Identification of a Common Epitope in Nucleocapsid Proteins of Euro-America Orthotospoviruses and Its Application for Tagging Proteins.

The NSs protein and the nucleocapsid protein (NP) of orthotospoviruses are the major targets for serological detection and diagnosis. A common epitope of KFTMHNQIF in the NSs proteins of Asia orthotospoviruses has been applied as an epitope tag (nss-tag) for monitoring recombinant proteins. In this study, a monoclonal antibody TNP MAb against the tomato spotted wilt virus (TSWV) NP that reacts with TSWV-serogroup members of Euro-America orthotospoviruses was produced. By truncation and deletion analyses of TSWV NP, the common epitope of KGKEYA was identified and designated as the np sequence. The np sequence was successfully utilized as an epitope tag (np-tag) to monitor various proteins, including the green fluorescence protein, the coat protein of the zucchini yellow mosaic virus, and the dust mite chimeric allergen Dp25, in a bacterial expression system. The np-tag was also applied to investigate the protein-protein interaction in immunoprecipitation. In addition, when the np-tag and the nss-tag were simultaneously attached at different termini of the expressed recombinant proteins, they reacted with the corresponding MAbs with high sensitivity. Here, we demonstrated that the np sequence and TNP MAb can be effectively applied for tagging and detecting proteins and can be coupled with the nss-tag to form a novel epitope-tagging system for investigating protein-protein interactions.

Development of a Platform for Noncovalent Coupling of Full Antigens to Tobacco Etch Virus-Like Particles by Means of Coiled-Coil Oligomerization Motifs.

Virus-like particles are excellent inducers of the adaptive immune response of humans and are presently being used as scaffolds for the presentation of foreign peptides and antigens derived from infectious microorganisms for subunit vaccine development. The most common approaches for peptide and antigen presentation are translational fusions and chemical coupling, but some alternatives that seek to simplify the coupling process have been reported recently. In this work, an alternative platform for coupling full antigens to virus-like particles is presented. Heterodimerization motifs inserted in both Tobacco etch virus coat protein and green fluorescent protein directed the coupling process by simple mixing, and the obtained complexes were easily taken up by a macrophage cell line.

A cell wall-localized NLR confers resistance to Soybean mosaic virus by recognizing viral-encoded cylindrical inclusion protein.

Soybean mosaic virus (SMV) causes severe yield losses and seed quality reduction in soybean (Glycine max) production worldwide. Rsc4 from cultivar Dabaima is a dominant genetic locus for SMV resistance, and its mapping interval contains three nucleotide-binding domain leucine-rich repeat-containing (NLR) candidates (Rsc4-1, Rsc4-2, and Rsc4-3). The NLR-type resistant proteins were considered as important intracellular pathogen sensors in the previous studies. In this study, based on transient expression assay in Nicotiana benthamiana leaves, we found that the longest transcript of Rsc4-3 is sufficient to confer resistance to SMV, and CRISPR/Cas9-mediated editing of Rsc4-3 in resistant cultivar Dabaima compromised the resistance. Interestingly, Rsc4-3 encodes a cell-wall-localized NLR-type resistant protein. We found that the internal polypeptide region responsible for apoplastic targeting of Rsc4-3 and the putative palmitoylation sites on the N terminus are essential for the resistance. Furthermore, we showed that viral-encoded cylindrical inclusion (CI) protein partially localizes to the cell wall and can interact with Rsc4-3. Virus-driven or transient expression of CI protein of avirulent SMV strains is enough to induce resistance response in the presence of Rsc4-3, suggesting that CI is the avirulent gene for Rsc4-3-mediated resistance. Taken together, our work identified a unique NLR that recognizes plant virus in the apoplast, and provided a simple and effective method for identifying resistant genes against SMV infection.

Responses of Passiflora spp. to cowpea aphid-borne mosaic virus reveal infection in asymptomatic plants and new species with probable immunity.

