Botrytis virus X ORF2 suppresses RNA silencing in a trigger-restricted manner and is associated with altered Dicer-like gene expression in Botrytis cinerea

RNA interference (RNAi) is a central antiviral defence mechanism in fungi, yet relatively few mycoviral suppressors of RNA silencing (VSRs) have been functionally characterized, particularly in phytopathogenic hosts. Botrytis virus X (BVX), a positive-sense RNA virus in the family Alphaflexiviridae , infects Botrytis cinerea and encodes five predicted ORFs, most of which have unknown functions. Here, we screened BVX ORFs 2–5 for RNA silencing suppressor activity using complementary GFP-based assays in Nicotiana benthamiana and examined the leading candidate in the fungal host B. cinerea . BVX ORF2 (X2) enhanced GFP transcript and protein accumulation in assays where silencing was triggered by sense RNA but failed to suppress silencing triggered by hairpin-derived small interfering RNAs or microRNA-guided targeting, indicating a trigger-restricted suppressor phenotype. In B. cinerea , transgenic expression of X2 was associated with reduced induction of the RNAi-associated genes BcDCL1 and BcDCL2 compared with empty-vector controls, with the strongest effect observed on BcDCL1 . In a virus-infected fungal background, X2 expression was also associated with increased viral RNA accumulation. Together, these results identify BVX X2 as a BVX-encoded, trigger-restricted suppressor of RNA silencing and link its expression to altered RNAi-related gene induction and increased viral RNA accumulation in B. cinerea .