The tomato calcium-permeable channel 4.1 (SlOSCA4.1) is a susceptibility factor for pepino mosaic virus.

The hyperosmolality-gated calcium permeable channel 4.1 (OSCA4.1) belongs to an evolutionarily conserved small family of mechano-sensitive channels. OSCA members may represent key players in plant resistance to drought and to pathogen infection but are scarcely studied. After screening for resistance to pepino mosaic virus (PepMV) a collection of 1000 mutagenized tomato families, we identified a mutant showing no symptoms and reduced virus accumulation. Resistance was mapped to chromosome 2 between positions 46 309 531 to 47 044 163, where a missense mutation caused the putative truncation of the OSCA4.1 protein. A CRISPR/Cas9 slosca4.1 mutant was resistant to PepMV, but not to tobacco mosaic virus or potato virus X. Inoculation of mutant and wild type tomato protoplasts showed that resistance was expressed in single cells, suggesting a role for SlOSCA4.1 in early viral function(s); congruently, SlOSCA4.1 re-localized to structures reminiscent of viral replication complexes. We propose that SlOSCA4.1 contributes to the correct regulation of the Ca2+ homeostasis necessary for optimal PepMV infection. PepMV is a pandemic virus that causes significant losses in tomato crops worldwide. In spite of its importance, no tomato-resistant varieties have been deployed yet; the mutant identified here has great potential to breed tomato varieties resistant to PepMV.

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.

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.