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

Abstract

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. Impact Statement RNA interference (RNAi) is a key antiviral defence in fungi, yet relatively few viral suppressors of RNA silencing (VSRs) have been characterized in mycoviruses, particularly in phytopathogenic hosts. This study identifies Botrytis virus X (BVX) ORF2 (X2) as a trigger-restricted suppressor that enhances GFP accumulation in plant-based assays initiated by sense RNA but does not suppress silencing initiated by defined hairpin-derived siRNA or miRNA-guided pathways. By linking this activity to altered induction of RNAi genes and increased viral RNA accumulation in Botrytis cinerea, this work provides one of the first experimental frameworks linking a candidate mycoviral VSR to measurable readouts of RNAi and viral RNA accumulation in a native fungal host context. These findings expand the diversity of known VSR phenotypes and establish a framework for connecting suppressor activity to RNAi-associated responses and viral outcomes in fungal systems.