Abstract
RNAi shows potential as an agricultural technology for insect control, yet, a relatively low number of robust lethal RNAi targets have been demonstrated to control insects of agricultural interest. In the current study, a selection of lethal RNAi target genes from the iBeetle (Tribolium castaneum) screen were used to demonstrate efficacy of orthologous targets in the economically important coleopteran pests Diabrotica virgifera virgifera and Meligethes aeneus. Transcript orthologs of 50 selected genes were analyzed in D. v. virgifera diet-based RNAi bioassays; 21 of these RNAi targets showed mortality and 36 showed growth inhibition. Low dose injection- and diet-based dsRNA assays in T. castaneum and D. v. virgifera, respectively, enabled the identification of the four highly potent RNAi target genes: Rop, dre4, ncm, and RpII140. Maize was genetically engineered to express dsRNA directed against these prioritized candidate target genes. T0 plants expressing Rop, dre4, or RpII140 RNA hairpins showed protection from D. v. virgifera larval feeding damage. dsRNA targeting Rop, dre4, ncm, and RpII140 in M. aeneus also caused high levels of mortality both by injection and feeding. In summary, high throughput systems for model organisms can be successfully used to identify potent RNA targets for difficult-to-work with agricultural insect pests.
Highlights
Worldwide, insect pests cause billions of U.S dollars of yield loss each year
We verified the lethality and dose response of the 50 genes selected from the iBeetle database by injecting a range of double-stranded RNA (dsRNA) concentrations (250 ng/μl, 1 ng/μl, 0.1 ng/μl and 0.01 ng/μl) into T. castaneum larvae (Table 1 and Supplementary Tables 1 and 3)
Of the 50 dsRNA targets tested in D. v. virgifera, 36 showed significant Growth Inhibition (Fig. 2B)
Summary
Insect pests cause billions of U.S dollars of yield loss each year. In past decades, the primary solution for insect control was the use of chemical pesticides. Coleopteran insects are known to exhibit a robust environmental RNAi response[14,15,16,17], and in 2007 Baum, et al.[15] successfully engineered the first insect-resistant dsRNA-expressing plant for protection from feeding damage by Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae). This milestone generated a high level of interest in RNAi-based control approaches for D. v. A pre-screen in a model organism was often proposed as a solution[37,38,39], but the application to orthologous RNAi targets in non-model pest insects is not well established
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