WO2003066900A2 - Mutations du gene eif4e et resistance aux potyvirus - Google Patents
Mutations du gene eif4e et resistance aux potyvirus Download PDFInfo
- Publication number
- WO2003066900A2 WO2003066900A2 PCT/FR2003/000397 FR0300397W WO03066900A2 WO 2003066900 A2 WO2003066900 A2 WO 2003066900A2 FR 0300397 W FR0300397 W FR 0300397W WO 03066900 A2 WO03066900 A2 WO 03066900A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- eif4e
- protein
- sequence
- plants
- potyviruses
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Definitions
- the present invention relates to a process for selecting or obtaining plants resistant to potyviruses.
- the method is particularly applicable to plants of the nightshade family, cucurbits, crucifers and compounds.
- the invention also includes the sequences making it possible to confer resistance to potyviruses and / or to mark the genes for resistance or sensitivity to these potyviruses.
- potyviruses whose type member is the potato virus Y or PVY for Potato Virus Y is the most important group of plant viruses. In fact, potyviruses are capable of infecting more than 30 families of plants currently listed. This group includes at least 180 members, which corresponds to a third of the currently known plant viruses. Transmission of potyviruses is carried out by aphids (for example, Myzus persicaé) in a non-persistent mode. Symptoms caused by potyviruses are abnormal coloring of the leaves (mosaics, yellowing of the veins), deformations of the leaves, necrosis of the nerves which can lead to necrosis of the whole plant, and significant reductions in the size of the diseased plant strongly influencing productivity.
- Solanaceae, cucurbits, crucifers and compounds are particularly sensitive to potyviruses.
- Solanaceae and more particularly tomatoes and peppers (or peppers) are infected with at least seven distinct potyviruses around the world: potato virus Y (PVY) is present in all areas of cultivation while the rest are confined to one continent (Tobacco Etch virus, Pepper Mottle Virus and Peru Tomato Virus on the American continent, Pepper Veinai Mottle Virus and Potyvirus E in Africa, and chili Veinai Mottle Virus in Asia).
- PVY potato virus Y
- This compartmentalisation is however no longer absolute, several potyviruses having been identified outside their area of origin. In France, and more generally in the Mediterranean basin, the predominant potyvirus is PVY.
- Potyviruses have a non-enveloped filamentous structure (Langenberg and Zhang, 1997) 680 to 900 nm long and 11-15 nm wide (Dougherty and Carrington, 1988; Riechmann et al., 1992).
- the viral genome consists of a single strand sense RNA with an approximate length of 10 kb.
- Single-stranded RNA has a poly A tail at its 3 'end and binds 5' to a viral protein called VPg (Murphy et al., 1990, Takahashi et al., 1997).
- Viral RNA codes for 10 proteins involved in the cleavage of polyproteins, genome replication, cell-to-cell movement and long distance movement, aphid transmission ...
- the fight against viruses is not feasible indirectly. Indeed, it is only possible to eliminate the vector of the disease (aphids in this case) or to cultivate varieties resistant to viral infection and / or vectors.
- the plant Faced with an attack by a pathogen (virus, bacteria, fungi or nematodes), the plant has several strategies to defend itself or resist infection. Among the defense strategies, the plant can implement:
- non-host resistance when all the entities of a species are resistant to a given pathogen
- host resistance when at least one entity of the species is sensitive to a strain of l 'pathogen.
- Host resistance the best known to date and the best characterized, is that involving a major, dominant gene. When the major gene is in the presence of a specific avirulence gene for the pathogen, the incompatibility between the plant and the pathogen is established and the plant is resistant. This interaction, described by Flor
- Point mutations in the viral gene encoding the protein VPg have been shown to be involved in the bypass of resistance to potyviruses in several host-pathogen pairs. This has been shown in TVMV / Nicotiana tabacum (va gene), PVY / tomato (pot-1 gene), LMV / Lettuce (mol gene) and PSbMV / pea (sbml gene) pairs (Keller et al, 1998, Morel , 2001, Redondo, 2001, Nicolas et al, 1997). This does not exclude that other viral genes can also intervene.
- the eIF4E gene codes for a eukaryotic factor initiating the translation of RNA.
- eIF4E corresponds to one of the eIF4F translation factor subunits (in wheat germ, it corresponds to the p26 subunit).
- the translation factor eIF4E binds to the mRNA cap at the level of m 7 G.
- the structure of eIF4E is characterized by a region rich in tryptophan residues (10 in Arabidopsis thaliana, 11 in wheat and 12 in mammals). These tryptophan residues are thought to be involved in binding to the functional group m 7 G (Rudd, K. Et al., 1998).
- the eIF4E translation factor is encoded by a multigene family.
- eIF4E For example in Arabidopsis thaliana, 4 copies of eIF4E have been identified (Rodriguez et al., 1998, Robaglia et al., Pers. Com.). These copies have, two by two, between 44 and 82% identity.
- - X 5 and X 14 represent a basic amino acid
- - Xi i represents an acidic amino acid
- - D, K, S, Q, A, W, G, R, Y, T, F, V, E, N, I, H, P, and L have their usual meaning in code 1 - letter.
- neutral amino acid any amino acid chosen from the following: alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine, threonine, tyrosine, cysteine, glutamine, asparagine.
- charged amino acid any amino acid chosen from the following: histidine, lysine, arginine, glutamate, aspartate. Among these charged amino acids, histidine, lysine or arginine are basic amino acids, and glutamate and aspartate are amino acids.
- sequence (I) is also represented in the annexed sequence list under the number SEQ ID NO: 1
- these mutations do not concern amino acids that are very conserved in eukaryotes, nor those involved in the essential functions of eIF4E, namely recognition of the cap or the interaction between eIF4E and eIF4G or 4E-binding protein.
- these mutations have an effect on the VPg / eIF4E interaction by modifying the structure of eIF4E at the level of the region or regions thereof involved in this interaction.
- This modification of the structure probably results from the substitution of amino acids by amino acids of different charge (replacement of neutral amino acids by charged amino acids, or conversely of charged amino acids by neutral amino acids, or amino acids of opposite charge) which constitutes the common point of all the mutations highlighted by the Inventors. It can therefore be reasonably assumed that other mutations of the same type in a plant eIF4E protein, at the level of the region defined by the sequence (I) will cause similar structural modifications, producing the same effect on the VPg / eIF4E interaction.
- the present invention relates more particularly to a method for selecting plants resistant to potyviruses, characterized in that it comprises detection in the plants to be tested:
- wild type eIF4E protein comprising a region defined by the sequence (I) above, or of a sequence coding for said protein
- mutant eIF4E protein comprising a region derived from that defined by the sequence (I) above, by substitution of at least one neutral amino acid of said sequence (I) with a charged amino acid, preferably a basic amino acid and / or substitution of at least one charged amino acid of said sequence (I) by a neutral amino acid or an amino acid of opposite charge, or of a sequence coding for said protein ;
- the subject of the present invention is also a process for selecting plants which can be used for obtaining plants resistant to potyviruses, characterized in that it comprises the detection in the plants to be tested of the presence or absence of a protein mutant eIF4E as defined above or of a sequence coding for said protein, and the selection of plants where said mutant eIF4E protein or a sequence coding for said protein is detected.
