BackgroundThe characterization of natural recessive resistance genes and Arabidopsis virus-resistant mutants have implicated translation initiation factors of the eIF4E and eIF4G families as susceptibility factors required for virus infection and resistance function.Methodology/Principal FindingsTo investigate further the role of translation initiation factors in virus resistance we set up a TILLING platform in tomato, cloned genes encoding for translation initiation factors eIF4E and eIF4G and screened for induced mutations that lead to virus resistance. A splicing mutant of the eukaryotic translation initiation factor, S.l_eIF4E1 G1485A, was identified and characterized with respect to cap binding activity and resistance spectrum. Molecular analysis of the transcript of the mutant form showed that both the second and the third exons were miss-spliced, leading to a truncated mRNA. The resulting truncated eIF4E1 protein is also impaired in cap-binding activity. The mutant line had no growth defect, likely because of functional redundancy with others eIF4E isoforms. When infected with different potyviruses, the mutant line was immune to two strains of Potato virus Y and Pepper mottle virus and susceptible to Tobacco each virus.Conclusions/SignificanceMutation analysis of translation initiation factors shows that translation initiation factors of the eIF4E family are determinants of plant susceptibility to RNA viruses and viruses have adopted strategies to use different isoforms. This work also demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. We have also developed a complete tool that can be used for both forward and reverse genetics in tomato, for both basic science and crop improvement. By opening it to the community, we hope to fulfill the expectations of both crop breeders and scientists who are using tomato as their model of study.

SUMMARYGenetic resistance to pathogens is important for sustainable maintenance of crop yields. Recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. Tomato (Solanum lycopersicum) and related species offer a model in which natural and TILLINGinduced potyvirus resistance alleles may be compared. For resistance based on translation initiation factor eIF4E1, we confirm that the natural allele Sh-eIF4E1 PI24 -pot1, isolated from the wild tomato species Solanum habrochaites, is associated with a wide spectrum of resistance to both potato virus Y and tobacco etch virus isolates. In contrast, a null allele of the same gene, isolated through a TILLING strategy in cultivated tomato S. lycopersicum, is associated with a much narrower resistance spectrum. Introgressing the null allele into S. habrochaites did not extend its resistance spectrum, indicating that the genetic background is not responsible for the broad resistance. Instead, the different types of eIF4E1 mutations affect the levels of eIF4E2 differently, suggesting that eIF4E2 is also involved in potyvirus resistance. Indeed, combining two null mutations affecting eIF4E1 and eIF4E2 re-establishes a wide resistance spectrum in cultivated tomato, but to the detriment of plant development. These results highlight redundancy effects within the eIF4E gene family, where regulation of expression alters susceptibility or resistance to potyviruses. For crop improvement, using loss-of-function alleles to generate resistance may be counter-productive if they narrow the resistance spectrum and limit growth. It may be more effective to use alleles encoding functional variants similar to those found in natural diversity.

Previous resistance analyses of Arabidopsis thaliana mutants knocked out for eukaryotic translation initiation factors showed that disruption of the At-eIF(iso)4E or both the AteIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against turnip mosaic virus (TuMV). This study selected TuMV virulent variants that overcame this resistance and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eIF(iso)4E and At-eIF(iso)4G1¾At-eIF(iso)4G2 knockout plants. As a VPg-eIF(iso)4E interaction has been shown previously to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMVs was performed. The results suggest that virulent TuMV variants may use an eIF4F-independent pathway.The eukaryotic translation initiation factors eIF4E and eIF4G play a key role during virus infection in plants ( Robaglia & Caranta, 2006). During mRNA translation, eIF4E provides the cap-binding function and is associated with eIF4G, the scaffold for the other components of the translation initiation complex, to form the eIF4F complex (Marcotrigiano et al., 1999). In plants, a second eIF4F complex called eIF(iso)4F is present, and results from the assembly of eIF(iso)4E and eIF(iso)4G isoforms (Browning, 2004). These two complexes perform essentially the same task in translation, but have different affinities for certain classes of mRNA substrates and are probably involved in different cellular events (Gallie & Browning, 2001). Plant genes encoding proteins from the eIF4F and eIF(iso)4F complexes belong to small gene families. In Arabidopsis thaliana, three genes encode the eIF4E subfamily proteins (eIF4E1, eIF4E2 and eIF4E3), one encodes eIF(iso)4E and one a non-canonical eIF4E-like protein, known as novel cap-binding protein (nCBP). A single gene encodes eIF4G, whilst two genes encode the eIF(iso)4G subfamily proteins [eIF(iso)4G1 and eIF(iso)4G2] ( Robaglia & Caranta, 2006). Viruses that depend on these factors to perform their infectious cycle include members of the genus Potyvirus, the largest and the most diverse genus of plant viruses (Adams et al., 2005;Shukla et al., 1994). Potyviruses are characterized by a positive-sense, single-stranded RNA genome of about 10 kb that encodes at least ten multifunctional proteins. The genome is 39-polyadenylated and covalently linked at its 59 end to a virus-encoded protein (VPg) (Murphy et al., 1991;Revers et al., 1999).Potyviruses may selectively use either the eIF4E or eIF(iso)4E isoform in A. thaliana to achieve a successful infection (Sato et al., 2005). We have demonstrated that potyviruses, including turnip mosaic virus (TuMV), selectively use members of the eIF4G family in a fashion that parallels the selective recruitment of eIF4E isoforms (Nicaise et al., 2007;Sato et al., 2005 ; data not shown).In order to confirm that each single amino acid change in the VPg cistron of TuMV determined its virulence on the At...

