FR2806095A1 - New polynucleotides for producing transgenic plants resistant to geminivirus infection comprising polynucleotides encoding proteins which interact with at least one of the products of the geminivirus genome - Google Patents

Abstract

A polynucleotide (N1) purified from a plant or yeast, encoding a protein (P1) which interacts with at least one of the six products of the geminivirus genome necessary for infection of a plant by the virus, is new. Independent claims are also included for the following: (1) a recombinant nucleic acid molecule comprising (N1); (2) a cloning vector comprising (N1); (3) a host cell transformed with (N1) or the vector; (4) a genetically modified plant resistant to geminivirus having stably in its genome a modified nucleic acid comprising (N1), and is: (i) modified to express a protein which blocks expression proteins encoded by the geminivirus genome; or (ii) modified to express a modified (P1) which compared to the native modifies or removes interactions with the viral proteins; (5) preparing a transgenic plant resistant to geminivirus, comprising transforming a plant cell with the above nucleic acid and regenerating a transformed plant; (6) a plant cell whose genome stably incorporates (N1); and (7) a protein susceptible to interaction with at least one of the geminivirus proteins, comprising one of the amino acid sequences fully defined in the specification or a fragment having a substantially similar sequence.

Description

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 PURIFIED POLYNUCLEOTIDE SEQUENCES OF PLANTS AND YEAST ENCODING PROTEINS THAT INTERACT WITH PRODUCTS OF THE GEMINIVIRUS GENOME.

 The present invention relates to purified polynucleotide sequences of plants and yeast encoding proteins which interact with products of the genome of geminiviruses and which are capable of being necessary for the infection of plants by this virus.

The invention also relates to the preparation of plants genetically modified to resist the geminivirus, family of Geminiviridae, and more particularly to the yellowing coiling virus of the tomato leaves, also designated TYLCV for tomato yellow leaf curl virus.

 TYLCV belongs to the genus Begomovirus and is transmitted by the whitefly Bemisia tabacs. Geminiviruses are responsible for many tomato diseases and TYLCV poses a serious economic problem since it causes up to 100% yield loss in many tropical and subtropical regions. This virus has now spread in the Mediterranean basin, including Italy and Spain, where the loss of yield in the field can reach 80%, as well as in America including recently Florida where it threatens the industry. tomato. The means of control against the whitefly Bemisia tabaci have not yet proved effective.

 Tolerant cultivars obtained by conventional improvement and showing attenuated or delayed symptoms have been proposed in the prior art, but not resistant cultivars. Transgenic plants have also been proposed in the prior art resistant to infections by geminiviruses based on the introduction and / or expression in plants of viral sequences. Mention may in particular be made of genetic constructs comprising genes

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 of geminiviruses proposed in the PCT international patent applications published under the numbers W097 / 42316 and W097 / 39110.

 The introduction of viral sequences into the genome of the tomato, however, raises a certain number of problems, in particular due to the risks of recombination or heteroencapsidation.

 The present invention aims precisely to overcome this drawback by proposing plant genes whose products interact with one or more proteins of TYLCV. These genes are therefore particularly useful for controlling viral infection in a plant without introducing viral genes into it.

 Viruses have been recognized for the first time as a pathogenic entity in a century. Since then, considerable efforts have been made to understand their biology: how they enter the cell, and once inside, how they exploit the host's processes to replicate and invade the body. It is now well established that viral replication in the infected cell requires the participation of cellular factors. This is particularly the case for viruses with small genomes that code for only a few proteins. Animal DNA viruses associated with tumors, for example, exploit cellular machinery to accomplish their transcription and replication processes. In addition, one or more proteins encoded by these viruses are capable of encroaching on the physiology of the infected cell in order to create a cellular environment suitable for viral replication. A typical example is that of the oncoproteins of these viruses, such as the SV40 T antigen, the ElA protein of the adenoviruses or the E7 protein of the human papilloma viruses, which activate the cycle.

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 cell in the infected cell by interfering with the retinoblastoma pathway, a tumor in the eye that affects young children.

 The situation seems to be similar in the case of Geminiviridae, a unique family of plant DNA viruses whose particles are in the form of two paired spheroids and whose genome consists of one or two small circular single-stranded DNA molecules (Lazarowitz, 1992). TYLCV belongs to the genus Begomovirus although it has only one genomic component (Kheyr-Pour & al., 1991; Navot & al., 1991).

 Figure 1 in the appendix represents the genome of TYLCV, which contains six open reading phases (ORF) which partially overlap (Kheyr-Pour & coll., 1991) - two on the strand of the virion (+), the gene for capsid CP and a gene necessary for the development of a systemic infection in the host, V2 (Wartig & coll., 1997), and - four on the complementary strand (-), the Cl (Rep) gene necessary for replication, the C2 gene considered to code for an activator of transcription of the genes located on the (+) strand of the virion (Noris & coll., 1996) and probably involved in the movement of the virus (Wartig & coll., 1997), the C3 gene which promotes the accumulation of viral DNA, and finally the C4 gene which is involved in the movement of the virus (Fig. 1) (Jupin & coll., 1994). These ORFs are separated by an intergenic region (IR) of 300 nucleotides which contains the key elements for the replication and transcription of the viral genome (Lazarowitz, 1992).

