Abstract
Double-stranded RNA (dsRNA) inducing RNA interference (RNAi) is expected to be applicable to management of agricultural pests. In this study, we selected a ladybird beetle, Henosepilachna vigintioctopunctata, as a model target pest that devours vegetable leaves, and examined the effects of feeding the pest sterilized microbes highly accumulating a target dsRNA for RNAi induction. We constructed an efficient production system for diap1*-dsRNA, which suppresses expression of the essential gene diap1 (encoding death-associated inhibitor of apoptosis protein 1) in H. vigintioctopunctata, using an industrial strain of Corynebacterium glutamicum as the host microbe. The diap1*-dsRNA was overproduced in C. glutamicum by convergent transcription using a strong promoter derived from corynephage BFK20, and the amount of dsRNA accumulated in fermented cells reached about 75 mg per liter of culture. In addition, we developed a convenient method for stabilizing the dsRNA within the microbes by simply sterilizing the diap1*-dsRNA-expressing C. glutamicum cells with ethanol. When the sterilized microbes containing diap1*-dsRNA were fed to larvae of H. vigintioctopunctata, diap1 expression in the pest was suppressed, and the leaf-feeding activity of the larvae declined. These results suggest that this system is capable of producing stabilized dsRNA for RNAi at low cost, and it will open a way to practical application of dsRNA as an environmentally-friendly agricultural insecticide.
Highlights
Electronic supplementary material The online version of this article contains supplementary material, which is available to authorized users.Many powerful chemical pesticides have been used to combat pests in agriculture
We indicated that the leaf-eating activity of H. vigintioctopunctata could be efficiently suppressed by feeding the insect double-stranded RNA (dsRNA) corresponding to part of the diap1 gene (Niimi et al 2015; Chikami et al 2019)
For the dsRNA overexpression, we adapted a convergent transcription system using a pair of strong F1 promoters from corynephage BFK20
Summary
Many powerful chemical pesticides have been used to combat pests in agriculture. They have brought great benefits to crop cultivation, they have various undesirable environmental consequences, for example, effects on beneficial insects and soil microbes and the emergence of pesticideresistant pests (Woodcock et al 2016; Alyokhin et al 2008). The development of environmentally-friendly pest control methods that replace conventional chemical pesticides is desired and required. RNA has attracted attention as a potential candidate for pest control. RNA molecules having such a function can be called RNA-based pesticides or RNA insecticides (Palli 2014; Gu and Knipple 2013)
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