulating the expression of the polynucleotide coding for the AKIN 11 polypeptide in the plant. According to a particular embodiment, the nucleic acid allowing the synthesis of the AKIN 11 polypeptide in a plant is inserted into an expression vector.

The invention also relates to methods for obtaining a plant transformed with a nucleic acid allowing the synthesis of the AKIN 11 polypeptide in this plant. The invention also relates to a plant resistant to a plant pathogen, characterized in that it has been transformed with a nucleic acid allowing the synthesis of the AKIN 11 polypeptide in this plant.

DETAILED DESCRIPTION OF THE INVENTION

It has been shown according to the invention that the gene coding for the protein AKIN11 makes it possible to induce resistance to a given pathogen in a plant initially sensitive to aggression by said pathogen.

The akin 11 gene was initially isolated by heterologous screening of a cDNA library using the yeast SNF1 gene. The akin 11 gene product had been shown to interact with the PRL1 protein, which is a pleiotropic regulator of genes induced in response to hormonal or "sugar-dependent" signals (NEMETH et al. 1998); BHALEARO et al. (1999) have shown that the akin11 gene is potentially involved in the metabolism of sugars in plants, since the AKIN 11 protein interacts with the yeast protein PRL1 which is involved in regulating the metabolism of sugar in response to a glucose regulating signal in this microorganism.

It has been shown for the first time according to the invention that the akin 11 gene is capable of inducing in a plant resistance to aggression by a pathogen. Based on a chemical mutagenesis operation at

Arabidopsis thaliana, the applicant has characterized a mutant, the mutant hxc-2, which exhibits a sensitive phenotype in response to infection with the avirulent strain of Xaηthomonas campestris pv.campestris strain 147. The applicant has also shown, by the use biochemical and molecular markers, that the mutation pleiotropically affected the defense responses of the plant. In addition, a first detailed genetic analysis carried out by the applicant has shown that the mutation is inherited as a recessive monogenic trait, the corresponding gene being located on chromosome III, between the markers mi 413 and atpox, in a region of the gene. about 2.1 cM, which roughly corresponds to a region of the chromosome 1 to 2 megabases long.

The absence of a recombination event in this region of

2.1 cM of chromosome III did not make it possible to envisage isolating the gene by a conventional approach of positional cloning. The applicant therefore created new physical markers in this region, then built a precise physical map of the HXC2 locus in order to construct a contiguous BAC clone between the markers mi413 and the new marker MXO21-LE before constructing a high map. resolution covering the HXC2 locus, then analyze the nucleotide sequences included in a defined interval between the marker mi413 and the new marker MXE2LE.

It is shown according to the invention that the interval defined by the markers MI413 and the new marker MXE2LE comprises the akin11 gene, which is mutated in hxc2 plants sensitive to a pathogen.

It has also been shown according to the invention that the complementation of the mutant hxc2 with the non-mutated akin11 gene makes it possible to restore, in the plant thus complemented, the phenotype of resistance to aggression by a pathogen. A first object of the invention consists in the use of a nucleic acid allowing the synthesis of the AKIN11 polypeptide in a plant to induce or increase, in this plant, resistance to aggression by a pathogen.

By "AKIN11 polypeptide" does not mean a polypeptide comprising the amino acid sequence SEQ ID No. 3 of the sequence listing.

According to the invention, any conventional technique of molecular biology, microbiology and recombinant DNA known to those skilled in the art can be used. Such techniques are described for example by SAMBROOK et al., (1989), GLOVER (1985), GAIT (1984),

HAMES and HIGGINS (1985), and AUSUBEL et al. (1989).

For the purposes of the present description, the expression “nucleotide sequence” can be used to denote either a polynucleotide or a nucleic acid. The expression "sequence nucleotide "includes the genetic material itself which is therefore not restricted to information regarding its sequence.

The terms “nucleic acid”, “polynucleotide”, “oligonucleotide” or “nucleotide sequence” include RNA, DNA, cDNA sequences or even RNA / DNA hybrid sequences of more than one nucleotide, indifferently in single strand form or in duplex form.

The term “nucleotide” designates both natural nucleotides (A, T, G, C) as well as modified nucleotides which comprise at least one modification such as (i) an analog of a purine, (ii) an analog of d 'a pyrimidine, or (iii) a similar sugar, such modified nucleotides being described for example in PCT application No. WO 95/04064.

A nucleic acid "allowing the synthesis" of the AKIN11 polypeptide consists of a nucleic acid whose sequence includes all of the codons necessary for the translation of this nucleic acid, or of a nucleic acid which is derived therefrom, to the AKIN11 polypeptide of sequence SEQ ID N ° 3. Thus, the nucleic acid allowing the synthesis of the AKIN11 polypeptide includes a nucleic acid comprising the eleven exons of the akin11 gene which is described in particular in the GENBANK database under the access number X99.279, or also by the sequence SEQ Sequence listing ID # 2. The respective positions of the first and last nucleotides of each of the exons of the akin11 gene, in the sequence SEQ ID No. 2, are respectively: Exon 1 (14-97); Exon 2 (393-587); Exon 3 (717-901); Exon 4 (996-1180); Exon 5 (1276-1406); Exon 6 (1504-1688); Exon 7 (1764-1854); Exon 8 (1940-2023); Exon 9 (2107-2246); Exon 10 (2383-2613) and Exon 11 (2899-3203). The base A of the ATG codon for initiating translation is the nucleotide at position 395 of the sequence SEQ ID No. 2. The T base of the translation stop TGA codon is the nucleotide at position 3005 of the sequence SEQ ID No. 2.

A nucleic acid allowing the synthesis of the polypeptide

AKIN11 also includes the nucleic acids comprising the region coding for the AKIN11 polypeptide which are in the form of an RNA or cDNA, the coding region of the cDNA coding for the polypeptide AKIN11 being included in the sequence SEQ ID N ° 1 of the sequence listing.

To obtain the cDNA sequence of the akin11 gene, a person skilled in the art can carry out an amplification by RT-PCR on the messenger RNAs extracted from Arabidopsis thaliana cells using the pair of primers AKIN 68 (SEQ ID No. 15) and AKIN1648 (SEQ ID No. 16). A cDNA sequence comprising the region coding for the AKIN11 polypeptide consists of the sequence SEQ ID No. 1. The base A of the translation initiation ATG codon is the nucleotide at position 1 of the sequence SEQ ID No. 1. The T base of the translation stop TGA codon is the nucleotide at position 1537 of the sequence SEQ ID No. 1.

To obtain the genomic nucleotide sequence of the akin11 gene, a person skilled in the art can carry out an amplification reaction using the pair of primers AKINF (SEQ ID No. 13) and AKINR (SEQ ID No. 14), from genomic DNA from Arabidopsis thaliana, for example from genomic DNA contained in a bank of BAC vectors, or from YAC vectors containing inserts of genomic DNA from this plant.

Preferably, the nucleic acid allowing the synthesis of the AKIN 11 polypeptide comprises a polynucleotide chosen from the nucleotide sequences SEQ ID No. 1 and SEQ ID No. 2, as well as their homologous sequences resulting from the degeneration of the genetic code.

By “coding region” of the AKIN11 polypeptide is meant a sequence which is transcribed and translated into the AKIN11 polypeptide in a cell, in vitro or in vivo, when this sequence is placed under the control of appropriate regulatory sequence (s) (s). The ends of the coding region are delimited on the 5 'side of this region by the translation initiation codon and, on the 3' side of this region, by the translation stop codon.

The objective pursued by the invention is:

- either to induce in a plant resistance to a pathogen to which it is naturally sensitive;

- or to increase in a plant its resistance to aggression by a pathogen. Consequently, it will be ensured that the nucleic acid allowing the synthesis of the AKIN11 polypeptide is expressed in an optimal and / or controlled manner in the plant for which resistance to aggression by a pathogen is sought. In a first preferred embodiment of a nucleic acid allowing the synthesis of the AKIN11 polypeptide according to the invention, said nucleic acid comprises, in addition to the coding region necessary for the synthesis of the polypeptide, a polynucleotide regulating the expression of the nucleotide sequence coding for AKIN11 in plants.

Within the meaning of the invention, the signals regulating the expression of the region coding for the AKIN11 polypeptide include sequences controlling transcription or translation, such as promoters, activator sequences ("enhancers"), terminator sequences and, the where appropriate, polyadenylation signals.

A “promoter” type regulatory sequence, within the meaning of the invention, is a regulatory region recognized by an RNA polymerase in the cell capable of initiating transcription of the coding region of the AKIN11 polypeptide. Preferably, a promoter sequence according to the invention comprises the site of initiation of transcription as well as the domains responsible for the binding of RNA polymerase.

The coding region of the AKIN11 polypeptide is placed "under the control" of a regulatory polynucleotide in a plant cell, when the RNA polymerase transcribes this coding region into messenger RNA, which can be subsequently spliced when the coding region is of origin. genomics, then translated into the AKIN11 protein.

