Truncated CMV2bN43 enhances virus-induced gene silencing in pepper by retaining systemic but not local silencing suppression

Pepper is an economically important crop. Owing to its recalcitrance to genetic transformation, virus-induced gene silencing (VIGS) is currently the major technique available for validating gene function in pepper. However, the low efficiency and difficulty of silencing genes in reproductive organs remain major challenges in pepper VIGS studies. To address these limitations, we developed an optimized VIGS system by structure-guided truncation of the Cucumber mosaic virus 2b (C2b) silencing suppressor. A silencing suppression assay revealed that the C2b^N43 mutant retained systemic silencing suppression while abrogated local silencing suppression activity in systemic leaves. The engineered TRV-C2b^N43 system significantly enhanced VIGS efficacy in pepper, providing a powerful tool for functional genomics studies in pepper. By leveraging transcriptomic profiles, we identified CaAN2, an anther-specific MYB transcription factor, whose suppression via TRV-C2b^N43 perturbation resulted in coordinated downregulation of structural genes in anthocyanin biosynthesis pathway and abolished anthocyanin accumulation in anthers establishing its essential regulatory role in pigmentation. This study validated and provided mechanistic insight for a further optimized VIGS system in pepper.