Passion fruit woodiness disease (PWD), caused by cowpea aphid-borne mosaic virus (CABMV), produces socioeconomic problems in Brazil. The objectives of this study were to i) evaluate the temporal progression of PWD, ii) identify Passiflora genotypes with resistance to CABMV, and iii) detect virus infection in asymptomatic plants by reverse transcription quantitative polymerase chain reaction (RT-qPCR) in cases where standard RT-PCR detection failed. The experiment was conducted in a greenhouse using 128 genotypes belonging to 12 species and three hybrids (inter- and intraspecific) of Passiflora, evaluated at five time points after inoculation. Progression rates and disease severity were lower in P. cincinnata, P. gibertii, P. miersii, and P. mucronata than in P. edulis, P. alata, Passiflora sp., and hybrids. Of the genotypes tested, 20.31% were resistant, especially the accessions of P. suberosa, P. malacophylla, P. setacea, P. pohlii, and P. bahiensis, which remained asymptomatic throughout the experiment. The absence of symptoms does not imply immunity of plants to the virus, since RT-qPCR analysis confirmed infection by the virus in asymptomatic plants of P. cincinnata, P. gibertii, P. miersii, P. mucronata, P. setacea, P. malacophylla, and P. suberosa. Even after four inoculations, the virus was not detected by RT-qPCR in the upper leaves in plants of the species P. pohlii and P. bahiensis, indicating that these species are probably immune to CABMV.

Gene expression profiling of papaya (Carica papaya L.) immune response induced by CTS-N after inoculating PLDMV.

Plant immune regulation is a defensive strategy of plants for protection against pathogen invasion, and Chitosan-N (CTS-N) can induce plant autoimmunity regulation mechanisms. CTS-N was found to induce an immunomodulatory response in papaya against Papaya leaf-distortion mosaic virus (PLDMV). To date, the gene expression profile of CTS-N-induced papaya immunomodulatory response has not been reported. Here, the transcriptional map of papaya leaf genes were subjected to three treatments, viz., non-viral inoculation without CTS-N treatment (CK), virus inoculation without CTS-N treatment (CG), and virus inoculation of 1 g/L treatment (B). These were studied by pot culture experiment. Comparison of the B group with the CG group revealed 732 upregulated and 510 downregulated genes. Comparison of the CG group with the CK group revealed 909 upregulated and 1024 downregulated genes. To determine gene function, gene ontology (GO) analysis was performed, where 480 biological process genes, 256 molecular function genes, and 343 cell composition genes were differentially expressed. Kyoto Encyclopedia of Genes and Genomes (KEGG) results revealed that the top three pathways were phenylpropane biosynthesis, starch and sucrose metabolism, and plant hormone signal transduction. Real-time Quantitative PCR (qPCR) results were consistent with the transcriptome results, with a correlation coefficient of 0.87. The results of the transcriptional group showed that genes associated with plant resistance were induced by CTS-N-treatment in papaya. The chitinase gene was related to the plant disease process. Related genes in plant hormone signal transduction pathways are associated with plant resistance, and six differentially expressed genes were correlated with enhanced immune resistance in papaya.

Differential Accumulation of Innate- and Adaptive-Immune-Response-Derived Transcripts during Antagonism between Papaya Ringspot Virus and Papaya Mosaic Virus.

Papaya ringspot virus (PRSV), a common potyvirus infecting papaya plants worldwide, can lead to either antagonism or synergism in mixed infections with Papaya mosaic virus (PapMV), a potexvirus. These two unrelated viruses produce antagonism or synergism depending on their order of infection in the plant. When PRSV is inoculated first or at the same time as PapMV, the viral interaction is synergistic. However, an antagonistic response is observed when PapMV is inoculated before PRSV. In the antagonistic condition, PRSV is deterred from the plant and its drastic effects are overcome. Here, we examine differences in gene expression by high-throughput RNA sequencing, focused on immune system pathways. We present the transcriptomic expression of single and mixed inoculations of PRSV and PapMV leading to synergism and antagonism. Upregulation of dominant and hormone-mediated resistance transcripts suggests that the innate immune system participates in synergism. In antagonism, in addition to innate immunity, upregulation of RNA interference-mediated resistance transcripts suggests that adaptive immunity is involved.

Soybean RNA interference lines silenced for eIF4E show broad potyvirus resistance.