Depending on the case, a constituent expression of the AKIN 11 polypeptide is sought in the plant in which an induction or an increase in resistance to aggression against a pathogen is sought, or an inducible expression of this polypeptide in response to external signals, for example biotic stress, that is to say an expression of the AKIN11 polypeptide which can be limited to one or more tissues of the plant, in particular in the case where the pathogen to which the plant is naturally sensitive specifically attacks only certain of its tissues or organs. According to another aspect, the localization of the AKIN11 polypeptide is sought in a given compartment of the cell, such as chloroplasts, mitochondria, endoplasmic reticulum, vacuoles or the cell wall. In this particular embodiment, the nucleic acid allows the synthesis of the AKIN11 polypeptide coding for a fusion polypeptide consisting of the AKIN11 polypeptide fused with a signal peptide allowing its localization in a given cell compartment or its secretion in the cell wall. By "pathogen" is meant a pathogen of bacterial, fungal or viral origin. Among the pathogens against which resistance is sought according to the invention, there may be mentioned, without limitation, Xanthomonas Erwinia, fusarium, Sclerotinia, mildew, Pvx ("Potatoe virus x"), Pvy ("Potatoe virus y ”), Cmc (“ Cucumber mosaic virus ”) or Tylcv (“ Tomatoe yellow leaf curl virus ”).

Most preferably, a pathogen against which the resistance of a plant is induced or increased according to the invention is a "necrotrophic pathogen, which mainly multiplies in the intercellular space and for which the development of cell death Early growth is favorable for its spread across all leaf tissues. Such necrotrophic pathogens are mainly bacterial and are often found in northern Europe. The Xanthomonas campestris strain is one such necrotrophic pathogen. which the resistance of a plant is induced or increased according to the invention are, by way of illustration but not limitation, the strains of Peronospora parasitica and Pseυdomonas syringae.

According to a first preferred embodiment, the nucleic acid allowing the synthesis of the AK1N11 polypeptide comprises, in addition to the coding region of the AKIN11 polypeptide, also a polynucleotide regulating the expression of this coding region in the plant. According to a second preferred embodiment of the invention, the nucleic acid allowing the synthesis of the AKIN11 polypeptide is inserted into a vector, said vector comprising one or more of the signals necessary for the control of expression of the coding region for the AKIN11 polypeptide in plants.

PREFERRED PROMOTERS ACCORDING TO THE INVENTION

In general, for the choice of a suitable promoter, a person skilled in the art can advantageously refer to the work by SAMBROOK et al. (1989) or to the techniques described by FULLER et al. (1996).

Constituent promoters

Promoters for the expression of a nucleic acid encoding the AKIN11 polypeptide in plants are for example: - the CaMV 35S promoter of the cauliflower mosaic virus, Me ELROY et al. (1991)

- the 35S promoter, or advantageously the constitutive double 35S promoter (pd35S) of CaMV, described in the article by KAY et al. (1987); - the rice actin promoter followed by the rice actin intron (PAR-

LAR) contained in the plasmid pAct-1-F4 described by McELROY et al. (1991);

- the promoter of the actin 1 gene from rice (Me ELROY et al., (1990) - or the promoter from the AdH gene from maize (DENNIS et al.,

1984) or the promoter of corn ubiquitin (CHRISTENSEN et al., 1996);

- the pUbii promoter of the gene coding for corn ubiquitin 1 (CHRISTENSEN et al., (1992) - chimeric promoters comprising activator sequences ("enhancer"), such as the 35S promoter described by HIGGINS et al. (1991 ).

Other promoters useful for the expression of a polynucleotide of interest in plants are described in particular in patents No. US 5,750,866 and US 5,633,363, all documents and articles mentioned above constituting references on which the person skilled in the art can rely on to construct recombinant vectors according to the invention.

Inducible promoters

Where appropriate, the regulatory sequence capable of controlling the nucleic acid coding for a polypeptide according to the invention can be a regulatory sequence inducible by a particular metabolite, such as: - a regulatory sequence inducible by glucocorticoids as described by AOYMA and al. (1997) or as described by McNELLYS et al. (1998);

- a regulatory sequence inducible by ethanol, such as that described by SALTER et al. (1998) or as described by KADDICK et al; (1998);

- a regulatory sequence inducible by tetracycline such as that sold by the company CLONTECH.

- a promoter sequence inducible by a pathogen or by a metabolite produced by a pathogen.

Specific tissue promoters

The regulatory sequence capable of controlling the nucleic acid coding for the AKIN11 polypeptide can also direct the synthesis of this polypeptide specifically in certain tissues, such as the seed, the leaf or the root. These may in particular be the following regulatory sequences:

- a specific tissue promoter such as the HMWG wheat or barley promoter, or the pCRU promoter of the radish cruciferin gene, which all allow expression of the protein of interest in the seeds (ROBERTS et al. ( 1989); ANDERSON OD et al. (1989); DEPIGNY-THIS et al. (1992);

- the promoters pGEA1 and pGEA6 corresponding to the 5 ′ non-coding region of the genes of the seed reserve protein, GEA1 and GEA6 respectively to Arabidopsis thaliana (GAUBIER et al., (1993) and allowing specific expression in seeds;

- the promoter of the corn zein gene (pzein) contained in the plasmid p63, and allowing the expression in the endosperm of corn seeds (REINA et al., 1990);

- specific promoters of particular regions or tissues of plants, and more particularly specific promoters of seeds (DATLA, R. et al., 1997), in particular the promoters of napine (EP 255 378), of phaseoline, of glutenin, heliantinin (WO 92/17580), albumin (WO 98/45460), oelosin (WO 98/45461), IΑTS1 or IΑTS3 (PCT / US 98/06978, filed October 20, 1998;

- specific promoters of leaf tissue or roots.

Advantageously, those skilled in the art may use the specific promoter of the light-inducible leaf tissue described by Terzaghi et al. (1995) or the specific root promoter described by Elmayan et al. (1995).

OTHER SEQUENCES CONTAINING REGULATORY SIGNALS

Advantageously, the nucleic acid allowing the synthesis of the AKIN11 polypeptide can also contain one or more other sequences containing regulatory signals for the expression of the region coding for AKIN11, or alternatively be placed under the control of such regulatory sequences.

Leader sequences

The other sequences containing regulatory signals include 5 'untranslated sequences called "leaders". Such sequences can increase the translation of the mRNA encoding AKIN11. Among these, we can cite, by way of illustration but not limitation:

- the leader EMCV (EncephaloMyoCarditis VIRUS 5 'non coding region) (ELROY-STEIN et al., 1989); - the leader TEV (Tobacco Etch Virus) (CARRINGTON AND FREED, 1990);

- the leader of the BiP gene coding for the heavy chain binding protein of human immunoglobulin (MACEJACK et al., 1991); - the leader AMV RNA 4 originating from the mRNA of the protein of the alfalfa mosaic virus (JOBLING et al. 1987);

- the leader of the tobacco mosaic virus (GALLIE et al., 1989).

"Terminator" sequences

The nucleic acid allowing the synthesis of the AKIN11 polypeptide, or the vector into which this nucleic acid is inserted can also comprise so-called "terminator" sequences. Among the terminators which can be used in the constructions of the invention, there may be mentioned, by way of illustration but not limitation:

- the polyA 35S of the cauliflower mosaic virus (CaMV), described in the article by FRANCK et al. (1980);

- the terminator nos corresponding to the 3 ′ non-coding region of the nopaline synthase gene of the Ti plasmid of Agrobacterium tumefaciens (DEPICKER et al., 1992).

- the terminator of the histone gene (EP 0 633 317).

Sequences encoding a signal peptide

The nucleic acid allowing the synthesis of the AKIN11 polypeptide, or the vector into which this nucleic acid is inserted can also comprise sequences of the vacuolar signal or apoplastic signal peptide type when they are not already present in the sequence of the gene of interest, to bring the protein encoded by the heterologous gene into specific compartments of plant cells.

Preferably, the signal peptide allows the localization of the AKIN11 polypeptide in the walls of the plant, in the cell wall, in conductive tissues such as the phloem, in the endoplasmic reticulum, in chloroplases or in the mitochondria. For the addition of sequences coding for such signal peptides, a person skilled in the art may advantageously refer to the following documents: Keegstra et al. (1995), Zoubenko et al. (1994), Jones et al. (1993), Nhakamura et al. (1993), Hemon et al. (1990), Yang et al. (14990), Thoma et al. (1994) or the PCT application published under No. Wθ 88/02402.

Sequences containing one or more heterologous introns.

When it is desired to express the sequence coding for the AKIN11 polypeptide in a plant of a species other than Brassica, a nucleotide sequence derived from an intron of a gene of the species in which the expression of akin11 is wanted. Preferably, the heterologous intronic nucleotide sequence will be placed on the 5 ′ side of the coding sequence.

According to another embodiment, one or more of the natural sequences of the introns of the akin11 gene will be replaced by such a heterologous intronic sequence specific for the species or specific for a dicotyledon or a monocotyledon.

By way of illustration, but not limitation, the intronic sequence of the Adh (Alcohol dehydrogenase) gene of maize will be used.

A nucleic acid comprising a coding region for the AKIN11 polypeptide and one or more of the regulatory signals as defined above constitutes an “expression cassette”, such an expression cassette constituting an object of the present invention. A particular embodiment of an expression cassette according to the invention is a cassette comprising a polynucleotide encoding a fusion polypeptide consisting of the AKIN11 polypeptide fused with a signal peptide. The invention also relates to a fusion polypeptide consisting of the AKIN11 polypeptide fused with a signal peptide allowing its targeted localization in a determined compartment of the plant cell. PREFERRED EXPRESSION VECTORS ACCORDING TO THE INVENTION

As indicated above, a nucleic acid allowing the synthesis of the AKIN1 1 polypeptide can be inserted into an appropriate vector.

By "vector" in the sense of the present invention, is meant a circular or linear DNA or RNA molecule which is either in single strand or double strand form.

