ZA200610874B - Method for selecting or obtaining plants which are resistant to PVMV - Google Patents

Method for selecting or obtaining plants which are resistant to PVMV Download PDF

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ZA200610874B
ZA200610874B ZA200610874A ZA200610874A ZA200610874B ZA 200610874 B ZA200610874 B ZA 200610874B ZA 200610874 A ZA200610874 A ZA 200610874A ZA 200610874 A ZA200610874 A ZA 200610874A ZA 200610874 B ZA200610874 B ZA 200610874B
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Carole Caranta
Sadrine Ruffel
Polloix Alain
Nicolas Fabre
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Genoplante Valor
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Description

Method for selecting or obtaining plants which are resistant to PVMV
The present invention relates to a method for selecting or obtaining plants which are resistant to a virus of the Potyvirus genus via the combination of mutations in the eIF4E and eIF(iso)4E genes.
Potyviruses form the largest group of phytoviruses. They represent approximately 1/3 of plant viruses and are capable of infecting more than 30 families of plants (SHUKLA et al., 1994, In Genome structure, variation and function in the Potyviridae, Ed. CAB
International, Wallingford, UK) . Potyviruses are transmitted by aphids (for example Myzus persicae) according to the non-persistant mode. The infection causes leaf coloration anomalies (mosaics, yellowing of the veins), leaf deformations, and veinal necroses that may result in necrosis of the whole plant and therefore in a considerable reduction in the size of the affected plant and a loss of productivity. The flowers, the seeds and the fruits are themselves also affected by many potyviruses, which results in a reduction in fertility, potyvirus transmission via the seed and fruit deformations, discolorations and necroses.
Potyviruses have a non-enveloped filamentous structure (LANGENBERG and ZHANG, J. Struct. Biol. 118(3) : 243-247, 1997) 680 to 900 nm long and 11 to 15 nm wide (DOUGHERTY and CARRINGTON, Ann. Rev. Phytopathol. 26: 123- 143, 1988; RIECHMANN et al., J. Gen. Virol. 73: 1-16, 1992). The viral genome consists of a single-stranded sense RNA approximately 10 kb in length. The single- stranded RNA has a poly A tail at its 3’ end and binds, in the 5’ position, to a viral protein called VPg for Viral genome-linked Protein (MURPHY et al., Virol. 178: 285-288, 1990; TAKAHASHI et al., Virus genes 14(3): 235-243, 1997).
The viral RNA encodes a polyprotein that is matured into 10 functional proteins involved in the various steps of the infectious cycle: polyprotein cleavage, viral genome replication, cell-to-cell movement, long distance movement, transmission by aphids and inhibition of "RNA silencing", etc.
Solanaceae, Cucurbitaceae, Fabaceae, crucifers and composites are particularly sensitive to potyviruses.
For example, Solanaceae, and more particularly tomato and pimento (or pepper) are infected with several different potyviruses: potato virus Y (PVY) 1s present on crop regions, whereas the others are confined to a continent (Tobacco etch virus (TEV), Pepper mottle virus on the
American continent, Pepper veinal mottle virus (PVMV) and
Potyvirus E in Africa, and Chili wveinal mottle virus in
Asia). This compartmentalization is no longer, however, absolute, several ©potyviruses having been identified outside their region of origin. PVMV was first of all isolated in 1971 on pimento and petunia in Ghana. Since then, it has been identified in several African countries, mainly in sub-Saharan regions, but also in Tunisia and
Ethiopia (BRUNT et al., Ann. Appl. Biol. 69: 235-243, 1971; BRUNT et al., Ann. Appl. Biol. 88: 115-119, 1978;
GORSANE et al., Plant Mol. Biol. Reptr. 17: 149-158, 1999). Its natural hosts are mainly Solanaceae, including pimento but also tomato, tobacco and aubergine.
At the current time, and contrary to fungal or bacterial infections, no means for directly controlling the viruses exist. The strategies used consist in eliminating the viral vectors such as aphids, and/or in selecting and cultivating plants which are resistant to the virus (es) most common in the crop region.
Due to the internationalization of the seed market, it 1s increasingly essential for selectors to create multiresistant varieties. More generally, considering the economic importance of potyvirus infections and the lack of direct means for controlling this type of infection, the search for multiresistant plant varieties constitutes one of the main lines for plant improvement.
CARANTA et al. (Phytopathol. 86(7): 739-743, 1996) have shown, in pimento, that the combination of certain loci makes it possible to broaden the spectrum of resistance by conferring resistances to new potyviruses.
Thus, plants which are resistant to PVMV have been identified in the doubled haploid progeny derived from crossing between the Capsicum annuum varieties Perennial and Florida VR2, which are both sensitive to PVMV. The genetic analyses have shown that the resistance to PVMV resulted from the association of the recessive gene pvrZz (derived from Florida VR2 and controlling resistance to
PVY pathotypes 0 and 1 and to TEV) with another recessive factor, called pvré, derived from Perennial.
Faced with an attack by a pathogenic agent (viruses, bacteria, fungi or nematodes), the plant has several strategies for defending itself or withstanding the infection. Non-host resistance (when all the entities of a species are resistant to a given pathogen) is distinguished from host resistance (when at least one entity of the species is sensitive to a strain of the pathogenic agent). It is possible to distinguish two methods of setting up host resistance depending on whether the genes involved are dominant or recessive.
The ‘gene-to-gene model" described by FLOR (Ann. Rev. Phytopathol. 9: 275-296, 1971) involves a major dominant plant gene. Resistance is conditioned by presence of this gene and of a corresponding specific avirulence gene, in the pathogenic agent. When these two genes are simultaneously present, the host plant's resistance is induced and is often associated with a hypersensitivity reaction (necrosis) localized at the site of infection.
The resistance may also be controlled by recessive plant genes. Strangely, it is estimated that close to half the potyvirus resistances are recessive, whereas, for the other pathogenic agents, this proportion reaches only 20% on average. FRASER (Euphytica 63: 175- 185, 1992) has put forward the hypothesis, known as the
"Fraser negative model", that recessive resistances would result from a deficiency or a specific impairment of the product of a host gene necessary for the accomplishment of the viral cycle in the plant.