SUMMARYArabidopsis thaliana represents a valuable and efficient model to understand mechanisms underlying plant susceptibility to viral diseases. Here, we describe the identification and molecular cloning of a new gene responsible for recessive resistance to several isolates of Watermelon mosaic virus (WMV, genus Potyvirus) in the Arabidopsis Cvi-0 accession. rwm1 acts at an early stage of infection by impairing viral accumulation in initially infected leaf tissues. Map-based cloning delimited rwm1 on chromosome 1 in a 114-kb region containing 30 annotated genes. Positional and functional candidate gene analysis suggested that rwm1 encodes cPGK2 (At1g56190), an evolutionary conserved nucleus-encoded chloroplast phosphoglycerate kinase with a key role in cell metabolism. Comparative sequence analysis indicates that a single amino acid substitution (S78G) in the N-terminal domain of cPGK2 is involved in rwm1-mediated resistance. This mutation may have functional consequences because it targets a highly conserved residue, affects a putative phosphorylation site and occurs within a predicted nuclear localization signal. Transgenic complementation in Arabidopsis together with virus-induced gene silencing in Nicotiana benthamiana confirmed that cPGK2 corresponds to rwm1 and that the protein is required for efficient WMV infection. This work uncovers new insight into natural plant resistance mechanisms that may provide interesting opportunities for the genetic control of plant virus diseases.

Molecular markers linked to phenotypically important traits are of great interest especially when traits are difficult and/or costly to be observed. In tomato where a strong focus on resistance breeding has led to the introgression of several resistance genes, resistance traits have become important characteristics in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications. Evaluation of disease traits in biological assays is not always straightforward because assays are often influenced by environmental factors, and difficulties in scoring exist. In this study, we describe the development and/or evaluation of molecular marker assays for the Verticillium genes Ve1 and Ve2, the tomato mosaic virusTm1 (linked marker), the tomato mosaic virus Tm2 and Tm22 genes, the Meloidogyne incognita Mi1-2 gene, the Fusarium I (linked marker) and I2 loci, which are obligatory traits in PBR testing. The marker assays were evaluated for their robustness in a ring test and then evaluated in a set of varieties. Although in general, results between biological assays and marker assays gave highly correlated results, marker assays showed an advantage over biological tests in that the results were clearer, i.e., homozygote/heterozygote presence of the resistance gene can be detected and heterogeneity in seed lots can be identified readily. Within the UPOV framework for granting of PBR, the markers have the potential to fulfil the requirements needed for implementation in DUS testing of candidate varieties and could complement or may be an alternative to the pathogenesis tests that are carried out at present.Electronic supplementary materialThe online version of this article (doi:10.1007/s00122-009-1183-2) contains supplementary material, which is available to authorized users.

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.

Potyviruses are important plant pathogens that rely on many plant cellular processes for successful infection. TOR (target of rapamycin) signalling is a key eukaryotic energy-signalling pathway controlling many cellular processes such as translation and autophagy. The dependence of potyviruses on active TOR signalling was examined. Arabidopsis lines downregulated for TOR by RNAi were challenged with the potyviruses watermelon mosaic virus (WMV) and turnip mosaic virus (TuMV). WMV accumulation was found to be severely altered while TuMV accumulation was only slightly delayed. In another approach, using AZD-8055, an active site inhibitor of the TOR kinase, WMV infection was found to be strongly affected. Moreover, AZD-8055 application can cure WMV infection. In contrast, TuMV infection was not affected by AZD-8055. This suggests that potyviruses have different cellular requirements for active plant TOR signalling.

SummaryA study was carried out on the linkage relationship between the Frl locus carrying resistance to Fusarium oxysporum f.sp. radicis‐lycopersici and the Tm‐2 locus carrying resistance to several races of tobacco mosaic virus in the tomato inbred line IRB‐301‐31. The inbred line Motelle (Frl+/Frl+, Tm‐2+/Tm‐2+) was crossed with the inbred line IRB‐301‐31 (Frl/Frl, Tm‐2/Tm‐l). The resulting 222 F2 plants were selfed, and from each F3 family groups of 15–60 seedlings were tested for resistance to either F. oxysporum f.sp. radicis‐lycopersici or tobacco mosaic virus race 0. Segregation data indicated a very tight linkage between Frl and Tm‐2, equal to 5.1 ± 1.07 map units.