A recombinant vector according to the invention is preferably an expression vector, or more specifically an insertion vector, a transformation vector or an integration vector.

It may especially be a vector of bacterial or viral origin.

In all cases, the nucleic acid allowing the synthesis of the AKIN11 polypeptide is placed under the control of one or more sequences containing signals for regulating its expression in the plant in question, in order to induce or increase the resistance of this plant under attack by a pathogen, either that the regulatory signals are all contained in the nucleic acid coding AKIN1 i, or that one, several of them, or even all the regulatory signals are contained in the container vector into which the nucleic acid coding for AKIN 11 has been inserted.

A recombinant vector according to the invention advantageously comprises appropriate sequences for initiating and stopping transcription.

In addition, the recombinant vectors according to the invention can include one or more origins of functional replication in plants in which their expression is sought, as well as, where appropriate, nucleotide selection marker sequences. The recombinant vectors according to the invention can also include one or more of the expression regulation signals as defined above in the description.

According to an advantageous aspect, a recombinant vector according to the invention is an integrative vector allowing the insertion of multiple functional copies of the nucleic acid allowing the synthesis of AKIN11 polypeptide in the genome of a plant, the induction or increase of resistance to aggression by a pathogen is sought.

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 pB! N19 (BEVAN et al.), sold by the company CLONTECH (Palo Alto, California, USA);

- vector pBI 101 (JEFFERSON, 1987), sold by the company CLONTECH;

- vector pBI121 (JEFFERSON, 1987), sold by the company CLONTECH;

- vector pEGFP; Yang et al. (1996), marketed by the company CLONTECH; - vector pCAMBIA 1302 (HAJDUKIIEWICZ et al., 1994)

PLANTS TRANSFORMED BY A NUCLEIC ACID FOR THE SYNTHESIS OF POLINPEPTIDE AKIN11 AND METHODS FOR OBTAINING THEM

The invention also relates to a transformed plant multicellular organism, characterized in that it comprises a transformed host cell or a plurality of host cells transformed by a nucleic acid allowing the synthesis of the AKIN11 polypeptide as defined in the present description or by a recombinant vector comprising such a nucleic acid.

The subject of the invention is also a transformed plant comprising, in a form integrated into its genome, a nucleic acid allowing the synthesis of the AKIN11 polypeptide, as defined in the present description.

Another object of the invention consists of a plant resistant to a pathogen, characterized in that it has been transformed with a nucleic acid allowing the synthesis of the AKIN11 polypeptide in this plant, or by a recombinant vector comprising such a nucleic acid. Among the plants capable of being transformed according to the invention, mention may be made, by way of examples, of cells of field crops (corn, wheat, rapeseed, sunflower, peas, soybeans, barley) or vegetable plants of flowers or fruit trees or the vine. Among the vegetable plants, non-limiting mention may be made of cucurbits, solanaceae, crucifers, legumes and in particular tomatoes, melons, peppers, zucchini, pumpkins, cucumbers, eggplant, potato, chilli, carrot or lettuce. Among the fruit trees, mention may be made, without limitation, of apple and apricot trees.

Preferably, a plant transformed according to the invention is a cereal and very preferably a corn, a wheat, a barley, a sorghum or a millet. In particular, it is possible to choose plants known to contain large reserves (protein, carbohydrate and lipid), in particular cereal plants or oil plants.

The hybrid plants obtained by crossing plants according to the invention also form part of the invention. The subject of the invention is also a seed or a seed of a plant of a transformed plant as defined above. Typically, such a transformed seed or such a transformed grain comprises one or more cells comprising in their genome one or more copies of a nucleic acid allowing the synthesis of the AKIN11 polypeptide.

According to another aspect, the invention also relates to a seed of a transformed plant as defined above, or even a fruit of such a transformed plant.

The invention also relates to a process for obtaining a transgenic plant as defined in this section, characterized in that it comprises the following steps: a) transfecting a plant host cell with a nucleic acid allowing synthesis AKIN11 polypeptide in a plant, or with a recombinant vector into which such a nucleic acid has been introduced; 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 nucleic acid as defined in the present description.

The subject of the invention is also a process for obtaining a transgenic plant having an induced or increased resistance to aggression by a pathogen, characterized in that it comprises the following steps: a) obtaining a host cell recombinant Agrobacterium tumefaciens transformed with a nucleic acid allowing the synthesis of the AKIN11 polypeptide, or with a recombinant vector into which such a nucleic acid has been introduced; b) transformation of a plant of interest by infection with the recombinant host cell obtained in step a); c) selection of plants which have integrated said nucleic acid into their genome.

In a first particular embodiment, any of the processes for obtaining a transformed plant described above may also include the following additional steps: d) crossing between them of two transformed plants as obtained at 'step c); e) selection of plants homozygous for the transgene.

In a second particular embodiment, any of the methods for obtaining a transformed plant described above can also comprise the following additional steps: f) crossing of 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 f) having conserved the transgene.

In a third embodiment, any of the methods for obtaining a transformed plant described above can also comprise the following steps: h) crossing a transformed plant obtained in step c) with a plant of an agronomic value line of the same species; i) selection of the plants resulting from the crossing of step h) having conserved the transgene; j) ret _. crossbreeding of a transformed plant obtained in step i) with the plant of an agronomic value line of the same species; k) repeat step j) at least twice;

I) selection of plants from backcrosses from step k) which have conserved the transgene and have a genetic background close to or identical to the genetic background of the plant of an agronomic value line of the same species.

Preferably, step j) is repeated two to twenty times, and most preferably three to ten times.

Another subject of the invention consists of a transgenic plant, as obtained according to any one of the methods for obtaining a transformed plant defined above, which exhibits a phenotype of resistance induced or increased to aggression by a pathogen.

The transformation of plant cells can be carried out by techniques known to those skilled in the art.

Mention may in particular be made of direct gene transfer methods such as direct microinjection into plant embryoids (NEUHAUS et al., 1987), vacuum infiltration (BECHTOLD et al., 1993) or electroporation (CHUPEAU and al., 1989) or direct precipitation by means of PEG (SCHOCHER et al., 1986) or the bombardment by cannon of particles covered with the plasmid DNA of interest (FROMM M. et al. 1990).

The plant can also be infected with a bacterial strain, notably Agrobacterium. According to one embodiment of the method of the invention, the plant cells are transformed by a vector according to the invention, said cell host being capable of infecting said plant cells by allowing the integration into the genome of these latter, sequences nucleotides of interest originally contained in the DNA of the above vector. Advantageously, the above-mentioned cell host used is Agrobacterium tumefaciens, in particular according to the method described in the article by AN et al., (1986), or even Agrobacterium rhizogenes, in particular according to the method described in the article by GUERCHE et al., (1987) or also in the PCT application No. WO 00 22.148. For example, the transformation of plant cells can be carried out by the transfer of the T region of the extrachromosomal circular plasmid inducing Ti tumors of Agrobacterium tumefaciens, using a binary system (WATSON et al. 1994). To do this, two vectors are constructed. In one of these vectors, the T region was deleted, with the exception of the right and left borders, a marker gene being inserted between them to allow selection in plant cells. The other partner of the binary system is a helper Ti plasmid, a modified plasmid which no longer has a T region but still contains the vir virulence genes, necessary for the transformation of the plant cell.

According to a preferred mode, the method described by ISH1DA et al. Can be used. (1996) for the transformation of Monocotyledons.

According to another protocol, the transformation is carried out according to the method described by FINER et al. (1992) using the tungsten or gold particle gun.

Those skilled in the art are capable of implementing numerous methods of the prior art in order to obtain plants transformed with a nucleic acid allowing the synthesis of the AKIN1 1 polypeptide. 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. Techniques using other types of vectors can also be used, such as the techniques described by BOUCHEZ et al. (1993) or by HORSCH et al. (1994).

By way of illustration, a transgenic plant according to the invention can be obtained by biolistic techniques such as those described by FINER et al. (1992) or those described by VAIN et al. (1993). Other preferred techniques for transforming a plant in accordance with the invention with Agrobacterium tumefaciens are those described by ISHIDA et al. (1996) or in the PCT application published under the number WO 95/06 722 in the name of JAPAN TOBACCO.

Another subject of the invention is a plant seed whose constituent cells comprise a nucleic acid allowing the synthesis of the AKIN11 polypeptide which has been artificially inserted into their genome.

The subject of the invention is also a seed of a transformed plant as defined above or also a fruit of such a transgenic plant. The invention also relates to any part of a transformed plant as defined above, and without implied limitation the leaves, roots or stems.

The invention also relates to a method for inducing or increasing the resistance of a plant to aggression by a pathogen, characterized in that it comprises a step according to which said plant is brought into contact with the AKIN11 polypeptide, or a composition comprising, as active compound, the AKIN1 1 polypeptide.

A subject of the invention is also a composition for inducing or increasing the resistance of a plant to a pathogen, said composition comprising an effective amount of the AKIN1 1 polypeptide as active principle.

PROBES AND PRIMERS ACCORDING TO THE INVENTION

The invention also relates to the use of probes and primers hybridizing with the genomic DNA or the cDNA of the akin11 gene to detect a trait of resistance to aggression by a pathogen in a plant. In other words, the invention also relates to the use of an akin 1 1 nucleic acid as a molecular marker of a phenotype of resistance to a pathogen in a plant.

The nucleic acids according to the invention, and in particular the nucleotide sequences SEQ ID No. 1, 2, 4 and 5, their fragments of at least 12 nucleotides, as well as the nucleic acids of complementary sequence, are useful for the detection of the presence of at least a copy of a nucleotide sequence of the akin11 gene or of a fragment or an allelic variant thereof in a sample.

