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.

No abstract

RNA-based pesticides exert their function by suppressing the expression of an essential gene in the target pest through RNA interference caused by double-stranded RNA (dsRNA). Here, we selected target genes for growth suppression of the solanaceous crop pests ladybird beetle (Henosepilachna vigintioctopunctata) and Colorado potato beetle (Leptinotarsa decemlineata)-the death-associated inhibitor of apoptosis protein 1 gene (diap1), and an orthologous gene of the COPI coatomer protein complex (copI), respectively. We constructed a cost-competitive overproduction system for dsRNA using Corynebacterium glutamicum as a host bacterium. The dsRNA expression unit was equipped with two sets of promoters and terminators derived from coliphage T7, and the convergent expression system was designed to be selectively transcribed by T7 RNA polymerase. This expression system efficiently overproduced both target dsRNAs. On culture in a jar fermentor, the yield of diap1-targeting dsRNA (approximately 360 bp) was > 1 g per liter of culture. Long-chain diap1-targeting dsRNAs (up to around 1 kbp) could be produced without a substantial loss of efficiency. dsRNA accumulated in C. glutamicum significantly suppressed larval growth of H. vigintioctopunctata. The dsRNA expression technology developed here is expected to substantially reduce dsRNA production costs. Our method can be applied for a wide range of industrial uses, including agricultural pest control. Key points • Overexpression of dsRNA was achieved in C. glutamicum using a coliphage T7 system. • The best strain produced > 1 g/L of the target dsRNA species, for use as an insecticide. • The developed system efficiently produced long dsRNA species, up to ~ 1 kbp.

25Selecting an appropriate target gene is critical to the success of feeding RNA interference 26 (f-RNAi)-based pest control. Gene targets have been chosen based on their ability to 27 induce lethality. However, lethality induction by f-RNAi is slow-acting and crop damage 28 can progress during this time. Here, we show that f-RNAi of death-associated inhibitor 29 of apoptosis protein 1 (diap1), but not two conventional targets vacuolar ATPase subunit 30 A and E, induces acute feeding cessation in the solanaceous pest, Henosepilachna 31 vigintioctopunctata during 24-48 hours. We also found that the feeding cessation by 32 diap1 f-RNAi has species-specificity and occurs with only 1.6 ng dsRNA. Our results 33 suggest that diap1 is an appropriate target in the context of rapid reduction of crop damage. 34 We propose that acute feeding disorder should be assessed as a novel criterion for 35 selecting appropriate target genes for RNAi-based pest control in addition to the 36 conventional criterion based on lethality. 37 38 dsRNA 5 on crops. Regardless of application method, the selection of an appropriate target 46 gene is essential to achieve effective pest control 6 . The most target genes reported so far 47 are housekeeping genes such as the v-ATPase genes and metabolic genes such as the 48 chitin synthase genes (representative targets listed in Baum et al. 2 ).49Conventionally, the criterion for selecting target genes is a combination of induction of 50 lethality or growth-inhibition by gene silencing with RNAi 6 . For example, silencing of v-51 ATPase subunit A (v-ATPase A) or v-ATPase subunit E (v-ATPase E) by oral delivery of 52 the dsRNA causes increased mortality in various pests of the order Coleoptera 7,8,9 , 53 Lepidoptera 7,10 , Diptera 7 , Hemiptera 11,12 and Orthoptera 13 . Such induction of lethality or 54 growth-inhibition is extremely effective in terms of pest reduction and eradication. 55However neither lethality nor growth-inhibition are phenotype that are quick to induce, 56 and it takes a certain amount of time before maximal effect of treatment is achieved. 57During the long-time span between treatment and phenotype, insect pests can continue to 58 damage crops. For example, it takes more than a week for the RNAi effect of v-ATPase 59 A or v-ATPase E to occur. Silencing of v-ATPase E requires 9 days to induce 100% 60 mortality through injected and 25 days to induce 100% mortality through oral RNAi in 61 Tribolium castaneum 14 . Therefore, in order to achieve effective RNAi-based control of 62 4 herbivorous pests, it would be desirable to evaluate and search for target genes from the 63 viewpoint of rapid termination of crop damage in addition to the induction of lethality 64 and growth-inhibition. 65In this regard, we found that the gene silencing of death-associated inhibitor of 66 apoptosis protein 1 (diap1) gene by oral-feeding RNAi (f-RNAi) causes acute feeding 67 disorder. diap1 is an insect homolog of the iap genes and is known as a suppressor of 68 apoptosis in the fruit ...