 To understand the molecular mechanisms that govern the biological processes of replication and movement of viruses, it is necessary to identify not only the actors of these processes, but also the

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 interactions that regulate them. For example, DNA replication of geminiviruses takes place in the nucleus of infected cells and, due to the absence of replication enzymes encoded by the viral genome, requires the functions of the S phase of the cell cycle. However, the majority of the cells of the leaf of a plant are completely differentiated and have left the cell division cycle and it is no longer possible to detect DNA replication enzymes or the nuclear proliferation antigen. the cell (PCNA) which associates with certain DNA polymerases and promotes their processivity. When the geminiviruses infect these differentiated cells, the Rep protein induces the expression of cellular proteins associated with the S phase of the cell cycle, such as PCNA (Nagar & coll., 1995). The rep protein of the geminiviruses is therefore capable of reactivating the host replication machinery in favor of replication of the viral DNA.

 The dependence of geminiviruses on host proteins is analogous to the situation of animal DNA viruses associated with tumors, such as SV40. This creates a cellular environment favorable to the replication of viral DNA thanks to the interaction of an oncoprotein encoded by the viral genome with the retinoblastoma protein (Rb), involved in the regulation of cell cycles. The Rep protein of the geminiviruses likewise forms a stable complex with a plant Rb protein (Xie & coll., 1995). The sequestration of the Rb protein of the host by the Rep protein of the virus confirms the role of the latter in the regulation of the cell cycle of the host. More recently, other cellular factors interacting with the Rep protein of geminiviruses, the GRAB proteins, have also been identified (Xie & coll., 1999). Although the function of these GRAB proteins has not yet been elucidated, these results

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 illustrate the importance and complexity of virus-host interactions necessary for the completion of the viral cycle.

 A particularly interesting observation is that the overexpression of the Rb protein or GRAB proteins in the plant cell inhibits viral replication (Xie & al., 1995, Xie & al., 1999). The identification of cellular factors that interact with the product of the viral genome therefore opens up new opportunities in the fight against viral diseases.

 As indicated above, the subject of the present invention is plant and yeast genes whose products are capable of interacting with one or more proteins of TYLCV and which are therefore particularly useful for developing new strategies for making plants resistant to 'viral infection.

 In fact, the work carried out within the framework of the present invention has made it possible to identify proteins of the yeast Schizosaccharomyces pombe and plants Arabidopsis thaliana, Nicotiana benthamiana and Lycopersicon esculentum which interact with at least one of the six proteins of the genome of the TYLCV: Rep, C2, C3, C4, CP and V2.

 The subject of the invention is therefore a purified plant or yeast polynucleotide sequence coding for a protein which interacts with at least one of the six products of the genome of a geminivirus necessary for the infection of a plant by this virus.

 Examples of polynucleotide sequences useful according to the invention consist of all or part of the genes or gene fragments reported in the table in FIG. 2 in the appendix which refers to the list of sequences in the appendix and the access numbers in Genbank. if they exist.

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 A first series of polynucleotide sequences encoding a protein necessary for one or more of the six proteins of the genome of a geminivirus to allow viral infection, according to the invention, consist of all or part of a gene chosen from the group comprising the gip3-C2, gip6-C2, gip7-C3, gip8-C3, gip9-C3, gipll-C3, gipl2-C4, gipl4-CP, gipl5-CP, gip16-V2, gipl8-V2, gipl9-V2 genes gip21-V2 and gip22-V2.

 The A. thaliana gip3-C2 gene fragment codes for the GIP3-C2 protein which interacts with the TYLCV protein C2. SEQ ID NO: 5 represents a nucleotide sequence of A. thaliana comprising the coding part of the gip3-C2 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 6 represents the amino acid sequence of the protein fragment GIP3-C2.

 The gip6-C2 gene fragment from N. benthamiana codes for the GIP6-C2 protein which interacts with the TYLCV protein C2. SEQ ID NO: 11 represents a nucleotide sequence of N. benthamiana comprising the coding part of the gip6-C2 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 12 represents the amino acid sequence of the GIP6-C2 protein fragment.

 The gip7-C3 gene from L. esculentum encodes the PCNA-tom protein which interacts with the C3 protein of TYLCV. SEQ ID NO: 13 represents a nucleotide sequence of L. esculentum comprising the coding part of the gip7C3 gene followed by the 3 'non-coding portion. SEQ ID NO: 14 represents the amino acid sequence of the PCNA-tom protein of L. esculentum.

 The gip 8-C3 gene fragment codes for the PCNA-ara protein of A. thaliana which interacts with the TYLCV protein C3. SEQ ID NO: 15 represents a nucleotide sequence of A. thaliana comprising the coding part of the gip8-C3 gene followed by the 3 'non-coding portion. SEQ

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 ID NO: 16 represents the amino acid sequence of the PCNA-ara protein of A. thaliana.

 The A. thaliana gip9-C3 gene fragment codes for a fragment of the coatomer delta subunit which interacts with the TYLCV protein C3. SEQ ID NO: 17 represents a nucleotide sequence of A. thaliana comprising the coding part of the gip9-C3 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 18 represents the amino acid sequence of the fragment of the delta subunit of the coatomer of A. thaliana.

 The gipll-C3 gene fragment from N. benthamiana codes for the DNAJ protein fragment which interacts with the TYLCV protein C3. SEQ ID NO: 21 represents a nucleotide sequence of N. benthamiana comprising the coding part of the gipl1-C3 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 22 represents the amino acid sequence of the DNAJ protein fragment of N. benthamiana.

 The A. thaliana gipl2-C4 gene fragment codes for the GIP12-C4 protein fragment which interacts with the TYLCV C4 protein. SEQ ID NO: 23 represents a nucleotide sequence of A. thaliana comprising the coding part of the gipl2-C4 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 24 represents the amino acid sequence of the protein fragment GIP12-C4 of A. thaliana.

