Cucumber necrosis virus p20 is a viral suppressor of RNA silencing

The p20 protein encoded by the tombusvirus, Cucumber necrosis virus has previously been shown to be involved in host pathogenicity and shares sequence similarity with the Tomato bushy stunt virus p19 suppressor of silencing. Using a virus-induced gene silencing (VIGS) assay, we show that p20 is a viral suppressor of RNA silencing (VSR) in infected plants. In addition, a CNV p20-knockout mutant showed a decline in viral RNA accumulation in infected plants, consistent with the role of p20 in suppression of RNA silencing. However, unexpectedly, all GFP transgenic plants co-infiltrated with p20 and GFP displayed RNA silencing using an Agrobacterium-mediated silencing assay. Detailed RNA analysis of GFP mRNA levels in p20 agro-infiltrated plants revealed that p20 did initially display suppressor activity but this was rapidly overcome by RNA silencing. p20 expression levels in agro-infiltrated plants were shown to be approximately 50-fold lower than that of the TBSV p19 silencing suppressor, consistent with the notion that p20 dosage levels are not sufficient to suppress RNA silencing in the Agrobacterium-mediated system. Our results suggest that a viral-based VIGS assay may be required for identifying VSRs encoded by some plant viruses. Based on bioinformatics studies the mechanism of suppression of silencing by p20 is predicted to be similar to that of the TBSV p19 suppressor.