Also part of the invention is the use of nucleotide probes and primers hybridizing, under conditions of high stringency hybridization, with a nucleic acid chosen from the sequences SEQ ID Nos. 1, 2, 4 and 5.

Probes or primers made from the sequences SEQ ID No. 1 or 2 are useful for detecting the presence of a trait of resistance to a pathogen. Probes or primers made from SEQ sequences

ID No. 4 or 5 are useful for detecting the presence of a susceptibility to a pathogen.

By high stringency hybridization conditions, within the meaning of the invention, is meant the following hybridization conditions:

Prehybridization: same conditions as for hybridization duration: 1 night.

Hybridization:

5 x SSPE (0.9 M NaCI, 50 mM sodium phosphate pH 7.7, 5 mM EDTA)

5 x Denhardt's (0.2% PVP, 0.2% Ficoll, 0.2% SAB)

100 μg / ml salmon sperm DNA 0.1% SDS duration: 1 night. washes:

2 x SSC, 0.1% SDS 10 min 65 ° C

1 x SSC, 0.1% SDS 10 min 65 ° C 0.5 x SSC, 0.1% SDS 10 min 65 ° C

0.1 x SSC, 0.1% SDS 10 min 65 ° C. The parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm). For sequences comprising more than 360 bases, Tm is defined by the relation:

Tm = 81.5 + 0.41 (% G + C) +16.6 Log (cation concentration) - 0.63 (% formamide) - (600 / number of bases) (SAMBROOK et al., (1989) , pages 9.54-9.62). For sequences of length less than 30 bases, Tm is defined by the relation: Tm = 4 (G + C) + 2 (A + T).

Under appropriate stringency conditions, in which the aspecific sequences do not hybridize, the hybridization temperature is approximately 5 to 30 ° C, preferably 5 to 10 ° C below Tm.

The hybridization conditions described above can be adapted according to the length of the nucleic acid whose hybridization is sought or 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 also in the work of AUSUBEL et al. (1989).

The nucleotide probes or primers used according to the invention comprise at least 12 consecutive nucleotides of a nucleic acid according to the invention, in particular of a nucleic acid of sequences SEQ ID No. 1, 2, 4 and 5 or of its complementary sequence or of a nucleic acid hybridizing under high stringency hybridization conditions with a sequence chosen from sequences SEQ ID No. 1, 2, 4 and 5. Preferably, nucleotide probes or primers according to the invention will have a length of at least 12, 15, .18, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000, 2000 or 3000 consecutive nucleotides of a nucleic acid according to the invention. Alternatively, a probe or a nucleotide primer according to the invention will consist and / or include fragments with a length of 12, 15, 18, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000, 2000 or 3000 consecutive nucleotides of a nucleic acid according to the invention. A probe of interest according to the invention consists of a probe which, under these given hybridization conditions, is capable of discriminating the sequences SEQ ID No. 1 or 2 from the sequences SEQ ID No. 4 or 5. Preferably, a such a probe will hybridize with a nucleic acid in a sequence comprising base C in position 1972 of the sequence SEQ ID No. 2 or in position 1003 of the sequence SEQ ID No. 1 and will not hybridize with a nucleic acid comprising base T in position 1972 of the sequence SEQ ID N ° 2 or in position 1003 of the sequence SEQ ID N ° 1.

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 the method to the phosphodiester of NARANG et al. (1979) or BROWN et al. (1979), the diethylphosphoramidite method of BEAUCAGE et al. (1980) or the solid support technique described in European patent n ° EP 0 707 592. Each of the nucleic acids according to the invention, including the oligonucleotide probes and primers described above, can be labeled, if desired, by incorporating a detectable molecule, that is to say a detectable marker, 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 the probes is preferably done by incorporating labeled molecules within the polynucleotides 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 French patent No. FR 78 10 975 or ^" also in the articles by 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 to the genomic DNA of the akin11 gene or else in hybridizations to the messenger RNA of this gene 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 comprise surfaces of the wells of microtitration plates, polystyrene beads, magnetic beads, nitrocellulose strips or even microparticles such as latex particles. Consequently, the subject of the invention is also the use of a nucleic acid of a nucleotide probe or primer, characterized in that that it comprises at least 12 consecutive nucleotides of a nucleic acid of nucleotide sequence SEQ ID No. 1, 2, 4 or 5.

The invention also relates to the use of a nucleic acid which can be used as a nucleotide probe or primer, characterized in that it consists of a polynucleotide of at least 12 consecutive nucleotides of a nucleic acid of sequence chosen from nucleotide sequences SEQ ID No. 1, 2, 4 or 5.

As described above, such a nucleic acid can further be characterized in that it is labeled with a detectable molecule. The present invention also relates to a method for detecting the presence of a nucleic acid of the akinll gene which is a marker for resistance to a pathogen in a sample, said method comprising the steps of:

1) bringing a probe or a plurality of nucleotide probes according to the invention into contact with the test sample;

2) detect 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 according to the invention 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 will be used to detect target sequences of interest of the akinll gene or alternatively to detect mutations in coding regions or non-coding regions of the akin 11 gene, more particularly nucleic acids of sequence SEQ ID No. 1, 2, 4 and 5 or nucleic acids of complementary sequence.

“Target sequence” is understood to mean, within the meaning of the invention, a nucleotic sequence included in a nucleic acid, said nucleotide sequence hybridizing, under the hybridization conditions specified in the description, with a probe or a nucleotic primer of the invention .

A preferred target sequence consists of a sequence included in the sequence SEQ ID No 1 or SEQ ID No 2 and comprising the base C in position 1972 of the sequence SEQ ID No 2 or in position 1003 of the sequence SEQ ID no. 1.

The nucleotide primers according to the invention can be used to amplify any nucleotide fragment (gDNA, cDNA, mRNA) of the akinll gene, and more particularly all or part of a nucleic acid of sequence SEQ ID No. 1, 2, 4 or 5, or a fragment or a variant of these sequences.

Another subject of the invention relates to a method for the amplification of a nucleic acid according to the invention, and more particularly of a nucleic acid of sequence SEQ ID No. 1, 2, 4 or 5 or a fragment or a allelic variant thereof 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 target nucleic acid of the akin11 gene whose amplification is sought, in the presence of the reagents necessary for the amplification reaction; and b) detection of the optionally amplified nucleic acid.

To carry out the amplification method as defined above, one will advantageously have recourse to any one of the nucleotide primers described below.

The subject of the invention is also a kit or kit for the amplification of a nucleic acid according to the invention, and more particularly all or part of a nucleic acid of sequences SEQ ID No. 1, 2, 4 or 5 , said kit or kit comprising: a) a pair of nucleotide primers according to the invention, the hybridization position of which is located respectively on the 5 ′ and 3 ′ side of the target nucleic acid of the akinll gene, of which l 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 described above.

USE OF TARGETED ANTISENSE OLIGONUCLEOTIDES ON THE AKIN11 GENE TO MODULATE THE RESISTANCE OF A PLANT TO AGGRESSION BY A PATHOGEN. The resistance of a plant to aggression by a pathogen can be modulated by acting on the level of expression and / or translation of the akin1 gene, for example by using antisense oligonucleotides. Preferably, an antisense polynucleotide according to the invention comprises a sequence complementary to a sequence located in the region of the 5 'end of the DNA of the akinl l gene, and very preferably the proximity of the codon initiation of translation (ATG) of the akinll gene.

According to a second preferred embodiment, an antisense polynucleotide according to the invention comprises a sequence complementary to one of the sequences located at the level of the exon / intron junctions of the akinl l gene and preferably sequences corresponding to a splicing site .

A preferred antisense polynucleotide according to the invention comprises at least 15 consecutive nucleotides of the cDNA of the akin11 gene having the nucleotide sequence SEQ ID No. 1 or of the genomic DNA of the akinl 1 gene of sequence SEQ ID No. 2.

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), and C and G.

In general, an antisense polynucleotide according to the invention has at least 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, 500, 1000 or 2000 consecutive nucleotides of the cDNA of AKIN11 of sequence SEQ ID N ° 1 or SEQ ID N ° 2.

By way of illustration, a preferred antisense polynucleotide according to the invention consists of the nucleic acid of the cDNA of AKIN1 1 of sequence SEQ ID No. 1. An antisense polynucleotide of the akin 11 gene according to the invention can be prepared by any suitable method well known to those skilled in the art, including by cloning and restriction enzyme action or by direct chemical synthesis according to techniques such as phosphodiester method of NARANG et al. (1979) or BROWN et al. (1979), the diethylphosphoramidite method of BEAUCAGE et al. (1980) or the solid support technique described in European patent No. EP-0 707 592.

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 akin-11 mRNA. Strategies for constructing the antisense polynucleotides are notably described by GREEN et al. (1986) and IZANT and WEINTRAUB (1984).

Methods for constructing antisense polynucleotides are also described by ROSSI et al (1991) as well as in PCT applications No. WO 947 / 23.026, WO 95/04141, WO 92 / L18.522 and in European patent application No. EP 0 572 287.

Other methods of implementing antisense polynucleotides are for example those described by SCZAKIEL et al. (1995) or those described in PCT application No. WO 95 / 24.223.

Other techniques for using antisense polynucleotides which can be used by those skilled in the art are those of SALE et al. (1995) as well as that of GAO et al. (1996).