- said mutant eIF4E protein comprises a region derived from that defined by the sequence (I) above, by: a) substitution of at least one of the amino acids X ⁇ ; X j X 3 or X 4> of said sequence
- the detection of the presence or absence of a wild-type or mutant eIF4E protein can be carried out in particular using antibodies specifically directed against the desired form of the eIF4E protein. They may in particular be antibodies directed either against the wild form or against the mutant form of the region of eIF4E defined by the sequence (I).
- a sequence coding for a wild type eIF4E protein or of a sequence coding for a mutant eIF4E protein may include polynucleotides derived from the sequence of the eIF4E gene and in particular from polynucleotides capable of hybridizing selectively either with a wild-type allele or with a mutant allele of eIF4E, as defined above or of polynucleotides allowing the amplification of the region of eIF4E containing the mutation sought; they may also be restriction enzymes recognizing a target sequence present in the wild form, but not in the mutated form (or vice versa).
- a subject of the present invention is therefore the use of a selection tool chosen from: a) a polynucleotide coding for a wild type or mutant eIF4E protein, as defined above; b) a polynucleotide complementary to polynucleotide a); c) a fragment of at least 10 bp of a polynucleotide a) or b); d) an antibody directed against a wild-type or mutant eIF4E protein, as defined above; for the selection of plants resistant to potyviruses.
- a selection tool chosen from: a) a polynucleotide coding for a wild type or mutant eIF4E protein, as defined above; b) a polynucleotide complementary to polynucleotide a); c) a fragment of at least 10 bp of a polynucleotide a) or b); d) an antibody directed against a wild-type or mutant eIF
- the invention relates to a method of selecting plants resistant to potyviruses characterized in that it comprises the implementation of at least one selection means chosen from the group of genetic (or related) tools comprising: * A / all or part of at least one of the sequences selected from the subgroup comprising:
- the selection means are selected from the subgroups of tools A / and / or B /, and more preferably still from the subgroup of tools A /.
- the process which is the subject of the invention is particularly applicable to solanaceae, cucurbits, crucifers and compounds and more specifically to plants of the genera Lycopersicon, Capsicum, Nicotiana, Solanum, Lactuca, Cucumis, Arabidopsis etc ...
- Potyviruses concerned are, for example, potato virus Y (PVY), tobacco burning virus (TEV) and / or lettuce mosaic virus (LMV), and / or virus yellow zucchini mosaic (ZYMV) and / or turnip mosaic virus (TuMV).
- PVY potato virus Y
- TMV tobacco burning virus
- LMV lettuce mosaic virus
- ZYMV virus yellow zucchini mosaic virus
- TuMV turnip mosaic virus
- nucleotide sequences and / or peptide sequences or restriction enzymes are used as detection means, probes or primers, to select plants resistant or sensitive to potyviruses.
- These detection means include in particular nucleotide probes or primers.
- Primer is understood to mean, within the meaning of the present invention, any polynucleotide sequence which can be used to amplify a sequence of an eIF4E gene capable of comprising a mutation associated with resistance to potyviruses. These are in particular polynucleotides which can be used to amplify all or part of the sequence of eIF4E coding for the region of eIF4E defined by the sequence (I), or of the mutant sequence which is derived therefrom.
- the term "probe” means any polynucleotide sequence which hybridizes with a wild type eIF4E gene or with a mutant elF4E gene as defined above. This notably includes the nucleotide sequences capable of hybridizing selectively either with an allele of the eIF4E gene associated with resistance to potyviruses, or with an allele of the elF4E gene associated with sensitivity to potyviruses. These probes and primers can be used as specific markers for plants resistant or sensitive to potyviruses.
- SEQ ID NO: 2, 4, 6, and 8 correspond to eIF4E genes of different solanaceae involved in resistance to potyviruses coding for a eukaryotic factor initiating the translation of RNA.
- SEQ ID NO: 8 corresponds to a recessive allele eIF4E of resistance to a potyvirus, while SEQ ID NO: 2, 4, and 6 represent dominant alleles eIF4E of sensitivity to a potyvirus.
- the selection process according to the invention can involve the two types of selection means or benchmarks separately or together.
- the invention also encompasses all the equivalents to these nucleotide sequences (A) SEQ ID NO :: 2, 4, 6, and 8, which retain the function of genetic benchmark eIF4E for sensitivity / resistance to potyviruses specific to the reference sequences .
- DNA these are in particular the analogs of genetic degeneration and cDNA sequences complementary to the reference sequences.
- the polynucleotide equivalents of the reference sequences (A) are also found among their transcription products (RNA) (B).
- Proteins (C) from (A) and (B) constitute other intracellular landmarks allowing the selection of plants resistant or sensitive to potyviruses.
- the selection means of the invention can also be nucleotide probes capable of hybridizing with complementary nucleotide targets (A) and (B), or even protein detection means (antibody D) capable of pairing with specific antigenic targets (C). It is possible to combine all these equivalent means (A), (B), (C) & (D) to form a selection tool (E).
- fragment is meant, according to the invention:
- polyamino acid of at least 3, 6, 10, 15, 30, 60, 100, 150, 200 contiguous amino acids of the reference sequence; preferred fragments are those which are capable of selectively hybridizing under stringent conditions with said reference sequence.
- the method is characterized in that:
- At least one detection means comprising at least one of the tools A, B, C, D, E according to claim 1 and / or at least one restriction enzyme, with at least one genomic extract and / or protein from a plant to be tested, - said genomic and / or protein extract, optionally paired and / or hybrid and / or digested, is subjected to at least one separation,
- any pairings and / or hybridizations and / or digestions likely to occur are revealed, - and the results are read to finally conclude on the presence or absence of a resistance allele (pvr2) or an allele of sensitivity (pvr + ) to at least one potyvirus.
- pvr2 resistance allele
- pvr + allele of sensitivity
- This process is part of known methodologies in the field of detection and recognition of genetic characteristics of plants.
- the method can respond to the following methodology:
- the coding sequence of the eIF4E gene is amplified by PCR, from the DNA of the plant to be tested, for example using primers SEQ ID NO: 18 and / or 19, the amplification product is digested with a suitable restriction enzyme;
- Any fragments obtained are separated, and resistant or sensitive plants are selected according to the restriction profile of said amplification product.
- plants susceptible to potyviruses can be detected by a restriction profile revealing the presence of a site of cleavage by the enzyme
- TspRI or one of its isoschizomers and plants resistant to potyviruses can be detected by a restriction profile showing the absence of said cleavage site by
- TspRI or one of its isoschizomers and the presence of a cleavage site by the enzyme Mvnl or one of its isoschizomers.
- the method preferably consists of:
- Hybridization of the single-stranded molecules of the probe and of the target is preferably carried out under stringent hybridization conditions allowing selective hybridization, which can be determined in a manner well known to those skilled in the art.
- the hybridization and washing temperature is at least 5 ° C. lower than the Tm of the reference sequence at a given pH and for a given ionic strength.
