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 defense 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 open reading frames (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 is triggered by sense RNA but failed to suppress silencing triggered by hairpin-derived siRNAs or miRNA-guided targeting, indicating a trigger-restricted suppressor phenotype. In B. cinerea , transgenic expression of X2 was associated with reduced induction of the RNAiassociated genes BcDCL1 and BcDCL2 compared to 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 .