Use of High-Throughput Sequencing and Two RNA Input Methods to Identify Viruses Infecting Tomato Crops.

We used high-throughput sequencing to identify viruses on tomato samples showing virus-like symptoms. Samples were collected from crops in the Iberian Peninsula. Either total RNA or double-stranded RNA (dsRNA) were used as starting material to build the cDNA libraries. In total, seven virus species were identified, with pepino mosaic virus being the most abundant one. The dsRNA input provided better coverage and read depth but missed one virus species compared with the total RNA input. By performing in silico analyses, we determined a minimum sequencing depth per sample of 0.2 and 1.5 million reads for dsRNA and rRNA-depleted total RNA inputs, respectively, to detect even the less abundant viruses. Primers and TaqMan probes targeting conserved regions in the viral genomes were designed and/or used for virus detection; all viruses were detected by qRT-PCR/RT-PCR in individual samples, with all except one sample showing mixed infections. Three virus species (Olive latent virus 1, Lettuce ring necrosis virus and Tomato fruit blotch virus) are herein reported for the first time in tomato crops in Spain.

Second generation of pepino mosaic virus vectors: improved stability in tomato and a wide range of reporter genes.

BACKGROUND: Vectors based on plant viruses are important tools for functional genomics, cellular biology, plant genome engineering and molecular farming. We previously reported on the construction of PepGFP2a, a viral vector based on pepino mosaic virus (PepMV) which expressed GFP efficiently and stably in plants of its experimental host Nicotiana benthamiana, but not in its natural host tomato. We have prepared a new set of PepMV-based vectors with improved stability that are able to express a wide range of reporter genes, useful for both N. benthamiana and tomato. RESULTS: We first tested PepGFPm1 and PepGFPm2, two variants of PepGFP2a in which we progressively reduced a duplication of nucleotides encoding the N-terminal region of the coat protein. The new vectors had improved GFP expression levels and stability in N. benthamiana but not in tomato plants. Next, we replaced GFP by DsRed or mCherry in the new vectors PepDsRed and PepmCherry, respectively; while PepmCherry behaved similarly to PepGFPm2, PepDsRed expressed the reporter gene efficiently also in tomato plants. We then used PepGFPm2 and PepDsRed to study the PepMV localization in both N. benthamiana and tomato cells. Using confocal laser scanning microscopy (CLSM), we observed characteristic fluorescent bodies in PepMV-infected cells; these bodies had a cytoplasmic localization and appeared in close proximity to the cell nucleus. Already at 3 days post-agroinoculation there were fluorescent bodies in almost every cell of agroinoculated tissues of both hosts, and always one body per cell. When markers for the endoplasmic reticulum or the Golgi apparatus were co-expressed with PepGFPm2 or PepDsRed, a reorganisation of these organelles was observed, with images suggesting that both are intimately related but not the main constituents of the PepMV bodies. Altogether, this set of data suggested that the PepMV bodies are similar to the potato virus X (PVX) "X-bodies", which have been described as the PVX viral replication complexes (VRCs). To complete the set of PepMV-based vectors, we constructed a vector expressing the BAR herbicide resistance gene, useful for massive susceptibility screenings. CONCLUSIONS: We have significantly expanded the PepMV tool box by producing a set of new vectors with improved stability and efficiency in both N. benthamiana and tomato plants. By using two of these vectors, we have described characteristic cellular bodies induced by PepMV infection; these bodies are likely the PepMV VRCs.

Stable and Broad Spectrum Cross-Protection Against Pepino Mosaic Virus Attained by Mixed Infection.