- the hybridization temperature is at least 30 ° C for a polynucleotide of 15 to 50 nucleotides and at least 60 ° C for a polynucleotide of more than 50 nucleotides.
- the level of the signal generated by the interaction between the sequence capable of selective hybridization and the reference sequences is generally 10 times, preferably 100 times more intense than that of the interaction of the other DNA sequences generating the background noise.
- a probe labeled for example with a radioactive element, such as 32 P, or a grafted enzyme, such as peroxidase hybridization is easily revealed qualitatively and quantitatively.
- the DNA used in the first or second mode of implementation can be either total DNA or cDNA.
- the method preferably consists in detecting the presence of a polypeptide partly consisting of all or part of one of the amino acid sequences described below and included in the invention.
- the method may consist in bringing the test sample into contact with an antibody as described above and then in detecting the antigen / antibody complex formed.
- the selection method according to the invention is reliable and sensitive.
- the present invention also relates to a polynucleotide coding for a mutant eIF4E protein comprising a region derived from that defined by the sequence (I) above, by substitution of at least one neutral amino acid of said sequence (I) with a charged amino acid, preferably a basic amino acid and / or substitution of at least one amino acid charged with said sequence (I) by a neutral amino acid or an amino acid of opposite charge.
- said polynucleotide codes for a mutant eIF4E protein which comprises a region derived from that defined by the sequence (I) above, by: a) substitution of at least one of the amino acids X ⁇ X 2 ⁇ X 3 or X > of said sequence (I) with a charged amino acid, and b) substitution of at least one of the other neutral amino acids of said sequence (I) with a charged amino acid and / or substitution of at least an amino acid charged with said sequence (I) by a neutral amino acid or an amino acid of opposite charge.
- Polynucleotides in accordance with the invention are, for example, those which code for the variants of the sequences SEQ ID NO: 22 or 23 associated with resistance to potyviruses.
- the invention relates to a nucleotide sequence characterized in that it is described by a sequence chosen from the group comprising all or part of the following sequences:
- the nucleotide sequence SEQ ID NO: 2 is a cDNA sequence obtained from tobacco DNA and corresponding to the tobacco gene.
- the nucleotide sequence SEQ ID NO: 4 is a sequence encoding an eIF4E protein from a variety of Lycopersicon esculentum sensitive to potyviruses.
- the sequences SEQ ID NO: 6 and 8 are sequences coding for proteins eIF4E of chilli (Capsicum annuum), varieties Yolo Wonder and Yolo Y respectively.
- the present invention also relates to primers allowing the amplification of an eIF4E gene, or of a portion thereof capable of containing at least one mutation, as defined above, associated with resistance to potyviruses; these are in particular primers allowing the amplification of the sequence of eIF4E coding for the region of eIF4E defined by the sequence (I), or of a mutant sequence which is derived therefrom.
- Primers according to the invention can be easily defined by a person skilled in the art, from the nucleotide or peptide sequences described in the present invention.
- the amplification primers constituted by the nucleotide primer sequences SEQ ID NO: 18 &19; the primers cloning SEQ ID NO: 10 to 17; the BAC library screening primers constituted by the nucleotide sequences SEQ ID NO: 20 & 21.
- SEQ ID NOs: 18 & 19 are primers derived from the coding sequence of eIF4E of the Yolo Wonder chili pepper, allowing in particular by PCR amplification then by enzymatic digestion, the detection of the nucleotide sequences carrying alleles of pvr2 resistance and of pvr + sensitivity to potyvirus.
- the degenerate SEQ ID NO: 10 & 11 cloning primers and the non-degenerate SEQ ID NO: 12 to 17 were defined on an alignment of the tobacco, tomato and Arabidopsis eIF4E sequences and used for probe synthesis (RACE) EIF4E detection cDNA in the tomato and chili genome.
- the primers SEQ ID NO: 20 & 21 for screening the BAC bank are not degenerate. These primers SEQ ID NO: 10 to 17, 20 & 21 can possibly be used directly or indirectly (construction of selection tools) in the detection of resistance or sensitivity characteristics to potyviruses.
- the subject of the present invention is also a mutant eIF4E protein comprising a region derived from that defined by the sequence (I) above, by substitution of at least one neutral amino acid of the said sequence (I) with a charged amino acid, preferably a basic amino acid and / or substitution of at least one amino acid charged with said sequence (I) by a neutral amino acid or an amino acid of opposite charge.
- said mutant eIF4E protein comprises a region derived from that defined by the sequence (I) above, by: a) substitution of at least one of the amino acids Xi, X 2 , X 3 or X > of said sequence (I) with a charged amino acid, and b) substitution of at least one of the other neutral amino acids of said sequence (I) with a charged amino acid and / or substitution of at least one charged amino acid of said sequence (I) with a neutral amino acid or an amino acid of opposite charge.
- the present invention also covers the translation products of the nucleotide sequences SEQ ID NO: 2, 4, 6, and 8, namely the polypeptides chosen from the group comprising all or part of the following sequences:
- the means for selecting resistance / sensitivity of plants to potyviruses, consisting of amino acid sequences, are preferably used as targets for locating. These are therefore indirect selection means which underlie the implementation of specific detection means for these peptide targets.
- These detection means are advantageously antibodies which constitute another object of the present invention.
- said antibodies are characterized in that they are specifically directed against all or part of at least one of the translation products C, and more particularly of the amino acid sequences SEQ ID NO: 3, 5, 7, 9, 22, or 23 or a fragment of at least 6 amino acids thereof.
- These antibodies can be monoclonal or polyclonal.
- Antibodies against the polypeptides as defined above can be prepared according to conventional techniques well known to those skilled in the art (for example, Kohler and Milstein, 1975; Kozbor et al. 1983, Martineau et al., 1998).
- An antibody according to the invention may comprise an isotopic or non-isotopic, for example fluorescent, detectable marker, or alternatively be coupled to a molecule such as biotin according to techniques well known to those skilled in the art.
- Another aspect of the invention relates to the selection means formed by probes for the detection of plants resistant to at least one potyvirus, these probes being taken in themselves.
- probes for the detection of plants resistant to at least one potyvirus we define probes for the detection of plants resistant to at least one potyvirus.
- a first category of probes is characterized in that each probe comprises at least one sequence corresponding to all or part of SEQ ID NOs: 2, 4, 6, and 8.
- the probes comprising at least one sequence corresponding to all or part of SEQ ID NO: 2, 4, 6, and 8, and in particular to all or part of the portion coding for the region of the eIF4E protein defined by the general sequence (I) are very particularly preferred.
- SEQ ID NO: 6 is a sensitivity probe from the Yolo Wonder pepper. It differs from SEQ ID NO: 8, which is a resistance probe derived from the Yolo Y pepper, by two nucleotide bases. These mutations shown on SEQ ID NO: 6 & 8, correspond to the restriction sites TspRI for SEQ ID NO: 6 and MnvI for SEQ ID NO: 8, respectively marking the sensitivity to potyviruses in Yolo Wonder and the resistance to potyviruses in Yolo Y .