Soybean mosaic virus (SMV), a potyvirus, is the most prevalent and destructive viral pathogen in soybean-planting regions of China. Moreover, other potyviruses, including bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), also threaten soybean farming. The eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in controlling resistance/susceptibility to potyviruses in plants. In the present study, much higher SMV-induced eIF4E1 expression levels were detected in a susceptible soybean cultivar when compared with a resistant cultivar, suggesting the involvement of eIF4E1 in the response to SMV by the susceptible cultivar. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that soybean eIF4E1 interacted with SMV VPg in the nucleus and with SMV NIa-Pro/NIb in the cytoplasm, revealing the involvement of VPg, NIa-Pro, and NIb in SMV infection and multiplication. Furthermore, transgenic soybeans silenced for eIF4E were produced using an RNA interference approach. Through monitoring for viral symptoms and viral titers, robust and broad-spectrum resistance was confirmed against five SMV strains (SC3/7/15/18 and SMV-R), BCMV, and WMV in the transgenic plants. Our findings represent fresh insights for investigating the mechanism underlying eIF4E-mediated resistance in soybean and also suggest an effective alternative for breeding soybean with broad-spectrum viral resistance.

Soybean antiviral immunity conferred by dsRNase targets the viral replication complex.

Eukaryotic positive-strand RNA viruses replicate their genomes in membranous compartments formed in a host cell, which sequesters the dsRNA replication intermediate from antiviral immune surveillance. Here, we find that soybean has developed a way to overcome this sequestration. We report the positional cloning of the broad-spectrum soybean mosaic virus resistance gene Rsv4, which encodes an RNase H family protein with dsRNA-degrading activity. An active-site mutant of Rsv4 is incapable of inhibiting virus multiplication and is associated with an active viral RNA polymerase complex in infected cells. These results suggest that Rsv4 enters the viral replication compartment and degrades viral dsRNA. Inspired by this model, we design three plant-gene-derived dsRNases that can inhibit the multiplication of the respective target viruses. These findings suggest a method for developing crops resistant to any target positive-strand RNA virus by fusion of endogenous host genes.

Immunocapture of virions with virus-specific antibodies prior to high-throughput sequencing effectively enriches for virus-specific sequences.

Virus discovery based on high-throughput sequencing relies on enrichment for virus sequences prior to library preparation to achieve a sufficient number of viral reads. In general, preparations of double-stranded RNA or total RNA preparations treated to remove rRNA are used for sequence enrichment. We used virus-specific antibodies to immunocapture virions from plant sap to conduct cDNA synthesis, followed by library preparation and HTS. For the four potato viruses PLRV, PVY, PVA and PYV, template preparation by virion immunocapture provided a simpler and less expensive method than the enrichment of total RNA by ribosomal depletion. Specific enrichment of viral sequences without an intermediate amplification step was achieved, and this high coverage of sequences across the viral genomes was important to identify rare sequence variations. Using this approach, the first complete genome sequence of a potato yellowing virus isolate (PYV, DSMZ PV-0706) was determined in this study. PYV can be confidently assigned as a distinct species in the genus Ilarvirus.

Over-expression of GmKR3, a TIR-NBS-LRR type R gene, confers resistance to multiple viruses in soybean.

KEY MESSAGE: That overexpression of GmKR3 enhances innate virus resistance by stimulating. Soybean mosaic virus (SMV) is found in many soybean production areas, and SMV infection is one of the prevalent viral diseases that can cause significant yield losses in soybean. In plants, resistance (R) genes are involved in pathogen reorganization and innate immune response activation. Most R proteins have nucleotide-binding site and leucine-rich repeat (NBS-LRR) domains, and some of the NBS-LRR type R proteins in dicots have Toll/Interleukin-1 Receptor (TIR) motifs. We report here the analysis of the over-expression of GmKR3, a soybean TIR-NBS-LRR type R gene on virus resistance in soybean. When over-expressed in soybean, GmKR3 enhanced the plant's resistance to several strains of SMV, the closely related potyviruses bean common mosaic virus (BCMV) and watermelon mosaic virus (WMV), and the secovirus bean pod mottle virus (BPMV). Importantly, over-expression of GmKR3 did not affect plant growth and development, including yield and qualities of the seeds. HPLC analysis showed that abscisic acid (ABA) content increased in the 35S:GmKR3 transgenic plants, and in both wild-type and 35S:GmKR3 transgenic plants in response to virus inoculation. Consistent with this observation, we found that the expression of two ABA catabolism genes was down-regulated in 35S:GmKR3 transgenic plants. We also found that the expression of Gm04.3, an ABA responsive gene encoding BURP domain-containing protein, was up-regulated in 35S:GmKR3 transgenic plants. Taken together, our results suggest that overexpression of GmKR3 enhanced virus resistance in soybean, which was achieved at least in part via ABA signaling.

