Allelic variation at the rpv1 locus controls partial resistance to Plum pox virus infection in Arabidopsis thaliana.

BACKGROUND: Sharka is caused by Plum pox virus (PPV) in stone fruit trees. In orchards, the virus is transmitted by aphids and by grafting. In Arabidopsis, PPV is transferred by mechanical inoculation, by biolistics and by agroinoculation with infectious cDNA clones. Partial resistance to PPV has been observed in the Cvi-1 and Col-0 Arabidopsis accessions and is characterized by a tendency to escape systemic infection. Indeed, only one third of the plants are infected following inoculation, in comparison with the susceptible Ler accession. RESULTS: Genetic analysis showed this partial resistance to be monogenic or digenic depending on the allelic configuration and recessive. It is detected when inoculating mechanically but is overcome when using biolistic or agroinoculation. A genome-wide association analysis was performed using multiparental lines and 147 Arabidopsis accessions. It identified a major genomic region, rpv1. Fine mapping led to the positioning of rpv1 to a 200 kb interval on the long arm of chromosome 1. A candidate gene approach identified the chloroplast phosphoglycerate kinase (cPGK2) as a potential gene underlying the resistance. A virus-induced gene silencing strategy was used to knock-down cPGK2 expression, resulting in drastically reduced PPV accumulation. CONCLUSION: These results indicate that rpv1 resistance to PPV carried by the Cvi-1 and Col-0 accessions is linked to allelic variations at the Arabidopsis cPGK2 locus, leading to incomplete, compatible interaction with the virus.

Evolution of plant eukaryotic initiation factor 4E (eIF4E) and potyvirus genome-linked protein (VPg): a game of mirrors impacting resistance spectrum and durability.

Polymorphism in the plant eukaryotic translation initiation factor 4E (eIF4E) and potyvirus genome-linked protein (VPg) determine, in many cases, the outcome of the confrontation between these two organisms: compatibility (i.e. infection of the plant by the virus) or incompatibility (i.e. resistance of the plant to the virus). The two interacting proteins eIF4E and VPg show strikingly similar evolution patterns. Most codon positions in their coding sequences are highly constrained for nonsynonymous substitutions but a small number shows evidence for positive selection. Several of these latter positions were shown to be functionally important, conferring resistance to the host or pathogenicity to the virus. Determining the mutational pathways involved in pepper eIF4E diversification revealed a link between an increase of the pepper resistance spectrum towards a panel of potyvirus species and an increase of durability of the resistance towards Potato virus Y. This relationship questions the interest of using more generally the spectrum of action of a plant resistance gene as a predictor of its durability potential.

Closely related poleroviruses depend on distinct translation initiation factors to infect Arabidopsis thaliana.

In addition to being essential for translation of eukaryotic mRNA, translation initiation factors are also key components of plant-virus interactions. In order to address the involvement of these factors in the infectious cycle of poleroviruses (aphid-transmitted, phloem-limited viruses), the accumulation of three poleroviruses was followed in Arabidopsis thaliana mutant lines impaired in the synthesis of translation initiation factors in the eIF4E and eIF4G families. We found that efficient accumulation of Turnip yellows virus (TuYV) in A. thaliana relies on the presence of eIF (iso)4G1, whereas Beet mild yellowing virus (BMYV) and Beet western yellows virus-USA (BWYV-USA) rely, instead, on eIF4E1. A role for these factors in the infectious processes of TuYV and BMYV was confirmed by direct interaction in yeast between these specific factors and the 5' viral genome-linked protein of the related virus. Although the underlying molecular mechanism is still unknown, this study reveals a totally unforeseen situation in which closely related viruses belonging to the same genus use different translation initiation factors for efficient infection of A. thaliana.

Identification of QTLs for Ralstonia solanacearum race 3-phylotype II resistance in tomato.