- probes are used to distinguish resistant and sensitive plants, either by selective hybridization and detection of the presence or absence of a hybridization signal, or by digestion with an appropriate restriction enzyme capable of differentially cleaving the allele.
- an appropriate restriction enzyme capable of differentially cleaving the allele.
- sensitivity and the resistance allele for example EcoRI, TspRI, or MnvI followed by hybridization of the probe with the restriction product.
- the distinction between sensitive plants and resistant plants is made in the latter case by the difference in size of the hybrid fragments.
- the present invention also provides tools making it possible to carry out another selection method in accordance with the first embodiment of the method according to the invention, as defined above.
- an amplification by PCR of the eIF4E sequence is first performed. Amplification is followed by selective digestion with a restriction enzyme.
- the tools involved are therefore of two types: restriction enzyme (s) and PCR primer (s) for amplifying the eIF4E sequence.
- the subject of the present invention is in particular a kit making it possible to detect an allele of eIF4E associated with resistance or sensitivity to potyviruses, characterized in that it comprises:
- At least one restriction enzyme chosen from: a) an enzyme recognizing a restriction site I present in at least one allele of eIF4E associated with sensitivity to potyviruses, and absent from the alleles of eIF4E associated with resistance to potyviruses; b) an enzyme recognizing a restriction site II present in at least one allele of eIF4E associated with resistance to potyviruses, and absent from the alleles of eIF4E associated with sensitivity to potyviruses; and - a pair of nucleotide primers making it possible to amplify eIF4E or a portion thereof comprising the restriction site I and / or the restriction site II.
- the restriction enzyme is TspRI, or one of its isoschizomers, recognizing a restriction site defined by the sense sequence: NNCASTGNN A and the antisense sequence ⁇ NNGTSACNN.
- the nucleotide primers are chosen so as to allow the amplification of the entire sequence of eIF4E or of at least a portion thereof comprising the TspRI site; - to detect an allele of eIF4E associated with resistance to potyviruses, such as that represented by the sequence SEQ ID NO: 8 the restriction enzyme is Mvnl, or one of its isoschizomers, recognizing a restriction site defined by the sense sequence: CG ⁇ CG and the antisense sequence: GC ⁇ GC.
- the nucleotide primers are chosen so as to allow the amplification of the entire sequence of eIF4E or of at least a portion thereof comprising the site
- primers SEQ ID NO: 18 and SEQ ID NO: 19 can be used, for example.
- the detection of plants sensitive or resistant to potyviruses can also be carried out by detection of the presence or absence of the wild or mutant form of the protein eIF4E.
- the present invention encompasses the use of a wild type or mutant eIF4E protein, as defined above, or of an antibody specific for one of said proteins, for the selection of plants resistant to potyviruses.
- said eIF4E protein is chosen from: - the protein represented by the polypeptide sequence SEQ ID NO: 3;
- a category of means for detecting resistance to potyviruses in accordance with the invention is characterized in that each of these means comprises at least one antibody specific for all or part of a mutant eIF4E protein in accordance with the invention, and in particular a fragment of at least 6 amino acids thereof carrying a mutation associated with resistance, as defined above.
- a means of detecting resistance can consist of an antibody specific for all or part of a polypeptide sequence as defined above, in particular of a fragment of at least 6 amino acids thereof carrying a mutation associated with resistance.
- the invention also relates to means for detecting sensitivity or resistance to potyviruses, each consisting of at least one amino acid sequence chosen from the group comprising the following sequences: - SEQ ID NO: 3;
- the invention relates more particularly to means for detecting sensitivity to potyviruses, each consisting of at least one antibody specific for an amino acid sequence chosen from the group comprising the following sequences:
- each aforementioned nucleotide probe or other means of detection is provided with at least one marker, useful as a witness to the nucleotide hybridization or the antigen / antibody pairing at the heart of the detection of the sensitive sequence.
- this marker is detectable by spectroscopic, photochemical, biochemical, immunochemical or even chemical means.
- a marker can consist of a radioactive isotope of P, H, a fluorescent molecule (5-bromodeoxyuridine, fluorescein, acetylaminofluorene) or also a ligand such as biotin.
- nucleotide probes their labeling is preferably done by incorporating labeled molecules within the polynucleotides by extension of primers or else by adding to the 3 ′ or 5 ′ ends.
- sequences used to detect plants resistant to potyviruses are used as nucleotide probes or primers.
- nucleic acids can be labeled, if desired, by incorporating a detectable molecule or label as set out above. 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 Urréa et al., (1988) or Sanchez-Pescador et al. (1988).
- the present invention relates to the use of the detection means defined above for the detection of plants resistant / sensitive to at least one potyvirus.
- oligonucleotide marker (s) of resistance / sensitivity to potyviruses the restriction sites Mvn1 and / or TspRI, which are, moreover, known, eIF4E sequences.
- restriction sites used as oligonucleotide reper (s) correspond: - to the sense sequence: CG ⁇ CG and to the antisense sequence: GC ⁇ GC
- restriction sites as benchmarks (or marks or labels) for resistance to potyviruses on expressed sequences, is to be compared to the first mode of implementation of the above-described detection method, in which recourse is had to restriction enzymes (for example: Mvnl and / or TspRI) and to primers for amplification of the sequence eIF4E, for example: SEQ ID NO 18 and / or 19.
- restriction enzymes for example: Mvnl and / or TspRI
- primers for amplification of the sequence eIF4E for example: SEQ ID NO 18 and / or 19.
- the present invention also encompasses the use, as oligonucleotide marker (s) of resistance / sensitivity to potyviruses, of the above-mentioned Mvnl & TspRI restriction sites, and, preferably , of the restriction site corresponding to the sense sequence: CG ⁇ CG and to the antisense sequence: GC ⁇ GC and / or of the restriction site corresponding to the sense sequence: NNCASTGNN ⁇ and to the antisense sequence: ⁇ NNGTSACNN.
- oligonucleotide marker (s) of resistance / sensitivity to potyviruses of the above-mentioned Mvnl & TspRI restriction sites, and, preferably , of the restriction site corresponding to the sense sequence: CG ⁇ CG and to the antisense sequence: GC ⁇ GC and / or of the restriction site corresponding to the sense sequence: NNCASTGNN ⁇ and to the antisense sequence: ⁇ NNGTSACNN.
- the present invention relates to a kit for selecting plants resistant / sensitive to potyviruses comprising at least one means of detection of antibody or polynucleotide type as defined above.
- the kit includes, where appropriate, the reagents necessary for carrying out a hybridization or amplification reaction.
- the subject of the invention is also the plants resulting from the process described above and / or from the implementation of the tools and / or from the use and / or from the selection kit defined above.
- these plants belong to the family of nightshades, cucurbits, crucifers and compounds. Even more preferably, they are chosen from tomatoes, peppers and / or lettuce.
- the inventors carry out the method which is the subject of the invention by following the RFLP (restriction fragment length polymorphism) analysis protocol.
- the inventors used a conventional RFLP protocol in which the probes which are the subject of the invention are labeled with 32 P and in which the DNA of the chilli plants to be analyzed is digested with the restriction enzyme EcoRI.
