Abstract
RNA interference (RNAi) is a regulatory mechanism of gene expression mediated by small RNAs. By using the RNAi technique, exogenous double-stranded RNA (dsRNA) designed to target mRNA, suppresses target gene expression levels in plants. In this study, we adopted the RNAi mechanism as a tool to protect plants from viruses. We designed and synthesized several dsRNAs targeting the pepper mottle virus (PepMoV) genes HC-Pro and NIb. When used on Nicotiana benthamiana plants, these dsRNAs protected the plant against viral infection over a specific period. By optimizing dsRNA and virus injection time, the protection efficiency of dsRNA by targeting virus genes could be maximized. It seems that exogenous dsRNA-derived RNA-induced silencing complex was able to defend the host against viral infection instantly. Furthermore, each dsRNA designed to target different regions within a transcript had varying levels of effects on virus survival in the host plants. When targeting the middle part of both the NIb and HC-Pro genes using the dsRNAs, the highest viral growth inhibitory effect was observed. An RLM-5′ RACE was performed using plant leaves infected with PepMoV after dsRNA treatment and it was observed that most of the mRNA cleavages occurred close to the 3′ part within the dsRNA target position on the mRNA. These results suggest that the dsRNA tool can be used as a plant vaccine platform for crop protection.
Highlights
Pepper mottle virus (PepMoV), a member of the genus Potyvirus which is the largest genus of plant RNA virus, infects most Capsicum sp. and causes significant economic losses
The HC-Pro and Nuclear Inclusion b (NIb) genes in pepper mottle virus (PepMoV) were selected and various double-stranded RNA (dsRNA) were designed to assess the efficacy of dsRNA in controlling PepMoV in N. benthamiana
For the 1557 nt NIb transcript, dsRNAs were designed for a target of 5′, central, and 3′ regions on the NIb transcript with 54 bp overlapped among these dsRNAs (Fig. 1b)
Summary
Pepper mottle virus (PepMoV), a member of the genus Potyvirus which is the largest genus of plant RNA virus, infects most Capsicum sp. and causes significant economic losses. RNA Ligase mediated‐5′ rapid amplification of cDNA ends (RLM‐ 5′RACE) RLM-5′RACE was performed to identify the cleavage positions within the target mRNA following dsRNA induced RNAi in N. benthamiana infected with PepMoV [26].
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