While recent pepino mosaic virus (PepMV; species Pepino mosaic virus, genus Potexvirus, family Alphaflexiviridae) epidemics seem to be predominantly caused by isolates of the CH2 strain, PepMV epidemics in intensive tomato crops in Spain are caused by both CH2 and EU isolates that co-circulate, representing a challenge in terms of control, including cross-protection. In this work, we hypothesized that mixed infections with two mild isolates of the EU and CH2 strains (PepMV-Sp13 and -PS5, respectively) may be useful in PepMV cross-protection in Spanish epidemics, providing protection against a broad range of aggressive isolates. Thus, we performed a range of field trials and an experimental evolution assay to determine the phenotypic and genetic stability of PepMV-Sp13 and -PS5 mixed infections, as well as their cross-protective efficiency. Our results showed that: (i) the phenotype of PepMV-Sp13 and -PS5 mixed infections was mild and did not change significantly when infecting different tomato cultivars or under different environmental conditions in Spain, (ii) PepMV-Sp13 and -PS5 mixed infections provided more efficient protection against two aggressive EU and CH2 isolates than single infections, and (iii) PepMV-Sp13 and -PS5, either in single or in mixed infections, were less variable than other two PepMV isolates occurring naturally in PepMV epidemics in Spain.

Pepino mosaic virus RNA-Dependent RNA Polymerase POL Domain Is a Hypersensitive Response-Like Elicitor Shared by Necrotic and Mild Isolates.

Pepino mosaic virus (PepMV) is an emerging pathogen that represents a serious threat to tomato production worldwide. PepMV-induced diseases manifest with a wide range of symptoms, including systemic necrosis. Our results showed that PepMV accumulation depends on the virus isolate, tomato cultivar, and environmental conditions, and associates with the development of necrosis. Substitution of lysine for glutamic acid at position 67 in the triple gene block 3 (TGB3) protein, previously described as a necrosis determinant, led to increased virus accumulation and was necessary but not sufficient to induce systemic necrosis. Systemic necrosis both in tomato and Nicotiana benthamiana shared hypersensitive response (HR) features, allowing the assessment of the role of different genomic regions on necrosis induction. Overexpression of both TGB3 and the polymerase domain (POL) of the RNA-dependent RNA polymerase (RdRp) resulted in necrosis, although only local expression of POL triggered HR-like symptoms. Our results also indicated that the necrosis-eliciting activity of POL resides in its highly conserved "palm" domain, and that necrosis was jasmonic acid-dependent but not salicylic acid-dependent. Altogether, our data suggest that the RdRp-POL domain plays an important role in PepMV necrosis induction, with necrosis development depending on the virus accumulation level, which can be modulated by the nature of TGB3, host genotype and environmental conditions.

Interfamilial recombination between viruses led to acquisition of a novel translation-enhancing RNA element that allows resistance breaking.

Many plant viruses depend on functional RNA elements, called 3'-UTR cap-independent translation enhancers (3'-CITEs), for translation of their RNAs. In this manuscript we provide direct proof for the existing hypothesis that 3'-CITEs are modular and transferable by recombination in nature, and that this is associated with an advantage for the created virus. By characterizing a newly identified Melon necrotic spot virus (MNSV; Tombusviridae) isolate, which is able to overcome eukaryotic translation initiation factor 4E (eIF4E)-mediated resistance, we found that it contains a 55 nucleotide insertion in its 3'-UTR. We provide strong evidence that this insertion was acquired by interfamilial recombination with the 3'-UTR of an Asiatic Cucurbit aphid-borne yellows virus (CABYV; Luteoviridae). By constructing chimeric viruses, we showed that this recombined sequence is responsible for resistance breaking. Analysis of the translational efficiency of reporter constructs showed that this sequence functions as a novel 3'-CITE in both resistant and susceptible plants, being essential for translation control in resistant plants. In conclusion, we showed that a recombination event between two clearly identified viruses from different families led to the transfer of exactly the sequence corresponding to a functional RNA element, giving rise to a new isolate with the capacity to infect an otherwise nonsusceptible host.

Genetic diversity and potential vectors and reservoirs of Cucurbit aphid-borne yellows virus in southeastern Spain.