The potyvirus VPg protein plays an important role in potyvirus/host plant interactions, and in particular in the virulence of certain potyvirus strains with respect to certain host plants. It has been shown that potyviruses carrying mutations in the VPg protein can bypass recessive resistance genes involved in mechanisms as varied as the inhibition of viral multiplication, the inhibition of cell-to-cell potyvirus migration or the inhibition of long-distance potyvirus migration. This has been shown in the pairs TVMV/Nicotiana tabacum (va gene),
PVY/tomato (pot-1 gene) , PVY/pimento (pvrz gene),
LMV/lettuce (mol gene) and PSbMV/pea (sbml gene) (KELLER et al., Mol. Plant-Microbe Interact. 11: 124-130 20, 1998;
MOURY et al., Mol. Plant-Microbe Interact. 17(3): 322-329, 2004; REDONDO et al., Mol. Plant-Microbe Interact. 14: 804-810, 2001; NICOLAS et al., Virol. 237: 452-459 35, 1997; MASUTA et al., Phytopathol. 89: 118-123, 1999).
Among the host plant factors capable of interacting with the potyvirus VPg protein are, in particular, the eukaryotic RNA translation initiation factor 4E (eIF4E), and in particular the isoforms called eIF4E and elIF(iso)4E (WITTMAN et al., Virol. 234: 84-92, 1997; SCHAAD et al., Virol. 273: 300-306, 2000; LELLIS et al., Curr. Biol. 12: 1046-1051, 2002; DUPRAT et al., Plant
J. 32: 927-934, 2002). In plants, eIF4E belongs to the elF4F complex (made up of eIF4E and eIF4G) which makes it possible to make the connection between the RNA cap (m7GpppN) and the translation initiation machinery (BROWNING, Plant Mol. Biol. 32(1-2): 107-143, 1996). Two eIF4F complexes (called eIF4F and eIF(iso)4F) have been purified from plant cells and their respective subunits have been named eIF4E (p26 subunit) and eIF(iso)4E (p28 subunit), for the subunit binding to the RNA cap, and eIF4G and eIF(iso)4G for the other subunit. eIF4E and eIF (iso) 4E, which appear to be involved in the translation of various RNA types, belong to a small multigene family in plants, comprising 4 genes in Arabidopsis thaliana, three of which encode eIF4E and one of which encodes the eIF (iso) 4E isoform (RODRIGUEZ et al., Plant J. 13(4): 465- 473, 1998).
The eIF4E and eIF(iso)4E proteins both possess the conserved domain called IF4E. This domain 1s referenced on the PFAM database (BATEMAN et al., Nucleic
Acids Res. 30: 276-280, 2002) under the number PFAM 01652.
However, the ‘eIF4E proteins belonging to the same subfamily (i.e., 4E or (iso)4E) and derived from different species exhibit a percentage identity (61% to 86% identity between the eIF4E proteins, 59% to 82% identity between the eIF(iso)4E) proteins) greater than that exhibited, with respect to one another, by the eIF4E and elF (iso) 4E proteins derived from the same species (42% to 51% identity) (RUFFEL et al., Gene, in press, 2004).
In the context of the disclosure that will follow, reference will be made, by way of proteins . representative, respectively, of the eIF4E subfamily and of the eIF(iso)4E subfamily, to the eIF4E protein and to the eIF(iso)4E protein of Capsicum annuum.
The cDNA sequence encoding the eIF4E protein of Capsicum annuum (GenBank No. AY122052) is represented in the sequence listing attached in the annex, under the number SEQ ID NO.: 1, and the deduced polypeptide sequence is represented under the number SEQ ID NO.: 2.
The cDNA sequencing coding the eIF(iso)4E protein of Capsicum annuum is represented in the sequence listing attached in the annex, under the number SEQ ID
NO. : 3, and the deduced polypeptide sequence is represented under the number SEQ ID NO.: 4. eIF4E protein is here defined as any protein comprising an IF4E domain, and the peptide sequence of which has at least 60%, preferably at least 65%, entirely preferably at least 70% overall identity with the elF4E protein of Capsicum annuum (SEQ ID NO.: 2). eIF (iso) 4E protein is defined as any protein comprising an IF4E domain, and the polypeptide sequence of which has at least 59%, preferably at least 655%, entirely preferably at least 70% identity with the eIF(iso)4E protein of Capsicum annuum (SEQ ID NO.: 4).
The identity percentages to which reference is made here are overall identity percentages, determined using the PILEUP program of GCG (Genetics Computer Group) on an aligment of sequences containing the entire reference sequence SEQ ID NO.: 2 or SEQ ID NO.: 4.
It has been reported that mutations that impair either eIF4E or eIF(iso)4E induce a recessive resistance in certain potyvirus species. For example, it has been shown that disruption of the gene encoding the elF (iso) 4E isoform in Arabidopsis thaliana induces : resistance to the Turnip mosaic virus (TuMV), to TEV and to the Lettuce mosaic virus (LMV), but has no effect on resistance to the Tomato black ring virus (TBRV, Nepovirus genus) or to the Cucumber mosaic virus (CMV, Cucumovirus genus) (DUPRAT et al., Plant J. 32: 927-934, 2002; LELLIS et al., Curr. Biol. 12: 1046-1051, 2002). NICAISE et al. (Plant Physiol., 132: 1272-1282, 2003) have shown that plants which are resistant to the Lettuce mosaic virus (LMV) carry point mutations in the eIF4E gene; on the other hand, the sequence of the eIF(iso)4E gene in the resistant lettuce plants does not differ from that of the sensitive plants.
RUFFEL et al. (Plant J. 32: 1067-1075, 2002) and also PCT application WO 03/066900 show that the recessive resistance to TEV and to PVY conferred by the pvr2 recessive gene is associated with sequence variations in eIF4E, and in particular in a region of this protein that is very conserved between the eIF4E proteins derived from various plant species.