Resistance against a Ralstonia solanacearum race 3-phylotype II strain JT516 was assessed in a F(2:3) and a population of inbred lines (RIL), both derived from a cross between L. esculentum cv. Hawaii 7996 (partially resistant) and L. pimpinellifolium WVa700 (susceptible). Resistance criteria used were the percentage of wilted plants to calculate the AUDPC value, and bacterial colonization scores in roots and stem (hypocotyl and epicotyl) assessed in two independent greenhouse experiments conducted during the cool and hot seasons in Réunion Island, France. Symptoms were more severe during the cool season trials. Heritability estimates in individual seasons ranged from 0.82 to 0.88, depending on resistance criterion. A set of 76 molecular markers was used for quantitative trait loci (QTL) mapping using the single- and composite- interval mapping methods, as well as ANOVA. Four QTLs, named Bwr- followed by a number indicating their map location, were identified. They explained from 3.2 to 29.8% of the phenotypic variation, depending on the resistance criterion and the season. A major QTL, Bwr-6, and a minor one, Bwr-3, were detected in each season for all resistance criteria. Both QTLs showed stronger effects in the hot season than in the cool one. Their role in resistance to R. solanacearum race 3-phylotype II was subsequently confirmed in the RIL population derived from the same cross. Two other QTLs, Bwr-4 and Bwr-8, with intermediate and minor effects, respectively, were only detected in the hot season, demonstrating that environmental factors may strongly influence the expression of resistance against the race 3-phylotype II strain JT516. These QTLs were compared with those detected in the RIL population against race 1-phylotype I strain JT519 as well as those detected in other previous studies in the same genetic background against other race 1-phylotype I and II strains. This comparison revealed the possible occurrence of some phylotype-specific resistance QTLs in Hawaii 7996.

The recessive potyvirus resistance gene pot-1 is the tomato orthologue of the pepper pvr2-eIF4E gene.

The translation initiation factor 4E (eIF4E) has been implicated in naturally occurring resistance to Potato virus Y (PVY) determined by the pvr2 locus in pepper (Capsicum annuum). Here, the molecular basis of the recessive resistance to PVY and Tobacco etch virus (TEV) controlled by the pot-1 locus in tomato (Lycopersicon esculentum; now Solanum lycopersicum) was investigated. On the basis of genetic mapping data that indicated that pot-1 and pvr2 are located in syntenic regions of the tomato and pepper genomes, the possible involvement of eIF4E in pot-1-mediated resistance was assessed. Genetic mapping of members of the eIF4E multigenic family in tomato introgression lines revealed that an eIF4E locus indeed maps in the same genomic region as pot-1. By comparing eIF4E coding sequences between resistant and susceptible Lycopersicon genotypes, a small number of polymorphisms that co-segregate with the pot-1 locus were identified, suggesting that this gene could be involved in resistance to potyviruses. Functional complementation experiments using Potato virus X-mediated transient expression of eIF4E from a susceptible genotype in a resistant pepper genotype confirmed that a small number of amino acid substitutions in the eIF4E protein indeed account for resistance/susceptibility to both the PVY and TEV, and consequently that pot-1 and pvr2 are orthologues. Taken together, these results support the role of this eIF4E gene as a key component of recessive resistance to potyviruses, and validate the comparative genomic approach for the molecular characterization of recessive resistance genes.

Comparative mapping of Phytophthora resistance loci in pepper germplasm: evidence for conserved resistance loci across Solanaceae and for a large genetic diversity.

Phytophthora capsici Leonian, known as the causal agent of the stem, collar and root rot, is one of the most serious problems limiting the pepper crop in many areas in the world. Genetic resistance to the parasite displays complex inheritance. Quantitative trait locus (QTL) analysis was performed in three intraspecific pepper populations, each involving an unrelated resistant accession. Resistance was evaluated by artificial inoculations of roots and stems, allowing the measurement of four components involved in different steps of the plant-pathogen interaction. The three genetic maps were aligned using common markers, which enabled the detection of QTLs involved in each resistance component and the comparison of resistance factors existing among the three resistant accessions. The major resistance factor was found to be common to the three populations. Another resistance factor was found conserved between two populations, the others being specific to a single cross. This comparison across intraspecific germplasm revealed a large variability for quantitative resistance loci to P. capsici. It also provided insights both into the allelic relationships between QTLs across pepper germplasm and for the comparative mapping of resistance factors across the Solanaceae.