Another subject of the invention is the use of an antisense poloyucleotide hybridizing with the nucleic acid of sequence SEQ ID No. 2 to modulate the resistance of a plant to aggression by a pathogen, and in particular to inhibit resistance from a plant to aggression by a pathogen.

The resistance of a plant to aggression by a pathogen can be modulated by acting on the expression of the akinl 1 gene, for example by using antisense oligonucleotides.

The present invention is further illustrated, without however being limited, to the following figures and examples.

DESCRIPTION OF THE FIGURES

Figure 1 illustrates Isolation of the ends of BAC from the TA&MU bank. The nuclear DNA inserts are cloned into the vector pBeloBACH which comprises, in its sequence, the unique Sphl sites, BamHI, Smal, Sacl, and EcoRI. Digestion of the clone by one of these restriction enzymes makes it possible to generate fragments comprising a portion of the vector and a portion of insert of variable size. After religation of these fragments on themselves, the right end can be amplified by reverse PCR. The insert fragment corresponding to the left end associated with the vector constitutes a functional plasmid comprising the origin of replication and the marker for resistance to chloramphenicol. The left end can thus be cloned by "plasmid rescue". Cm ^r , resistance to chloramphenicol; Ori, origin of replication.

FIG. 2 illustrates the search for ORFs in the region delimited by the markers mi413 and MXE2RE.

(AT). The sequenced portion of the clone T7I19 (corresponding to the portion covered by the clone MXE2) is represented on this clone. The vertical black bars indicate the position of the EcoRI sites.

(B) Cosmids C7l 19-66 and C7119-49 covering the 18 kb region between the markers atpox and mi413 are shown in gray. (C) ° The presence of two ORFs was detected by the Genscan program in the interval delimited by the markers atpox and mi413. One corresponds to the coding sequence of the Akinl 1 gene which codes for a protein kinase of SNFI type; the other for a protein of 171 amino acids showing no homologies with the sequences present in the databases. The solid squares represent the coding sequence of the two identified genes. The first exon with the translation initiation codon is shown.

FIG. 3 illustrates the genomic structure of the Akinl 1 gene from Arabidopsis and position of the hxc-2 mutation: The Akinl 1 gene comprises 11 exons, represented by gray rectangles, the size of which is indicated in kb. The exon containing the translation initiation codon is shown. The position of the bases, bordering each exon is indicated on the MXE2 clone. Fragments B, C, D, E, F, G and H amplified by PCR and cloned for the sequencing of the hxc-2 allele are represented by lines. The hxc-2 mutation, which results from a C -> T inversion at position 77578 of the clone MXE2, in exon 8 of the Akinl 1 gene is indicated.

Figure 4 illustrates the complementation of the hxc-2 mutation.

Above: Cosmids used to transform mutant plants. The cosmids C7119-29, C7119-32, C7119-66 and C7119-49 were used to transform, via Agrobacterium, mutant plants. Below: Phenotype observed, in response to inoculation with strain 147, in a primary transformant, T66, obtained with the clone C7119-66, compared to the mutant hxc-2, 5 days after inoculation.

FIG. 5 illustrates the construct of a recombinant expression vector containing a cDNA or genomic DNA coding for the AKIN11 polypeptide, or the corresponding antisense sequence.

35S Pro: 35 S promoter of CaMV; NOS Ter: Terminator of the Nopaline synthase gene from A. Tumefaciens; EF: blunt ends. NPIII: kanamycin resistance gene. AKINF: SEQ ID N ° 13; AKINR: SEQ ID N ° 14.

FIG. 6 illustrates the construction of a recombinant expression vector containing a sequence of the CDNA coding for the AKIN11 polypeptide; or the corresponding antisense sequence.

35S Pro: 35 S promoter of CaMV; NOS Ter: Terminator of the Nopaline synthase gene from A. Tumefaciens; EF: blunt ends ,. AKIN68: SEQ ID N ° 15. AKIN1648; SEQ ID N ° 16. EXAMPLES

EXAMPLE 1: POSITIONAL CLONING OF THE CAUSAL GENE OF THE SENSITIVITY PHENOTYPE IN THE HXC2 MUTANT PLANT: GLOBAL MAPPING ON DIFFERENT CHROMOSOMES OF ARABIDOPSIS THALIANA.

Following the phenotypic characterization of the hxc-2 mutant, the mapping of the locus had been undertaken by the analysis of the phenotype of 132 F2 plants from the cross between a Co-gl-1-hxc-2 plant and the wild polymorphic ecotype , Ws-0. A first phenotypic screen was carried out on this population in response to inoculation with strain 147 of Xanthomonas campestris pv. campestris and 78 individuals, including 28 plants of mutant phenotype, were selected and self-fertilized. The analysis of the segregation of the hxc-2 character in each F3 subfamily resulting from self-fertilization made it possible to determine, in a co-dominant fashion, the genotype of the F2 individuals at the HXC2 locus. The integration, in a genetic map previously carried out on the same population, of the genotype of the F2 plants at the HXC2 locus made it possible to position the locus in the centromeric position, on chromosome III, between the RFLP markers mi413 and atpox (Table 1).

This interval, defined by the presence of a recombination event to the right of the mi413 telomeric marker and of a recombination event to the left of the centromeric marker, atpox, represents a physical distance of approximately 1.2 Mb, evaluated on the basis physical mapping data offered by the arrangement of YACs ("Yeast Artificial Chromosomes") clones from the Bank of Versailles in the region. These data are available on the TAIR website (WWW.arabidopsis.org). The absence of additional recombination events in this interval made it impossible to envisage isolating the gene by a positional cloning approach. The pursuit of chromosomal walking therefore consisted, firstly, in isolating recombinants in the interval defined by the flanking markers (mi413 and atpox), and secondly, in increase the number and therefore the resolution of molecular markers in this interval.

Table 1: Frequency of genetic recombination between the hxc2 locus and molecular markers of the Arabidopsis genome.

EXAMPLE 2: POSITIONAL CLONING OF THE CAUSAL GENE OF THE SENSITIVITY PHENOTYPE IN THE HXC2 MUTANT PLANT: RESTRICTION OF THE INTERVAL DEFINED BY THE mi413 and atpox MARKERS.

I. Isolation of recombinants between the markers mi413 and aήαox:

The positioning of a mutated locus by a positional cloning approach is only defined by the existence of genetic recombination events between the genotype of F2 individuals at the locus considered, accessible by the analysis of the segregation of the mutant character in the F3 lines, and the genotype of these same individuals for molecular markers linked to the mutation. The restriction of the physical interval defining the position of a mutation therefore implies the identification of recombination events sufficiently close to the mutated locus. The isolation of these random recombinants can only be done by increasing the size of the segregated population.

The mapping of the hxc2 locus had been carried out on a small population (78 individuals); therefore, the need to isolate new recombinants for the positional cloning of the gene was considered long before its location between the markers

and atpox. The isolation of random recombinants was therefore approached by an inverse genetics approach, after having located the HXC2 locus in a 13 cM window, between the GL-1 and m249 markers. The hxc-2 mutant was isolated from a Co \ -gl-1 genetic background. The recessive mutation, gl-1, located in the centromeric position on chromosome III is responsible for the lack of differentiation of epidermal cells into trichomes and confers the glabrous phenotype. This phenotypic marker, inherited from the mutant parent in the population, co-aggregates with the hxc-2 mutation and constitutes one of the upper limits of the locus. The presence of this phenotypic marker was used to isolate recombinants between the gl-1 and m249 markers. Within an F2 population resulting from the cross between a mutant plant Co \ -gl-1 -hxc-2 and a wild plant Ws-0, 367 individuals of hairless phenotype (having inherited this trait from the mutant parent) were selected. The DNA of these individuals, having a homozygous Col-0 genotype at the GL-1 locus, was isolated, digested with EcoRI and subjected to an RFLP analysis by the marker m249 which constituted the lower limit of the HXC2 locus. 61 individuals with a Ws-0 homozygous or heterozygous profile for this marker, resulting respectively from a double and a simple recombination event between the gl-1 and m249 markers were selected, self-fertilized and subjected to a second screen by the markers m \ 413 and atpox. Only 8 recombinant individuals between these last two markers were preserved and the phenotype of the corresponding F3 lines was tested in response to inoculation with the strain 147. This phenotypic screen made it possible to determine the genotype of each recombinant individual, at the HXC2 locus , co-dominantly. The recombination events of these 8 individuals, R80, R139, R148, R269, R273, R284, R287 and R313 added to those of the H25 and Wh72 lines, from the initial mapping population, were used to specify the position of the locus HXC2.

