Knockdown of Genes Involved in Transcription and Splicing Reveals Novel RNAi Targets for Pest Control

Abstract

RNA interference (RNAi) is a promising next generation technology for the development of species-specific pest management. The key to successful RNAi based-plant protection is dependent in part on data-driven target gene selection, a challenging task due to the absence of laboratory strains and the seasonality of most pest species. In this study, we aimed to identify novel target genes by performing a knowledge-based approach in order to expand the spectrum of known potent RNAi targets. Recently, the protein-coding genes ncm, Rop, RPII-140, and dre4 have been identified as sensitive RNAi targets for pest control. Based on these potent RNAi targets, we constructed an interaction network and analyzed a selection of 30 genes in the model beetle Tribolium castaneum via injection of dsRNA synthesized by in vitro transcription. Nineteen of these targets induced significant mortality of over 70%, including six that caused 100% lethality. Orthologs of active T. castaneum RNAi targets were verified in the economically important coleopteran pests Diabrotica virgifera virgifera and Brassicogethes aeneus. Knockdown of D. v. virgifera genes coding for transcription factor Spt5, Spt6, and RNA polymerase II subunit RPII-33 caused over 90% mortality in larval feeding assays. Injection of dsRNA constructs targeting RPII-215 or the pre-mRNA-processing factor Prp19 into adult B. aeneus resulted in high lethality rates of 93 and 87%, respectively. In summary, the demonstrated knowledge-based approaches increased the probability of identifying novel lethal RNAi target genes from 2% (whole genome) to 36% (transcription- and splicing-related genes). In addition, performing RNAi pre-screening in a model insect increased also the probability of the identification essential genes in the difficult-to-work-with pest species D. v. virgifera and B. aeneus.

Effective target genes for RNA interference-based management of the cabbage stem flea beetle

“…Counterintuitively, the proteasome is known to be a stable complex, at least in the rat liver, with a half-life of up to 15 days (Tanaka & Ichihara, 1989;Pack et al, 2014;Marshall & Vierstra, 2019). This is in contrast to the general assumption that the protein of a good RNAi target should be unstable for it to be sufficiently susceptible to mRNA depletion (Knorr et al, 2021). Hence, other factors such as mRNA turnover and the proteasome's vital function might be making the proteasome a very effective RNAi target.…”

Gene silencing in the aedine cell lines C6/36 and U4.4 using long double-stranded RNA

“…The potential of RNAi as a control strategy against insect pests was first shown in beetles [ 21 ] and moths [ 22 ]. Prominent targets include nucampholin (NCM), Ras opposite (ROP), RNA polymerase II subunit-140 (RPII-140), and dre4, which were effective for the control of the red flour beetle Tribolium castaneum [ 23 , 24 ]. In Aedes albopictus , potential target genes for RNAi-based control in mosquitoes include chitin synthase, β-tubulin, and the inhibitor of apoptosis (see [ 25 ] for a review).…”

RNAi Targets in Agricultural Pest Insects: Advancements, Knowledge Gaps, and IPM

“…Since these two landmark studies, researchers have continued to demonstrate potential applications for RNAi technology in crop protection, through targeted downregulation of genes in other agricultural pest insects. While some studies have examined the use of species as screening models for identifying sensitive RNAi targets across crop pest taxa (Knorr et al, 2018(Knorr et al, , 2021Mehlhorn et al, 2021), sensitivity of a target gene in one species may not necessarily apply to another species, given the physiological and ecological differences between species, especially between distantly related taxa.…”