EP2900814A1 - Potyvirus- and/or potexvirus-resistant plants expressing a mutant chloroplastic phosphoglycerate kinase - Google Patents

Abstract

The invention relates to methods for obtaining potyvirus- and/or potexvirus- resistant plants. Said plants express, instead of a wild type chloroplastic phosphoglycerate kinase (cPGK) wherein the N-terminal sequence of the mature protein is MAKKSVG, a mutant cPGK wherein the serine residue in position 5 of said N-terminal sequence is replaced by an amino-acid residue other than serine.

Description

POTYV IRUS- AND/OR POTEXVIRIJS-RESISTANT PLANTS EXPRESSING A MUTANT CHLOROPLAST1C PHOSPHOGLYCERATE KINASE.

The present invention relates to the obtaining of potyvirus- and/or potcxvirus-resistant plants.

The potyvirus group, the standard member of which is PVY, for potato virus Y, is the largest plant virus group. In fact, potyviruses are capable of infecting more than 30 plant families currently registered. This group comprises at least 180 members, which corresponds to a third of the plant viruses currently known. Potyvirus transmission is effected by aphids (for example Myzus persicae) via the nonpersistent mode. The symptoms caused by potyviruses are abnormalities in leaf coloration (mosaics, yellowing of veins), leaf deformities, vein necroses which can result in necrosis of the entire plant, and considerable decreases in size o the sick plant, greatly influencing productivity.

The Solanaceae, Cucurbitaceae, Cruciferae, Fabacae, Compositae, and Rosaceae are particularly sensitive to potyviruses. The Solanaceae, and more particularly the tomato and the capsicum (or pepper), are infected with at least seven different potyviruses throughout the world, including for instance PVY, Tobacco etch virus (TEV), Pepper mottle virus (PMV), Pepper veinal mottle virus (PVMV) Potyvirus E, and Chili veinal mottle virus (CVMV). The potyvirus plum pox vims (PPV), is responsible of a devastating disease of stone fruit from the genus Primus.

Potyviruses have a nonenveloped filamentous structure, from 680 to 900 nm long and from 1 1 to 15 nm wide. 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. The viral RNA encodes 10 proteins involved in polyproteins cleavage, genome replication, cell-to-cell movement and longdistance movement, transmission via aphids, etc.

The potexvirus group, represented by the type species POTato Virus X (PVX), belongs to the family Alphaflexiviridae and the order Tymovirales. Transmission occurs mechanically, mainly by aphids. Potexviruses cause mosaic and ringspot symptoms. Many crop plants from various families are sensitive to potexviruses. For instance, Pepino mosaic virus (PepMV) has been reported to infect greenhouse tomato crops in many countries around the world.

Potexviruses are non-enveloped filamentous viruses, 470- 1000 nm or more long and 12-13 nm in diameter. The viral genome is a single-stranded sense RNA of 5,9-7 kb in size. Its 5' end is capped and its 3' end is polyadenylated; it encodes for 5 proteins.

At the current time, the strategies used for controlling viral plant diseases 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.

To invade plants, pathogens have developed highly specialized tactics to acquire nutrients, to bypass or overcome host defenses, and to reroute host cellular functions for their own benefits. These features also apply to viruses that are obligatory parasites. Because the repertoire of the viral gene products is limited, viruses completely rely on the use of host cellular factors to perform infection, In this scheme, absence or mutation of a single host factor, also called susceptibility factor, may lead to plants fully or partially resistant to viruses (Truniger and Aranda, 2009), The characterization of such susceptibility factors is therefore of great interest to shed light on molecular mechanisms underlying plant-virus interactions and also to identify new targets to improve plant resistance to viruses (Pavan et al.. 2010).

The characterization of host factors required for viral infections has greatly benefited from the fact that numerous viruses can efficiently infect the model plant Arabidopsis t Indiana under standard laboratory conditions (Carr and Whit ham, 2007). It offers the unique opportunity to exploit the facile genetics, efficient transformation, genome sequence, molecular and genomic resources to decipher the molecular dialogue between plants and viruses, The use of reverse genetic approaches in Arabidopsis based on the screening of mutagenized host populations for altered infection phenotypes, the assessment of specific virus-host protein interactions or the phenotypic characterization of mutants for a particular candidate gene, has resulted in the identification of several host factors required for various key steps of viral life cycles such as replication, translation or movement. These susceptibility factors include the plant Heat shock cognate 70-3 protein (Dufresne et al., 2008), the RNA helicase RH8 (Huang et al., 2010), the transmembrane proteins TOM1 and TOM3 (Yamanaka et al., 2002), a cystcine-rich protein called potyvirus interacting protein (PVIP) (Dunoyer et al., 2004), and several translation factors including eIF4E, eIF(iso)4E, PABP and eEFIA (Wittmann et al, 1997; Leonard et al, 2000; Dufresne et al., 2008; Thivierge et al., 2008).

Insights into plant factors required for successful viral infections can also be obtained by the molecular characterization of naturally-occurring recessive resistance genes. To date, eight recessive resistance genes have been cloned from a variety of crop species and all were demonstrated to correspond to mutated forms of translation initiation factors eIF4E or eIF4G (Le Gall et al., 201 1). Data from several studies showed that eIF4- mediated resistance involves few amino acid changes in translation initiation factors that impair interactions with viral factors, thereby interfering with the ability of viruses to complete their infectious cycles (Leonard et al., 2000; Yearn et al., 2007; Charron et al.. 2008; Ala-Poikela et al., 201 1). In Arabidopsis, the characterization of loss-of-lunction mutants for the eIF4E or eIF4G genes has provided evidence supporting the key role of these factors in the life cycle of numerous plant RNA viruses (Le Gall et al., 201 1). However, eIF4-mediated resistance has never been identified in the natural diversity of Arabidopsis.

In parallel, several studies have identified recessive resistance genes from diverse Arabidopsis accessions that are distinct from translation initiation factors. For instance, the rpvl gene in the Cape verde island (Cvi) accession associated with the inhibition of Plum pox potyvirus accumulation at the systemic level, and the dsiml gene responsible for a delay in systemic Tobacco mosaic tobamoviriis movement in the Columbia (Col) accession were physically mapped to genomic regions that do not contain any eIF4E- or eIF4G- encoding genes (Decroocq et al., 2006; Serrano et al., 2008). Similarly, the rim I gene restricting Lettuce mosaic potyvirus to inoculated leaves in the Col accession does not involve translation initiation factors as indicated by the absence of polymorphism in the Arabidopsis eIF4E genes between susceptible and resistant genotypes (Re vers et al., 2003). The sequencing of eIF4E genes in a core collection of 54 accessions maximizing genetic diversity in Arabidopsis failed to identify signature amino-acid substitutions previously demonstrated to be responsible for virus resistance in crops (Charron, 2007).

The inventors have now identified and characterized rwml, a recessive gene that controls Arabidopsis resistance to Watermelon Mosaic Virus, a member of the genus Potyvirus. rwml confers broad-spectrum resistance against WMV infection by impairing virus accumulation in inoculated leaves. Using map-based cloning in combination with a candidate gene approach, a chloroplast phosphoglycerate kinase (cPGK) was identified as the candidate gene for new /-mediated resistance. Efficient virus-induced silencing of cPGK in tobacco resulted in decreased accumulation of WMV RNA and coat protein,

These findings of the inventors make it possible to propose new tools, in particular genetic tools, for screening and/or for obtaining plants resistant or sensitive to potyviruses.

