Abstract
BackgroundRNA interference (RNAi) is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. However, its usefulness in delivering resistance against plant DNA viruses belonging to family Geminiviridae is still being debated. Although the RNAi approach has been shown, using a transient assay, to be useful in countering monocotyledonous plant-infecting geminiviruses of the genus Mastrevirus, it has yet to be investigated as a means of delivering resistance to dicot-infecting mastreviruses. Chickpea chlorotic dwarf Pakistan virus (CpCDPKV) is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan.ResultsHere a hairpin (hp)RNAi construct containing sequences encompassing part of replication-associated protein gene, intergenic region and part of the movement protein gene of CpCDPKV under the control of the Cauliflower mosaic virus 35S promoter has been produced and stably transformed into Nicotiana benthamiana. Plants harboring the hairpin construct were challenged with CpCDPKV. All non-transgenic N. benthamiana plants developed symptoms of CpCDPKV infection within two weeks post-inoculation. In contrast, none of the inoculated transgenic plants showed symptoms of infection and no viral DNA could be detected by Southern hybridization. A real-time quantitative PCR analysis identified very low-level accumulation of viral DNA in the inoculated transgenic plants.ConclusionsThe results presented show that the RNAi-based resistance strategy is useful in protecting plants from a dicot-infecting mastrevirus. The very low levels of virus detected in plant tissue of transgenic plants distal to the inoculation site suggest that virus movement and/or viral replication was impaired leading to plants that showed no discernible signs of virus infection.
Highlights
RNA interference (RNAi) is a homology-dependent mechanism that involves the specific degradation of cellular RNA by a complex of enzymes
The key role in RNAi is played by small RNAs [known as short interfering RNA and micro RNA], which act as effectors of silencing [2]
The RNA transcribed from such a transgene hybridizes with itself to form a hairpin structure comprising a single-stranded loop region, encoded by the spacer region/intron, and a base-paired stem encoded by the inverted repeats, which mimics the dsRNA structure that induces RNAi
Summary
RNA interference (RNAi) is a homology-dependent mechanism that involves the specific degradation of cellular RNA by a complex of enzymes. One of the first studies investigating DNA-directed RNAi in plants compared the ability of constructs expressing transcripts of sense, antisense and both polarities to yield resistance to an RNA virus in tobacco and silence an endogenous GUS reporter gene in rice [6] In both cases it was shown that duplex RNA (expression of both polarities simultaneously) was more effective than expression of either sense or antisense RNA alone. RNA interference (RNAi) is a homology-dependant gene silencing mechanism and has been widely used to engineer resistance in plants against RNA viruses. Chickpea chlorotic dwarf Pakistan virus (CpCDPKV) is a legume-infecting mastrevirus that affects chickpea and other leguminous crops in Pakistan
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