II. Production of molecular markers in the interval defined by the markers mi 413 and atpox. Beyond the identification of recombinants in the interval defined by the two markers flanking the locus of interest, the precise positioning of the locus in relation to these recombination events also requires increasing the number and therefore the resolution of the molecular markers in this range. Several sources of molecular markers are available from Arabidopsis. First of all, the generation of recombinant lines between the Col-0 and La-er ecotypes made it possible to bring together all of the pre-existing markers (cloned genes, fragments of genomic DNA, ESTs, microsatellite markers ...) in one unique genetic map (Lister and Dean, 1993), which currently specifies the relative position of more than 300 of them on the 5 linking groups of the Arabidopsis genome. However, despite its exhaustive nature, this map did not provide new markers to limit the interval defined by m \ 413 and atpox. Furthermore, the production of resolving markers can be ensured by the creation of a precise physical map covering the locus considered. Several genomic libraries produced by the cloning of partial digests of nuclear DNA into BAC (Bacterial Artificial Chromosome) type vectors or resulting from the packaging of inserts of defined size in phage particles (P1 clones (Liu et al., 1995)) were produced in Arabidopsis. The arrangement of these contiguous clones constitutes the preliminary stage of the sequencing programs. It offers the advantage of defining overlapping zones between these clones and thus makes it possible to considerably reduce the number of BAC clones to be sequenced to ensure total coverage of a chromosomal region. Unlike chromosomes I, II, IV, and V for which very precise physical maps were usable at many points in the genome, for the centromeric region of chromosome III, at the start of this study, only an approximate and discontinuous physical map, matched some sequence data was available. Although incomplete, this map resulting from the screening of P1 (Liu et al., 1995) and BAC clones by ends of YACs (Yeast Artificial Chromosomes) clone inserts (Creusot et al., 1995) served as support for the positional cloning of the HXC2 gene: the sequence data of the P1 MOD1, MX021 and MT024 clones positioned in the region (Table 2), were used to amplify genomic fragments by PCR, which served as RFLP markers.

Table 2:

Sequence of oligonucleotides used for the generation of new RFLP markers

Generation of RFLP markers from sequences of BAC and P1 clones positioned within the interval defined by the mi413 and atpox markers.

The production of an RFLP marker from a given sequence requires the prior identification of a restriction polymorphism between the parents of the crossing from which the segregated individuals come. All the RFLP probes used in this study were therefore hybridized to a parental membrane resulting from the digestion of genomic DNA from the two parental ecotypes (Co \ -gl-1-hxc-2 and Ws-0) by 19 restriction enzymes. The identification of 3 enzyme / probe pairs (MODI / EcoRV, MT024 / Λ / del MX021LE / Spel) showing a polymorphic hybridization profile between the parents, and making it possible to detect unambiguous heterozygous individuals, made it possible to define 3 markers usable for chromosomal walking (Table 2 below above). Their use for mapping the mutated locus required the digestion of the DNA of all F2 individuals (or pools of F3 DNA resulting from these F2 individuals) with the specific enzyme, and the hybridization of these digests. to their respective probe.

III. Interval restriction by MOD1 markers. MTQ24 and MXO21-LE.

The clones MOD1, MT024 and MX021, anchored on the physical map by their respective hybridization to the markers CIC8D2RE, CIC10A4LE and CIC10A8RE (corresponding to the ends of YAC clones located in the region (Creusot et al., 1995)) were completely sequenced in as part of the chromosome III sequencing program. The use of the fragments amplified from these clones as RFLP markers made it possible to reduce the interval defined by the markers mi413 and atpox. The recombination events corresponding to the lines R313, R287 and R139, phenotypic mutant at the hxc2 locus and homozygous mutant for the marker MOD1, were eliminated by the use of this marker. Intervaiie was thus reduced to 400 kb separating MOD1 from mi413. Finally, the use of markers MT024 and MX021 made it possible to reduce to 2, the number of recombinants on either side of the locus, now limited to the left, by the marker mi413 and to the right, by the marker MX021LE. The absence of overlap between the MXE2 and MX021 clones does not allow precise evaluation of the physical distance separating the new flanking markers, mi413 and MX021LE, it was evaluated by the construction of a precise physical map around the HXC2 locus.

EXAMPLE 3: POSITIONAL CLONING OF THE CAUSAL GENE OF THE SENSITIVITY PHENOTYPE IN THE HXC2 MUTANT PLANT: CONSTRUCTION OF A PRECISE PHYSICAL MAP OF THE HXC2 LOCUS.

The construction of a contig of BACs covering the HXC2 locus required the screening of the TA&MU bank, (http://http.tamu.edu:8000/~creel/TOC.html) available on high density filter, using markers bordering the locus.

I.- Screening of the BAC TA&MU bank (Texas A&M University). The high density filter, generated from the BAC bank

TA&MU is a 21 x 14 cm membrane on which approximately four equivalents of the haploid genome have been attached. The filter comprising the 12288 clones of the bank is divided into 4 fields each containing 384 squares. Each square has 16 points corresponding to the transplanting of 8 clones in duplicate. The position of the hybridization signals which appear twice in the square of a given field therefore provides unambiguous information on the origin of the clone turned on by the hybridization.

The screening of this filter, using the marker corresponding to a YAC clone end, CIC12D2RE which constituted the first telomeric limit of the HXC2 locus used for physical mapping, enabled the clones T16014, T25B14 and T8B20 to be identified. A second screening, carried out using the mi413 marker, made it possible to isolate the clones T7I19 and T24F18. In addition, alignment of the m \ 413 marker sequence with all of the nuclear sequences available from Arabidopsis made it possible to identify a P1, MXE2 clone from another genomic library and sequence in Kazuza, Japan, in as part of the chromosome III short arm sequencing program. The mi413 marker was thus able to be anchored between the bases of the MXE2 clone. In order to ensure the junction between clones T8B20, T25B14, T16014, isolated using the marker CIC12D2RE, and the clone MXE2, identified using the marker mi413, a third screening of the library, carried out using of a 1.5 kb fragment corresponding to the left end of the clone MXE2, allowed the isolation of the clones T2H8 and T3N15.

II. Construction of a contiguous BAC clone between the markers mi413 and MX021-LE.

The contiguous organization of the BAC clones identified by screening from the TA&MU bank was allowed by the structure of the vector. pBeloBACH in which the nuclear DNA fragments, with an average size of 100 kb, were cloned (Figure 1).

The vector facilitates the isolation and cloning of the ends of the inserts which can subsequently be hybridized to the other BACs. These hybridizations make it possible, on the one hand, to specify the zones of overlap between BACs and, on the other hand, the orientation of the clones with respect to each other, steps necessary for the development of the contiguous.

The isolation of the right and left ends of these inserts, defined by the arms of the vector, can be carried out respectively by reverse PCR and "plasmid rescue": the unique sites Sph \, BamH \, Sma \, Sac \ and EcoR \ present in the sequence of pBeloBACH allow, via the digestion of a clone by one of these enzymes and the religation of the fragments generated on themselves, amplification by reverse PCR of the right end. The religation products also generate a plasmid comprising the origin of replication of the vector, the chloramphenicol resistance marker, and a fragment of variable size corresponding to the left end of the insert (Figure 1).

Hybridization of all of the clones isolated at the end of the three successive screenings with a 3.7 kb fragment corresponding to HindIII I BamH \ digestion of the left end of the clone T8B20 (T8B20LE) made it possible to detect a signal d hybridization corresponding to clone T16014. Furthermore, the alignment of 129 bp of the sequence of this end to all of the available Arabidopsis sequences made it possible to anchor this fragment between the bases 11191 and 11319 of the clone P1 MXE2. The overlap between the clones T8B20 and MXE2 isolated respectively using the markers CIC12D2RE and ml413 shows that the physical distance which separates these two markers is sufficiently small to overcome the clone T2H8, initially identified using the marker MXE2LE for ensure the junction between these two clones.

The EcoRI digestion of the T8B20 clone made it possible, after religation of the fragments on themselves and reverse PCR, the isolation of 700 bp corresponding to its right end (T8B20RE). The labeling of this sequence revealed a hybridization signal corresponding to the clone T25B14. This hybridization signal, absent from the clone T16014, identified with

I use the same marker CIC12D2RE, shows that T25B14 extends further to the left of the HXC2 locus than the clones T8B20 and T16014. Furthermore, the signal detected on 4 of the 5 clones isolated by the markers CIC12D2RE and MXE2LE after labeling the right end of T16014 made it possible to confirm their presence in the region. On the other hand, the absence of signals corresponding to the clone T3N15 for all the probes tested in the region as well as its digestion profiles EcoRI, KpN \ and BamH \ distinct from the other clones present in the region have led to considering it as a screening artifact.

The "gap" present between the sequenced P1 clones, MXE2 and MX021, was filled by the clone T7119, isolated using the marker mi413 (Figure, 1). The sequence of the right and left ends of this clone available in Genbank with the accession numbers al091267 and al091268, made it possible to anchor T7I19 between the base 52265 of the clone MXE2 and the base 34747 of the clone MX021. The marker MX021LE which constitutes the centromeric limit of the HXC2 locus was amplified between bases 351 and 1510 of the clone MX021; this marker is therefore present in the sequence of the clone T7I19 which overlaps the clone MX021 up to base 34747. Hybridization of the clone T7I19 with the two markers bordering the HXC2 locus provides proof that the HXC2 gene is present in the clone T7I19 . The isolation of this clone, the size of which was evaluated by EcoRI digestion and migration on agarose gel at 80 kb therefore offered on the one hand, the possibility of evaluating the physical distance separating the two flanking markers (45 kb) and on the other hand, the possibility of constructing a high resolution map around the locus so as to produce smaller fragments for the complementation of the mutation.

III. Construction of a high resolution map covering the HXC2 locus.

The BAC banks produced in Arabidopsis were made from the wild Col-0 ecotype. The BAC clones isolated by screening from the TA&MU bank therefore provided the elements necessary for the complementation of the hxc-2 mutation, isolated in a genetic background. Col-g. However, these clones were not generated in binary vectors allowing the transformation of Arabidopsis. Complementation of the mutation therefore required the production of smaller fragments, in a binary vector allowing the stable transformation of mutant plants. A high-resolution map of the HXC2 locus was therefore developed by partial EcoRI digestion of the clone T7I19 covering the locus, and by cloning of these fragments into a binary-type cosmid vector (pSLJ75515) making it possible to integrate fragments of a medium size. 20 kb. The analysis of approximately 160 cosmids from this bank made it possible to select 10 of them, ensuring total coverage of the T7I19 clone. The arrangement of these contiguous clones was facilitated by the knowledge of the sequence, and therefore of the restriction map of the 30 kb telomeric common to the clone, MXE2 and the 34 kb centromeric corresponding to the clone MX021.