 The gipl4-CP gene fragment of N. benthamiana codes for the protein fragment 14-3-3 which interacts with the CP protein of TYLCV. SEQ ID NO: 27 represents a nucleotide sequence of N. benthamiana comprising the coding part of the gipl4-CP gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 28 represents the amino acid sequence of the protein fragment 14-3-3 of N. benthamiana.

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 The N. benthamiana gipl5-CP gene fragment codes for the GIP15-CP protein which interacts with the TYLCV CP protein. SEQ ID NO: 29 represents a nucleotide sequence of N. benthamiana comprising the coding part of the gipl5-CP gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 30 represents the amino acid sequence of the GIP15-CP protein fragment from N. benthamiana.

 The N. benthamiana gip16-V2 gene fragment codes for the GIP16-V2 protein which interacts with the TYLCV V2 protein. SEQ ID NO: 31 represents a nucleotide sequence of N. benthamiana comprising the coding part of the gipl6-V2 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 32 represents the amino acid sequence of the protein fragment GIP16-V2 from N. benthamiana.

 The A. thaliana gip18-V2 gene fragment codes for the GIP18-V2 protein fragment which interacts with the TYLCV V2 protein. SEQ ID NO: 35 represents a nucleotide sequence of A. thaliana comprising the coding part of the gipl8-V2 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 36 represents the amino acid sequence of the protein fragment GIP18-V2 from A. thaliana.

 The A. thaliana gip19-V2 gene fragment codes for the GIP19-V2 protein fragment which interacts with the TYLCV V2 protein. SEQ ID NO: 37 represents a nucleotide sequence of A. thaliana comprising the coding part of the gip19-V2 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 38 represents the amino acid sequence of the protein fragment GIP19-V2 from A. thaliana.

 The N. benthamiana gip21-V2 gene fragment codes for the GIP21-V2 protein which interacts with the TY2V V2 protein fragment. SEQ ID N0: 41 represents

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 a nucleotide sequence of N. benthamiana comprising the coding part of the gip21-V2 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 42 represents the amino acid sequence of the protein fragment GIP21-V2 N. benthamiana.

 The gip22-V2 gene fragment N. benthamiana codes for the protein fragment GIP22-V2 which interacts with the V2 protein of TYLCV. SEQ ID NO: 43 represents a nucleotide sequence of N. benthamiana comprising the coding part of the gip22-V2 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 44 represents the amino acid sequence of the protein fragment GIP22-V2 N. benthamiana.

 It should be noted that with regard to the genes gip3-C2, gip9-C3, gipl6-V2 and gipl9-V2, the sequences of the chromosomes containing the sequence of these genes, interrupted by introns, are found in the bases of data (AB005240, AB005245, AB007651, AC026636 respectively as shown in the table in Figure 2). Consequently, the work carried out within the framework of the present invention has made it possible to define the ORFs of these genes which have therefore never been described in the prior art.

 Similarly, with regard to the gipl8-V2 gene, the chromosome sequence containing the sequence of this gene is found in the databases (AC003027 as indicated in the table in Figure 2). However, the ORF prediction described in the prior art has inaccuracies and the sequence of the gip18-V2 gene is different from the sequence of the predicted gene because certain parts of the sequence are considered to be introns when they do not. are not.

 The subject of the present invention is therefore a purified polynucleotide sequence of plant encoding a protein whose amino acid sequence is chosen from

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 the group comprising the amino acid sequences represented in the sequence list in the appendix under the numbers SEQ ID NO: 6, SEQ ID N0: 12, SEQ ID N0: 14, SEQ ID N0: 16, SEQ ID N0: 18, SEQ ID N0: 22, SEQ ID N0: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID N0: 38, SEQ ID N0: 42 or SEQ ID N0 : 44, a substantially similar fragment or sequence thereof.

 The invention therefore relates very particularly to a polynucleotide sequence consisting of all or part of a polynucleotide sequence chosen from those represented in the sequence list in the appendix under the numbers SEQ ID NO: 5, SEQ ID N0: 11, SEQ ID N0: 13, SEQ ID N0: 15, SEQ ID N0: 17, SEQ ID N0: 21, SEQ ID N0: 23, SEQ ID N0: 27, SEQ ID N0: 29, SEQ ID N0: 31, SEQ ID N0: 35, SEQ ID NO: 37, SEQ ID NO: 41 or SEQ ID NO: 43, a sequence complementary or substantially similar thereto.

 The research carried out in the context of the present invention also made it possible to identify genes which, although already described in the prior art, had never been envisaged as capable of coding for proteins capable of interacting with geminivirus proteins and therefore useful for making plants resistant to these viruses. A second series of polynucleotide sequences useful according to the invention consist of all or part of a gene chosen from the group comprising the genes SY 0487 (N accession Genbank D89128), T8K22.14 (N * accession Genbank AC004136), ajhl ( N accession Genbank AF087413), T26F17.15 (N accession Genbank AC013482), F9Ll.33 (N accession Genbank AC007591), F6H11.170 (N accession Genbank AL021684), F2103.7 (N accession Genbank AC003027), and Cs26 (N accession Genbank AB003041).

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 The gene SY 0487 of S. pombe encodes the protein similar to the NDP-hexose pyrophosphorylase of S. cerevisiae which interacts with the Rep protein of TYLCV. SEQ ID NO: 1 represents a nucleotide sequence of S. pombe comprising the coding part of the gene SY 0487. SEQ ID NO: 2 represents the amino acid sequence of the protein of S. pombe similar to the NDP-hexose pyrophosphorylase of S. cerevisiae.

