Properties of post-transcriptional gene silencing suppression proteins of Benyviruses

Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) belong to the Benyvirus genus, possess a multipartite genome formed by four ssRNAs(+) and are both transmitted by the plasmodiophorid Polymyxa betae. BSBMV and BNYVV are closely related since they possess the same host range, vector and genome organization. The innate mechanism that plants use to protect themselves against viral infections is called Post Transcriptional Gene Silencing (PTGS). PTGS is triggered by the presence of aberrant RNA or dsRNA generated during the replication of viral genomes and leads to their degradation. To counteract this innate mechanism, viruses co-evolved with their hosts and express viral suppressors of RNA silencing (VSR) that inhibit the PTGS. In this work we investigated properties of the Benyvirus VSRs. Such VSR consist of cysteine-rich proteins (CRP) of 14kDa expressed from RNA2 of both BNYVV and BSBMV species used in this study. We demonstrated that P14s have a zinc-finger domain (Znf) able to bind nucleic acids. Agroinfection of Nicotiana benthamiana plants demonstrated that these proteins are able to suppress the PTGS downstream of the Dicer proteins action, without interfering with the transitivity. Sequence motifs essential for the nucleolus targeting of the protein (NoLS) and cysteine residues essential to the Znf structure folding, have been also identified. Both p14s localize in the nucleolus, form homodimers and bind the “coremin” sequence, a stretch of 20 nucleotides present in the RNAs-3 sequence of Benyviruses and required for their systemic spread in the plant. Moreover, the coremin sequence is able to complement defective BNYVV P14 mutants in long distance movement illustrating an obvious link between P14, suppression of RNA silencing activity, RNAs-3 coremin sequence and long distance movement