Abstract
BackgroundRNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.Methodology/Principal FindingsIn this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.Conclusions/SignificanceSimilar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.
Highlights
RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms [1]
We found that the expression of both M. persicae C002 (MpC002) and Rack-1 is knocked down when M. persicae are fed from transgenic plants that transiently (N. benthamiana) and stably (A. thaliana) express double-stranded RNA (dsRNA) corresponding to MpC002 and Rack-1
Expression profiles of RNAi target genes C002 and MpC002 are predominantly expressed in the salivary glands of A. pisum and M. persicae [10,12,13], and Rack-1 in aphid gut tissues [19]
Summary
RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms [1]. With the RNAi method, double-stranded RNA (dsRNA) can lower the transcript abundance of a target gene when injected into an organism or introduced into cultured cells [3]. RNAi involves the cleavage of dsRNA precursors into smallinterfering RNA (siRNA) of approximately 21 to 23 nucleotides by the enzyme Dicer [6]. These siRNAs are incorporated into a RNA-induced silencing complex (RISC). RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs
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