Construction of Corynebacterium glutamicum cells as containers encapsulating dsRNA overexpressed for agricultural pest control

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.Electronic supplementary materialThe online version of this article (10.1007/s00253-019-10113-9) contains supplementary material, which is available to authorized users.

Novel approaches for efficient in vivo fermentation production of noncoding RNAs

“…Their roles in normal physiology and disease should be fully understood towards the development of new therapeutic or agricultural strategies. While chemo-engineered ncRNA mimics continue to dominate ncRNA research and development, recent efforts have led to breakthroughs in novel biotechnologies for fermentation production of RNA molecules (Chen et al 2015;Hashiro et al 2019a;Hashiro et al 2019b;Ho et al 2018;Ponchon et al 2009;Ponchon and Dardel 2007). Among them, the tRNA/pre-miRNA carrier-based technology has proven to be a robust and versatile platform to achieve a consistent, efficient, large-scale, and cost-effective production of a wide variety of biologic RNA agents, including miRNAs, siRNAs, aptamers, and sRNAs, all of which are biologically active in vitro and in vivo.…”

“…Overexpression of diap1*-dsRNA was achieved in C. glutamicum 2256LΔrnc strain by convergent transcription under two strong F1 promoters (Fig. 4b) (Hashiro et al 2019a). The target diap1*-dsRNA and U1A*-RNA was estimated to reach up to 75 and 300 mg per liter of bacterial fermentation, respectively (Table 1), although the purification and overall yield were not reported.…”

“…A most recent advance is the development and application of RNase III-deficient C. glutamicum bacterium for in vivo production of target RNA molecules (Table 1) (Hashiro et al 2019a;Hashiro et al 2019b). C. glutamicum has been widely used in industrial settings for large-scale production of various biochemical compounds such as amino acids and lipids (Kalinowski et al 2003).…”

“…The use of stable RNA scaffolds or carriers (e.g., hybrid tRNA/pre-miRNA) to assemble various types of target RNAs (e.g., aptamers, miRNAs, siRNAs, sRNAs) has proven to be the most efficient approach to achieve a large-scale production of novel ncRNA molecules (e.g., tens of milligrams of pure ncRNAs from 1 L of bacterial culture) for research and development (Chen et al 2015;Ho et al 2018;Petrek et al 2019). Direct heterogenous expression in particular bacterial strains remains a challenge, yielding only nano-to micrograms of target RNAs per liter of fermentation (Kikuchi and Umekage 2018), until the very recent demonstration of efficient overexpression of two specific RNAs in RNase III-deficient bacteria (Hashiro et al 2019a;Hashiro et al 2019b). Therefore, following a brief introduction of in vitro enzymatic production approaches, we focus on the review and discussion of novel fermentation-based in vivo production of ncRNAs, including the use of particular RNAs as carriers and unique bacterial strains to avoid degradation and achieve efficient overexpression and accumulation.…”

Functional divergence of white genes in Henosepilachna vigintioctopunctata revealed by RNA interference

“…Molecular resources for H. vigintioctopunctata have only recently been developed (Guo et al, 2019;Zhang et al, 2018). Moreover, it has been documented that dsRNA can successfully knock down target genes in H. vigintioctopunctata (Hashiro et al, 2019;Ohde et al, 2009;Wu et al, 2020). This offers a great opportunity to explore the molecular mechanisms of white gene(s) by RNAi.…”