- EcoRI restriction enzyme
- the invention does not only concern the selection of plants resistant or sensitive to potyviruses. Indeed, since the inventors have been able to identify the eIF4E gene determining recessive resistance to potyviruses, it is now possible to obtain new varieties of genetically modified plants, resistant (or sensitive) to at least one potyvirus.
- the invention therefore relates to a non-biological process for obtaining new varieties of genetically modified plants, resistant (or sensitive) to at least one potyvirus, characterized in that it consists essentially in ensuring that appears and / or introduce an allele of eIF4E associated with resistance (or sensitivity) to said potyvirus in the genome of these plants.
- the appearance of the resistance allele is caused by the implementation of a method selected from the group comprising:
- the present invention thus relates to any genetic unit constructed comprising a polynucleotide in accordance with the invention coding for a mutant eIF4E protein, placed under the control of appropriate elements for controlling transcription, and optionally translation.
- Said mutant eIF4E protein can advantageously be chosen from variants of sequences SEQ ID NO: 22 or 23 associated with resistance to potyviruses.
- the present invention also relates to any constructed genetic unit characterized in that it comprises: - at least one genetic tool A / and / or B / as defined above,
- nucleotide sequence chosen from the group comprising all or part of the following sequences:
- Another genetic transformation tool covered by the invention consists of any plant cell transformation vector comprising at least one genetic unit constructed as referred to above. It can be any known and appropriate cloning vector (phages / plasmids / cosmids). Plant cells and microorganisms transformed using at least one vector or at least one constructed genetic unit as defined above, are also targeted by the invention.
- the invention encompasses plants transformed with at least one vector and / or at least one constructed genetic unit and / or transformed plant cells and / or transformed microorganisms, such as they have have been described above.
- FIG. 1 shows the gel from a southern blot analysis and showing the differences in profiles of the eIF4E marker for resistance to potyviruses, observed for different sensitive or resistant peppers.
- Pepper genomic DNA is digested with the EcoRI enzyme and hybridized with tobacco eIF4E cDNA - SEQ ID NO: 1
- - Figure 2 shows the gel showing the PCR amplifications of the eIF4E gene involved in resistance to potyviruses in chilli and highlights a differential Mvnl restriction site between sensitive and resistant, (Example 4).
- - Figure 3 represents the alignment of the protein sequence of eIF4E of different varieties of chilli, sensitive or resistant to potyviruses.
- Figure 4 represents the alignment of the protein sequence of eIF4E of different varieties of tomato, sensitive or resistant to potyviruses.
- the tomato and chili cDNAs were obtained by the 3 'and 5' RACE technique (System for Rapid Amplification of cDNA Ends sold by the company Invitrogen TM) from the extraction of total RNA from tomato and chili and using degenerate primers defined on an alignment of the eIF4E sequences of tobacco, tomato and Arabidopsis.
- the 3 'part of the cDNA was cloned by 3'RACE. Primers defined between the TAG and the polyA tail of the sequences obtained by 3 'RACE were used to obtain the complete cDNAs by 5' RACE.
- the protocol followed uses 2.5U of enzyme / ⁇ g of DNA.
- the enzymatic volume must be less than 10% of the reaction volume.
- the reaction volume is calculated according to the size of the well: it depends on the type of tank and comb used and on the volume of the gel (300 ml in general).
- the volume of the specific buffer of the enzyme and of spermidine must each represent 10% of the reaction volume:
- Digestion is carried out at 37 ° C overnight.
- samples of phage ⁇ digested with Hind III are prepared: 0.5 ⁇ g / well.
- proper digestion of DNA is checked on 1% agarose gel, TAE IX with 1 ⁇ l of digestion product. If the digestion is correct, the loading buffer is then added.
- the charge buffer must represent at least 10% of the total volume (or 20%).
- the deposition is then carried out on a 300 ml gel, NEB IX, 1% agarose containing 10 ⁇ l of BET. The migration takes place at 25V for 24 hours in NEB IX buffer (migration stops 2 cm from the edge of the gel). Transfer on nylon membrane
- a Hybond N + membrane and 1 Wattman paper the size of the gel are cut.
- the gel is soaked for 30 min. under stirring (blue becomes yellow).
- the blotteur is prepared by:
- the gel is rinsed in a tank containing distilled H 2 O, then placed on the cover of the jogger, avoiding bubbles and checking the tightness of the system.
- the blotter is started at 50 mb max. A little 0.4N soda is poured onto the gel. Two sponges soaked in soda are placed on the gel which will be covered with soda until saturation.
- the transfer takes 2 to 3 hours.
- the membranes are rinsed in a SSC 2X bath for 10 to 15 minutes, then dried in the open air and cooked for 2 hours at 80 ° C.
- Probe preparation
- the preparation of the probes by PCR labeling with 32 P relates to probes of 3 kb maximum, amplified by PCR or directly on plasmids, making it possible to reveal the major bands for a probe, of concentration between 1 and 5 ng / ⁇ l.
- the labeling of the probes is done during 30 PCR cycles of:
- the probes are then denatured according to the following protocol: add each probe to a tube containing 160 ⁇ l of 0.8 N NaOH + 2 to 5 ⁇ l of labeled Lambda (by random priming) incubate for 5 min
- hybridization buffer * 20 ml are used per tube for 2 to 6 half blots. 25 ml of buffer beyond, without exceeding 10 half blots per tube.
- the membranes are moistened in a box containing hybridization buffer before being slightly drained then rolled (all together) and put in the tube. Check during prehybridization that the tubes are tight and that the membranes are going well, otherwise change their direction.
- composition of the pre-hybridization and hybridization buffer For 500 ml: 21.91 g NaCl; 18.38 g Na Citrate; 380 mL H2O; 15 mL SDS 20%; 25 mL NaPO4 IM pH 7.5; 25 mL Denhardt 100X; 5 mL 0.25M EDTA; 50 mL Dextran sulfate 50%.
- the temperature of the tubes is allowed to decrease before opening them to avoid wetting the thread.
- the denatured probe is added (5 min in 0.8 M NaOH then cessation of denaturation Tris-HCl IM). Under these conditions, the hybridization can last 48 or 72 hours.
- the viral material used in these infection tests are the PVY N-605 isolates obtained from Solanum tuberosum (Jakab et al., 1997), or PVY-LYE84, or PVY-LYE240r for tomatoes (Légnani et al. , 1995) and the PVY-To72, PVY-Si 15 isolates for chilli (Dogimont et al., 1996) as well as the CAA-10 isolate from TEV (Légnani et al, 1996).
- the same protocol is used for all other PVY and VTE isolates that are controlled by the pvr2 / pvrl / pvr5 and / or pot-1 loci.
- the isolates are maintained according to the Bos procedure (Bos, 1969) and propagated on plants of Nicotiana tabacum cv. Xanthii before inoculation of tomato or pepper plants in the cotyledon stage with two spreading leaves.
- the viral inoculum is prepared as described in the articles by Légnani et al. (1995, 1996) and de Dogimont et al. (1996).
- the cotyledons and the first two leaves of the plants are inoculated mechanically.
