Abstract
RNA interference (RNAi) is a promising method for controlling pest insects by silencing the expression of vital insect genes to interfere with development and physiology; however, certain insect Orders are resistant to this process. In this study, we set out to test the ability of in planta-expressed dsRNA synthesized within the plastids to silence gene expression in an insect recalcitrant to RNAi, the lepidopteran species, Manduca sexta (tobacco hornworm). Using the Manduca vacuolar-type H+ ATPase subunit A (v-ATPaseA) gene as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the in vitro-synthesized dsRNAs to M. sexta larvae. We found that a long dsRNA of 2222 bp was the most effective in inducing lethality and silencing the v-ATPaseA gene, when delivered orally in a water droplet. We further transformed the plastid genome of the M. sexta host plant, Nicotiana tabacum, to produce this long dsRNA in its plastids and performed bioassays with M. sexta larvae on the transplastomic plants. In the tested insects, the plastid-derived dsRNA had no effect on larval survival and no statistically significant effect on expression of the v-ATPaseA gene was observed. Comparison of the absolute quantities of the dsRNA present in transplastomic leaf tissue for v-ATPaseA and a control gene, GFP, of a shorter size, revealed a lower concentration for the long dsRNA product compared to the short control product. We suggest that stability and length of the dsRNA may have influenced the quantities produced in the plastids, resulting in inefficient RNAi in the tested insects. Our results imply that many factors dictate the effectiveness of in planta RNAi, including a likely trade-off effect as increasing the dsRNA product length may be countered by a reduction in the amount of dsRNA produced and accumulated in the plastids.
Highlights
Utilization of the RNA interference (RNAi) pathway, first discovered in Caenorhabditis elegans (Fire et al, 1998) and later observed in a wide variety of species (Bellés, 2010), to knockdown gene expression is a popular tool for biological research involving insects (Dietzl et al, 2007)
Reverse transcription-qPCR indicated that v-ATPaseA expression was reduced significantly, by 68.9%, in insects fed long vATPaseA double-stranded RNA (dsRNA) product compared to insects fed water (F = 3.70, p = 0.042) (Figure 2B)
We showed that targeting of the M. sexta v-ATPaseA gene using an in vitro synthesized long dsRNA (2222 bp) induces higher mortality and gene knockdown compared to a short dsRNA (259 bp)
Summary
Utilization of the RNA interference (RNAi) pathway, first discovered in Caenorhabditis elegans (Fire et al, 1998) and later observed in a wide variety of species (Bellés, 2010), to knockdown gene expression is a popular tool for biological research involving insects (Dietzl et al, 2007). The RNAi pathway can be activated through environmental RNAi, whereby exogenous double-stranded RNA (dsRNA) molecules taken up from the environment, trigger the post-transcriptional silencing of endogenous mRNA molecules (Whangbo and Hunter, 2008). This process is accomplished by uptake of ingested dsRNA molecules from the midgut lumen into the insect cells through SID-1 channels and receptor-mediated endocytosis (Shih and Hunter, 2011; Cappelle et al, 2016). Oral consumption is considered an effective tool for introducing dsRNA into insect cells for developing RNAi-based pest control strategies
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