Towards the saturation of the pepper linkage map by alignment of three intraspecific maps including known-function genes.

Three populations composed of a total of 215 doubled haploid lines and 151 F2 individuals were used to design an intraspecific consensus map of pepper (Capsicum annuum L.). The individual maps varied from 685 to 1668 cM with 16 to 20 linkage groups (LGs). The alignment of the three individual maps permitted the arrangement of 12 consensus major linkage groups corresponding to the basic chromosome number of pepper and displaying a complex correspondence with the tomato map. The consensus map contained 100 known-function gene markers and 5 loci of agronomic interest (the disease-resistance loci L, pvr2, and Pvr4; the C locus, which determines capsaicin content; and the up locus, controlling the erect habit of the fruits). The locations of three other disease-resistance loci (Tsw, Me3, and Bs3) and the y locus, which determines the yellow fruit colour, were also found on this consensus map thanks to linked markers. Here we report on the first functional detailed map in pepper. The use of candidate gene sequences as genetic markers allowed us to localize four clusters of disease-resistance gene analogues and to establish syntenic relationships with other species.

QTLs involved in the restriction of cucumber mosaic virus (CMV) long-distance movement in pepper.

Partial restriction of cucumber mosaic virus (CMV) long-distance movement originating from the Capsicum annuum inbred line 'Vania' was assessed in a doubled-haploid progeny using two screening methods: the first allowed one to assess the resistance of adult plants decapitated above the fourth leaf and inoculated on the third leaf using a common CMV strain, and the second allowed one to assess CMV resistance to long-distance movement on seedlings inoculated using an atypical CMV strain. For both resistance tests, the behavior of the F(1) hybrid between 'Vania' and the susceptible line 'H3' indicated that partial resistance is inherited as a dominant trait. Phenotypic data from the two screening methods were correlated but the one performed on seedlings was much more severe. A subset of 184 molecular markers well-distributed over the pepper genome was selected for QTL mapping using the composite interval mapping (CIM) method. A total of seven genomic regions, including one major effect and several minor effect QTLs, were shown to be associated with partial restriction of CMV long-distance movement. These results are compared with those already obtained in pepper and also in other solanaceous crops, potato and tomato.

Recessive resistance genes against potyviruses are localized in colinear genomic regions of the tomato ( Lycopersicon spp.) and pepper ( Capsicum spp.) genomes.

Resistance against both Potato virus Y (PVY) and Tobacco etch virus (TEV) was identified in the wild tomato relative Lycopersicon hirsutum PI247087. Analysis of the segregation ratio in F(2)/F(3) and BC(1) interspecific progenies indicated that a single recessive gene, or two very tightly linked recessive loci, are involved in resistance to both potyviruses. This locus was named pot-1. Using amplified fragment length polymorphism markers and a set of L. hirsutum introgression lines, pot-1 was mapped to the short arm of tomato chromosome 3, in the vicinity of the recessive py-1 locus for resistance to corky root rot. Because of the occurrence of phenotypically similar genes in pepper ( Capsicum spp.), the comparative genetics of resistance to potyviruses between tomato and pepper was investigated. Unlike most of the comparative genetic studies on resistance genes, pot-1 was tightly flanked by the same restriction fragment length polymorphism (RFLP) markers than the pvr2/pvr5 locus for resistance to PVY and TEV from pepper. These results may indicate that recessive resistance genes against potyviruses evolve less rapidly than the majority of the dominant genes cloned so far, and consequently may belong to a different family of resistance genes.

Disease resistance gene analogs as candidates for QTLs involved in pepper-pathogen interactions.

