Discovery of midgut genes for the RNA interference control of corn rootworm

Abstract

RNA interference (RNAi) is a promising new technology for corn rootworm control. This paper presents the discovery of new gene targets -dvssj1 and dvssj2, in western corn rootworm (WCR). Dvssj1 and dvssj2 are orthologs of the Drosophila genes snakeskin (ssk) and mesh, respectively. These genes encode membrane proteins associated with smooth septate junctions (SSJ) which are required for intestinal barrier function. Based on bioinformatics analysis, dvssj1 appears to be an arthropod-specific gene. Diet based insect feeding assays using double-stranded RNA (dsRNA) targeting dvssj1 and dvssj2 demonstrate targeted mRNA suppression, larval growth inhibition, and mortality. In RNAi treated WCR, injury to the midgut was manifested by "blebbing" of the midgut epithelium into the gut lumen. Ultrastructural examination of midgut epithelial cells revealed apoptosis and regenerative activities. Transgenic plants expressing dsRNA targeting dvssj1 show insecticidal activity and significant plant protection from WCR damage. The data indicate that dvssj1 and dvssj2 are effective gene targets for the control of WCR using RNAi technology, by apparent suppression of production of their respective smooth septate junction membrane proteins located within the intestinal lining, leading to growth inhibition and mortality.

Control of western corn rootwormviaRNAi traits in maize: lethal and sublethal effects ofSec23dsRNA

“…RNAi has become well established as an effective technology to control WCR with several lethal targets documented. 17,23,24,26,27 The first commercial maize product expressing insecticidal dsRNA for WCR management will have three modes of action, dvSnf7 dsRNA and two Bt proteins Cry3Bb1 and Cry34/35Ab1. 27,70 The The root damage rating data estimated on 0-1.0 NIS scale was converted into binomial categorical data and the pass frequency (≤0.50 root rating) for each construct was calculated using a generalized linear model (JMP ® Pro 12.2.0).…”

“…One should also note that known potent RNAi targets such as v-ATPase A are enriched in the midgut epithelium. 26,75 The loss of Sec23 in Drosophila perturbs epidermal adherents and septate junctions. 73 As described by Hu et al, 26 the depletion of adherent junction transcripts, mesh, and snakeskin, via dsRNA feeding in WCR also leads to strong lethal phenotypes.…”

“…17 While the sensitivity of insects to dsRNA varies greatly among insect orders, [18][19][20] rootworms are very sensitive to dsRNA applied to artificial diet 17,[21][22][23][24] or transgenically expressed in maize plants. 18,[23][24][25][26] Target sequences that have demonstrated plant protection against WCR include the vacuolar proton pump, V-ATPase A, 17 the Snf7/Vps32 subunit of the ESCRT-III (endosomal sorting complex required for transport III), 27 septate junction proteins snakeskin (dvssj1) and mesh (dvssj2), 26 Troponin I, 24 SNARE-binding protein Ras opposite/Sec1, RNA polymerase II subunit RpII140, and FACT complex proteins dre4/spt16. 23 The most common method to initially assess the efficacy of RNAi targets is by applying dsRNA to artificial diet to determine if the insect is susceptible to orally administered dsRNA and the efficacy of the selected target gene.…”

“…To date, RNAi target identification has been performed using larvae. 17,21,23,25,26,28 However, WCR adults are also sensitive to orally fed dsRNA. 22,29,30 Feeding of V-ATPase A dsRNA to adults via artificial diet resulted in mortality and reduction of transcript levels suggesting that adult WCR bioassays may serve as an alternative screening method to identify novel RNAi targets.…”

Implementing the sterile insect technique with RNA interference – a review

“…For aquatic larvae, dissolving dsRNA in solution and bathing the larvae within it, is the most common method of effecting gene silencing via eRNAi (Figueira-Mansur et al, 2013;Singh et al, 2013;Whyard et al, 2015;Bona et al, 2016). dsRNA can be delivered to non-aquatic larvae: (1) topically via droplet feeding (Toprak et al, 2013), (2) by inducing the larvae to feed upon dsRNA-expressing transgenic plants (Xiong et al, 2013;Mamta et al, 2015;Tian et al, 2015;Hu et al, 2016), (3) by feeding larvae dsRNA-expressing transgenic bacteria (Zhu et al, 2011;Yang & Han, 2014;Li et al, 2015c), and (4) by feeding larvae naked dsRNA overlaid onto an artificial diet (Asokan et al, 2014;Yang & Han, 2014;Hu et al, 2016). Non-aquatic larvae, or those that develop in relatively anoxic conditions, can also be bathed in dsRNA solution, but the timing of exposure is critical to avoid drowning (Whyard et al, 2009).…”