Targeting siRNA hotspots : a novel strategy for controlling viral diseases

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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.

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