- the lines are evaluated under controlled conditions in a culture chamber (14 hours day, 18 ° C night and 24 ° C day) to monitor their reaction after inoculation.
- the genomic DNA of chilli is digested with the enzyme EcoRI and hybridized with the tobacco cDNA eIF4E - SEQ ID NO: 2 - (the same RFLP profiles are obtained by hybridization with the tomato cDNA or the chili cDNA).
- the first point mutation corresponds to a restriction site
- TspRI (or its isoschizomers) existing only at Yolo Wonder.
- This differential restriction site was validated by PCR on the cDNA of Yolo Wonder and Yolo Y: definition of primers 5 ′ and 3 ′ of cDNA and digestion of the PCR amplifier by the enzyme
- the second point mutation corresponds to an Mvnl restriction site (or its isoschizomers) existing only in Yolo Y.
- This differential restriction site was validated by PCR on the cDNA of Yolo Wonder and Yolo Y: definition of primers in 5 ′ and 3 'to cDNA and digestion of the PCR amplifier with the enzyme Mvnl.
- PCR reaction on cDNA :
- Florida VR2 Florida / pvr2 2
- C69 HD line from the FI hybrid between CM334 and Yolo Wonder / pvr5
- CM334 Criollo de Morelos 334 / pvr5
- Per Perennial / partial resistance (QTL) at the pvr2 locus
- Highlighted black mutation specific to the PMI 008 genotype.
- b RFLP markers are obtained using random tomato genomic DNA
- TG tomato leaf epidemic cDNA probes
- CD and CT tomato leaf epidemic cDNA probes
- DNA samples from 6 F2 plants (from the self-fertilization of the FI hybrid between Lycopersicon esculentum Mospomorist and L. hirsutum PI247087) (pot- l + / pot-l + ) having generated F3 families completely sensitive to PVY strain N 605 and the DNA samples of 9 F2 plants which have generated F3 families completely resistant to potyvirus are pooled for a bulk segregating analysis and for an AFLP labeling of pot-1.
- AFLP markers are generated according to the protocol of Vos et al. (1995) with the restriction enzymes EcoRl, HindIII, and Msel.
- the first amplification is carried out using a combination of primers with a single selective nucleotide and a second combination with 3 selective nucleotides.
- AFLP markers linked to pot-1 are mapped on the introgression lines of L. hirsutum in L. esculentum (Montforte and Tanksley, 2001) in order to assign pot-1 to a tomato chromosome.
- the assignment is validated by mapping RFLP markers located on the target chromosome.
- the RFLP procedure is described by Saliba-Colombani et al. (2000). The screening of the polymorphism between Lycopersicon esculentum Mospomorist (sensitive to potyviruses) and L.
- hirsutum PI247087 (resistant to potyviruses) is carried out with 3 restriction enzymes (EcoRI, HindIII and Xbal) and RFLP markers previously mapped in tomatoes (CT, cDNA of tomato derived from mRNA of epidermal tissue from tomato, TG, clones of genomic DNA from tomato; the CAB3 probe coding for a polypeptide linked to chlorophyll a and b, Tanksley et al., 1992)
- CT cDNA of tomato derived from mRNA of epidermal tissue from tomato
- TG clones of genomic DNA from tomato
- CAB3 probe coding for a polypeptide linked to chlorophyll a and b Tanksley et al., 1992
- Segregation analysis for molecular markers (AFLP, RFLP) and for resistance data are analyzed by Mapmaker / Exp v software. 3.0 with a minimum Lod of 4.0 and a maximum recombination percentage of 0.3. The percentage of recombination is then converted into mapping distance in centiMorgans (cM) using the Kosambi mapping function (Kosambi, 1944).
- the tomato cDNA eIF4E was mapped by the RFLP method described previously on the introgression lines of L. pennellii in L. esculentum (Eshed and Zamir, 1995). This work made it possible to locate 5 copies of the eIF4E gene in tomatoes. One of these copies was located on chromosome 3, in the same genomic region as the pot-1 gene, thus confirming the synteny between chilli and tomato for resistance to potyviruses and therefore the possibility of using eIF4E as markers and tools for resistance selection.
- the coding sequence of this gene (variety 'Mospomorist' from. Esculentum, sensitive to PVY and TEV) is represented in the sequence list under the number
- the eIF4E protein of L. hirsutum PI 134417 and that of L. hirsutum PI247087 are also represented by the sequence SEQ ID NO: 23
- Example 6 Screening of the BAC library of the chili genome with primers defined on the eIF4E coding sequence of the genotype Yolo Wonder, demonstration of co-segregation with resistance and determination of the genomic structure of the eIF4E gene which co-segregates with pvr2.
- a BAC chili bank was constructed from an HD208-lined haploid line derived from the FI hybrid of a cross between Capsicum annuum
- the BAC chili library is made up of 239,232 clones with an average insert size of 125kb, which corresponds to a theoretical representativeness of 10 genome equivalents (size of the genome of chilli 3000 Mpb). This BAC library was organized into 623 DNA pools for PCR screening (1 pool corresponds to the DNA mixture of 384 clones).
- Example 7 Experiment of transient expression of cDNA eIF4E from Yolo Wonder in a resistant pepper genotype (carrying the pvr21 allele) for validation of the role of eIF4E in sensitivity to Potyviruses.
- the cDNA eIF4E derived from the sensitive Yolo Wonder genotype is cloned in an oriented manner into an expression vector PVX-CES-35S at the Clal cloning site and
- Yolo Y is co-inoculated with this recombinant plasmid and with Potato Virus Y pathotype 0 (PVY).
- PVY Potato Virus Y pathotype 0
- the transient expression of the eIF4E gene from the sensitive Yolo Wonder genotype via the recombinant PVX vector allows the PVY to multiply in the resistant genotype.
- PVY is detected by the ELISA or RT-PCR method (Legnani et al., 1995, 1996, Dogimont et al., 1996).
- Example 8 Search for mutants in the eIF4E gene and in the genes of the RNA translation initiation complex for the creation of plants resistant to potyviruses.
- eIF4E multi-gene family belong to a complex of at least 8 proteins forming the translation initiation complex in eukaryotic cells (Browning 1996).
- Example 9 Creation of plants resistant to potyviruses by methods which may involve the transgenesis. Alternatively to example 8, the resistance allele of the eIF4E gene
- any other allele of eIF4E which confers resistance to potyviruses can be transferred in planta by methods of the directed mutagenesis type (Hohn et al., 1999) , homologous recombination (Kempin et al., 1997) or by overexpression methods.
- the allele of eIF4E which confers resistance is expressed under a strong promoter of the 35S type of the CaMV virus by transgenesis in planta (Jones et al., 1992; Bevan 1984).
- Resistance plants can also be created by knockout of the endogenous eIF4E gene by methods of the “gene silencing” type (Post Transcriptional Gene Silencing) and the simultaneous expression by transgenesis of the form of eIF4E conferring resistance to potyviruses.
- a specific knockout by PTGS can be carried out by digesting it against the 5 'UTR of the endogenous eIF4E gene; the form of eIF4E which confers the resistance expressed by transgenesis will not carry the 5 ′ UTR sequence of the endogenous eIF4E. This specificity of the PTGS knockout against the 5 'UTR is based on new data resulting from the understanding of the mechanism of PTGS (Nishikura 2001).