RNA decay is an antiviral defense in plants that is counteracted by viral RNA silencing suppressors.

Exonuclease-mediated RNA decay in plants is known to be involved primarily in endogenous RNA degradation, and several RNA decay components have been suggested to attenuate RNA silencing possibly through competing for RNA substrates. In this paper, we report that overexpression of key cytoplasmic 5'-3' RNA decay pathway gene-encoded proteins (5'RDGs) such as decapping protein 2 (DCP2) and exoribonuclease 4 (XRN4) in Nicotiana benthamiana fails to suppress sense transgene-induced post-transcriptional gene silencing (S-PTGS). On the contrary, knock-down of these 5'RDGs attenuates S-PTGS and supresses the generation of small interfering RNAs (siRNAs). We show that 5'RDGs degrade transgene transcripts via the RNA decay pathway when the S-PTGS pathway is disabled. Thus, RNA silencing and RNA decay degrade exogenous gene transcripts in a hierarchical and coordinated manner. Moreover, we present evidence that infection by turnip mosaic virus (TuMV) activates RNA decay and 5'RDGs also negatively regulate TuMV RNA accumulation. We reveal that RNA silencing and RNA decay can mediate degradation of TuMV RNA in the same way that they target transgene transcripts. Furthermore, we demonstrate that VPg and HC-Pro, the two known viral suppressors of RNA silencing (VSRs) of potyviruses, bind to DCP2 and XRN4, respectively, and the interactions compromise their antiviral function. Taken together, our data highlight the overlapping function of the RNA silencing and RNA decay pathways in plants, as evidenced by their hierarchical and concerted actions against exogenous and viral RNA, and VSRs not only counteract RNA silencing but also subvert RNA decay to promote viral infection.

Genome-wide identification of the auxin/indole-3-acetic acid (Aux/IAA) gene family in pepper, its characterisation, and comprehensive expression profiling under environmental and phytohormones stress.

Auxin is an essential phytohormone that plays a crucial role in the growth and development of plants in stressful environments. Here, we analysed the auxin/indole-3-acetic acid (Aux/IAA) gene family, which produces auxin in pepper, and succeeded in identifying 27 putative members containing four conserved domains (I. II. III and IV) in their protein sequences. Sequence analysis, chromosomal mapping and motif prediction of all identified CaAux/IAA genes were performed. It was observed that these genes contained four conserved motifs divided into nine different groups and distributed across nine chromosomes in pepper plants. RNA-seq analysis revealed the organ specific expression of many CaAux/IAA genes. However, the majority of genes were expressed with high expression levels in the early stages of fruit development. However, the maximum expression level of the CA03g34540 gene was observed in the breaker stage. Moreover, thirteen CaAux/IAA genes were labelled as early responsive genes to various phytohormone and abiotic stresses. Furthermore, RNA-seq analysis in response to pathogen inoculation (PepMoV, TMV strains P0/P1, and Phytophthora capsici) showed distinct expression profiles of all identified genes, suggesting the diverse expression nature of genes under these stress conditions. Overall, this study provides insight into the dynamic response of CaAux/IAA genes under environmental and phytohormones stress conditions, providing bases to further explore the importance of these genes through mutant/transgenic analysis in pepper.

Guanosine tetraphosphate modulates salicylic acid signalling and the resistance of Arabidopsis thaliana to Turnip mosaic virus.

Chloroplasts can act as key players in the perception and acclimatization of plants to incoming environmental signals. A growing body of evidence indicates that chloroplasts play a critical role in plant immunity. Chloroplast function can be regulated by the nucleotides guanosine tetraphosphate and pentaphosphate [(p)ppGpp]. In plants, (p)ppGpp levels increase in response to abiotic stress and to plant hormones which are involved in abiotic and biotic stress signalling. In this study, we analysed the transcriptome of Arabidopsis plants that over-accumulate (p)ppGpp, and unexpectedly found a decrease in the levels of a broad range of transcripts for plant defence and immunity. To determine whether (p)ppGpp is involved in the modulation of plant immunity, we analysed the susceptibility of plants with different levels of (p)ppGpp to Turnip mosaic virus (TuMV) carrying a green fluorescent protein (GFP) reporter. We found that (p)ppGpp accumulation was associated with increased susceptibility to TuMV and reduced levels of the defence hormone salicylic acid (SA). In contrast, plants with lower (p)ppGpp levels showed reduced susceptibility to TuMV, and this was associated with the precocious up-regulation of defence-related genes and increased SA content. We have therefore demonstrated a new link between (p)ppGpp metabolism and plant immunity in Arabidopsis.