 The T8K22.14 gene codes for the putative TBP binding protein which interacts with the TYLCV C2 protein. SEQ ID NO: 3 represents a nucleotide sequence of A. thaliana comprising the coding part of the T8K22.14 gene followed by the 3 'non-coding portion. SEQ ID NO: 4 represents the amino acid sequence of the putative TBP binding protein.

 The ajhl gene codes for the AJH1 protein which interacts with the C2 protein of TYLCV. SEQ ID NO: 7 represents a nucleotide sequence of A. thaliana comprising the coding part of the ajhl gene followed by the 3 'non-coding portion. SEQ ID NO: 8 represents the amino acid sequence of the AJH1 protein.

 The T26F17.15 gene fragment from A. thal i ana codes for the protein T26F17.15 which interacts with the protein C2 of TYLCV. SEQ ID NO: 9 represents a nucleotide sequence of A. thaliana comprising the coding part of the gip5-C2 gene fragment followed by the 3 'non-coding portion. SEQ ID NO: 10 represents the amino acid sequence of the protein fragment T26F17.15.

 The F9L1.33 gene codes for the protein of the glyoxalase family which interacts with the C3 protein of TYLCV. SEQ ID NO: 19 represents a nucleotide sequence of A. thaliana comprising the coding part of the F9L1.33 gene followed by the 3 'non-coding portion. SEQ ID NO: 20 represents the amino acid sequence of the protein of the glyoxalase family.

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 The F6H11.170 gene codes for the kinase receptor type protein which interacts with the C4 protein of TYLCV. SEQ ID NO: 25 represents a nucleotide sequence of A. thaliana comprising the coding part of the F6H11.170 gene followed by the 3 'non-coding portion. SEQ ID NO: 26 represents the amino acid sequence of the receptor kinase protein.

 The F2103.7 gene codes for the hypothetical protein that interacts with the TY2V protein V2.

SEQ ID NO: 33 represents a nucleotide sequence of A. thaliana comprising the coding part of the F2103 gene. 7 followed by the non-coding 3 'portion. SEQ ID NO: 34 represents the amino acid sequence of the hypothetical protein.

 The Cs26 gene codes for the protein acetylserine (thiol) lyase which interacts with the V2 protein of TYLCV. SEQ ID NO: 39 represents a nucleotide sequence of A. thaliana comprising the coding part of the Cs26 gene followed by the 3 'non-coding portion. SEQ ID NO: 40 represents the amino acid sequence of the protein o-acetylserine (thiol) lyase.

 The subject of the present invention is therefore a purified plant or yeast polynucleotide sequence encoding a protein whose amino acid sequence is chosen from the group comprising the amino acid sequences represented in the sequence list in the appendix under the numbers SEQ ID NO : 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID N0: 10, SEQ ID N0: 20, SEQ ID N0: 26, SEQ ID N0: 34 or SEQ ID N0: 40, a fragment or a sequence substantially similar to it.

 The invention therefore relates very particularly to a polynucleotide sequence consisting of all or part of a polynucleotide sequence chosen from those represented in the sequence list in the appendix under the numbers SEQ ID NO: 1,

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 SEQ ID N0: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID N0: 19, SEQ ID N0: 25, SEQ ID N0: 33 or SEQ ID N0: 39, a sequence complementary or substantially similar to that -this.

 The term substantially similar to a protein or a protein fragment is understood to mean an amino acid sequence exhibiting one or more modifications such as the substitution, deletion or insertion of one or more amino acids which do not affect the functional properties of the protein or protein fragment from which it is derived.

 The term “substantially similar” to a polynucleotide sequence according to the invention is also understood to mean a sequence having one or more modifications of nucleotides which lead to the substitution of one or more amino acids without affecting the functional properties of the protein or of the protein fragment. encoded by the polynucleotide sequence from which it is derived. Alterations in a gene which result in the expression of an amino acid chemically equivalent to a given site, but which do not affect the functional properties of the protein encoded by the gene, are well known to those skilled in the art. . For example, a codon coding for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon coding for another less hydrophobic residue, such as glycine, or another more hydrophobic residue, such as valine, leucine or isoleucine. Likewise, changes that result in the substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or a positively charged residue for another, such as lysine for arginine, are presumed to produce a functionally equivalent protein. In addition, those skilled in the art know that the polynucleotide sequences which are the subject of the present invention can also be defined by their

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 ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65 C), with the reference sequences given in the sequence list in the appendix. Advantageously, the substantially similar sequences which are the subject of the present invention are those which share at least 80% identity with the reference sequences given in the sequence list in the appendix, preferably at least 90% identity, more preferably at least 95% identity.

 The term substantially similar is also understood to mean a polynucleotide sequence having one or more modifications of nucleotides which in no way affect the capacity of the polynucleotide sequence thus modified to alter the expression of genes by the antisense or co-expression technique. . The term “plant or modified yeast polynucleotide sequence” also means an antisense polynucleotide sequence. It is well known to those skilled in the art that the suppression by antisense or the co-suppression of gene expression can be carried out using a nucleotide sequence representing only a portion of the whole of a coding gene, as well as by a nucleotide sequence sharing less than 100% identity with the gene whose expression must be modified.

 The invention also relates to a nucleic acid molecule comprising at least one polynucleotide sequence as defined above. These are recombinant nucleic acid molecules comprising at least one purified plant or yeast polynucleotide sequence encoding a protein whose amino acid sequence is chosen from the group comprising the amino acid sequences represented in the sequence list. annex under the numbers:

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 - SEQ ID NO: 6, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID N0: 16, SEQ ID N0: 18, SEQ ID N0: 22, SEQ ID N0: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID N0: 38, SEQ ID N0: 42 or SEQ ID N0: 44, or - SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 34 or SEQ ID NO: 40, a substantially similar fragment or sequence thereof.