The present invention relates to a method for selecting a potyvirus- and/or potexvirus-resistant plant, characterized in that it comprises the detection, in plants to be tested:

- of the presence or of the absence of an allele of a cPGK gene encoding a chloroplastic phosphoglycerate kinase (cPGK) (hereinafter called: "wild-type cPGK allele"), wherein the mature form of the cPGK encoded by said allele (hereinafter called: "wild-type cPGK protein") has the N-terminal sequence MAK SVG (SEQ ID NO: 1 );

- of the presence or of the absence of a mutant allele of said cPGK gene

(hereinafter called: "mutant cPGK allele"), wherein the mature form of the cPGK encoded by said allele (hereinafter called: "mutant cPG protein") has the N-terminal sequence MAKKXVG (SEQ ID NO: 2), wherein X represents an amino-acid residue other than serine, preferably a non-phosphorylatable amino acid residue, and still more preferably a glycine residue;

and the selection of a plant in which the mutant cPGK allele is detected, and in which the wild-type cPGK allele is not detected.

A subject of the present invention is also a method for selecting a plant which can be used a a progenitor for obtaining a potyvirus- and/or potexvirus-resistant plant, characterized in that it comprises the detection, in plants to be tested, of the presence or of the absence of the mutant cPGK allele as defined above, and the selection of the plants in which said mutant cPGK allele is detected.

According to a preferred embodiment of the invention, said cPGK gene is a cPGK2 gene, such as the Arabidopsis thaliana gene Atl g56190 (or SEQ ID NO: 3) or an ortholog thereof. Complete inactivation of this cPGK2 gene in a plant induces an albino phenotype and is lethal. However, the plants expressing a mutant cPGKl allele of the invention are viable and no phenotypical change other than the increased virus resistance has been observed.

The detection of the presence or of the absence of a wild-type or mutant cPGK allele may be carried out indirectly by detection of the wild type or the mutant cPGK protein, for instance by use antibodies specifically directed against either the wild-type form or the mutant form of cPGK.

However, it is generally more convenient to carry out this detection by searching directly for the allelic form of the cPGK gene present in the plant to be tested.

The detection of the allelic form of the cPGK gene 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 a mutant allele of cPGK as defined above, or amplification primers for the amplification of cPGK or of the portion of this gene 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.

Primers and probes designed for carrying out these methods are also part of the present invention.

Another object of the present invention is an isolated mutant cPGK protein as defined above, as well as an isolated polynucleotide encoding said mutant cPGK protein. The present invention also encompasses recombinant vectors, in particular recombinant expression vectors, containing a polynucleotide of the invention encoding a cPGK protein, under control of an appropriate promoter. It also encompasses host-cells, prokaryotic or eukaryotic, transformed by an expression vector of the invention.

A subject of the present invention is also a method for obtaining plants which are resistant to potyvirus and/or potexvirus. Said method comprises the expression of a mutant allele of the cPGK gene as defined above, either in replacement or in supplement of the wild-type allele that is present in a potyvirus- and/or pot ex virus-sensitive plant.

The expression "replacement of the wild-type allele" of the cPGK 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 replacement of the wild-type allele of the cPGK gene with the desired mutant allele may be carried out by techniques known in themselves, making use of transgenesis, such as homologous recombination, or of mutagenesis, for instance TILLING (Targeting Induced Local Lesions IN Genomes; McCALLUM et ah, Plant Physiol. 123: 439- 442, 2000).

Resistant plants can also be created by inactivation of the endogenous wild- type cPGK gene associated with the expression by transgenesis, of the mutant cPGK allele of the invention which confers potyvirus resistance. Inactivation of the wild-type cPGK gene can be obtained for instance by deletion, or by transposon insertion, or by epigenetic inactivation resulting from blocking of the transcription (TGS for "Transcriptional Gene Silencing") or from specific degradation of the mR As (PTGS for "Post-Transcriptional Gene Silencing") means of gene silencing-type methods (post transcriptional gene silencing). By way of example, a specific knock-out by PTGS can be carried out by directing it against the 5' UTR of the endogenous cPGK gene; the mutant cPGK allele which confers resistance, expressed by transgenesis, will not carry the 5' UTR sequence of the endogenous cPGK.

The expression "supplement of the wild-type allele" of the cPGK gene is here intended to mean any genetic modification, regardless of the method used to carry out this modification, resulting in the introduction of the mutant sequence in the host plant which keeps the initial coding sequence of the gene concerned with the desired mutant sequence. It has been shown that such an expression of a recessive resistance gene in a transgenic plant may confer to said plant dominant resistance properties (Kang et al., 2007).

A subject of the present invention is also plants which are resistant to potyvirus and/or potexvirus, and which can be obtained by means of a method in accordance with the invention.

The methods for selecting or for obtaining plants which arc resistant to potyvirus and/or potexvirus in accordance with the invention arc applicable to all plants that it is desired to protect against potyvirus and/or potexvirus, for instance to plants of the Solanaceae family, such as pimento, tomato or aubergine, of the Cucurbitaceae family, such as melon, of the Cruciferae family, of the family Fabaceae, such as pea, of the Compositae family, and of Rosaceae family, such as those of the genus Primus.

The present invention will be understood more clearly from the further description which follows, which refers to non-limitative examples illustrating the involvement of cPGK in resistance to poty viruses.

LEGENDS OF THE DRAWINGS :

Figure 1. Phenotypic characterization of WMV resistance in Cvi-0. (A) Pictures of WMV-inoculated Ler-1 and Cvi-0 plants taken two weeks post-inoculation. Non-inoculated plants are shown on the left. Scale bar. 5cm. (B) Representative gel image showing presence/absence of WMV RNA accumulation in inoculated and systemic leaves of Ler- 1 and Cvi-0 plants, as determined by conventional RT-PCR at 9 and 19 dpi, respectively. AP I L internal control; NI = non-inoculated control; -RNA = no template control. (C) Quantitative RT-PCR analysis of WMV RNA accumulation in inoculated leaves of Ler- 1 and Cvi-0. Virus RNA levels were normalized to APT I and expressed as fold-change relative to Ler-1. Results shown arc mean values ± SDs of four measurements from two independent experiments. * indicates a significant difference i Cvi-0 compared to Ler-1 (Student's t-test, P< 0.05).

Figure 2. Map-based cloning of rwml. Key near-isogenic lines (A) and F3 progenies (B) used for rwml genetic and physical mapping. Cvi-0 and Ler-1 segments are represented in black and white columns, respectively. Gray columns indicate heterozygous segment and dotted areas are regions of unknown genotype. DNA markers are represented based on the sequence position in the Arabidopsis genome. Responses to WMV were determined by DAS- ELISA in whole plants at 19 dpi. Data are from two to three independent experiments, each done with at least 10 plants per line.

Figure 3, cPG 2 protein sequence showing the single amino acid mutation distinguishing resistant and susceptible rwml alleles. The predicted N-terminal chlorop!ast targeting sequence is highlighted in bold and the putative bipartite NLS motif is underlined; it starts with two basic residues followed by a 10-residiie spacer and ends with three basic residues in the next five positions. The arrowhead points to residue 78, which is a glycine or serine in alleles of rwml conferring WMV resistance or susceptibility, respectively.