Isolation and validation of a candidate Rsv3 gene from a soybean genotype that confers strain-specific resistance to soybean mosaic virus.

Soybean mosaic virus (SMV), a member of the genus Potyvirus, significantly reduces soybean production worldwide. Rsv3, which confers strain-specific resistance to SMV, was previously mapped between the markers A519F/R and M3Satt in chromosome 14 of the soybean [Glycine max (L.) Merr.] genotype L29. Analysis of the soybean genome database revealed that five different NBS-LRR sequences exist between the flanking markers. Among these candidate Rsv3 genes, the full-length cDNA of the Glyma.14g204700 was successfully cloned from L29. Over-expression of Glyma.14g204700 in leaves inoculated with SMV inhibited viral infection in a soybean genotype lacking Rsv3. In addition, the transient silencing of the candidate gene caused a high accumulation of an avirulent strain in L29 carrying Rsv3. Our results therefore provide additional line of evidence to support that Glyma.14g204700 is likely Rsv3 gene that confers strain-specific resistance to SMV.

Rapid detection of fifteen known soybean viruses by dot-immunobinding assay.

A dot-immunobinding assay (DIBA) was optimized and used successfully for the rapid detection of 15 known viruses [Alfalfa mosaic virus (AMV), Bean pod mottle virus (BPMV), Bean yellow mosaic virus (BYMV), Cowpea mild mottle virus (CPMMV), Cowpea severe mosaic virus (CPSMV), Cucumber mosaic virus (CMV), Peanut mottle virus (PeMoV), Peanut stunt virus (PSV), Southern bean mosaic virus (SBMV), Soybean dwarf virus (SbDV), Soybean mosaic virus (SMV), Soybean vein necrosis virus (SVNV), Tobacco ringspot virus (TRSV), Tomato ringspot virus (ToRSV), and Tobacco streak virus (TSV)] infecting soybean plants in Oklahoma. More than 1000 leaf samples were collected in approximately 100 commercial soybean fields in 24 counties of Oklahoma, during the 2012-2013 growing seasons. All samples were tested by DIBA using polyclonal antibodies of the above 15 plant viruses. Thirteen viruses were detected, and 8 of them were reported for the first time in soybean crops of Oklahoma. The highest average incidence was recorded for PeMoV (13.5%) followed by SVNV (6.9%), TSV (6.4%), BYMV, (4.5%), and TRSV (3.9%), while the remaining seven viruses were detected in less than 2% of the samples tested. The DIBA was quick, and economical to screen more than 1000 samples against 15 known plant viruses in a very short time.

Engineering an auto-activated R protein that is in vivo activated by a viral protease.

Autonomous hypersensitive responses (self-HRs) are caused by constitutively active R proteins. In this study, we identified an auto-activated form of the R gene Pvr9 (autoPvr9); the auto-activation results from an amino acid substitution between its NBS and LRR domains. Self-HR was strongly reduced or completely inhibited by fusion of an extra peptide to the autoPvr9 N-terminal or C-terminal, respectively. When an NIa recognition site was placed between autoPvr9 and the extra peptide, the fusion construct could trigger an NIa-dependent HR. Several C-terminal fusions were tested, but only those that maintained detectable protein expression were capable of an NIa-dependent HR. Our results suggest the potential for transforming malfunctioning and auto-activated R proteins into a new construct targeting potyviral NIa proteases.

Inheritance, fine-mapping, and candidate gene analyses of resistance to soybean mosaic virus strain SC5 in soybean.