 Said purified plant polynucleotide sequences forming part of the constitution of the nucleic acid molecules of the invention consist of all or part of a polynucleotide sequence chosen from those represented in the sequence list in the appendix under the numbers.

 - SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID N0: 15, SEQ ID N0: 17, SEQ ID N0: 21, SEQ ID N0: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID N0: 37, SEQ ID N0: 41 or SEQ ID N0: 43, or - SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID N0: 19, SEQ ID N0: 25, SEQ ID N0: 33 or SEQ ID N0: 39, a sequence complementary or substantially similar to these.

 Recombinant nucleic acid molecules according to the invention are in particular chimeric genes or expression cassettes comprising said polynucleotide sequence placed under the control of regulatory elements in position 5 'and / or 3' thereof which can function in a host organism, especially in plant cells.

 Regulatory elements in the 5 ′ and 3 ′ position used in the composition of the recombinant nucleic acid molecules according to the invention, the use of which depends on the host organism are well known to the

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 job. They include in particular promoter sequences, transcription activators, terminator sequences, including start and stop codons. The means and methods for identifying and selecting the regulatory elements are well known to those skilled in the art.

 As promoter regulatory sequence in plants, any promoter sequence of a gene expressing itself naturally in plants can be used, for example a promoter of bacterial, viral or plant origin such as, for example, the 35S promoter of the virus. cauliflower mosaic (CaMV), or a promoter inducible by pathogens such as tobacco PR-la, any suitable known promoter that can be used.

 According to the invention, it is also possible to use, in association with the promoter regulatory sequence, other regulatory sequences, which are located between the promoter and the coding sequence, such as transcription activators (enhancer), such as, for example. example, the activator of the tobacco mosaic virus (TMV) described in US Patent 5,891,665.

 As a terminating or polyadenylation regulatory sequence, any corresponding sequence of bacterial origin, such as, for example, the terminator nos of Agrobacterium tumefaciens, or of plant origin, such as, for example, a histone terminator, can be used. as described in patent EP 633,317.

 The polynucleotide sequence can also be associated in the nucleic acid molecule of the invention with a selection marker adapted to the transformed host organism. Such selection markers are well known to those skilled in the art. It could be an antibiotic resistance gene, or a herbicide tolerance gene for plants. Such genes

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 herbicide tolerance are described in particular in patents EP 115 673, EP 337 899, WO 96/38567 or WO 97/04103.

 The present invention also relates to a cloning or expression vector for the transformation of a host organism constituting or containing at least one recombinant nucleic acid molecule defined above. Such vectors of transformation as a function of the host organism to be transformed are well known to those skilled in the art and widely described in the literature. The vectors according to the invention can be used to transform any type of cell host such as mono- or multicellular organisms, lower or higher, in particular plant cells or plants. For the transformation of plant cells or plants, it will in particular be a virus containing its own elements of replication and expression. Preferably, the vector for transforming plant cells or plants according to the invention is a plasmid.

 As indicated above, the polynucleotide sequences of the invention are useful for preparing plants resistant to geminiviruses. Indeed, these polynucleotide sequences code for proteins which are necessary for the six proteins of the genome of the geminivirus to infect the plant. Consequently, these polynucleotide sequences make it possible to prepare modified nucleic acids which, once integrated in a stable manner into the genome of the plant, will oppose the interaction of the proteins encoded by the polynucleotide sequences of the invention with the six geminivirus genome products and thereby prevent viral multiplication.

 The invention therefore relates to a genetically modified plant resistant to geminiviruses stably possessing at least one acid in its genome

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 modified nucleic acid comprising or consisting of a polynucleotide sequence of the invention encoding a protein necessary for one or more of the six proteins of the genome which is modified to: - block the expression of said plant protein necessary for one or more of the six proteins of the genome of a geminivirus, - expressing a modified plant protein, the modification of which compared to the natural protein modifies or suppresses the interaction with at least one of the six viral proteins.

 The invention is remarkable in that it makes it possible to prepare transgenic plants: - either, no longer expressing a plant protein necessary for one or more of the six proteins of the genome of a geminivirus to allow viral infection, - either, expressing a modified plant protein, and the modification of which compared to the natural protein modifies or suppresses the interaction with at least one of the six viral proteins and thus prevents the multiplication of the virus.

 The term “modified plant protein” is intended to mean a protein which, relative to the natural protein, is: - overexpressed thanks to a particular promoter or due to a large number of copies of the polynucleotide sequence encoding said protein, - modified at the level of one or more of these amino acids so that even if the interaction with a viral protein is retained, the modified protein is no longer functional for viral infection; advantageously the protein thus modified is overexpressed so that it is the modified protein which interacts with the viral protein rather than the natural protein,

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 - modified at the level of one or more of these amino acids so that the interaction with a viral protein is suppressed, - truncated, it is then a fragment of the natural protein which, even if l interaction with a viral protein is conserved, no longer functional for viral infection; advantageously the protein thus modified is overexpressed so that it is the modified protein which interacts with the viral protein rather than the natural protein.

 Modified plant proteins are expressed in a genetically modified plant according to the invention thanks to the modified plant polynucleotide sequence compared to the natural polynucleotide sequence. The term “modified plant polynucleotide sequence” is thus understood to mean a polynucleotide sequence which, with respect to the polynucleotide sequence has one or more modifications of nucleotides or else consists of a fragment of the natural polynucleotide sequence which codes for a modified protein.