Figure 4. Alignment of the amino acid sequences of the N-terminal domain of cPGK proteins from several dicot (At, Arabidopsis thaliana; Kb, Nicotiana benthamiana; Nt, Nicoiiana tabacum; Si, Solarium lycopersicum; St, Solarium tuberosum; and Pt, Populus trichocarpa) and monocot plants (Sb, Sorghum bicolor; Os, Oryza sativa; Ta, Triticum aestivum; and Zm, Zea mays). Sequences were retrieved from the TAIR (At- 1 , At3g 12780 and At-2, Atlg56190), the SON (SI. SGN-U580583) or the NCBI databases (Nb. ADR71054; Nt, CAA88841; St- 1. ABB72817 and St-2, ABB86255; Pt-1 , XP_00231 5066 and Pt-2, XP 002312238; Sb. XP_002441 31 3; Os-1 , AAT07576 and Os-2, NP_ 001 172606; Ta. CAA51931 ; and Zm. NP_001 147628). At- 1 : SEQ ID NO: 4; At-2: SEQ ID NO: 5; Nb: SEQ ID NO: 6; Nt: SEQ ID NO: 7; SkSEQ ID NO: 8; St- 1 : SEQ ID NO: 9; St-2: SEQ ID NO: 10; Pt- 1 : SEQ ID NO: 1 1 ; Pt-2: SEQ ID NO: 12; Sb: SEQ ID NO: 13; Os-1 : SEQ ID NO: 14; Os-2: SEQ ID NO: 15; Ta: SEQ ID NO: 16; Zm: SEQ ID NO: 17). The arrowhead points to the highly conserved serine residue mutated in the Arabidopsis cPGK2 protein encoded by the rwml resistance allele.

Figure 5. cPGK2 transcript accumulation levels in the WMV-susceptible (Ler-1) and resistant (Cvi-0) accessions in the absence of WMV challenge. Total RNAs extracted from three-week old leaves were subjected to semi-quantitative RT-PCR using primers amplifying a 743 bp cPGK2 cDNA fragment. APT 1 was used as an internal control. The number of PCR cycles is indicated.

Figure 6. Virus-induced silencing of Nb-cPGK compromises WMV infection. ( A) Leaf mosaic symptom development in N. benthamiana plants infiltrated with the TRV:PGK5 and TRV:PGK3 constructs compared to the TRV:mCherry empty vector control. Pictures were taken 12 days post-infiltration. (B) Quantitative RT-PCR analysis of Nb-cPGK mRNA and WMV RNA accumulation in inoculated leaves of control plants (mock and empty vector), and TRV:PG 5 and TRV:PG 3-infiltrated plants. RNA levels were normalized to EF1 and expressed as fold-change relative to the mock control. Results shown are mean values ± SDs from two independent experiments, each involving three plants per treatment. (C) WMV CP accumulation assessed by DAS-ELISA using the same samples as in B. Results shown are average absorbance values ± SDs, The horizontal line depicts the average value of four healthy control samples (i.e., Α ₀₅ = 0,35). * indicate significant differences between TRV:PGK plants and the mock control (Student's t-test, P< 0.05).

EXAMPLES

Materials and Methods

Plant materials and growth conditions

Seeds of Arabidopsis accessions, the Ler/Cvi recombinant inbred line population (Alonso-Blanco et al, 1998) and the F2 progeny derived from the Bur-0 x Cvi-0 cross were provided by the Versailles Biological Resource Centre for Arabidopsis (http://dbsgap.versailles.inra.fr/vnat/). Cvi-0 plants were crossed with Ler-1 plants and the resulting Fl hybrids were self- fertilized to generate F2 populations. The Ler/Cvi near isogenic line population was kindly provided by J. Keurentjes from the Wageningen Agricultural University (The Netherlands) (Keurentjes et ah, 2007). Arabidopsis and N. benthamiana plants were grown in growth chambers with a 14-h photoperiod at 24°C day/18°C night and a 16-h photoperiod at 28°C day/24°C night, respectively.

Virus strains, inoculation and infectivity assays

WMV strains (FR, LL1, LL2, AUST89 and C05-270) were provided by H. Lecoq (INRA, Montfavet, France). Their geographic origin, serological and molecular properties have been described in Desbiez et al. (2007) and Desbiez and Lecoq (2008). Strains were maintained at 4°C according to the BOS (1969) procedure and multiplied on zucchini squash {Cucurbita pepo) prior to inoculation. Plants at rosette stage were mechanically inoculated with virus inocula prepared as described previously (Duprat et al., 2002). WMV infectivity assays were conducted using double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA) with ant i- WMV CP serum (S EDI AG, France). Plants were considered infected when the absorbance value of the sample was at least three times greater than the mean value of the healthy controls.

RT-PCR and quantitative real-time RT-PCR

Total RNA was isolated using TRI Reagent® (Sigma Aldrich, St-Louis, USA), treated with RQ1 RNase-free DNase (Promega, Madison, WI, USA), and ^g was reverse transcribed using ΙΟΟμΜ oligodTIS primers and AMV Reverse Transcriptase (Promega, Madison, WI, USA). Conventional RT-PCR for WMV was performed with primers specific for the VPg-coding sequence using GoTaq® DNA polymerase (Promega, Madison, WI, USA). Quantitative real-time RT-PCR for WMV was performed with primers specific for the CP-coding sequence using a MESA Green PCR kit from Eurogentec on the MX3005P System (Stratagene, Amsterdam, The Netherlands). Relative WMV RNA accumulation levels were calculated following the comparative delta-C method (Pfafil 2001) and normalized to the adenine ribosylphosphotransferase 1 and the elongation factor 1 gene levels in Arabidopsis and N. benthamiana, respectively (APT1-F/R and EF1-F/R; Rotenberg et al, 2006; Guenin et al.. 2009).

Marker development and rwml fine mapping

To fine map rwml, 12 markers polymorphic between her- 1 and Cvi-0, were developed from the Arabidopsis Bacterial Artificial Chromosome (BAG) sequences (www.arabidopsis.org). SSR motifs were retrieved from F7A10, F14C21 , F14J 16, F20N2 and T6H22 BAG sequences using the Sputnik SSR finder software (http://www.cbib.u~ bordeaux2.fr/outils/Pise/sputnik.html). Primers flanking the repeat sequences were generated with Primer 0.5 by selecting a minimal Tm of 57.5°C and amplicon sizes ranging from 100 to 200bp. SNPs and SSLPs mapping within the F6D8, F14G9, F25P12, T8L23 and T18A20 BAG regions were identified from the Arabidopsis polymorphism database available online (ftp://ftp.arabidopsis.org/Polymorphisms/), Flanking sequences were retrieved by BLAST analysis and primers were designed as described above. The ISBP N°16 marker was generated by subjecting a 458-Kb long interval (20963719bp-21422460bp) encompassing the rwml locus to the ISBP Finder software (Paux et al, 2010). They were used to score > 1,700 F2 plants issued from a cross between LCN 1 - 12 (WMV resistant) and Ler-1 (susceptible). Five recombinants were selected and F3 progenies were evaluated for their response to WMV infection.

cPGK2 sequence analysis

Genomic DNA was extracted from Arabidopsis leaf tissues as described previously (Simon et al. 2008). Specific amplification of the cPGK2 gene (2025-bp) was performed using six primer pairs (PGK-glF/R, PGK-g2F/R₅ PGK-g3F/R, PGK-g4F/R, PGK- g5F/R, and PGK-g6F/R). Specific amplification of a cPGK2 cDNA fragment (743-bp) encompassing the region of the missense mutation in Cvi-0 was performed using primers based on exons one and four. Each amplification product was sequenced twice (Gcnoscreen). Nucleotide and amino acid sequences were analyzed using multalin (Corpet. 1988).

