During a plant viral infection, host-pathogen interactions are critical for successful replication and propagation of the virus through the plant. RNA silencing suppressors (RSSs) are key players of this interplay, and they often interact with different host proteins, developing multiple functions. In the Potyviridae family, viruses produce two main RSSs, HCPro and type B P1 proteins. We focused our efforts on the less known P1b of cucumber vein yellowing virus (CVYV), a type B P1 protein, to try to identify possible factors that could play a relevant role during viral infection. We used a chimeric expression system based on plum pox virus (PPV) encoding a tagged CVYV P1b in place of the canonical HCPro. We used that tag to purify P1b in Nicotiana-benthamiana-infected plants and identified by mass spectrometry an importin-β-like protein similar to importin 7 of Arabidopsis thaliana. We further confirmed the interaction by bimolecular fluorescence complementation assays and defined its nuclear localization in the cell. Further analyses showed a possible role of this N. benthamiana homolog of Importin 7 as a modulator of the RNA silencing suppression activity of P1b.

Cucumber vein yellowing virus (CVYV) causes severe yield losses in cucurbit crops across Mediterranean countries. The control of this virus is based on cultural practices to prevent the presence of its vector (Bemisia tabaci) and breeding for natural resistance, which requires the identification of the loci involved and the development of molecular markers for linkage analysis. In this work, we mapped a monogenic locus for resistance to CVYV in cucumber by using a Bulked Segregant Analysis (BSA) strategy coupled with whole-genome resequencing. We phenotyped 135 F3 families from a segregating population between a susceptible pickling cucumber and a resistant Long Dutch type cucumber for CVYV resistance. Phenotypic analysis determined the monogenic and incomplete dominance inheritance of the resistance. We named the locus CsCvy-1. For mapping this locus, 15 resistant and 15 susceptible homozygous F2 individuals were selected for whole genome resequencing. By using a customized bioinformatics pipeline, we identified a unique region in chromosome 5 associated to resistance to CVYV, explaining more than 80% of the variability. The resequencing data provided us with additional SNP markers to decrease the interval of CsCvy-1 to 625 kb, containing 24 annotated genes. Markers flanking CsCvy-1 in a 5.3 cM interval were developed for marker-assisted selection (MAS) in breeding programs and will be useful for the identification of the target gene in future studies.

The P1a protein of the ipomovirus Cucumber vein yellowing virus is one of the self-cleavage serine proteases present in Potyviridae family members. P1a is located at the N-terminal end of the viral polyprotein, and is closely related to potyviral P1 protease. For its proteolytic activity, P1a requires a still unknown host factor; this might be linked to involvement in host specificity. Here we built a series of constructs and chimeric viruses to help elucidate the role of P1a cleavage in host range definition. We demonstrate that host-dependent separation of P1a from the remainder of the polyprotein is essential for suppressing RNA silencing defenses and for efficient viral infection. These findings support the role of viral proteases as important determinants in host adaptation.