The inventors have now demonstrated, in pimento, cosegregation between the pvré locus and the eIF(iso)4E gene. They have noted that plants exhibiting a resistance to PVMV have, in addition to the mutated allele encoding the eIF4E translation factor (pvr2 resistance allele), as described in patent application WO 03/066900, a mutation in the sequence encoding eIF(iso)4E.
This mutation consists of a deletion of 82 bp in the coding sequence, incorporating a stop codon at the 51st amino acid out of the 202 of the wild-type protein.
Only the first 29 amino acids are conserved between the wild-type eIF(iso)4E protein and the mutated protein, indicating that the mutated protein 1s no longer functional.
The pvré recessive factor, previously described as not conferring, on its own, resistance to
PVMV, but capable of conferring this resistance when it is associated with the pvr2 recessive factor (CARANTA et al., 1996, mentioned above), therefore corresponds to the eIF(iso)4E gene. This correspondence between pvré and eIF(iso)4E is unexpected, insofar as, in the case of the eIF4E or eIF(iso)4E mutants identified up until now, the mutation of just one of these two genes was sufficient to confer a recessive resistance to one or more specific potyviruses.
It therefore appears that certain potyviruses, of which PVMV is the first example, behave differently, with respect to the use of eIF4E and of eIF (iso) 4E for accomplishing their infectious cycle, from that which had been observed up until now. Whereas the potyviruses studied up until now showed a specalization in terms of the use, in a host plant, either of elIF4E or of eIF (iso) 4E, PVMV appears to be capable of using both eIF4E and elIF(iso)4E.
The present invention relates to a method for selecting plants which are resistant to PVMV, characterized in that it comprises the search, in the plants to be tested, for the forms of the eIF4E and eIF (iso)4E proteins present in said plants, and the selection of the plants which: a) do not express any eIF4E protein (hereinafter referred to as: "wild-type eIF4E protein") comprising a region defined by the following general ] sequence (I):
DX; X,X3X4KSXsQXcAWGS SX RX eX YTFSX10VEX 11 FWX12X13YNNIHX, 4 PSKLX 5X16
GAD in which: - Xi, Xa, X3, Xq4, Xe, X97, Xs, Xo, Xio, X12, X13, Xis and X,¢ each represent a neutral amino acid; - X¢ and Xj; represent a basic amino acid; - Xi, represents an acidic amino acid; - D, XK, S, Q, A, W, G, R, ¥, T, FF, V, E, N, I, H, Pp, and L have their usual l-letter-code meaning; and express 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 charged amino acid of said sequence (I) with a neutral amino acid or an amino acid of opposite charge; and b) do not express any functional eIF(iso)4E protein.
A "neutral amino acid" is here defined as any amino acid chosen from the following: alanine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, methionine, glycine, serine, threonine, tyrosine, cysteine, glutamine and asparagine. A "charged amino acid" is defined as any amino acid chosen from the following: histidine, lysine, arginine, glutamate and aspartate.
Among these charged amino acids, histidine, lysine or arginine are basic amino acids, and glutamate and aspartate are acidic amino acids.
A "functional eIF(iso)4E protein" is here defined as an elIF(iso)4E protein capable of forming a complex with eIF(iso4G), and of binding to the RNA cap-
Plants that do not express any functional eIF (iso)4E protein may either express no eIF(iso)4E protein, or express only a nonfunctional eIF(iso)4E protein.
The absence of expression of an eIF(iso)4Ek protein may result, for example, from the deletion of the sequence encoding this protein, or from the absence of transcription and/or of translation of the eIF(iso)4E gene. The nonfunctionality of an eIF(iso)4E protein may result, for example, from a mutation in the sequence encoding this protein.
As regards this nonfunctional eIF (iso) 4E protein, plants in which said nonfunctional eIF (iso)4E protein is devoid of a portion of the wild-type eIF (iso)4E protein comprising one or more of the 8 tryptophan residues of the IF4E domain that are conserved between eukaryotic elF (iso) 4E proteins are preferentially selected. These tryptophan residues correspond to those located, in the sequence SEQ ID NO.: 4, at positions 34, 37, 50, 67, 96, 107, 121 and 156. They can be readily located by those skilled in the art in any other eIF (iso) 4E protein, based on a sequence alliance.
Advantageously, the deleted portion comprises one or more of the regions of the IF4E domain corresponding to positions 31-36, 47-52, 64-69, 93-98, 118-123, and 153-158 of the sequence SEQ ID NO.: 4.
Particularly advantageously, the deleted portion comprises the residues of the IF4E domain corresponding to positions 30-202 of the reference sequence SEQ ID NO.: 4.
This deletion in the eIF(iso)4E protein may result from a deletion in the coding portion of the eIF(iso)4E gene; it may also result from a point mutation in the eIF(iso)4E gene, or from the insertion of an exogenous sequence, introducing a premature stop codon and/or a reading frame shift. Depending on the type of mutation concerned, and the position of this mutation, the nonfunctional eIF(iso)4E protein that results therefrom may comprise, in place of the deleted portion, an unrelated sequence.
As regards the eIF4E protein, the plants preferentially selected are the plants containing a mutant eIF4E protein comprising a region derived from that defined by the sequence (I) above, by substitution: - of at least one of the amino acids Xi, Xz, X3 or X,4 of said sequence (I) with a charged amino acid; and - of at least one of the other neutral amino acids of said sequence (I) with a charged amino acid; and/or - of at least one of the charged amino acids of said sequence (I) with a neutral amino acid or an amino acid of opposite charge.
Particularly preferably, the plants that are selected are the plants containing a mutant eIF4E protein comprising a region derived from that defined by the sequence (I) above, by substitution: - of the neutral amino acid X; of the sequence (I) with a basic amino acid; - of the neutral amino acid X,; of the sequence (I) with a basic amino acid; - of the aspartate residue in the C-terminal position of the sequence (I) with a neutral amino acid.
The detection of the form of the eIF4E and eIF (iso)4E proteins that is present in the plant to be tested may be carried out, for example, using antibodies specifically directed either against the wild-type form or against the mutant form of the eIF4E or of the eIF(iso)4E protein.