Plasmid construction and plant transformation

The full-length ORF of the cPGK2 gene was amplified from total RNA of Ler-1 and Cvi-0 by RT-PCR using specific primers bearing attBl and attB2 recombination sites (PGK-tF/R) and cloned into pDONR207. The resulting plasmids were sequenced to check the integrity of the cloned fragments and inserts were subsequently recombined using the LR Clonase reaction (Invitrogen) into pMDC32 (Curtis et al, 2003) to generate binary vectors containing the cauliflower mosaic virus 35S promoter driving transgene expression. Cvi-0 plants were transformed using the floral dip method (Clough & Bent, 1998) with Agrobacierium t mefaciens strain C58 (pMP90). Primary transformants (Tl) were obtained after selection on Murashige skoog medium supplemented with 15 mg/L hygromycin (Kalys). To confirm the presence of the transgene, PGR was performed on genomic DNA using primers specifically designed to distinguish between endogenous and transgenic cPGK2 sequences. T2 seeds collected on individual Tl trans formants were sown on hygromycin- containing plates and seedlings transferred to soil were used for infectivity assays.

V1GS assay

The TRV-mediated VIGS system was used to knock down the expression of Nb-cPGK in N. benthamiana. The pTRV-1 and the Gateway-compatible pTRV2 vectors have been described (Liu et al., 2002a; Liu et al., 2002b). Two cDNA fragments, corresponding to nucleotides 54-535 and nucleotides 1039-1509 of the Nb-cPGK coding sequence were amplified from total RNA using the primer pairs PG 5-F/R and PGK3-F/R, respectively. A 215-bp fragment of rnCherry was amplified by PCR with primers mCherry-F/R from a synthetic plasmid construct (GenBank accession number AY678264). All fragments were cloned into pDONR™/Zeo. The resulting plasmids were verified by sequencing and then recombined using the LR Clonase reaction (Invitrogen) into pTRV2 to generate the pTRV2 PG 5, pTRV2_PGK3 and pTRV2_mCherry vectors. Cultures of A. tumefaciens strain C58PGV2260 (OD₆oo = containing pTRVl and the pTRV2 derivatives were mixed at a 1 : 1 ratio and infiltrated by syringe onto three young, fully expanded leaves of three- week old plants. Quantitative real-time RT-PCR for Nb-cPGK was performed as described above using primers designed outside the regions targeted for gene silencing.

Example 1: Identification of an Arabidopsis accession exhibiting broad-spectrum resistance to WMV

To identify genes controlling resistance to Watermelon Mosaic Virus, a set of 53 Arabidopsis accessions were evaluated for their ability to support systemic WMV infection. This set contains a core collection of 48 lines capturing over 90% of Arabidopsis genetic diversity (McKhann et al, 2004), and few additional lines previously reported as resistant against plus-strand RNA viruses (Simon et al., 1992; Takahashi et al., 1994; Martin Martin et al.. 1997; Revers et al, 2003; Decroocq et al., 2006). Infectivity assays were performed using two strains differing in their aggressiveness on susceptible cucurbit cultivars, WMV-FR and WMV-LL1 (Lecoq et al., 201 1) Because poty viruses do not always induce obvious symptoms in Arabidopsis, DAS-ELISA was performed to discriminate between WMV-susceptible and -resistant accessions. The responses of Arabidopsis accessions were classified according to the percentage of infected plants. The results are shown in Table I below. Table I

aResponse to WMV was determined by DAS-ELiSA in whole plants at 19 days post-inoculation (dpi). S+++: 100% of infected plants; S++: 55-85% of infected plants; S+: 15-45% of infected plants; R: complete resistance; nd: not determined. Data are from two to three independent experiments, each done with at least five plants per accession-strain combination.

Of the 97 accession-strain combinations tested, 62 (63.9%) were fully susceptible with WMV CP accumulation detected in all inoculated plants (referred to as S^++T). The majority of the remaining accession-strain combinations presented intermediate susceptibility phenotypes. Among these, 1 1 (1 1.3%) were classified as susceptible (S++) with WMV CP accumulation being detected in 55 to 85% of the inoculated plants, and 21 (21.6%) were considered as partially resistant (S+) with WMV accumulation in 15 to 45% of inoculated plants.

Only one accession showed resistance to both strains: Cvi-0 plants inoculated with WMV-FR or WMV-LLl did not present viral CP accumulation in any of the plants tested.

Cvi-0 was further challenged with three other WMV strains including LL2,

AUST89 and C05-207. Plants were considered infected when the DAS-ELISA absorbance value detemiined at 19 dpi was at least 3 times greater than the mean value of the healthy controls. Results are shown in Table II below.

Table II

These confirmed that whereas the Ler- 1 accession is fully susceptible to infection, Cvi-0 displays broad-spectrum resistance to WMV, although systemic viral accumulation could be detected at low frequency. ^ ^

Example 2: Local WMV RNA accumulation is impaired in Cvi-0

To determine which step of WMV infection is impaired in Cvi-0, RT-PCR experiments were performed on inoculated and non-inoculated upper systemic leaves from infected plants. The results are shown in Figure 1

Plants of the fully susceptible control Ler-l accumulated WMV RNA both locally and systemically, and displayed a stunted growth phenotype. By contrast, no visible symptom developed in Cvi-0 nor did plants accumulate detectable amounts of viral RNA in systemic leaves. However, several Cvi-0 inoculated plants presented WMV RNA accumulation in inoculated leaves (10 plants among 25) (Figure IB). Quantitative detection of WMV using real-time RT-PCR demonstrated that the amount of viral RNA in the infected inoculated leaves of Cvi-0 was 2.5-fold lower than in the inoculated leaves of Ler-l (Figure 1C). These results suggest that WMV infection is suppressed in Cvi-0 as a result of reduced or abolished viral accumulation in initially infected leaf tissues.

Example 3: Resistance to WMV is controlled by a recessive gene located on chromosome 1

The genetic basis of WMV resistance in Cvi-0 was analyzed (Table 3). The results are shown in Table III. WMV infection was evaluated by DAS-ELI SA in whole plants at 19 dpi (susceptible: A₄os > 0.39; resistant: A405 < 0.39). The results are shown in Table III.

Table III

Expected ratio

Crosses Generation Resistant Susceptible Chi-square

(R:S)

Bur-0 x Cvi-0 F₂ 21 58 1 :3 0.1 1 0,75

Ler-1 x Cvi-0 F₈ (RIL) 15 22 1 :1 2.19 0.25

''When P > 5%, the hypothesis that the observed segregation ratio is consistent with the expected ratio cannot be rejected.

The Fl progeny derived from a cross between Cvi-0 and Ler-1 was found to accumulate high levels of WMV CP, indicating that resistance is inherited as a recessive trait.

Of the 1 12 F2 plants, 21 were resistant and 91 susceptible. This result fits a 3 :3 ratio (χ²=2.61 and =0. 1 ) for a single recessive gene involved in resistance. Segregation analysis of Ler/Cvi recombinant inbred lines and F2 progenies of Cvi-0 crossed with Bur-0, another WMV-susceptible accession, gave similar results. We therefore conclude that a single recessive gene named rwml for resistance to watermelon mosaic virus i determines WMV resistance in Cvi-0.