 The invention also contemplates, as a modified plant polynucleotide sequence, short fractions of the gene which it is desired to alter. These short nucleotide fractions are useful for carrying out gene interventions without the addition of an external gene, using techniques of the chimeraplasty type, in order to carry out a very precise and efficient mutation in a gene. These sequences have a particular structure which activates their integration in the right place.

 The invention also envisages, as a modified polynucleotide sequence, antisense sequences capable of blocking the transcription or the translation of the natural polynucleotide sequence and therefore the expression of plant proteins interacting with the six products of the genome of the geminivirus.

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 The examples of polynucleotide sequences or of modified proteins above are given without implied limitation, since the person skilled in the art is capable of carrying out other modifications from the polynucleotide sequences given below making it possible to prevent viral infection . Those skilled in the art are able, using molecular biology techniques described in the literature, to prepare transgenic plants and to test their resistance to viruses.

 Thus, in certain advantageous embodiments of the invention, the modified plant polynucleotide sequence is placed under the control of a permanent or inducible promoter.

 For the purposes of the present invention, the term “plant” means any differentiated multicellular organism capable of photosynthesis, in particular monocots or dicots, more particularly crop plants intended or not for animal or human consumption, such as tomatoes, corn, wheat , rapeseed, soybeans, rice, sugar cane, beets, cotton, etc.

 The term “genetically modified plants” within the meaning of the present invention means a transgenic plant obtained by transformation, with at least one nucleic acid molecule comprising a polynucleotide sequence as defined above, or the progeny of such a plant if the latter contains the say nucleic acid molecules in its seeds.

 The invention also relates to a process for preparing a transgenic plant resistant to geminiviruses consisting in: - transforming a plant cell with at least one nucleic acid comprising or consisting of a modified polynucleotide sequence defined above,

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 - regenerating a plant from said transformed cell.

 There are several techniques for the transformation and regeneration of plants described in the literature, in particular in the following patent applications to which a person skilled in the art may refer: US 4,459,355, US 4,536,475, US 5,464,763, US 5,177,010, US 5,187,073, EP 267 159, EP 604 662, EP 672 752, US 4, 945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5,179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,518, 5,510 , US 5,204,253, US 5,405,765, EP 442 174, EP 486 233, EP 486 234, EP 539 563, EP 674 725, W091 / 02071 and W095 / 06128.

 Mention may more particularly be made of the methods consisting in bombarding cells, protoplasts or tissues with particles to which the nucleic acid molecules are attached. Other methods consist in using as a means of transfer into the plant a Ti plasmid of A. tumefaciens or Ri of Agrobacterium rhizogenes into which is inserted a recombinant nucleic acid molecule. Still other methods can be used, such as microinjection or electroporation, or even transformation by means of PEG. Either of these techniques may be more or less suited to the nature of the host organism, in particular the plant cell or plant.

 The invention also relates to the transformed plants resulting from the culture and / or from the crossing of the regenerated plants above, as well as the seeds of said transformed plants resistant to the diseases caused by the viruses, preferentially resistant to the diseases caused by the geminiviruses.

 The invention therefore also relates to a plant cell transformed in the genome of which is

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 stably incorporated a modified nucleic acid defined above.

 The term “plant cell” is understood to mean more particularly any cell originating or originating from a plant and which may constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, or seeds.

 The work carried out in the context of the present invention has therefore made it possible to identify new genes coding for proteins capable of interacting with at least one of the six proteins of the genimiviruses. As indicated above, these sequences consist of all or part of a gene chosen from the group comprising the genes gip3-C2, gip6-C2, gip7-C3, gip8-C3, gip9-C3, gipll-C3, gipl2 -C4, gipl4-CP, gipl5-CP, gipl6-V2, gipl8-V2, gipl9-V2, gip21-V2 and gip22-V2. The invention relates more particularly to proteins or fragments of proteins encoded by these genes. The subject of the invention is therefore very specifically a protein capable of interacting with at least one of the six proteins of the genimiviruses, characterized in that it comprises or consists of an amino acid sequence chosen from the sequences represented in the sequence list in annex under the numbers SEQ ID NO: 6, SEQ ID N0: 12, SEQ ID N0: 14, SEQ ID N0: 16, SEQ ID N0: 18, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID N0: 42 or SEQ ID N0: 44, a substantially similar fragment or sequence of these.

 Other advantages and characteristics of the invention will appear on reading the examples below concerning the gipl-Rep gene of S. pombe, the product of which

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 interacts with the TYLCV Rep protein and its use for tomato processing.

 Example 1. Isolation of the gipl-Rep gene from S. pombe, the product of which interacts with the Rep protein of TYLCV.

 Using the yeast double-hybrid method (Fields and Song, 1989), a S. pombe cDNA library was screened with the TYLCV rep protein fused to the DNA binding domain of GAL4. Two yeast clones, called GIPl-Rep.ll and GIPl-Rep.13, were able to form colonies on selection medium and gave a positive result in the LacZ test. Analysis of the sequence of these two clones showed that they were identical except for the 3 'noncoding end. The gipl-Rep gene, contained in the two clones GIP1-Rep.l1 and GIPl-Rep.13, is a homolog of the PSA1 gene from the yeast Saccharomyces cerevisiae. This gene seems to be involved in the regulation of the cell cycle in S. cerevisiae (Benton & coll., 1996). The expression of the PSA1 gene is at its maximum just before entering the S phase of the cell cycle (Benton & al., 1996).

 Several deletions of the Rep gene have been constructed and cloned into the vector pAS2. These clones were used to test the capacity of the deleted Rep proteins to interact with the GIP1-Rep protein. These analyzes suggest that the N-terminal region of the Rep protein is essential for interaction with GIP1-Rep.