However, it 1s generally more convenient to carry out this detection by searching for the allelic forms of the eIF4E and eIF(iso)4E denes present in the plant to be tested.
The present invention relates to a method for selecting plants which are resistant to PVMV, characterized in that it comprises the search, in the plants to be tested, for the allelic forms of the eIF4E and eIF(iso)4E genes present or expressed in said plants, and the selection of the plants which do not exhibit or do not express any allele of the eIF4E gene encoding a wild- type eIF4E protein, as defined above, and which express an allele of the eIF4E gene encoding a mutant eIF4E protein, as defined above, and which also do not exhibit or do not express any allele of the eIF(iso)4E gene encoding a functional eIF(iso)4E protein, as defined above, and, where appropriate, express an allele of the eIF(iso)4E gene encoding a nonfunctional eIF(iso)4E protein, as defined above.
The detection of the allelic forms of the eIF4E and eIF(iso)4E genes present in the plant to be tested may be carried out by conventional methods, known in themselves to those skilled in the art. Use may, for example, be made of polynucleotide probes capable of hybridizing selectively either with the wild-type allele or with the mutant allele of eIF4E or eIF(iso)4E, or amplification primers for the amplification of eIF4E or of : eIF (iso) 4E or of the portion of these genes containing the mutation being sought; the presence or the absence of this mutation can then be detected on the amplification product, for example by sequencing the latter, or, depending on the nature of the mutation sought, by determination of the size of the amplification product, or digestion with a restriction enzyme that recognizes a target sequence present, exclusively, either in the wild- type allele or in the mutant allele.
A subject of the present invention is also methods for obtaining plants which are resistant to PVMV,
According to a first variant of a method in accordance with the invention, it comprises:
- replacement of the alleles of the eIF4E gene that are present in said plant, with alleles encoding a mutant eIF4E protein as defined above; and - replacement of the alleles of the eIF(iso)4E gene that are present in said plant, with alleles encoding a nonfunctional mutant eIF(iso)4E protein as defined : above, or direct or epigenetic inactivation of said eIF (iso) 4E gene.
According to a second variant of a method in accordance with the invention, it is implemented using a plant in which all the alleles of the eIF4E gene encode a mutant eIF4E protein as defined above, and comprises replacement of the alleles of the eIF(iso)4E gene that are present in said plant, with alleles encoding a nonfunctional eIF(iso)4E protein as defined above, or direct or epigenetic inactivation of said eIF(iso)4E gene.
According to a third variant of a method in accordance with the invention, it is implemented using a plant in which all the alleles of the eIF(iso)4E dene encode a mutant eIF(iso)4E protein as defined above, or using a plant in which said eIF(iso)4E gene has been inactivated, and comprises replacement of the alleles of the eIF4E gene that are present in said plant, with alleles encoding a mutant eIF4E protein as defined above.
The expression "replacement of the alleles" of the eIF4E gene or of the eIF(iso)4E gene is here intended to mean any genetic modification resulting in the substitution of the initial coding sequence of the gene concerned with the desired mutant sequence, regardless of the method used to carry out this modification.
The term "inactivation" of the eIF(iso)4E gene is here intended to mean any genetic modification resulting in the absence of expression of an eIF(iso)4E protein. This inactivation is defined as being "direct" if it results from a modification of the eIF(iso)4E gene (for example, in the coding sequence or in the promoter "sequence of said gene), and as being "epigenetic" 1f it does not result from a modification of the eIF(iso)4E gene. The epigenetic inactivation, also called "silencing", can result in particular from blocking of the transcription (TGS for "Transcriptional Gene Silencing") or from specific degradation of the mRNAs (PTGS for "Post-
Transcriptional Gene Silencing").
The replacement of the alleles of the eIF4E or eIF(iso)4E genes with the desired mutant alleles may be carried out by techniques known in themselves, making use of transgenesis, such as homologous recombination, Or mutagenesis, for instance TILLING (Targeting Induced Local
Lesions IN Genomes; McCALLUM et al., Plant Physiol. 123: 439-442, 2000) or, in the case of the eIF(iso)4E gene, insertional mutagenesis.
The direct inactivation of the eIF(iso)4E gene can also be carried out by the same techniques.
The silencing of the eIF(iso)4E gene can be carried out by sense or antisense suppression, or using
RNAi (NISHIKURA, Cell 107: 415-418, 2001; TENLLADO et al.,
Virus Res. 102: 85-96, 2004; ZAMORE, Methods Mol. Biol. 252: 533-543, 2004).
A subject of the present inveniton is also plants which are resistant to PVMV, and which can be obtained by means of a method in accordance with the invention.
This includes, in particular: - the plants in which the alleles of the eIF4E denes present are mutant alleles as defined above, and the alleles of the eIF(iso)4E genes present are nonfunctional mutant alleles as defined above, with the exception of the pvr2 pvré double mutants described in the publication by CARANTA et al. (1996, mentioned above) ; - the plants in which the alleles of the eIF4E dene that are present are mutant alleles as defined above, and the alleles of the eIF(iso)4E gene have been directly inactivated;
- the plants in which the alleles of the eIF4E gene that are present are mutant alleles as defined above, and which are transformed with a sequence (sense, antisense, or that may be transcribed into RNAi), capable of silencing the expression of the eIF(iso)4E gene.
The methods for selecting or for obtaining plants which are resistant to PVMV in accordance with the invention are applicable to all plants that it is desired to protect against PVMV, and more particularly to plants of the Solanaceae family, such as pimento, tomato or aubergine, of the Cucurbitaceae family, such as melon, of the crucifer family, of the family Fabaceae, such as pea, and of the composite family.
In addition, they may also be applicable for the protection of plants against other potyviruses, which, like PVMV, can use eIF4E or eIF(iso)4E without distinction for accomplishing their infectious cycle; by way of example, mention will be made of the BCMV (Bean common mosaic virus) potyvirus, or the AzMV (Azukini mosaic virus) potyvirus (FISHER and KYLE, Theor. Appl. Genet. 92: 204-212, 1996).