The mapping of rwml was initiated using near-isogenic lines containing Cvi-0 segments of variable lengths introgressed into a Ler- l genetic background. Infectivity assays on a core set of 25 lines providing an almost complete genome-wide coverage (>90%) of the Arabidopsis genome identified a single line, LCN 1-22, with resistance to WMV (Figure 2A and data not shown). Therefore, rwml was located on chromosome 1 between the amplified fragment length polymorphism markers CC.82C-Col (map position 65.9cM) and DF.408C-Col (map position 87cM). In order to confirm and further delimit the rwml- containing interval, three additional introgression lines were challenged with WMV. Results obtained for LCN 1 -6 and LCN 1-12, containing Ler-1 and Cvi-0 alleles within the [CC.82C- Col - DF.408C-Col] interval respectively, confirmed the rwml map location, whereas analysis of LCN 1 -17, exhibiting recombination between CC.82C-Col and DF.408C-Col. allowed to further delimit rwml into a genomic region of 3-Mb from C1W1 to DF.408C-Col (Figure 2A). To obtain a higher resolution mapping of rwml, 12 additional markers including five simple sequence repeat (SSR) markers, four single nucleotide polymorphism (SNP) markers, two simple sequence length polymorphism (SSLP) markers and one insertion site based polymorphism (ISBP) marker that distinguish between Ler-1 and Cvi-0 within the [C1W1 - DF.408C-COI] interval were developed and used to screen an F2 population derived from a cross between LCN 1-12 and Ler-1. Subsequent analysis of F3 progenies exhibiting recombination between CI W l and DF.408C-Col revealed complete co-segregation between the SSR marker T6F122-SSR2 and the resistance locus, thereby assigning rwml to a 1 14-kb region between the F 14J 16-2 SSR marker and the F14G9-1 SNP marker (Figure 2B).

Example 4: A chloroplast phosphoglycerate kinase fePGK) is a candidate gene for rwml

According to The Arabidopsis Information Resource database (TAIR; www.arabidopsis.org), 30 annotated genes are present in the rwml interval. No gene encoding translation initiation factors was found within this region, but we identified a gene encoding a chloroplast phosphoglycerate kinase (cPGK2, Atl 561 0) belonging to a small gene family already demonstrated to be involved in host-virus interactions. The cPGK protein from Nicotiana henthamiana was shown to interact with the viral RNA of Bamboo mosaic virus (BaMV, genus Potexvirus) and to be required for efficient BaMV multiplication (Lin et al., 2007). In mammals, PGK has been reported to control Sendai virus replication (Ogino et al, 1999). Based on these findings, the role of cPGK2 in rwml -mediated resistance was further investigated.

The cPGK2 gene is 2,052-bp long, contains six exons, and encodes a predicted protein of 478 amino acids (Figure 3 and SEQ ID NO: 3). The deduced cPGK2 protein sequence exhibits several features that could influence functional properties; (/^*) a consensus sequence of a bipartite nuclear localization signal (NLS) is found between amino acids 76 and 92 (Dingwall and Laskey, 1991); (/?^') a putative serine phosphorylation site is present at position 78 (Reiland et al, 2009); and (Hi) the N-terminal region ranging from amino acids I to 73 is recognized as an import signal sequence of nucleus-encoded chloroplast-targeted proteins (Apweiler et al, 2004). A comparison of the genomic DNA region covering the full-length cPGK2 coding sequence from Let - 1 and Cvi-0 identified four single point mutations among which three silent mutations (G171 A, C458T and T2046C) and one missense mutation(A232G) resulting in an amino acid change from serine to glycine at position 78 (Figure 3). Interestingly, the S78G mutation occurs within the NLS motif and affects the putative serine phosphorylation site. It also targets an amino acid residue that is highly conserved across monocot and dicot cPGK proteins (Figure 4 and SEQ ID NOs 4-17).

No obvious differences in the abundance of the cPGK2 transcript were observed between non-inoculated Lcr- 1 and Cvi-0 plants, indicating that the S78G mutation does not affect the level of cPGK2 transcript accumulation (Figure 5). To investigate further the possible involvement of the S78G substitution in nv/n /-mediated resistance, sequencing of a 743 -bp fragment of the cPGK2 cDNA encompassing the mutation was performed on the Arabidopsis 48-core collection. Consistent with the results from the primary screen showing that Cvi-0 is the only accession displaying resistance to WMV infection, the S78G substitution could not be identified in any of the other accessions (data not shown).

Example 5: rwml encodes a chloroplast phosphogiycerate kinase required for efficient

WVM infection

Transgenic complementation was performed to provide a direct link between cPGK2 and rwtnl. The cauliflower mosaic virus 35S promoter was used to drive expression of the full-length cPGK2 open reading frame (ORF) from Ler- 1 that contains Ser 78, (P35S-cPGK2L) in the resistant Cvi-0 accession. As a control, Cvi-0 was transformed with a construct overexpressing its own cPGK2 ORF (P35S-cPGK2C), Tl plants were selected by resistance to hygromycin and the presence of the transgenes was checked by DNA genotyping. WMV tests was carried out on T2 plants using WMV-Fr strain. Non-transgenic Lcr- 1 and Cvi-0 plants, together with T2 progeny derived from hygromycin-resistant Tl transformants were inoculated with WMV. DAS-ELISA analysis was used to determine resistance/susceptibility to WMV infection at 19 dpi In non-transgenic and transgenic plants.

Three independent transgenic lines (T2) with 6 plants each were used for the assay. The results are shown in Table IV.

Table IV

These results show that the majority of Cvi-0 plants and P35S-cPGK2C transgenic plants displayed resistance (12 and 14 plants among 1 , respectively) whereas all Ler-1 plants and most ?35S-cPGK2L transformants (14 plants among 18) became systemically infected. Variability in the cPGK2 transgene expression levels among individual transgenic plants and incomplete penetrance of WMV resistance in Cvi-0 most likely explain the partial complementation phenotype. These results demonstrate that ectopic expression of the dominant susceptible cPGK2 allele restores the ability of WMV to infect plants containing the recessive rwml resistance allele, and therefore identify cPGK2 as rwml.

Natural recessive resistance to plant viruses has been associated with mutations in host factors required for infection. Thus, the involvement of chloroplast phosphoglycerate kinase in WMV accumulation was investigated by analyses of loss-of- function for the cPGK2 to see whether the lack of functional cPGK would result in resistance to WMV. In an initial attempt, we obtained an Arabidopsis mutant line, SALK_071724, containing a T-DNA tag inserted within the fifth exon of the cPGK2 gene (ABRC; www.arabidopsis.org/abrc). Consistent with previous findings (Myouga et ah, 2010), homozygous mutants lacking cPGK2 transcript accumulation exhibited a seedling-lethal albino phenotype that could not be used for WMV inoculation assays (data not shown).