 Southern blot analysis shows that the gipl-Rep gene is present in a single copy in S. pombe. A Northern Blot analysis, performed on a culture of synchronous S. pombe cells, shows that the level of expression of the gipl-Rep gene varies during the cycle

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 cell and is at its maximum during the Gl phase, which precedes phase S of the cell cycle.

 Example 2. Genetic characterization of gip1Rep.

 A fragment of the gipl-Rep gene corresponding to the complete coding part of the gene was cloned into the expression vector pREP3x to give the plasmid pREP3xGipl. This plasmid was constructed to overexpress GIPl-Rep using a promoter whose expression is regulated by the presence of thiamine in the medium. Wild cells as well as various mutants of the cell cycle were transformed by the plasmid pREP3x-Gipl. The cells were spread on minimum medium and checked by electron microscopy. No effect of overexpression of the gipl-Rep gene was observed.

 To carry out the gene disruption, a genomic fragment containing the coding part of the gipl-Rep gene was used. This fragment was flanked by two PstI restriction sites which made it possible to clone it in pBS + to give the plasmid pBIG4. The latter was then digested with EcoRV to release almost all of the coding phase of gip1-Rep which was replaced by a fragment containing the ura4 gene to give the plasmid pBIG45. The ura4 gene is therefore framed at its 5 ′ and 3 ′ ends of, respectively, 1380 and 570 nucleotides of the genomic sequence of the gipl-Rep gene. A culture of a diploid strain of S. pombe auxotrophic for uracil and leucine was transformed with the PstI restriction fragment obtained from the plasmid pBIG45 and spread on medium allowing selection of the ura + colonies. Three independent colonies were selected and their sporulation was induced. Analysis of the tetrads carried out on one of the diploids showed that only two spores of

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 the tetrad are viable. These spores do not contain the ura4 gene because they are ura-. As with the PAS1 gene in S. cerevisiae (Benton & al., 1996), disruption of the gipl-Rep gene in S. pombe therefore proved to be lethal. These results indicate that gipl-Rep is an essential gene.

 To obtain a conditional mutant, the diploid cells containing the disruption of gipl-Rep were transformed with the plasmid pREP3x-Gipl, carrying the leucine marker, and the leu + colonies were selected. Sporulation was induced in some of these colonies and the spores were spread on minimum medium containing adenine and uracil. The phenotype of the haploid colonies was then analyzed.

All colonies were leu + and about half were ura + indicating that disruption of gipl-Rep was complemented by expression of gipl-Rep from the plasmid. This result was confirmed by spreading these cells on a medium with thiamine to repress the expression of gipl-Rep. Under these conditions, the cells were unable to grow. Analysis of the culture under a microscope revealed that most of the cells had two nuclei separated by a septum. This indicates that the gipl-Rep gene is essential at a particular stage in the cell cycle.

 Example 3. Creation of a vector of A. tumefaciens containing the construction of the gene coding for GIPl-Rep.

 The gipl-Rep gene contained in the plasmid pACTgipl-Rep is amplified by PCR. The amplified DNA fragment is purified after electrophoresis on agarose gel, it is then digested with SalI and KpnI and linked in the plasmid pJC2EN2 digested with the same enzymes. This gives the vector pJC2EN2gipl-Rep containing the

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 sequence coding for GIP1-Rep surrounded by the CaMV 35S promoter and the OCS terminator. The DNA fragment containing the GIP1-Rep expression cassette is released from the vector by the restriction enzymes HindIII and XbaI and purified. It is then linked to the vector pBINPLUS (van Engelen & coll., 1995) digested with the same enzymes. A vector of A is thus obtained. tumefaciens, pBINPLUSgipl-Rep, containing the sequence coding for GIPl-Rep which leads to the expression of this protein in the plant.

 Example 4. Genetic transformation of the tomato.

 The vector pBINPLUSgipl-Rep is introduced into the strain of A. tumefaciens LBA4404 (Hoekema & coll., 1983).

The transformation technique is based on the procedure of McCormick & coll. (1986) on the one hand and that of Fillati & al. (1987) on the other hand.

 The regeneration of the tomato from leaf explants is carried out on a Murashige and Skoog (MS) base medium comprising 30 g / l of sucrose as well as 500 mg / l of carbenicillin and 75 mg / ml of kanamycin. When the buds are well developed, they are placed on a medium allowing rooting. Well rooted, the plants are acclimatized in the greenhouse. Molecular evidence for the integration of the GIP1-Rep expression cassette is provided by molecular hybridization techniques of the Southern and Northern type, as well as by PCR. All the transformed plants are then used in various experiments to verify whether the expression of GIP1-Rep makes them resistant to viral aggressions. Transformed plants, as well as non-transformed control plants, are, for example, infected with the TYLCV virus by agroinoculation. The development of symptoms and the presence of viral DNA are then analyzed.

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 BIBLIOGRAPHICAL REFERENCES - Benton & coll. (1996) Curr. Broom. 29,106-113.

- Fields and Song (1989) Nature 340,245-246.

- Fillati & coll. (1987) Bio / Technology 5,726-730.

- Hoekema & coll. (1983) Nature 303, 179-180.

- Jonsson and Hubscher (1997) BioEssays 19,967-975 - Jupin & coll. (1994) Virology 204, 82-90.

- Kelman, Z. (1997) Oncogene 14,629-640 - Kheyr-Pour & coll. (1991) Nuc. Acids Res. 19.6763-6769.

- Lazarowitz, S (1992) Crit. Rev. Plant Sci., 11,327-349.

- McCormick & coll. (1986) Plant Cell Rep. 5.81-84.

- Nagar & coll. (1995) Plant Cell 7,705-719.