The present invention will be understood more clearly from the further description which follows, which refers to examples illustrating the involvement of eIF4E and eIF(iso)4E in resistance to PVMV, and the use of methods in accordance with the invention for selecting and for obtaining plants which are resistant to PVMV.
EXAMPLE 1: OBTAINING THE eIF(ISO)4E (pvr6') cDNA IN
PIMENTO
The total RNA derived from the potyvirus- sensitive pimento Yolo Wonder genotype was isolated from 100 to 200 mg of leaves using the TRI-Reagent (Sigma-
Aldrich, St Louis).
The 3’ end of the pimento eIF(iso)4E cDNA was obtained using the 3’RACE PCR technique (GIBCO/BRL Life technologies 3'RACE system kit, version 2.0) using a primer 5' -AAGTGGACTGTTACGAGCAGCAG-3"! (SEQ ID NO.: 5) designed based on the alignment of c¢DNA sequences of elF(iso)4E of Arabidopsis thaliana (EMBL accession No.:
Y10547), of Lactuca sativa (GenBank accession No.:
AF530163) and of Lycopersicon esculentum (TIGR: TC126316) .
The full-length cDNA of the pimento eIF(iso)4E gene was amplified by RT-PCR with a primer designed based on the sequence of the 3’'RACE product (5' -ATTGCTGGAACTTGGGGAGGG-3’, SEQ ID NO.: 6) and a primer designed based on the 5’ UTR end of the tomato eIF (iso)4E sequence (TIGR accession No. TC126316) (5' -AAAACAATGGCCACCGAAGCA-3’, SEQ ID NO.: 7). The RT-PCR is carried out under the following conditions: 5 min at 94°C then 30 cycles (30 sec at 94°C, 30 sec at 53°C, 1 min at 72°C) and 5 min at 72°C.
The pGEM-T Easy vector system (Promega,
Madison, USA) was used to «clone the cDNA after amplification.
At least 3 independent positive clones were sequenced, from the two ends, by Genome express (Grenoble,
France). The PileUp program from GCG (Genetics Computer
Group, Madison, USA) was used for the nucleotide and peptide sequence analysis.
The pimento eIF(iso)4E cDNA sequence thus obtained has a length of 660 nucleotides (SEQ ID NO.: 3) and a single open reading frame of 609 nucleotides encoding 202 amino acids (SEQ ID NO.: 4).
The greatest sequence identity percentages, determined using the BLAST software on a comparison window comprising the entire pimento eIF(iso)4E cDNA, were obtained for the elIF(iso)4E cDNA from lettuce (AF530163,
E=1.10"%%), from pea (AY423377, E=2.10"%), from maize (AF076955, E=4.10%°), from A. thaliana (Y10547, E=6.107°) and from wheat (M95818, E=9.107°).
The greatest sequence identity percentages, determined using the BLAST software on a comparison window
. © 16 comprising the entire predicted peptide sequence of pimento eIF(iso)4E, were obtained for the eIF (iso) 4E protein from lettuce (AAP86603, E=8.10""") and from wheat (AAA34296, E=91.10"%"3), confirming that the Yolo Wonder cDNA obtained clearly corresponds to the eIF(iso)4E cDNA.
The coding regions of the pimento eIF4E gene (GenBank accession No.: AY122052, RUFFEL et al., 2002) and the pimento eIF(iso)4E gene exhibit 57.2% sequence identity (and 48.3% sequence identity at the level of the corresponding peptide sequences).
EXAMPLE 2: COMPARISON OF THE SEQUENCES OF THE pvré” AND pvré GENOTYPES IN PIMENTO: DEMONSTRATION OF MUTATIONS IN eIF(ISO)4E
To determine whether the sequence variations may be associated with the pvré® or pvré allele, the corresponding nucleotide and amino acid sequences of the full-length eIF(iso)4E cDNA of 5 different pimento lines were aligned: - the Yolo Wonder (YW) variety 1s sensitive to potyviruses; - the Yolo Y (YY) variety has the pvr2' resistance allele (resistance to PVY pathotype 0), and is sensitive to PVMV; - the Florida VR2 (F) variety has the pvr2®’ resistance allele (resistance to PVY (0) and (1) and to TEV), and is sensitive to PVMV; - the Perennial (P) variety has the pvr2® allele for partial resistance to PVY and to potyvirus E and the pvré allele, and is sensitive to PVMV; - the DH801 doubled haploid line derived from the F1 [Perennial x Florida VR2] has the pvr2’ and pvrs alleles, and is resistant to PVMV.
The results of the alignment of the open reading frames of eIF(iso)4E, and of the corresponding amino acid sequences, of the 5 pimento lines described above are represented in Figure 1.
Legend of Figure 1: (A) = alignment of the nucleotide sequences of the pvré' and pvré genotypes (coding portion); (B) = alignment of the corresponding amino acid sequences of the pvré® and pvré genotypes.
The nucleotide alignments show that the coding portion of the pvré* alleles of the YW, YY and F genotypes are 100% identical to one another. The nucleotide alignments show that the coding portion of the pvré alleles of the P and DH801 genotypes are also 100% identical to one another, but different from the first group by virtue of the deletion of nucleotides 89 to 170, and by virtue of a subtitution of a G with an A at position 268, of a C with an A at position 483, and of a C with a T at position 537 (Figure 1A). The deletion of 82 nucleotides modifies the open reading frame and introduces a stop codon after amino acid 51. The alignment of the eIF(iso)4E protein sequences of the pvre® and pvré genotypes shows that only the first 29 amino acids (out of 202 that the protein has) are conserved (Figure 1B).