The approximately 15 kb non-sequenced interval of the clone T7I19, deduced from the total length of the BAC, could contain the hxc2 gene. In order to test this hypothesis, an RFLP marker corresponding to the right end of the MXE2 clone was generated by amplification of a 675 bp fragment between bases 86114 and 86789 of this clone. The amplified product made it possible to detect an EcoRV polymorphism between the parents of the cross. The use of this marker on the population of recombinants isolated between the atpox and mi413 markers made it possible to eliminate the recombination event corresponding to the individual R284. Maintaining a single recombinant, R269, between the marker MXE2RE and the locus made it possible to limit the interval to the few 20kb sequences corresponding to the right end of the clone MXE2, comprised between the markers mi413 and MXE2RE. In order to identify the candidate genes present in this region, a prediction analysis of "ORFs" was carried out in the region.

EXAMPLE 4 POSITIONAL CLONING OF THE CAUSAL GENE OF THE SENSITIVITY PHENOTYPE IN THE HXC2 MUTANT PLANT: IDENTIFICATION OF THE CAUSAL GENE. The use of the Genscan program (http: // CCR- 081.mit.edu/GENSCAN.htmπ on the sequence of the clone MXE2 made it possible to identify two ORFs in the interval defined by the markers mi4 3 and MXE2LE: a protein 171 amino acids with no homologies with the sequences present in the databases and a serine threonine kinase type SNF1, homologous to the yeast protein SNF1 (Figure 2). The predicted coding sequence of the protein kinase is 100% identical to that of the cDNA of the Akinl 1 gene from Arabidopsis (Accession No. X99279), initially isolated by heterologous screening of a cDNA library using the yeast SNF1 gene and interacting with PRL1 , a pleiotropic regulator of genes induced in response to hormonal or "sugar-dependent" signals (Németh et al., 1998, Bhalerao et al., 1999). The strict identity revealed by the alignment of the predicted sequence with that of the protein Akinl 1, characterized in this The experiment was confirmed by mapping the Akinl 1 cDNA on the population of Col-0 / La-er recombinant lines, in the centromeric position of the mι413 marker (Bhalerao et al., 1999).

Sequencing of the Akinll αene in the genetic background Col-αl-1-hxc-2

The strategy adopted for sequencing the Akinl 1 gene in the hxc-2 mutant therefore consisted in amplifying overlapping fragments of the genomic region containing this gene, and in cloning these fragments into a vector of the pGEM-T type (PROMEGA). The sequencing of several independent clones, originating from each of the amplified fragments (FIG. 3) made it possible to identify the hxc-2 mutation. This method, which uses PCR, generates point deletions or base mismatches. These errors appear with a frequency, evaluated in this study at 10 ^"5 , which brings to 10 ^{" 25} the probability of occurrence of an error on the same basis in two independent clones. The detection of a C / T inversion at the base 77578 of the fragment E in 8 clones resulting from 2 independent amplifications is therefore, most probably, the result of the actual presence of this mutation in the gene sequence and not of 'an artifact due to an amplification error. This point mutation is responsible for the change of arginine 335 from the predicted protein to tryptophan. The genomic nucleotide sequence of the mutated akinl 1 gene is sequence SEQ ID No. 4 from the sequence listing.

The nucleotide sequence of the cDNA of the mutated akinl 1 gene is sequence SEQ ID No. 5 of the sequence listing. Relative to the wild-type sequence, the mutated nucleotide sequence of the akinl 1 gene involves replacing an initial C base with a T base, which constitutes the nucleotide at position 1972 of the sequence SEQ ID No. 4 and the nucleotide at position 1003 of the sequence

SEQ ID N ° 5. The deduced amino acid sequence corresponding to the mutated AKIN11 polypeptide is the sequence SEQ ID No. 6 of the sequence listing.

Relative to the sequence of the wild-type AKIN11 polypeptide, the mutated polypeptide of sequence SEQ ID No. 6 has the substitution of the initial arginine residue at position 335 with a tryptophan residue on the mutated peptide.

EXAMPLE 5 USE OF A NUCLEIC ACID FOR THE SYNTHESIS OF THE AKIN11 POLYPEPTIDE TO INDUCE RESISTANCE TO AGGRESSION BY A PATHOGEN

In order to induce a phenotype of resistance to a pathogen in the sensitive mutant hxc2 of Arabidopsis thaliana, complementation experiments were carried out, using the cosmids obtained by partial digestion of the clone T7I19, cloned beforehand in a binary vector, the vector pSLJ5515 derived from vector pRK290. The mutant plants were therefore transformed with the cosmids C7119-66 and C7I19-49 which cover the Akinl 1 gene. At the end of two independent transformation events 12 and 5 primary transformants were obtained, respectively, for the cosmids C7I19- 49 and C7I19-66. Control transformations were also carried out using the cosmids C7119-29 and C7119-32 and the binary vector itself (Figure 4, top).

Inoculation of the transformants corresponding to clones C7119-49 and C7119-66 with strain 147 of Xanthomonas shows that the transformation by clones 66 and 49 made it possible to restore the wild phenotype (FIG. 4, bottom).

EXAMPLE 6 CONSTRUCTION OF A RECOMBINANT EXPRESSION VECTOR CONTAINING A NUCLEIC ACID ALLOWING THE SYNTHESIS OF THE AKIN11 POLYPEPTIDE IN A PLANT.

1. Construction of a recombinant expression vector containing the genomic DNA of the Akinl 1 gene, or a corresponding antisense sequence.

The plasmid pRK290 (John Innés Center transfer) containing the genomic DNA of the Akinl 1 gene is prepared by mini-preparation and used as template for a PCR experiment with the oligonucleotides AKINF (SEQ ID No. 13) and AKINR (SEQ ID No. 14 ) .. The use of this pair of primers makes it possible to insert, by point mutagenesis, a Nael restriction site at the two ends of the amplified fragment of 2684 bp. This fragment is purified and cloned into the vector pGEM®-T (Promega). The insertion is verified by Nael / Drdl digestion. The sequence of the fragment is entirely determined on several clones to test for the presence of mutations induced by PCR. Nael / Drdl digestion of the plasmid makes it possible to purify on agarose gel a fragment of 2674 bp: ORF Akinl 1 of the retained clone (without mutation). The diagram of the construction method is shown on the left of Figure 5.

The plasmid pBI121 (Clontech) is subjected to a double Sacl / Smal digestion. The end digested with Sac1 is made blunt by the action of the Kleenow fragment of the DNA polymerase I enzyme. The ends of the fragment are dephosphorylated.

The ORF fragment Akinl 1 is ligated into the vector pBI121 dephosphorylated.

The diagram of the construction method is shown on the right of Figure 5. 2. Construction of a recombinant vector containing the cDNA of the Akinl 1 gene, or a corresponding antisense sequence.

The plasmid pBlueScript SK- containing the cDNA of the Akinl 1 gene is prepared by mini-preparation and used as template for a PCR experiment with the oligonucleotides AKIN68 (SEQ ID No. 15) and AKIN1648 (SEQ ID No. 16). The use of this pair of primers makes it possible to insert, by point mutagenesis, a Nael restriction site at the two ends of the 1602 bp amplifiate. This fragment is purified and cloned into the vector pGEM®-T (Promega). The insertion is verified by Nael digestion. The sequence of the fragment is entirely determined for several clones to test for the presence of mutations induced by PCR. Nael digestion of the plasmid makes it possible to purify on agarose gel a fragment of 1578 bp: cDNA Akinl 1.

The construction diagram is shown on the left of Figure 6.

The Akinl 1 cDNA fragment is ligated into the dephosphorylated vector pBI121.

The construction diagram is shown on the right of Figure 6.

EXAMPLE 7 OBTAINING TRANSGENIC PLANTS TRANSFORMED BY A NUCLEIC ACID ALLOWING THE SYNTHESIS OF THE AKIN11 POLYPEPTIDE IN THIS PLANT

The transformation of a plant in order to obtain a stable expression of the polynucleotide of interest in the transformed plant is necessary to ensure a lasting modification of the phenotype of resistance to pathogens in this plant. Tests are carried out with a vector construction described in Example 6 above.

I. PARTICLE CANON The method used is based on the use of a particle cannon identical to that described by FINER (1992).

Target cells are undifferentiated cells in rapid divisions which have retained the ability to regenerate whole plants. This type of cell makes up the embryogenic callus (called type II) of corn. These calluses are obtained from immature Hill genotype embryos according to the method and on the media described by ARMSTRONG (1994). Fragments of such calluses with an area of 10 to 20 mm ² were placed, 4 h before bombardment, at the rate of 16 fragments per dish in the center of a Petri dish containing a culture medium identical to the initiation medium , supplemented with 0.2M mannitol + 0.2M sorbitol. The plasmids described in the previous examples and carrying the genes to be introduced are purified on a Qiagen ^R column according to the manufacturer's instructions. They are then precipitated on tungsten particles (M 10) following the protocol described by KLEIN (1987). The particles thus coated are projected towards the target cells using the cannon and according to the protocol described by J. FINER (1992). The callus boxes thus bombarded are then sealed using Scellofrais ^R and then grown in the dark at 27 ° C. The first subculture takes place 24 hours later, then every fortnight for 3 months on medium identical to the initiation medium added with a selective agent. Calls are obtained after 3 months or sometimes earlier, calluses whose growth is not inhibited by the selective agent, usually and mainly composed of cells resulting from the division of a cell having integrated into its genetic heritage one or more copies of the selection gene. The frequency of obtaining such calluses is approximately 0.8 cal per bombarded box.

