A hallmark of antiviral RNA interference (RNAi) is the production of viral small interfering RNA (vsiRNA). Profiling of vsiRNAs indicates that certain regions of viral RNA genome or transcribed viral RNA, dubbed vsiRNA hotspots, are more prone to RNAi-mediated cleavage for vsiRNA biogenesis. However, the biological relevance of hotspot vsiRNAs to the host innate defence against pathogens remains to be elucidated. Here, we show that direct targeting a hotspot by a synthetic vsiRNA confers host resistance to virus infection. Using Northern blotting and RNAseq, we obtained a profile of vsiRNAs of the African cassava mosaic virus (ACMV), a single-stranded DNA virus. Sense and anti-sense strands of small RNAs corresponding to a hotspot and a coldspot vsiRNA were synthesised. Co-inoculation of Nicotiana benthamiana with the double-stranded hotspot siRNA protected plants from ACMV infection, where viral DNA replication and accumulation of viral mRNA were undetectable. The sense or anti-sense strand of this hotspot vsiRNA, and the coldspot vsiRNA in both double-stranded and single-stranded formats possessed no activity in viral protection. We further demonstrated that the hotspot vsiRNA-mediated virus resistance had a threshold effect and required an active RDR6. These data show that hotspot vsiRNAs bear a functional significance on antiviral RNAi, suggesting that they may have the potential as an exogenous protection agent for controlling destructive viral diseases in plants.
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