Knockout of the HaREase Gene Improves the Stability of dsRNA and Increases the Sensitivity of Helicoverpa armigera to Bacillus thuringiensis Toxin.

Ruobing Guan,Guirong Wang,Xuexia Miao,Bin Yang,Haichao Li,Shaoru Hu,Qiuyan Chen

doi:10.3389/fphys.2019.01368

Abstract

Double-stranded RNA (dsRNA)-induced genes are usually related to RNA interference (RNAi) mechanisms and are involved in immune-related pathways. In a previous study, we found a lepidopteran-specific nuclease gene REase that was up-regulated by dsRNA and that affected RNAi efficiency in Asian corn borer (Ostrinia furnacalis). In this study, to verify the function of REase, the homologous gene HaREase in cotton bollworm (Helicoverpa armigera) was knocked out using CRISPR/Cas9 system. We found that the midgut epithelium structure was apparently not affected in the ΔHaREase mutant [Knock out (KO)]. Transcript sequencing results showed that most of the known insect immune-related genes were up-regulated in KO. When second instar larvae were fed artificial diet with Cry1Ac, a protoxin from Bacillus thuringiensis (Bt), in sublethal doses (2.5 or 4 μg/g), the growth rate of KO was repressed significantly. The dsRNA stability was also enhanced in midgut extraction of KO; however, RNAi efficiency was not obviously improved compared with the wild type (WT). The KO and WT were injected with dsEGFP (Enhanced green fluorescent protein) and subjected to transcriptome sequencing. The results showed that the expression levels of 14 nuclease genes were enhanced in KO after the dsRNA treatment. These findings revealed that HaREase expression level was not only related with dsRNA stability, but also with Bt resistance in cotton bollworm. When HaREase was knocked out, other immune- or nuclease-related genes were enhanced significantly. These results remind us that insect immune system is complex and pest control for cotton bollworm is an arduous task.

Highlights

RNA interference (RNAi) technology is a potential strategy for crop protection against insect pests by double-stranded RNA spraying or feeding, or via transgenic plants (Nandety et al, 2015; Joga et al, 2016; Zhang J. et al, 2017; Whitten, 2019)
The Knock out (KO) mutants that we obtained using CRISPR/Cas9 technology were used to study the function of HaREase in cotton bollworm
The results indicate that HaREase is a lepidopteranspecific gene that can be induced by Double-stranded RNA (dsRNA) and is

Summary

Introduction

RNA interference (RNAi) technology is a potential strategy for crop protection against insect pests by double-stranded RNA (dsRNA) spraying or feeding, or via transgenic plants (Nandety et al, 2015; Joga et al, 2016; Zhang J. et al, 2017; Whitten, 2019). Many factors affect RNAi efficiency in insects, including the absence of RNA-dependent RNA polymerase (RdRP)-mediated synthesis of secondary small interfering RNAs (siRNAs) (Sijen et al, 2001), the rate of dsRNA processing into siRNAs (Guan et al, 2018a), the dsRNA uptake and transport mechanism (Luo et al, 2012), and the degradation rate of dsRNA (Christiaens et al, 2014; Spit et al, 2017; Song et al, 2019). We identified an RNAi efficiency-related nuclease gene REase that was induced by dsRNA from Asian corn borer (Ostrinia furnacalis). Overexpression of REase decreased the RNAi efficiency in Drosophila, and knocking down the expression level of REase improved RNAi efficiency in Asian corn borer (Guan et al, 2018b). Further studies of dsRNA-induced genes will help to better understand the RNAi and immune-related pathways, and guide the theory and practice of using RNAi in pest control

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Conclusion