Soybean mosaic virus (SMV) is one of the most devastating pathogens for soybeans in China. Among the country-wide 22 strains, SC5 dominates in Huang-Huai and Changjiang valleys. For controlling its damage, the resistance gene was searched through Mendelian inheritance study, gene fine-mapping, and candidate gene analysis combined with qRT-PCR (quantitative real-time polymerase chain reaction) analysis. The parents F1, F2, and RILs (recombinant inbred lines) of the cross Kefeng-1 (Resistance, R) × NN1138-2 (Susceptible, S) were used to examine the inheritance of SC5-resistance. The F1 was resistant and the F2 and RILs segregated in a 3R:1S and 1R:1S ratio, respectively, indicating a single dominant gene conferring the Kefeng-1 resistance. Subsequently, the genomic region conferring the resistance was found in "Bin 352-Bin353 with 500 kb" on Chromosome 2 using the phenotyping data of the 427 RILs and a high-density genetic map with 4703 bin markers. In the 500 kb genomic region, 38 putative genes are contained. The association analysis between the SNPs in a putative gene and the resistance phenotype for the 427 RILs prioritized 11 candidate genes using Chi-square criterion. The expression levels of these genes were tested by qRT-PCR. On infection with SC5, 7 out of the 11 genes had differential expression in Kefeng-1 and NN1138-2. Furthermore, integrating SNP-phenotype association analysis with qRT-PCR expression profiling analysis, Glyma02g13495 was found the most possible candidate gene for SC5-resistance. This finding can facilitate the breeding for SC5-resistance through marker-assisted selection and provide a platform to gain a better understanding of SMV-resistance gene system in soybean.

Evaluation of the Immunogenicity of a Potyvirus-Like Particle as an Adjuvant of a Synthetic Peptide.

Improvement of current vaccines is highly necessary to increase immunogenicity levels and protection against several pathogens. Virus-like particles (VLPs) are promising approaches for vaccines because they emulate infectious virus structure, but lack any genetic material needed for replication. Plant viruses have emerged as a potential framework for VLP design, mainly because there is no preexisting immunity in mammals. In this study, we evaluated the scaffold of the papaya ringspot virus (PRSV) as a VLP adjuvant for a short synthetic peptide derived from the Hemagglutinin protein of AH1 N1 influenza virus-hemagglutinin (VLP-HA). Our results demonstrated that the adjuvant property of this VLP is highly similar to the trivalent influenza vaccine, showing comparable levels of IgG- and IgA-specific antibodies to HA-derived peptide in serum and feces of vaccinated mice, respectively. Furthermore, VLP-HA-immunized mice showed Th1-biased immune response as suggested by measuring IgG subclasses in comparison with the predominance of Th2-biased immune response in trivalent influenza vaccine dose-vaccinated mice. VLP-HA administration in mice induced comparable levels of activated CD4+- and CD8+-specific T lymphocytes for the HA-derived peptide. These results suggest the potential adjuvant capacity of the PRSV-VLP as a carrier for short synthetic peptides.

Self-assembly of hexahistidine-tagged tobacco etch virus capsid protein into microfilaments that induce IgG2-specific response against a soluble porcine reproductive and respiratory syndrome virus chimeric protein.

BACKGROUND: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. FINDINGS: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2-specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. CONCLUSIONS: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-µm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen.

A new eIF4E1 allele characterized by RNAseq data mining is associated with resistance to potato virus Y in tomato albeit with a low durability.

Allele mining on susceptibility factors offers opportunities to find new sources of resistance among crop wild relatives for breeding purposes. As a proof of concept, we used available RNAseq data to investigate polymorphisms among the four tomato genes encoding translation initiation factors [eIF4E1 and eIF4E2, eIFiso4E and the related gene new cap-binding protein(nCBP)] to look for new potential resistance alleles to potyviruses. By analysing polymorphism among RNAseq data obtained for 20 tomato accessions, 10 belonging to the cultivated type Solanum lycopersicum and 10 belonging to the closest related wild species Solanum pimpinellifolium, we isolated one new eIF4E1 allele, in the S. pimpinellifolium LA0411 accession, which encodes a potential new resistance allele, mainly due to a polymorphism associated with an amino acid change within eIF4E1 region II. We confirmed that this new allele, pot12, is indeed associated with resistance to potato virus Y, although with a restricted resistance spectrum and a very low durability potential. This suggests that mutations occurring in eIF4E region II only may not be sufficient to provide efficient and durable resistance in plants. However, our study emphasizes the opportunity brought by RNAseq data to mine for new resistance alleles. Moreover, this approach could be extended to seek for putative new resistance alleles by screening for variant forms of susceptibility genes encoding plant host proteins known to interact with viral proteins.