- Navot & coll. (1991) Virology 185, 151-161.

- Noris & coll. (1996) Virology 217,607-612.

- van Engelen & coll. (1995) Transgenic Res. 4,288-290.

- Wartig & coll. (1997) Virology 228,132-140.

- Xie & coll. (1995) EMBO J. 14, 4073-4082.

- Xie & coll. (1999) Plant Mol. Organic. 39,647-656.

Claims (17)

1) A purified plant or yeast polynucleotide sequence coding for a protein which interacts with at least one of the six products of the genome of a geminivirus necessary for the infection of a plant by this virus.  2) A polynucleotide sequence according to claim 1, characterized in that it codes for a protein whose amino acid sequence is chosen from the group comprising the amino acid sequences represented in the sequence list in the annex under the numbers SEQ ID NO : 6, SEQ ID N0: 12, SEQ ID N0: 14, SEQ ID N0: 16, SEQ ID N0: 18, SEQ ID N0: 22, SEQ ID N0: 24, SEQ ID N0: 28, SEQ ID N0: 30 , SEQ ID NO: 32, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 42 or SEQ ID NO: 44, a fragment or sequence substantially similar thereto.  3) A polynucleotide sequence according to claim 2, characterized in that it consists of all or part of a polynucleotide sequence chosen from those represented in the sequence list in the annex under the numbers SEQ ID NO: 5, SEQ ID NO : 11, SEQ ID N0: 13, SEQ ID N0: 15, SEQ ID N0: 17, SEQ ID N0: 21, SEQ ID N0: 23, SEQ ID N0: 27, SEQ ID N0: 29, SEQ ID N0: 31 , SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 41 or SEQ ID NO: 43, a sequence complementary or substantially similar thereto.  4) A polynucleotide sequence according to claim 1, characterized in that it codes for a protein whose amino acid sequence is chosen from the group comprising the amino acid sequences represented in the sequence list in the annex under the numbers SEQ ID NO : 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID <Desc / Clms Page number 29>  NO: 10, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 34 or SEQ ID N0: 40, a substantially similar fragment or sequence thereof.  5) A polynucleotide sequence according to claim 4, characterized in that it consists of all or part of a polynucleotide sequence chosen from those represented in the sequence list in the annex under the numbers SEQ ID NO: 1, SEQ ID NO : 3, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID N0: 19, SEQ ID N0: 25, SEQ ID N0: 33 or SEQ ID N0: 39, a sequence complementary or substantially similar thereto.  6) Recombinant nucleic acid molecule, characterized in that it comprises at least one polynucleotide sequence according to any one of claims 1 to 5.  7) Recombinant nucleic acid molecule according to claim 6, characterized in that said polynucleotide sequence is placed under the control of regulatory elements in position 5 'and / or 3' thereof which can function in a host organism.  8) Recombinant nucleic acid molecule according to one of claims 6 or 7, characterized in that said polynucleotide sequence is associated in the recombinant nucleic acid molecule with a selection marker.  9) Cloning or expression vector for the transformation of a host organism constituting or containing at least one recombinant nucleic acid molecule according to any one of claims 6 to 8. <Desc / Clms Page number 30>  10) A cellular host transformed by a nucleic acid molecule according to one of claims 6 to 8 or by a vector according to claim 9.  11) A genetically modified plant resistant to geminiviruses stably possessing in its genome at least one modified nucleic acid comprising or consisting of a polynucleotide sequence according to any one of claims 1 to 5 which is modified to: - block the expression of said plant protein necessary for one or more of the six proteins of the genome of a geminivirus, or - expressing a modified plant protein, and the modification of which relative to said protein modifies or suppresses the interaction with at least one of the six viral proteins.  12) A genetically modified plant resistant to geminiviruses according to claim 11, characterized in that the modified plant protein is a protein defined in one of claims 1 to 5 which is: - over-expressed, - modified at the level of one or more of these amino acids so that even if the interaction with a viral protein is retained, the modified protein is no longer functional for viral infection, - truncated in the form of a fragment of said protein which, even if the interaction with a viral protein is preserved, is no longer functional for viral infection.  - modified at the level of one or more of these amino acids so that the interaction with a viral protein is suppressed, <Desc / Clms Page number 31> 13) A genetically modified plant resistant to geminiviruses according to claim 12, characterized in that said modified polynucleotide sequence is placed under the control of a permanent or inducible promoter.  14) A genetically modified plant resistant to geminiviruses according to claim 11, characterized in that said modified polynucleotide sequence is an antisense sequence capable of blocking the transcription or the translation of a polynucleotide sequence according to any one of claims 1 to 5.  15) Method for preparing a transgenic plant resistant to geminiviruses, characterized in that it comprises the steps consisting in: - transforming a plant cell with at least one modified nucleic acid defined in one of claims 11 to 14, - regenerating a plant from said transformed cell.  16) A plant cell in the genome of which is stably incorporated a modified nucleic acid defined in one of claims 11 to 14.  17) A protein capable of interacting with at least one of the six genimivirus proteins, characterized in that it comprises or consists of an amino acid sequence chosen from the sequences represented in the sequence list in the annex under the SEQ ID NO: 6, SEQ ID N0: 12, SEQ ID N0: 14, SEQ ID N0: 16, SEQ ID N0: 18, SEQ ID N0: 22, SEQ ID N0: 24, SEQ ID N0: 28, SEQ ID N0: 30, SEQ ID N0: 32, SEQ ID N0: 36, SEQ ID <Desc / Clms Page number 32>  NO: 38, SEQ ID NO: 42 or SEQ ID NO: 44, a substantially similar fragment or sequence thereof.