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Botany (AREA)
- Mycology (AREA)
- Virology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2003222893A AU2003222893B2 (en) | 2002-02-08 | 2003-02-07 | eIF4E gene mutations and potyvirus resistance |
US10/502,795 US7919677B2 (en) | 2002-02-08 | 2003-02-07 | eIF4E gene mutations and potyvirus resistance |
JP2003566248A JP2006503544A (ja) | 2002-02-08 | 2003-02-07 | eIF4E遺伝子の突然変異とポティウイルス(potyvirus)抵抗性 |
MXPA04007718A MXPA04007718A (es) | 2002-02-08 | 2003-02-07 | Mutaciones del gen eif4e y resisntencia a los potyvirus. |
EP03718850A EP1472374A2 (fr) | 2002-02-08 | 2003-02-07 | Mutations du gene eif4e et resistance aux potyvirus |
NZ534545A NZ534545A (en) | 2002-02-08 | 2003-02-07 | EIF4E gene mutations and potyvirus resistance |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR02/01583 | 2002-02-08 | ||
FR0201583A FR2835849B1 (fr) | 2002-02-08 | 2002-02-08 | Procede de selection ou d'obtention de plantes resistantes aux potyvirus et sequences "marquant" ou "conferant" cette resistance |
FR0213678A FR2835698B1 (fr) | 2002-02-08 | 2002-10-31 | Procede de selection ou d'obtention de plantes resistantes aux potyvirus et sequences marquant ou codant cette resistance |
FR02/13678 | 2002-10-31 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2003066900A2 true WO2003066900A2 (fr) | 2003-08-14 |
WO2003066900A3 WO2003066900A3 (fr) | 2004-04-08 |
Family
ID=27624217
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2003/000397 WO2003066900A2 (fr) | 2002-02-08 | 2003-02-07 | Mutations du gene eif4e et resistance aux potyvirus |
Country Status (8)
Country | Link |
---|---|
US (1) | US7919677B2 (fr) |
EP (1) | EP1472374A2 (fr) |
JP (1) | JP2006503544A (fr) |
AU (1) | AU2003222893B2 (fr) |
FR (1) | FR2835698B1 (fr) |
MX (1) | MXPA04007718A (fr) |
NZ (1) | NZ534545A (fr) |
WO (1) | WO2003066900A2 (fr) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004057941A2 (fr) * | 2002-12-17 | 2004-07-15 | Cornell Research Foundation, Inc. | Resultats de resistance de plante recessive issus de mutations du facteur d'initiation de la traduction eif4e |
FR2870856A1 (fr) * | 2004-05-25 | 2005-12-02 | Genoplante Valor Soc Par Actio | Procede de selection ou d'obtention de plantes resistantes au pvmv |
WO2009037369A1 (fr) * | 2007-09-21 | 2009-03-26 | Consejo Superior De Investigaciones Cientificas | Manipulation de la fonction du gène atdbp1 afin de générer une résistance au potyvirus |
US8927808B2 (en) | 2010-10-18 | 2015-01-06 | J.R. Simplot Company | Potyvirus resistance in potato |
US8933298B2 (en) | 2008-10-22 | 2015-01-13 | Cornell University | Mutated eIF4E sequences from potato which impart resistance to potato virus Y |
US9913439B2 (en) | 2012-08-23 | 2018-03-13 | Seminis Vegetable Seeds, Inc. | Multiple-virus-resistant melon |
CN108350045A (zh) * | 2015-05-22 | 2018-07-31 | 首尔大学校产学协力团 | 具有马铃薯Y病毒属抗性的Pvr4基因及其用途 |
CN109371056A (zh) * | 2018-09-16 | 2019-02-22 | 云南省烟草农业科学研究院 | 一种选育对辣椒脉斑驳病毒抗性的烟草植物的方法 |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2878532B1 (fr) * | 2004-11-26 | 2007-03-02 | Genoplante Valor Soc Par Actio | Methode d'adressage d'acides nucleiques vers des plastes |
GB201010740D0 (en) * | 2010-06-25 | 2010-08-11 | Univ Warwick | A plant |
CA2809573A1 (fr) * | 2010-09-03 | 2012-03-08 | Philip Morris Products S.A. | Reduction des metaux lourds dans les plantes |
US10729082B2 (en) * | 2017-10-16 | 2020-08-04 | Benchbio Pvt. Ltd. | Papaya with novel traits and methods for producing such papaya plants |
US11814639B2 (en) | 2018-02-02 | 2023-11-14 | Kikkoman Corporation | Virus-resistant plant and method for producing the same |
CN112094942B (zh) * | 2020-10-23 | 2023-09-12 | 贵州省烟草科学研究院 | 与烟草pvy抗性紧密连锁的分子标记、引物及应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999043820A1 (fr) * | 1998-02-25 | 1999-09-02 | E.I. Du Pont De Nemours And Company | FACTEUR DE TRANSLATION eIF-4E DE VEGETAUX |
WO2001040490A2 (fr) * | 1999-12-02 | 2001-06-07 | Institut National De La Recherche Scientifique | Inhibiteurs du facteur d'initiation 4e eucaryotique et utilisation en tant qu'herbicides |
-
2002
- 2002-10-31 FR FR0213678A patent/FR2835698B1/fr not_active Expired - Fee Related
-
2003
- 2003-02-07 JP JP2003566248A patent/JP2006503544A/ja active Pending
- 2003-02-07 MX MXPA04007718A patent/MXPA04007718A/es unknown
- 2003-02-07 US US10/502,795 patent/US7919677B2/en not_active Expired - Fee Related
- 2003-02-07 NZ NZ534545A patent/NZ534545A/en not_active IP Right Cessation
- 2003-02-07 AU AU2003222893A patent/AU2003222893B2/en not_active Ceased
- 2003-02-07 EP EP03718850A patent/EP1472374A2/fr not_active Withdrawn
- 2003-02-07 WO PCT/FR2003/000397 patent/WO2003066900A2/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999043820A1 (fr) * | 1998-02-25 | 1999-09-02 | E.I. Du Pont De Nemours And Company | FACTEUR DE TRANSLATION eIF-4E DE VEGETAUX |
WO2001040490A2 (fr) * | 1999-12-02 | 2001-06-07 | Institut National De La Recherche Scientifique | Inhibiteurs du facteur d'initiation 4e eucaryotique et utilisation en tant qu'herbicides |
Non-Patent Citations (4)
Title |
---|
LELLIS A ET AL: "Loss-of-susceptibility mutants of Arabidopsis thaliana reveal an essential role for eIF(iso)4E during Potyvirus infection" CURRENT BIOLOGY, vol. 12, no. 12, 25 juin 2002 (2002-06-25), pages 1046-51, XP002217535 * |
LEONARD SIMON ET AL: "Complex formation between potyvirus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity" JOURNAL OF VIROLOGY, THE AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 74, no. 17, septembre 2000 (2000-09), pages 7730-7737, XP002176524 ISSN: 0022-538X * |
REVERS F ET AL: "NEW ADVANCES IN UNDERSTANDING THE MOLECULAR BIOLOGY OF PLANT/POTYVIRUS INTERACTIONS" MOLECULAR PLANT-MICROBE INTERACTIONS, APS PRESS, ST. PAUL, MN, US, vol. 12, no. 5, 1999, pages 367-376, XP001009714 ISSN: 0894-0282 * |