EXAMPLE 3: SELECTION OF PLANTS HAVING BOTH AN ALLELE
MUTATED AT THE pvr2 LOCUS (eIF4E SEQUENCES CORRESPONDING
TO THE pvr2® OR pvr2? ALLELES) AND THE pvr6 MUTATED ALLELE (MUTATED eIF(ISO)4E SEQUENCE) IN THE HOMOZYGOUS STATE
From the pimento plants having the pvrz! (SEQ
ID NO.: 8 of PCT application WO 03/066900) or pvr2z? (SEQ
ID NO.: 22 of PCT application WO 03/066900) alleles in the homozygous state (the methods for selecting these mutants are described in PCT application WO 03/066900), the plants also having the pvré allele corresponding to mutated eIF(iso)4E, and therefore resistant to PVMV, are selected as follows:
Selection by RT PCR
The plant RNA extraction follows the standard extraction protocols based on the protocol provided by
: | 18
Sigma-Aldrich (St. Louis, USA) with the TRI-Reagent product sold by this same company.
The reverse transcription (RT) of the RNA to cDNA is carried out as follows: a mixture of 7.5 pul of
H,0, 1 pl of oligoDT primer (100 ng/pl), 2.5 pl of dNTP (each 4 mM) and 2 pl of RNA is incubated for 5 min at 65°C, and then for 2 min in ice. 4 pl of Invitrogen RT buffer (5X) and 2 pul of DTT (0.1M) are added, and the mixture is then incubated for 2 min at 42°C. 1 pul of
SuperScript RT II (Invitrogen, 200 U/ul) is added, and the mixture is then incubated for 50 min at 42°C. The reaction is inactivated for 15 min at 70°C, and then the cDNA produced is stored at 4°C.
The PCR is carried out by mixing 17 pl of HO, 2.5 pl of Hifi PCR buffer (10X) (GIBCO/BRL Life
Technologies), 1 pul of MgSO4 (50 mM), 1 pl of ANTP (each 4 mM), 0.5 ul of the sense primer 5’ -ATGGCCACCGAAGCACCACCACCGG-3' (SEQ ID NO.: 8) (10 um), 0.5 ul of the antisense primer 5 ~
TCACACGGTGTATCGGCTCTTAGCT-3’ (SEQ ID NO.: 9) (1ouM) (the position of these primers is indicated in Figure 1A), 0.5 pl of High Fidelity Platinium Tag Polymerase (5 U/ul) (GIBCO/BRL Life Technologies) and 2 pl of RT product. The
PCR is carried out under the following conditions: 5 min at 94°C, then 30 cycles (30 sec at 94°C, 30 sec at 64°C, 1 min at 72°C) and 5 min at 72°C.
The PCR products are migrated on a 1% agarose gel with 1X TAE buffer, and the amplified fragments are visualized by ethidium bromide (ETB) staining.
The results are presented in Figure 2.
Legend of Figure 2:
Lane 1 corresponds to the RT-PCR carried out on the RNA of the cultivar Perennial (pvré allele), 2 and 3 correspond to the cultivars Florida VR2 and Yolo Wonder (pvré+ allele). Lanes 5, 6, 7, 8, 11, 12, 13, 14, 15, 19, 20, 22, 23 and 25 correspond to the RT-PCR carried out in
PVMV-resistant progeny and lanes 4, 9, 10, 16, 17, 18, 21 and 24 correspond to the RT-PCR carried out in PVMV- sensitive progeny.
The results show that the plants carrying the mutated eIF(iso)4E allele (pvreé) exhibit a 527 bp amplification product (corresponding to the deletion of 82 pb), whereas the plants carrying the wild-type eIF(iso)4E allele (pvrée+) exhibit a 609 bp amplification product.
Selection by RFLP 1) Digestion with EcoRV and separation of the restriction fragments
The DNA is extracted from the plants according to the microextraction protocol described by FULTON et al. (Plant Mol. Biol. Reptr. 13(3): 207-209, 1995).
It is then digested with 2.5 U of EcoRV enzyme/ug of DNA, at 37°C overnight. The digestion product is loaded onto a 1% agarose, 1X NEB gel containing 10 pl of ETB, in order to separate the restrition fragments. The migration is carried out at 25V for 24 h in 1X NEB buffer.
After migration, the fragments are transferred onto a nylon membrane. The membranes are rinsed in a 2X SSC bath for 10 to 15 min and then dried in the open air and baked for 2 h at 80°C. 2) Preparation of probes
The eIF(iso)4E (pvré*) cDNA of the Yolo Wonder genotype, obtained as described in Example 1, is used as a probe.
The 3?P-labeled probes are prepared by PCR under the following conditions:
i . | 20
Ea
HO 25.6 pl
Tp Promega 10 X 4 ul 1X
MgCl, Promega 2.4 yl
Mix (ATG 50 uM+dCTP 5 uM)* 2p ATG 2.5 uM; dCTP 0.25 uM
Taq 2U/pl iy
Primer (5pM) 1 ul a32P-dCTP (1000Ci/mmol, 10uCi/ul 3ul TemplateONA [aw |] Finalreactionvolume | ao *: Mix (50 uM ATG + 5 uM dCTP) for labeling of the RFLP probes by PCR.
Dilution of dATP, dTTP and dGTP to 10 mM, from the stock solutions at 100 mM: - 5 pul ANTP at 100 mM - 45 pl HO
Dilution of dCTP to 1 mM, from the stock solution at 100 mM: - 0.5 pl ACTP at 100 mM - 49.5 pl H0 :
Mix ATG + ACTP: - 2.5 pul dATP at 10 mM final concentration: 50 uM - 2.5 pl 4ATTP at 10 mM final concentration: 50 uM - 2.5 pl 4GTP at 10 mM final concentration: 50 uM - 2.5 pul dCTP at 1 mM final concentration: 5 uM - 490 pl HO.
The labeling of the probes is carried out over the course of 30 PCR cycles: 30 sec at 94°C, 45 sec at 52°C, 1 min 30 at 72°C.
Once labeled, the probes are denatured, and then hybridized with the membranes obtained as described in 1). The hybridization is carried out at 65°C for at least 16 hours in the following buffer: for 500 mL: 21.91 g NaCl; 18.38 g Na citrate; 380 mL HO; 15 mL 20%
SDS; 25 mL 1M NaPOs, pH 7.5; 25 mL 100X Denhardt's; 5 mL 0.25M EDTA; 50 mL 50% dextran sulfate.
The membranes are then washed in 1% SDS (Serva), 40mM NaPi buffer, preheated to 65°C.