Example 6: ePGK is required for efficient WMV multiplication in tobacco

Interrogation of the NCB1 protein database revealed a single cPGK ortholog in N. benthamiana that shares 84% amino acid sequence identity with Arabidopsis cPGK.2. Therefore, and also because N. benthamiana is a compatible host for WMV (Lecoq et al., 201 1), we performed Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) to assess whether knockdown of Nb-cPGK influences WMV accumulation. Three- week old N. benthamiana plants were agro-infiltrated with VIGS constructs targeting the 5' region (TRV:PGK5) or the 3' region (TRV:PG 3) of the Nb-cPGK coding sequence (Genbank accession number HQ450764). Plants that received infiltration buffer only or a TRV vector expressing a non-endogenous sequence (TRV:mCherry) served as mock and empty vector controls, respectively. Approximately one week post-infiltration . TRV:PGK plants started to display a mosaic of dark and light green patches onto the leaves above the infiltrated ones whilst control plants remained symptomless (Figure 6A). This phenotype is consistent with abnormal chloroplast functioning and indicated that Nb-cPGK was silenced. The upper leaves of TRV:PGK plants showing the mosaic pattern and the corresponding leaves of control plants were inoculated with WMV at 13 days post-infiltration, and comparative analysis were conducted six days later (Figure 6B). Quantitative RT-PCR showed that, compared to control plants, the Nb-cPGK mRNA level was reduced to about 12 and 5.5% for TRV:PGK5 and TRV:PGK3 plants, respectively. For TRV:PGK5 plants, WMV RNA accumulation was reduced to 6 and 12% of levels in the mock and empty vector controls, respectively, whereas it was reduced to 14 and 26% for TRV:PGK3 plants compared to the mock and empty vector controls, respectively. No significant differences were found in the control plants, indicating that decreased WMV infectivity was not due to agrobacterium or TRV infection. In parallel, DAS-ELISA experiments confirmed the reduced WMV accumulation in TRV:PG 5 and TRV:PG 3 plants (Figure 6C). Collectively, these data show that reduction in Nb-cPGK expression levels correlates with decreased accumulation of

WMV and demonstrate that cPGK is required for efficient WMV infection.

Discussion:

The above results reveal a major and yet undiscovered role of cPGK proteins in potyvirus infection, and point to a new function for natural virus resistance genes, clearly distinct from the well-known eIF4- mediated resistance.

Another key result from the present study was the demonstration that WMV resistance could be achieved by knocking down the expression of the cPGK ortholog in N. henthamiana, This indicates that the involvement of cPGK in WMV infection is conserved among different plant species. Correlated with this finding is that PPV resistance in the Arabidopsis Cvi accession is conferred by the recessive gene rpvl which maps into a genomic region overlapping the nvml map location (Decroocq et al, 2006). These data, together with those from an earlier work showing that Nb-cPGK is required for BaMV infection (Lin et al., 2007) indicate that recruitment of cPGK may be a strategy used by a range of RNA plant viruses representing different genera.

REFERENCES:

Ala-Poikela M, Goytia E, Haikonen T, Rajamaki ML, Valkonen JP (201 1) Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif. J Virol 85: 6784-6794

Alonso-BIanco C, Peeters AJ, Koornnecf M, Lister C, Dean C, van den Bosch N, Pot J, Kuiper MT ( 1998) Development of an AFLP based linkage map of Ler, Col and Cvi Arabidopsis thaliana ecotypes and construction of a Lcr/Cvi recombinant inbred line population. Plant J 14: 259-271

Anderson LE, Bryant JA, Carol AA (2004) Both chioroplastic and cytosolic phosphoglycerate kinase isozymes are present in the pea leaf nucleus. Protoplasma 223: 103- 1 10

Anderson LE, Wang X, Gibbons JT (1995) Three enzymes of carbon metabolism or their antigenic analogs in pea leaf nuclei. Plant Physiol 108: 659-667

Apwciler R, Bairoch A, Wu CH, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Hwang Ft, Lopez R, Magrane M, Martin MJ, Natale DA, O'Donovan C, Redaschi N, Yeh

LS (2004) UniProt: the Universal Protein knowledgebase. Nucleic Acids Res 32: D 15- 19 Bae MS, Cho EJ, Choi EY, Park OK (2003) Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J 36: 652-663

Brice DC, Bryant JA, Dambrauskas G, Drury SC, Littlechild JA (2004) Cloning and expression of cytosolic phosphoglycerate kinase from pea (Pisum sativum L.). J Exp Bot 55: 955-956

Bryant JA, Brice DC, Fitchett PN, Anderson LE (2000) A novel DNA-binding protein associated with DNA polymerase-alpha in pea stimulates polymerase activity on infrequently primed templates. J Exp Bot 51: 1945-1947

Burton S , Van 't Ho J, Bryant JA (1997) Novel DNA-binding characteristics of a protein associated with DNA polymerase-alpha in pea. Plant J 12: 357-365

Carr T, Whifham SA (2007) An emerging model system: Arabidopsis as a viral host plant. Plant Cell Monogr. 7: 359-183

Charron C (2007) Caracterisation fonctionnelie et evolution moleculaire des genes codant pour les facteurs d'initiation de la traduction eIF4E: des facteurs cles dans la resistance des plantes aux Potyvirus. Thesis, Universite Aix-Marseille II, Faculte des Sciences de Luminy Charron C, Nicolai M, Gallois JL, Robaglia C, Moury B, Palloix A, Caranta C (2008) Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg. Plant J 54: 56-68

Corpet F ( 1988) Multiple sequence alignement with hierarchical clustering. Nucleic Acids Res 16: 10881-10890

Curtis MD, Grossniklaus U (2003) A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol 133: 462-469. C lough SJ, Bent AF (1998) Floral dip; a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16; 735-743.

Dardick C (2007) Comparative expression profiling of Nicotiana benthamiana leaves systemically infected with three fruit tree viruses. Mol Plant Microbe Interact 20: 1004-1017 Decroocq V, Sicard 0, Alamillo JM, Lansac M, Eyquard JP, Garcia JA, Candresse T, Le Gall O, Revers F (2006) Multiple resistance traits control Plum pox virus infection in Arabidopsis thaliana. Mol Plant Microbe Interact 19: 541-549

Desbiez C, Costa C, Wipf-Scheibel C, Girard M, Lecoq H (2007) Serological and molecular variability of watermelon mosaic virus (genus Potyvirus). Arch Virol 152: 775-781 Desbiez C, Lecoq H (2008) Evidence for multiple intraspecific recombinants in natural populations of Watermelon mosaic virus (WMV, Potyvirus). Arch Virol 153: 1749-1754 Dingwall C, Laskey RA (1991) Nuclear targeting sequences— a consensus? Trends Biochem Sci 16: 478-481

Dufresne PJ, Thivierge K, Cotton S, Beauchemin C, Ide C, Ubalijoro E, Laliberte .IF, Fortin MG (2008) Heat shock 70 protein interaction with Turnip mosaic virus RNA- dependent RNA polymerase within virus-induced membrane vesicles. Virology 374: 217-227 Dufresne PJ, Ubalijoro E, Fortin MG, Laliberte JF (2008) Arabidopsis thaliana class II poly(A)-binding proteins are required for efficient multiplication of turnip mosaic virus. J Gen Virol 89: 2339-2348

Dun oyer P, Thomas C, Harrison S, Revers F, Manie A (2004) A cysteine-rich plant protein potentiates Potyvirus movement through an interaction with the virus genome-linked protein VPg. J Virol 78: 2301 -2309

Duprat A, Caranta C, Revers F, Menand B, Browning KS, Robaglia C (2002) The Arabidopsis eukaryotic initiation factor (iso)4E is dispensable for plant growth but required for susceptibility to potyviruses. Plant J 32: 927-934