WHITMAN S ET AL: "Selectable viruses and altered susceptibility mutants in Arabidopsis thaliana" PROC. NATL. ACAD. SCI. USA, vol. 96, janvier 1999 (1999-01), pages 772-77, XP002217536 * |
Cited By (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004057941A3 (fr) * | 2002-12-17 | 2005-02-10 | Cornell Res Foundation Inc | Resultats de resistance de plante recessive issus de mutations du facteur d'initiation de la traduction eif4e |
US7772462B2 (en) | 2002-12-17 | 2010-08-10 | Cornell Research Foundation, Inc. | Recessive plant viral resistance results from mutations in translation initiation factor eIF4E |
WO2004057941A2 (fr) * | 2002-12-17 | 2004-07-15 | Cornell Research Foundation, Inc. | Resultats de resistance de plante recessive issus de mutations du facteur d'initiation de la traduction eif4e |
FR2870856A1 (fr) * | 2004-05-25 | 2005-12-02 | Genoplante Valor Soc Par Actio | Procede de selection ou d'obtention de plantes resistantes au pvmv |
WO2005118850A1 (fr) * | 2004-05-25 | 2005-12-15 | Genoplante-Valor | Procede de selection ou d'obtention de plantes resistantes au pvmv. |
JP2008500036A (ja) * | 2004-05-25 | 2008-01-10 | ジュノプラント−ヴァロール | Pvmvに対する抵抗性を有する植物の選別又は取得方法 |
WO2009037369A1 (fr) * | 2007-09-21 | 2009-03-26 | Consejo Superior De Investigaciones Cientificas | Manipulation de la fonction du gène atdbp1 afin de générer une résistance au potyvirus |
ES2316298A1 (es) * | 2007-09-21 | 2009-04-01 | Consejo Superior De Investigaciones Cientificas | Manipulacion en la funcion de atdbp1 para generar resistencia a potyvirus. |
US8933298B2 (en) | 2008-10-22 | 2015-01-13 | Cornell University | Mutated eIF4E sequences from potato which impart resistance to potato virus Y |
US8927808B2 (en) | 2010-10-18 | 2015-01-06 | J.R. Simplot Company | Potyvirus resistance in potato |
US9913439B2 (en) | 2012-08-23 | 2018-03-13 | Seminis Vegetable Seeds, Inc. | Multiple-virus-resistant melon |
US11019777B2 (en) | 2012-08-23 | 2021-06-01 | Seminis Vegetable Seeds, Inc. | Multiple-virus-resistant melon |
CN108350045A (zh) * | 2015-05-22 | 2018-07-31 | 首尔大学校产学协力团 | 具有马铃薯Y病毒属抗性的Pvr4基因及其用途 |
CN108350045B (zh) * | 2015-05-22 | 2021-12-07 | 首尔大学校产学协力团 | 具有马铃薯Y病毒属抗性的Pvr4基因及其用途 |
CN109371056A (zh) * | 2018-09-16 | 2019-02-22 | 云南省烟草农业科学研究院 | 一种选育对辣椒脉斑驳病毒抗性的烟草植物的方法 |
CN109371056B (zh) * | 2018-09-16 | 2022-04-08 | 云南省烟草农业科学研究院 | 一种选育对辣椒脉斑驳病毒抗性的烟草植物的方法 |
Also Published As
Publication number | Publication date |
---|---|
FR2835698B1 (fr) | 2004-08-06 |
AU2003222893B2 (en) | 2008-05-15 |
JP2006503544A (ja) | 2006-02-02 |
WO2003066900A3 (fr) | 2004-04-08 |
FR2835698A1 (fr) | 2003-08-15 |
NZ534545A (en) | 2008-06-30 |
EP1472374A2 (fr) | 2004-11-03 |
AU2003222893A1 (en) | 2003-09-02 |
MXPA04007718A (es) | 2005-06-08 |
US20050255455A1 (en) | 2005-11-17 |
US7919677B2 (en) | 2011-04-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Junghans et al. | Resistance to rust (Puccinia psidii Winter) in Eucalyptus: mode of inheritance and mapping of a major gene with RAPD markers | |
RU2562864C2 (ru) | Генетические локусы, связанные с устойчивостью к пыльной головне у маиса | |
Pavli et al. | Achievements and prospects in breeding for rhizomania resistance in sugar beet | |
WO2003066900A2 (fr) | Mutations du gene eif4e et resistance aux potyvirus | |
US20220248620A1 (en) | Downy mildew resistant spinach and genes conferring resistance to downy mildew | |
JP2008289481A (ja) | 線虫及び/又はアリマキに対する耐性 | |
JP2008289481A6 (ja) | 線虫及び/又はアリマキに対する耐性 | |
Michel et al. | A complex eIF4E locus impacts the durability of va resistance to Potato virus Y in tobacco | |
EP1753879B1 (fr) | Procede de selection ou d'obtention de plantes resistantes au pvmv | |
AU2016352829A1 (en) | Resistance to ToLCNDV in squash | |
EP1585759A1 (fr) | Gene de resistance a aphis gossypii | |
FR2835849A1 (fr) | Procede de selection ou d'obtention de plantes resistantes aux potyvirus et sequences "marquant" ou "conferant" cette resistance | |
EP1185708B1 (fr) | Moyens pour l'identification d'une nouvelle classe de genes de resistance au virus de la panachure jaune du riz et du locus d'un gene majeur de la resistance au virus, et leurs applications | |
WO2020239186A1 (fr) | Plants de melon résistants au mildiou | |
Johnson | Identification and marker-assisted selection of a major gene for Phytophthora resistance, its origin and effect on agronomic characters in tobacco | |
WO2020185663A2 (fr) | Maîtriser l'auto-incompatibilité chez les plantes diploïdes pour la sélection et la production d'hybrides par la modulation du ht | |
WO2011080329A2 (fr) | Gene gpav de resistance aux nematodes chez les solanacees | |
WO2003016564A2 (fr) | Utilisation d'associations entre polymorphismes du gene sh2 et des caracteristiques de qualite de la graine pour la selection de plantes | |
Hu | Analysis of fine structure and necrotic mutation of Rp1 rust resistance genes in maize |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A2 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SC SD SE SG SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A2 Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 163338 Country of ref document: IL |
|
WWE | Wipo information: entry into national phase |
Ref document number: 534545 Country of ref document: NZ |
|
WWE | Wipo information: entry into national phase |
Ref document number: PA/a/2004/007718 Country of ref document: MX Ref document number: 2003566248 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2003718850 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2003222893 Country of ref document: AU |
|
WWP | Wipo information: published in national office |
Ref document number: 2003718850 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10502795 Country of ref document: US |