The washing conditions are as follows: - 1 wash of 20 min at 65°C with shaking;
’ } . | 21 - 1 rinse of 2-3 min in fresh buffer heated to 65°C.
The RFLP results are presented in Figure 3.
Legend of Figure 3:
The figure shows the difference in RFLP profile observed for the eIF(iso)4E marker, between the
Perennial genotype with the pvré allele (lane P), the
Florida VR2 genotype with the pvré+ allele (lane F), the
Perennial X Florida VR2 Fl hybrid (lane F1) and PVMV- resistant (lane R) and PVMV-sensitive (lane S) progeny.
The results show that the sensitive plants with the pvré+ allele possess the restriction fragment H, whereas the resistant plants with the pvré allele (in addition to the pvr2? allele) possess the restriction fragment B.
EXAMPLE 4: TEST FOR RESISTANCE TO PVMV AND TRANSIENT
EXPRESSION OF eIF(iso)4E (pvré6') OR eIF4E (pvr2') IN A
PVMV-RESISTANT PIMENTO GENOTYPE
Test for the resistance of pimento to PVMV
This test is used to verify that the plants carrying the pvr2! (or pvr2?) and pvré resistance alleles are indeed resistant to PVMV.
The viral material used in the inoculation tests corresponds to the PVMV-IC isolate isolated in Ivory
Coast and provided by J.C. THOUVENEL (IRD, Montpellier,
France) and to the isolates described in the publication by CARANTA et al. (1996, mentioned above). The isolates are maintained according to the BOS procedure (BOS, Meded.
Fac. Landbouwwet Gent. 34: 875-887, 1969) and multiplied on Capsicum annuum CV. Yolo Wonder plants before mechanical inoculation of the pimento plants at the cotyledons or 2-spread-leaf stage.
The viral inoculum is prepared as described in
CARANTA et al. (1996, mentioned above). The inoculation tests are carried out under controlled conditions in "insect-proof" climatized chambers. 4 weeks after inoculation, all the plants are evaluated individually for the presence or absence of the PVMV capsid protein antigen by means of a DAS-ELISA assay as described by CARANTA et al. (1996, mentioned above). Other protocols that are entirely known to those skilled in the art may also be used for the mechanical inoculation and the detection of
PVMV in the pimento plants.
This resistance test makes it possible to follow the total cosegregation between the PVMV resistance and the presence of the pvrz! (or pvr2?) alleles corresponding to mutated forms of eIF4E and the presence of the pvré allele corresponding to the mutated form of eIF(iso)4E.
Transient expression of the Yolo Wonder eIF(iso)4E or eIF4E cDNAs in a PVMV-resistant pimento genotype (pvr2’ + pvr6 combinant) for validation of the independent role of the eIF(iso)4E and eIF4E proteins in PVMV sensitivity
In order to validate the hypothesis that PVMV is capable of using, independently, either the pvr2® allele (corresponding to the eIF4E wild-type allele) or the pvré® allele (corresponding to the eIF(iso)4E wild- type allele) for accomplishing its infectious cycle, experiments for transient expression of the Yolo Wonder eIF4E or elIF(iso)4E cDNAs via a PVX (Potato virus X) viral - 25 vector (CHAPMAN et al., Plant J. 2(4): 549-557, 1992;
BAULCOMBE et al., Plant J. 7: 1045-1053, 1995) were carried out on the resistant genotype DH801, carrying the pvr2? and pvré alleles.
The eIF4E ORFs of the Yolo Wonder (pvr2+) and
Florida VR2 (pvr2’) genotypes were cloned into the expression vector pPVX201l (BAULCOMBE et al., 1995, mentioned above) at Clal and Sall cloning site, so as to obtain the vectors pPVXeYW and pPVXeF, respectively. In the same manner, the eIF(iso)4E ORFs of the Yolo Wonder (pvré+) and Perennial (pvré) genotypes were cloned into pPVX201 =]o} as to obtain the expression vectors
. . 2s pPPVX (iso) eYW and pPVX(iso)eP, respectively. These plasmids are inoculated mechanically onto Nicotiana benthamiana.
Ten days after inoculation, the leaves inoculated with N. benthamiana are used as inoculum for the transient expression tests in the Yolo Wonder and DH801 pimento genotypes. Ten days after inoculation of the pimento plants with the expression vector PVX, the same leaves are inoculated with PVMV-IC as described above.
The accumulation of the PVX vector and of PVMV is evaluated by DAS-ELISA and RT-PCR on the inoculated leaves, 10 days after the inoculation with PVMV. The RT-
PCR (conventional conditions known to those skilled in the art) that makes it possible to detect the accumulation of
PVX is carried out with the following primers: sense 5’ -CCGATCTCAAGCCACTCTCCG-3’ (SEQ ID NO.: 10) and antisense 57 -CCTGAAGCTGTGGCAGCGAGTTG-3’ (SEQ ID NO.: 11). The RT-PCR (conventional conditions known to those skilled in the art) that makes it possible to detect the accumulation of
PVMV is carried out with the following degenerate primers: sense (defined in the Nib gene) 5’'-GGNAARGCNCCNTAYAT-3’' (SEQ ID NO.: 12) and antisense (defined in the CP gene) 5’ -CGCGCTAATGACATATCGGT-3’ (SEQ ID NO.: 13).
The PVMV-resistant genotype DH801 is coinoculated with a recombinant plasmid (possessing eIF4E or eIF(iso)4E) and with the PVMV-IC isclate. Control experiments are carried out in parallel, expressing, in
DH801, either the pvr2? allele (of the Florida VR2 genotype) or the pvré allele (of the Perennial genotype) so as to verify that the mutated forms do not restore the sensitivity to PVMV.
The results are summarized in the table below, indicating the ratio of the number of leaves infected with
PVMV to the number of leaves inoculated with PVMV in the transient expression experiments.
