Hotspot siRNA Confers Plant Resistance against Viral Infection

Abstract

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.

Exogenous Application of dsRNA for Protection against Tomato Leaf Curl New Delhi Virus

“…A certain level of protection against geminiviruses was obtained until now with dsRNAs, mainly when they were applied together with the viral inoculum (this study; [ 20 , 39 , 41 ]); no protection was observed when the virus is delivered through viruliferous whiteflies on plants treated with dsRNAs 24 h before [ 24 ]. The contrasting results could be partially related to the poor systemic translocation and movement of the naked dsRNAs in plants.…”

“…In this case, only a limited protection against viral infection would be guaranteed. The use of selected exogenous ds-sRNAs targeting the viral sRNAs hot spots could be evaluated as a possible more efficient alternative, as highlighted in the pathosystem N. benthamiana /African cassava mosaic virus [ 39 ]. Indeed, it seems that the vast majority of sRNAs generated by external dsRNAs have non-canonical AGO-incompatible sizes and that only a small fraction of external dsRNAs is processed by DCL2 and DCL4 into AGO2 and AGO1-compatible siRNA duplexes [ 38 , 40 ].…”

Analysis of Virus-Derived siRNAs in Strawberry Plants Co-Infected with Multiple Viruses and Their Genotypes

“…Analyzing the vsiRNA pattern of virus-infected plants has shown that specific genomic regions give rise to greater numbers of vsiRNAs, termed RNA silencing 'hotspots' [7]. In practice, the characteristics of virus-specific siRNAs can be used for developing induced virus resistance [7][8][9], with hotspot regions being first choice of template for the artificial production of vsiRNAs against selected pathogens [10].…”