The genetic variability of a Cucurbit aphid-borne yellows virus (CABYV) (genus Polerovirus, family Luteoviridae) population was evaluated by determining the nucleotide sequences of two genomic regions of CABYV isolates collected in open-field melon and squash crops during three consecutive years in Murcia (southeastern Spain). A phylogenetic analysis showed the existence of two major clades. The sequences did not cluster according to host, year, or locality of collection, and nucleotide similarities among isolates were 97 to 100 and 94 to 97% within and between clades, respectively. The ratio of nonsynonymous to synonymous nucleotide substitutions reflected that all open reading frames have been under purifying selection. Estimates of the population's genetic diversity were of the same magnitude as those previously reported for other plant virus populations sampled at larger spatial and temporal scales, suggesting either the presence of CABYV in the surveyed area long before it was first described, multiple introductions, or a particularly rapid diversification. We also determined the full-length sequences of three isolates, identifying the occurrence and location of recombination events along the CABYV genome. Furthermore, our field surveys indicated that Aphis gossypii was the major vector species of CABYV and the most abundant aphid species colonizing melon fields in the Murcia (Spain) region. Our surveys also suggested the importance of the weed species Ecballium elaterium as an alternative host and potential virus reservoir.

Melon RNA interference (RNAi) lines silenced for Cm-eIF4E show broad virus resistance.

Efficient and sustainable control of plant viruses may be achieved using genetically resistant crop varieties, although resistance genes are not always available for each pathogen; in this regard, the identification of new genes that are able to confer broad-spectrum and durable resistance is highly desirable. Recently, the cloning and characterization of recessive resistance genes from different plant species has pointed towards eukaryotic translation initiation factors (eIF) of the 4E family as factors required for the multiplication of many different viruses. Thus, we hypothesized that eIF4E may control the susceptibility of melon (Cucumis melo L.) to a broad range of viruses. To test this hypothesis, Cm-eIF4E knockdown melon plants were generated by the transformation of explants with a construct that was designed to induce the silencing of this gene, and the plants from T2 generations were genetically and phenotypically characterized. In transformed plants, Cm-eIF4E was specifically silenced, as identified by the decreased accumulation of Cm-eIF4E mRNA and the appearance of small interfering RNAs derived from the transgene, whereas the Cm-eIF(iso)4E mRNA levels remained unaffected. We challenged these transgenic melon plants with eight agronomically important melon-infecting viruses, and identified that they were resistant to Cucumber vein yellowing virus (CVYV), Melon necrotic spot virus (MNSV), Moroccan watermelon mosaic virus (MWMV) and Zucchini yellow mosaic virus (ZYMV), indicating that Cm-eIF4E controls melon susceptibility to these four viruses. Therefore, Cm-eIF4E is an efficient target for the identification of new resistance alleles able to confer broad-spectrum virus resistance in melon.

Development of expression vectors based on pepino mosaic virus.

BACKGROUND: Plant viruses are useful expression vectors because they can mount systemic infections allowing large amounts of recombinant protein to be produced rapidly in differentiated plant tissues. Pepino mosaic virus (PepMV) (genus Potexvirus, family Flexiviridae), a widespread plant virus, is a promising candidate expression vector for plants because of its high level of accumulation in its hosts and the absence of severe infection symptoms. We report here the construction of a stable and efficient expression vector for plants based on PepMV. RESULTS: Agroinfectious clones were produced from two different PepMV genotypes (European and Chilean), and these were able to initiate typical PepMV infections. We explored several strategies for vector development including coat protein (CP) replacement, duplication of the CP subgenomic promoter (SGP) and the creation of a fusion protein using the foot-and-mouth disease virus (FMDV) 2A catalytic peptide. We found that CP replacement vectors were unable to move systemically and that vectors with duplicated SGPs (even heterologous SGPs) suffered from significant transgene instability. The fusion protein incorporating the FMDV 2A catalytic peptide gave by far the best results, maintaining stability through serial passages and allowing the accumulation of GFP to 0.2-0.4 g per kg of leaf tissue. The possible use of PepMV as a virus-induced gene silencing vector to study gene function was also demonstrated. Protocols for the use of this vector are described. CONCLUSIONS: A stable PepMV vector was generated by expressing the transgene as a CP fusion using the sequence encoding the foot-and-mouth disease virus (FMDV) 2A catalytic peptide to separate them. We have generated a novel tool for the expression of recombinant proteins in plants and for the functional analysis of virus and plant genes. Our experiments have also highlighted virus requirements for replication in single cells as well as intercellular and long-distance movement.