Targeting siRNA hotspots : a novel strategy for controlling viral diseases

Abstract

RNA silencing is a plant antiviral defence mechanism. A hallmark of RNA silencing is the production of small interfering (si) RNAs of 21-25 nucleotides that are generated from the viral genome. Moreover, profiles of viral siRNAs suggest that certain regions, namely hotspots, of the viral genome are more prone to RNA silencing-mediated degradation. African cassava mosaic virus (ACMV) causes cassava mosaic disease which is considered one of the greatest threats to the production of cassava in Africa and throughout the world. Geminiviruses such as ACMV are believed to be both inducers and targets of RNA silencing. The hypothesis of this research is that by targeting siRNA hotspots in the viral genome a novel strategy can be developed to control viral infection. This project has three main objectives. First, it aims to profile siRNAs associated with cassava-infecting DNA viruses via hybridisation and deep sequencing technology. Second, it aims to test the interaction between hotspot vsiRNAs and ACMV. Third, it aims to induce resistance to ACMV, and assess the level of resistance developed, by direct delivery of hotspot vsiRNA in plants. As a preliminarily study, it was demonstrated that synthetic exogenous siRNA targeting one of the siRNA hotspots in the genome of Tomato mosaic virus (ToMV) had a negative impact on virus infection in terms of RNA accumulation and symptom severity in Nicotiana benthmiana plants. In further experiments, different sized siRNAs associated with ACMV infection was detected by both northern blotting and high throughput 454 sequencing of samples from infected plants. Based on the 454 sequencing data obtained sense and anti-sense strand siRNAs have been produced corresponding to siRNA hotspot and coldspot regions. The effects of these exogenous siRNAs on ACMV infection in N. benthamiana plants were investigated. The results revealed that the co-inoculation of the hotspot siRNAs with ACMV reduced viral DNA replication and disease development. ACMV/siRNA co-inoculated plants remained symptomless throughout the 3-month course of the experiments. On the other hand, when ACMV was coinoculated with coldspot targeted siRNA, the viral DNA accumulation was comparable to infection with ACMV alone, and plants displayed typical ACMV infection symptoms. The effect of hotspot siRNA in inducing viral resistance was dose-dependent. The results also revealed that the effectiveness of hotspot siRNA to elicit durable resistance to ACMV infection requires a functional host RNAdependent RNA polymerase 6 (RDR6). In conclusion, these data demonstrate that hotspot vsiRNA could offer a novel strategy for controlling major destructive viral diseases in plants