Fu DY, Hong J, Chen JS, Wu JX (2004) Accumulation of coat protein of Turnip mosaic virus in host chloroplasts and its effect on PS II activity. Journal of Plant Physiology and Molecular Biology 30: 34-40 (In Chinese)

Gadh IP, Hari V ( 1986) Association of tobacco etch virus related RNA with chloroplasts in extracts of infected plants. Virology 150: 304-307

Guenin S, Mauriat M, Pelloux J, Van Wuytswinkel O, Bellini C, Gutierrez L (2009) Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references. J Exp Bot 60: 487-493

Gunasinghe UB, Berger PH ( 1991 ) Association of Potato virus Y gene products with chloroplasts in tobacco. Molecular Plant Microbe Interactions 4: 452-457

Guo DP, Guo YP, Zhao JP, Liu H, Peng Y, Wang QM, Chen JS, Rao GZ (2005) Photosynthetic rate and chlorophyll fluorescence in leaves of stem mustard (Brassica juncea var. tsatsai) alter turnip mosaic virus infection. Plant Science 1 8: 57-63 Haldeman-Cahill R, Daros JA, Carrington JC (1998) Secondary structures in the capsid protein coding sequence and 3' nontranslated region involved in amplification of the tobacco etch virus genome, J Virol 72: 4072-4079

Heazlewood JL, Tonti-FIlippini JS, Gout AM, Day DA, Whelan J, Millar AH (2004) Experimental analysis of the Arabidopsis mitochondiral proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins. , Plant Cell 16: 241256

Ho MY, Tang SJ, Ng WV, Yang W, Leu SJ, Lin YC, Feng CK, Sung JS, Sun KH (2010) Nucleotide-binding domain of phosphogly cerate kinase 1 reduces tumor growth by suppressing COX-2 expression. Cancer Sci 101 : 241 1 -2416

Huang TS, Wei T, Lalibertc JF, Wang A (2010) A host RNA helicase-like protein. AtRHS, interacts with the potyviral genome-linked protein, VPg, associates with the virus accumulation complex, and is essential for infection. Plant Physiol 152: 255-266

ang BC, Yearn I, Li H, Perez KW, Jahn MM. (2007) Ectopic expression of a recessive resistance gene generates dominant potyvirus resistance in plants. Plant Biotechnol J. 5: 526-36.

Keurentjes J.J, Bentsink L, Alonso-Blanco C, Hanhart CJ, Blankestijn-De Vries H, EfFgen S, Vreugdenhil D, Koornneef M (2007) Development of a near-isogenic line population of Arabidopsis thaliana and comparison of mapping power with a recombinant inbred line population. Genetics 175: 891 -905

Krishnan P, Gullen EA, Lam W, Dutschman GE, Grill SP, Cheng YC (2003) Novel role of 3-phosphogIycerate kinase, a glycolytic enzyme, in the activation of L-nucleoside analogs, a new class of anticancer and antiviral agents. J Biol Chem 278: 36726-36732

Kruskal H, Wallis WA ( 1952) Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47: 583-621

Lay AJ, Jiang XM, Kisker O, Flynn E, Underwood A, Condron R, Hogg PJ (2000) Phosphoglycerate kinase acts in tumour angiogenesis as a disulphide reductase. Nature 408: 869-873

Le Gall O, Aranda M, Caranta C (2011) Plant resistance to viruses mediated by translation initiation factors. In Recent advances in plant virology, pp 177-194. Edited by Caranta C, Aranda MA, Tepfer M, Lopez-Moya JL, Caister Academic Press

Lecoq H, Fab re F, Joannon B, Wipf-Scheibel C, Chandeysson C, Schoeny A, Desbiez C

(2011) Search for factors involved in the rapid shift in Watermelon mosaic virus (WMV) populations in South-eastern France. Virus Res 159: 1 15-123

Lee DW, Lee S, Lee GJ, Lee KH, Kim S, Cheong GW, Hwang 1 (2006) Functional characterization of sequence motifs in the transit peptide of Arabidopsis small subunit of rubisco. Plant Physiol 140: 466-483 Leonard S, Plante D, Wittmann S, Daigneault N, Fortin MG, LaJiberte JF (2000) Complex formation between poty irus VPg and translation eukaryotic initiation factor 4E correlates with virus infectivity. J Virol 74; 7730-7737

Lin JW, Ding MP, Hsu YH, Tsai CH (2007) Chloroplast phosphoglycerate kinase, a gluconeogenetic enzyme, is required for efficient accumulation of Bamboo mosaic virus. Nucleic Acids Res 35: 424-432

Liu Y, Schiff M, Dinesh-Kumar SP (2002a) Virus-induced gene silencing in tomato. Plant J 31: 777-786

Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP (2002b) Tobacco Rail, EDS! and NPRl NIMl like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30: 415-429

Martin Martin A, Martinez Herrera D, Cabrera y Poch IIL, Ponz F ( 1997) Variability in the interactions between Arabidopsis thaliana ecotypes and Oilseed Rape Mosaic Tobamo virus. Australian Journal of Plant Physiology 24: 275-2 1

Mayers CN, Lee KC, Moore CA, Won SM, Carr .IP (2005) Salicylic acid-induced resistance to Cucumber mosaic virus in squash and Arabidopsis thaliana: contrasting mechanisms of induction and antiviral action. Mol Plant Microbe Interact 18: 428-434

McKhann HI, Camilleri C, Berard A, Bataillon I , David JL, Reboud X, Le Corre V, Caloustian C, Gut IG, Brunei D (2004) Nested core collections maximizing genetic diversity in Arabidopsis thaliana. Plant J 38: 193-202

Mitra SK, Clouse SD, Goshe MB (2009) Enrichment and preparation of plasma membrane proteins from Arabidopsis thaliana for global proteomic analysis using liquid chromatography-tandem mass spectrometry. Methods Mol Biol 564: 341355

Myouga F, Akiyama K, Motohashi R, Kuromori T, Ito T, lizumi H, Ryusui R, Sakurai T, Shinozaki K (2010) The Chloroplast Function Database: a large-scale collection of Arabidopsis Ds/Spm- or T-DNA-tagged homozygous lines for nuclear-encoded chloroplast proteins, and their systematic phenotype analysis. Plant J 61 : 529-542

Ogino T, Iwama M, Kinouchi J, Shibagaki Y, Tsukamoto T, Mizumoto K (1999)

Involvement of a cellular glycolytic enzyme, phosphoglycerate kinase, in Sendai virus transcription. J Biol Chem 274: 35999-36008

Ogino T, Yamadera T, Nonaka T, Imajoh-Ohnii S, Mizumoto K (2001) Enolase, a cellular glycolytic enzyme, is required for efficient transcription f Sendai virus genome. Biochem Biophys Res Comraun 285: 447-455

Paux E, Faure S, Choulet F, Roger D, Gauthier V, Martinant .IP, Sourdine P, Balfourier F, Le Paslier MC, Chauveau A, Cakir M, Gandon B, Feuillet C (2010) Insertion site- based polymorphism markers open new perspectives for genome saturation and marker- assisted selection in wheat. Plant Biotechnol J 8: 196-210 Pavan S, Jacobsen E, Visser RG, Bai Y (2010) Loss of susceptibility as a novel breeding strategy for durable and broad -spectrum resistance. Mol Breed 25: 1 -12

Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45

Popanda O, Fox G, Thielmann HW (1 98) Modulation of DNA polymerases alpha, delta and epsilon by lactate dehydrogenase and 3-phosphoglycerate kinase. Biochim Biophys Acta 1397: 102-1 17

Rciland S, Messerli G, BaerenfalJer K, Gerrits B, Endler A, Grossmann J, Gruissem W,

Baginsky S (2009) Large-scale Arabidopsis phosphoproteome profiling reveals novel chloroplast kinase substrates and phosphorylation networks. Plant Physiol 150: 889-903

Rev ers F, Guiraud T, Houvcnaghel MC, Mauduit T, Le Gall O, Candresse T (2003) Multiple resistance phenotypes to Lettuce mosaic virus among Arabidopsis thaliana accessions. Mol Plant Microbe Interact 16: 608-616

Rotenberg D, Thompson TS, German TL, Willis DK (2006) Methods for effective real- time RT-PCR analysis of virus-induced gene silencing. J Virol Methods 138: 49-59

Sekiguchi H, Tacahashi Y, Uyeda I (2003) The 3' terminal region is strictly required for clover yellow vein virus genome replication. Arch Virol 148: 759-772

Serrano C, Gonzalez-Cruz J, Jauregui F, Medina C, Mantilla P, Mens JT, Arce- Johnson P (2008) Genetic and histological studies on the delayed systemic movement of Tobacco Mosaic Virus in Arabidopsis thaliana. BMC Genetics 9: 59

Shah N, Bradbecr JW (1994) The occurrence of chloroplastic and cytosolic isoenzymes of phosphoglycerate kinase in a range of plant species, Planta 193: 232-237

Shancr NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomelic red, orange and yellow fluorescent proteins derived from Disco so ma sp. red fluorescent protein. Nat Biotechnol 22: 1567-1572

Shetty S, Muniyappa H, Halady PK, Idell S (2004) Regulation of urokinase receptor expression by phosphoglycerate kinase. Am J Respir Cell Mol Biol 31: 100-106

Simon AE, Li XH, Lew JE, Stange R, Zhang C, Folacco M, Carpenter CD (1992) Susceptibility and resistance of Arabidopsis thaliana to Turnip Crinkle Virus. Molecular Plant Microbe Interactions 5: 496-503

Simon M, Loudet O, Durand S, Berard A, Brunei D, Sennesal FX, Durand-Tardif M, Pelletier G, Camilleri C (2008) Quantitative trait loci mapping in five new large recombinant inbred line populations of Arabidopsis thaliana genotyped with consensus single- nucleotide polymorphism markers. Genetics 178: 2253-2264

Stoimenova E, Marinova G, Garcia-Luque I (2005) Influence of virus concentration on expression of cucumber mosaic virus resistance in pepper. Biotechnology and Biotechnol ogical Equipment. Supplement Virology 19: 36-41 Takahashi H, Goto N, Ehara Y (1994) Hypersensitive response in cucumber mosaic virus- inoculated Arabidopsis thaliana. The Plant Journal 6: 369-377

Tccsi LI, Smith AM, Manie AJ, Leegood RC (1996) A Spatial Analysis of Physiological Changes Associated with Infection of Cotyledons of Marrow Plants with Cucumber Mosaic Virus. Plant Physiol 111: 975-985

Thivierge K, Cotton S, Dufresne PJ, Mathieu I, Beauchemin C, We C, Fortin MG, Laliberte JF (2008) Eukaryotic elongation factor 1 A interacts with Turnip mosaic virus RNA-dependent RNA polymerase and VPg-Pro in virus-induced vesicles. Virology 377: 216- 225

Troncoso-Ponce MA, Rivoal J, Venegas-Caleron M, Dorion S, Sanchez R, Cejudo FJ, Garces R, Martinez-Force E (2012) Molecular cloning and biochemical characterization of three phosphoglycerate kinase isoforms from developing sunflower (Helianthus annuus L.) seeds. Phytochemistry 79: 27-38

Truniger V, Aranda MA (2009) Recessive resistance to plant viruses. Adv Virus Res, 1 19- 159

Vish anatha JK, Jindal HK, Davis RG ( 1992 ) The role of primer recognition proteins in DNA replication: association with nuclear matrix in HeLa cells. J Cell Sci 101 ( Pt 1 ): 25-34 Wei T, Huang TS, McNeil J, Laliberte JF, Hong J, Nelson RS, Wang A (2010) Sequential recruitment of the endoplasmic reticulum and chloroplasts for plant poty virus replication. J Virol 84: 799-809

Wittmann S, Chatel H, Fortin MG, Laliberte JF ( 1997) Interaction of the viral protein genome linked of turnip mosaic poty virus with the translational eukaryotic initiation factor (iso) 4E of Arabidopsis thaliana using the yeast two-hybrid system. Virology 234: 84-92

Yamanaka T, Imai T, Satoh R, Kawashima A, Takahashi M, Tomita K, Kubota K, Meshi T, Naito S, Ishikawa M (2002) Complete inhibition of tobamovirus multiplication by simultaneous mutations in two homologous host genes. J Virol 75: 2491-2497

Yang C, Guo R, Jie F, Nettleton D, Peng J, Carr T, YeakJey JM, Fan JB, Whitham SA (2007) Spatial analysis of arabidopsis thaliana gene expression in response to Turnip mosaic virus infection. Mol Plant Microbe Interact 20: 358-370

Yearn I, Cavatorta JR, Ripa DR, Kang BC, Jahn MM (2007) Functional dissection of naturally occurring amino acid substitutions in eIF4E that confers recessive poty virus resistance in plants. Plant Cell 19: 2913-2928

Claims

CLAMS

1) Method for selecting a potyvirus- and/or potexvirus-resistant plant, characterized in that it comprises the detection, in plants to be tested:

- of the presence or of the absence of an allele of a cPGK gene encoding a chloroplastic phosphoglycerate kinase (cPGK) (hereinafter called: "wild-type cPGK allele"), wherein the mature form of the cPGK encoded by said allele (hereinafter called: "wi ld-type cPGK protein") has the N-terminai sequence MAK SVG (SEQ ID NO: 1);

- of the presence or of the absence of a mutant allele of said cPGK gene (hereinafter called: "mutant cPGK al lele"), wherein the mature form of the cPGK encoded by said allele (hereinafter called: "mutant cPGK protein") has the N-terminal sequence MAKKXVG (SEQ ID NO: 2), wherein X represents an amino-acid residue other than serine, preferably a non-phosphory atablc amino acid residue, and still more preferably a glycine residue;

and the selection of a plant in which the mutant cPGK allele is detected, and in which the wild-type cPGK allele is not detected.

2) Method for selecting a plant which can be used a a progenitor for obtaining a potyv irus-and/or potexvirus-resistant plant, characterized in that it comprises the detection, in plants to be tested, of the presence or of the absence of the mutant cPGK allele as defined in claim 1 , and the selection of the plants in which said mutant cPGK allele is detected.

3) Method for obtaining a plant which is resistant to a potyvirus and/or to a potexvirus, characterized in that it comprises the expression in said plant of a mutant cPGK protein as defined in claim 1 .

4) The method as claimed in any one of claims 1 to 3, characterized in that it is carried out on a plant chosen from Solanaceae, Cucurbitaceae, Cruciferae, Fabaceae, Compositae, and Rosaceae,

5) The method as claimed in any one of claims 1 to 4 wherein the potyvirus is selected among the Watermelon Mosaic Virus and the Plum Pox Virus.