These calluses are identified, individualized, multiplied and then cultivated so as to regenerate seedlings, by modifying the hormonal and osmotic balance of the cells according to the method described by VAIN et al. (1989). These plants are then acclimatized in the greenhouse where they can be crossed to obtain hybrids or self-fertilized.

II. TRANSFORMATION BY AGROBACTERIUM

Another transformation technique which can be used in the context of the invention uses Agrobacterium tumefaciens, according to the protocol described by ISHIDA et al. (1996), in particular from immature embryos 10 days after fertilization. All the media used are referenced in the cited reference. The transformation begins with a coculture phase where the immature embryos of the corn plants are brought into contact for at least 5 min with Agrobacterium tumefaciens LBA 4404 containing the superbinary vectors. The superbinary plasmid is the result of homologous recombination between an intermediate vector carrying the T-DNA containing the gene of interest and / or the selection marker derived from the plasmids described in the previous examples, and the vector pSB1 from Japan. Tobacco (EP 672 752) which contains: the virB and virG genes of the plasmid pTiBo542 present in the supervirulent strain A281 of Agrobacterium tumefaciens (ATCC 37349) and a homologous region found in the intermediate vector allowing this homologous recombination. The embryos are then placed on LSAs medium for 3 days in the dark and at 25 ° C. A first selection is made on the transformed calluses: the embryogenic calluses are transferred to LSD5 medium containing phosphinotricin at 5 mg / l and cefotaxime at 250 mg / l (elimination or limitation of contamination by Agrobacterium tumefaciens). This stage is carried out for 2 weeks in the dark and at 25 ° C. The second selection step is carried out by transfer of the embryos which have developed on LSD5 medium, on LSD10 medium (phosphinotricin at 10 mg / l) in the presence of cefotaxime, for 3 weeks under the same conditions as above. The third selection step consists in excising the type I calluses which remain white (fragments of 1 to 2 mm) and in transferring them 3 weeks in the dark and at 25 ° C on LSD 10 medium in the presence of cefotaxime. The regeneration of the seedlings is carried out by excising the type I calluses which have proliferated and. by transferring them to LSZ medium in the presence of phosphinotricin at 5 mg / l and cefotaxime for 2 weeks at 22 ° C and under continuous light.

The plants which have regenerated are transferred to RM + G2 medium containing 100 mg / l of Augmentin for 2 weeks at 22 ° C. and under continuous illumination for the development stage. The plants obtained are then transferred to the phytotron for their acclimatization.

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Claims

claims

1. Use of a nucleic acid allowing the synthesis of the AKIN1 1 polypeptide in a plant to induce or increase, in this plant, resistance to aggression by a pathogen.

2. Use according to claim 1, characterized in that the nucleic acid allowing the synthesis of the AKIN 11 polypeptide comprises a polynucleotide coding for the AKIN11 polypeptide chosen from the sequences SEQ ID N ° 1 and 2 and their homologous sequences resulting from the degeneration of the genetic code.

3. Use according to one of claims 1 or 2, characterized in that the nucleic acid allowing the synthesis of the polypeptide

AKIN11 comprises a polynucleotide regulating the expression of the polynucleotide encoding the AKIN11 polypeptide in the plant.

4. Use according to claim 3, characterized in that the nucleic acid allowing the synthesis of the AKIN11 polypeptide codes for a fusion polypeptide consisting of the AKIN11 polypeptide fused with a signal peptide allowing its localization in a given cellular compartment or its secretion in the cell membran.

5. Use according to claim 3 or 4, characterized in that the polynucleotide regulating the expression of the polynucleotide encoding the AKIN11 polypeptide in the plant is a constitutive promoter.

6. Use according to claim 5, characterized in that the constitutive promoter is chosen from the 35S CaMV promoter, the rice actin promoter or the pUbil promoter of the maize ubiquitin 1 gene.

7. Use according to claim 3 or 4, characterized in that the polynucleotide regulating the expression of the polynucleotide encoding the AKIN11 polypeptide in the plant is an inducible promoter.

8. Use according to claim 7, characterized in that the inducible promoter is chosen from a promoter inducible by glucocorticoids or a promoter inducible by ethanol.

9. Use according to claim 3 or 4, characterized in that the polynucleotide regulating the expression of the polynucleotide encoding the AKIN11 polypeptide in the plant is a specific tissue promoter.

10. Use according to claim 9, characterized in that the tissue-specific promoter is chosen from the HMWG wheat or barley promoter, the pCRU promoter of the radish cruciferin gene, the PGEA1 promoter, the PGEA6 promoter, the corn zein gene promoter, napine promoter, phaseoline promoter, glutenin promoter, heliantinin promoter, albumin promoter, oleosin promoter, promoter of IΑTS1, the promoter of IΑTS3.

11. Use according to one of claims 1 to 10, characterized in that the nucleic acid allowing the synthesis of the AKIN11 polypeptide in a plant comprises an untranslated "leader" sequence allowing to increase the translation of this polypeptide in the plant .

12 Use according to claim 11 characterized in that the "leader" sequence is chosen from the leader EMCV, the leader TEV, the leader BiP, the leader AMV RNA 4 and the leader of the tobacco virus mosaic.

13 Use according to one of claims 1 to 12 characterized in that the nucleic acid allowing the synthesis of the AKIN11 polypeptide in a plant is inserted into an expression vector.

14 Use according to claim 13 characterized in that the expression vector is chosen from the vectors pBIN19, pBI101, pBI121, pEGFP, pCAMBIA 1302 and L127a5.

15 Use according to one of claims 1 to 14 characterized in that the plant is chosen from field crops, vegetable plants and flowers.

16 Use according to one of claims 1 to 15 characterized in that the pathogen is a bacterium, a virus, a fungus or a nematode.

17. Use according to claim 16, characterized in that the pathogen is a necrotrophic pathogen.

18. Expression cassette characterized in that it comprises a promoter chosen from the promoters defined in claims 5 to 10, said promoter controlling the expression of a polynucleotide allowing the synthesis of the Akin 11 polypeptide of sequence SEQ ID No. 1 or SEQ ID No. 3, said expression cassette also comprising a terminator sequence.

19. Expression cassette characterized in that it comprises a promoter chosen from the promoters defined in claims 5 to 10, said promoter controlling the expression of a polynucleotide chosen from the sequences SEQ ID No. 1, SEQ ID N ° 2, SEQ ID No.3 or SEQ ID No.4, said expression cassette also comprising a terminator sequence.

20. Method for obtaining a transformed plant possessing resistance to a pathogen, characterized in that it comprises the following steps: a) transfecting a plant host cell with a nucleic acid allowing the synthesis of the AKIN 11 polypeptide in a plant , or with a recombinant vector into which such a nucleic acid has been introduced; 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 said nucleic acid.

21. Process for obtaining a transformed plant possessing resistance to a pathogen, characterized in that it comprises the following stages: a) obtaining a host cell of Agrobacterium tumefaciens transformed with a nucleic acid allowing the synthesis of AKIN11 polypeptide in a plant; or with a recombinant vector into which such a nucleic acid has been introduced; b) Transformation of a plant of interest by infection with the recombinant host cell obtained in step a); c) Selection of plants which have integrated said nucleic acid into their genome.

22. Method for obtaining a transformed plant according to any one of claims 20 or 21, characterized in that it comprises the following additional steps: d) crossing between them of two transgenic plants as obtained from step c); e) selection of plants homozygous for the transgene.

23. Method for obtaining a transformed plant according to any one of claims 20 or 21, characterized in that it comprises the following additional steps: f) crossing a transgenic 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. • ^•

24. Method for obtaining a transformed plant according to any one of claims 20 or 21 characterized in that it comprises the following additional steps:

h) crossing a transformed plant obtained in step c) with a plant of an agronomic value line of the same species; i) selection of the plants resulting from the crossing of step h) having preserved the transgene; j) backcrossing of a transformed plant obtained in step i) with the plant of an agronomic value line of the same species; k) repeat step j) at least twice;

25. Plant transformed according to the method according to any one of claims 20 to 24.

26. Plant resistant to a plant pathogen, characterized in that it has been transformed with a nucleic acid allowing the synthesis of the AKIN 11 polypeptide in this plant.

27. Part of a plant transformed according to one of claims 25 or 26.

28. Use of a probe or a nucleotide primer hybridizing with the sequence SEQ ID No. 1 or 2 to detect the presence of a trait of resistance to a pathogen in a plant.

29. Use of an antisense polynucleotide hybridizing with the nucleic acid of sequence SEQ ID No. 2 to modulate the resistance of a plant to aggression by a pathogenic agent.

30. A method for inducing or increasing the resistance of a plant to a pathogen, characterized in that it comprises a step according to which said plant is brought into contact with the AK1 11 polypeptide.

31. Use of the AKIN11 polypeptide as an active compound to induce or increase the resistance of a plant to a pathogen.

32. Fusion polypeptide consisting of the AKIN11 polypeptide fused with a signal peptide allowing its targeted localization in a determined compartment of the plant cell.