: . | 24 -—
Pimento genotypes
Leaves inoculated with Yolo Wonder DH801
PVMV 20/20 0/20 pPVX201 + PVMV 20/20 0/20 pPVXeYW + PVMV 20/20 13/30 pPVXeF + PVMV 20/20 0/30 pPVX (iso) eYW + PVMV 20/20 5/30 pPVX (iso)eP + PVMV 20/20 0/30
These experiments show that both the expression of the eIF4E gene and, independently, that of the eIF(iso)4E gene (genes derived from the sensitive genotype Yolo Wonder), allow PVMV to multiply in the resistant genotype DH801. Based on these experiments, we can conclude that PVMV is capable of using either the eIFAE wild-type protein or the eIF(iso)4E wild-type protein, in order to perform its infectious cycle, and that mutations in the two proteins are necessary to render the plant resistant.
EXAMPLE 5: OBTAINING PLANTS WHICH ARE RESISTANT TO PVMV BY
INACTIVATION OF eIF(iso)4E
The allele of the eIF(iso)4E gene that confers the resistance to PVMV (pvré), in plants carrying the pvr2' (or pvr2®) resistance alleles, can be transferred in planta by methods of the site-directed mutagenesis type (HOHN et al., Proc. Natl. Acad. Sci. USA. 96: 8321-8323, 1999) or homologous recombination type (KEMPLIN et al.,
Nature 389: 802-803, 1997; JONES et al., Transgen. Res. 1: 285-297, 1992; BEVAN, Nucleic Acid research. 12: 8711- 8721, 1984).
Plants carrying the pvr2! (or pvr2®) resistance alleles, which are resistant to PVMV, can also be obtained by knock-out of the endogenous eIF(iso)4E gene by methods of the "gene silencing" type (Post Transcriptional Gene
Silencing or PTGS). A specific knock-out by PTGS can be carried out by directing it against the 5’ or 3’ UTR of the endogenous eIF(iso)4E gene. This specificity of the knock-out by PTGS against the 5’ or 3’UTR is based on the new data derived from the understanding of the mechanism of PTGS (NISHIKURA, Cell 107: 415-418, 2001).

Claims (8)

  1. WHAT IS CLAIMED IS 1) A method for selecting plants which are resistant to PVMV, charcterized in that it comprises the search, in the plants to be tested, for the forms of the eIF4E and eIF (iso)4E proteins present in said plants, and the selection of the plants which:
    a) do not express any eIF4E protein (hereinafter referred to as: "wild-type eIF4E protein") comprising a region defined by the following general sequence (I):
    DX, X5X3X,KSXsOXcAWGSSX RX eX oY TF SX; oVEX 11 FWX 12X13 YNNIHX; s PSKLX 15X16 GAD in which: - Xi, Xi, X3, X4, Xe, X79, Xs, Xo, X10, X12, X13, Xis and X,¢ each represent a neutral amino acid; - Xs and Xs represent a basic amino acid; - X,, represents an acidic amino acid; - D, XK, $s, Q, A, W, G, R, ¥Y, T, F, Vv, E, N, I, H, P, and L have their usual 1l-letter-code meaning;
    and express 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 charged amino acid of said sequence (I) with a neutral amino acid or an amino acid of opposite charge;
    and b) do not express any functional eIF(iso)4E protein.
  2. 2) The method as claimed in claim 1, characterized in that it comprises the selection of the plants expressing a nonfunctional eIF(iso)4E protein deleted of at least one portion of the sequence of a wild- type eIF(iso)4E protein, said deletion comprising one or more of the tryptophan residues of the IF4E domain of said protein corresponding to positions 34, 37, 50, 67, 096, 107, 121 and 156 of the sequence SEQ ID NO.: 4.
  3. 3) The method as claimed in either one of claims 1 and 2, characterized in that it comprises the selection of the plants expressing a mutant eIF4E protein comprising a region derived from that defined by the sequence (I) above, by substitution: - of at least one of the amino acids Xj, Xa, X; or X, of said sequence (I) with a charged amino acid; and - of at least one of the other neutral amino acids of said sequence (I) with a charged amino acid; and/or - of at least one of the charged amino acids of said sequence (I) with a neutral amino acid or an amino acid of opposite charge.
  4. 4) The method as claimed in claim 3, characterized in that it comprises the selection of the plants expressing a mutant eIF4E protein comprising a region derived from that defined by the sequence (1) above, by substitution: - of the neutral amino acid X; of the sequence (I) with a basic amino acid; - of the neutral amino acid X, of the sequence (I) with a basic amino acid; - of the aspartate residue in the C-terminal position of the sequence (I) with a neutral amino acid.
  5. 5) A method for obtaining a plant which is resistant to PVMV, characterized in that it comprises: replacement of the alleles of the eIF4E gene that are present in said plant, with alleles encoding a mutant eIF4E protein as defined in any one of claims 1, 3 or 4; and replacement of the alleles of the eIF(iso)4E gene that are present in said plant, with alleles encoding a mutant elIF (iso)4E protein as defined in either one of claims 1 and 2, or direct or epigenetic inactivation of said eIF(iso)4E gene.
  6. 6) A method for obtaining a plant which is resistant to PVMV, characterized in that it is implemented using a plant in which all the alleles of the eIF4E gene encode a mutant eIF4E protein as defined in any one of claims 1, 3 or 4, and in that it comprises replacement of the alleles of the eIF(iso)4E gene that are present in said plant, with alleles encoding a mutant eIF(iso)4E protein as defined in either one of claims 1 or 2, or direct or epigenetic inactivation of said eIF(iso)4E gene.
  7. 7) A method for obtaining a plant which is resistant to PVMV, characterized in that it is implemented using a plant in which all the alleles of the eIF(iso)4E gene encode a mutant eIF(iso)4E protein as defined in either one of claims 1 and 2, or using a plant in which said eIF(iso)4E gene has been inactivated, and that it comprises replacement of the alleles of the eIF4E gene that are present in said plant, with alleles encoding a mutant eIF4E protein as defined in any one of claims 1, 3 or 4.
  8. 8) The method as claimed in any one of claims 1 to 7, characterized in that it is carried out on a plant chosen from Solanaceae, Cucurbitaceae, crucifers, Fabaceae and composites.
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