Whereas resistance genes (R-genes) governing qualitative resistance have been isolated and characterized, the biological roles of genes governing quantitative resistance (quantitative trait loci, QTLs) are still unknown. We hypothesized that genes at QTLs could share homologies with cloned R-genes. We used a PCR-based approach to isolate R-gene analogs (RGAs) with consensus primers corresponding with conserved domains of cloned R-genes: (i) the nucleotide binding site (NBS) and hydrophobic domain, and (ii) the kinase domain. PCR-amplified fragments were sequenced and mapped on a pepper intraspecific map. NBS-containing sequences of pepper, most similar to the N gene of tobacco, were classified into seven families and all mapped in a unique region covering 64 cM on the Noir chromosome. Kinase domain containing sequences and cloned R-gene homologs (Pto, Fen, Cf-2) were mapped on four different linkage groups. A QTL involved in partial resistance to cucumber mosaic virus (CMV) with an additive effect was closely linked or allelic to one NBS-type family. QTLs with epistatic effects were also detected at several RGA loci. The colocalizations between NBS-containing sequences and resistance QTLs suggest that the mechanisms of qualitative and quantitative resistance may be similar in some cases.

Development of a CAPS marker for the Pvr4 locus: a tool for pyramiding potyvirus resistance genes in pepper.

The Pvr4 resistance gene in pepper confers a complete resistance to the three pathotypes of potato virus Y (PVY) and to pepper mottle virus (PepMoV). In order to use this gene in a marker-assisted selection (MAS) program and to permit the pyramiding of several potyvirus resistance genes in the same cultivar, tightly linked amplified fragment length polymorphism (AFLP) markers were obtained by the bulked segregant analysis method. Eight linked AFLP markers were mapped in an interval from 2.1 +/- 0.8 to 13.8 +/- 2.9 cM around this locus. The closest codominant AFLP marker was converted into a codominant CAPS (cleaved amplified polymorphic sequence) marker using data from the alignment of the two allele sequences. We have further characterized the relevance of the CAPS marker for MAS programs in different pepper breeding lines.

Both common and specific genetic factors are involved in polygenic resistance of pepper to several potyviruses.

Absolute resistance to potato virus Y pathotype 0 (PVY 0), potyvirus E and chili veinal mottle virus (CVMV) and a partial resistance to potato virus Y pathotype 1,2 (PVY 1,2) were found in an Indian pepper line, 'Perennial'. In the doubled haploid (DH) progeny from the F1 of a cross 'Perennial' by 'Yolo Wonder', resistance to CVMV was confered by two independent genes, one with a clear dominant effect. Resistance to PVY and potyvirus E was quantitatively expressed and controlled by several recessive genetic factors. Genetic analysis showed that fewer resistance factors were necessary to explain resistance to PVY (0) and potyvirus E than resistance to PVY(1,2). Genetic correlations between resistances to the different potyviruses in the DH progeny showed that most of genetic factors involved in PVY(0) resistance appear to be also involved in potyvirus E resistance, and some of these polyvalent factors may be also involved in PVY(1,2) resistance but, in this case, additional specific genes were necessary. One of the two CVMV resistance genes seems to be implicated in potyvirus E resistance. Thus, the polygenic resistance of 'Perennial' to these potyviruses was due both to polyvalent genetic factors, i.e. factors that apparently interact with several viruses, and strain-specific genetic factors.

Construction of an intraspecific integrated linkage map of pepper using molecular markers and doubled-haploid progenies.

An integrated molecular linkage map of pepper (Capsicum annuum L.), including mainly RFLP and RAPD markers, has been constructed by alignment of three intraspecific linkage maps generated by segregating doubled-haploid progenies. A total of 85 markers covered approximately 820 cM in 14 linkage groups. Four linkage groups were assigned to 4 chromosomes. Two new genes of agronomic interest were located: L controlling hypersensitive resistance to TMV and up controlling the erect habit of the fruits. The C gene controlling the fruit pungency was more precisely located. This map is estimated to represent from 36 to 59% of the total pepper genome. An examination of segregation data has revealed several genomic regions with aberrant segregation ratios often favouring the agronomic big-fruited parents, particularly in crosses involving the exotic parent CM334, suggesting that these genome regions are subjected to selection during the process of doubled-haploid production. The suitability of doubled-haploid progenies for mapping projects and the differences observed between this intraspecific integrated map with earlier published interspecific pepper maps are discussed.