Optimization of recombinant bacteria expressing dsRNA to enhance insecticidal activity against a lepidopteran insect, Spodoptera exigua

Mohammad Vatanparast,Yonggyun Kim,Erjun Ling

doi:10.1371/journal.pone.0183054

Mohammad Vatanparast, Yonggyun Kim + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0183054

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Journal: PloS one	Publication Date: Aug 11, 2017
Citations: 43	License type: CC BY 4.0

Affiliation: Bu-Ali Sina University, Andong National University

Abstract

Double-stranded RNA (dsRNA) has been applied to control insect pests due to its induction of RNA interference (RNAi) of a specific target gene expression. However, developing dsRNA-based insecticidal agent has been a great challenge especially against lepidopteran insect pests due to variations in RNAi efficiency. The objective of this study was to screen genes of chymotrypsins (SeCHYs) essential for the survival of the beet armyworm, Spodoptera exigua, to construct insecticidal dsRNA. In addition, an optimal oral delivery method was developed using recombinant bacteria. At least 7 SeCHY genes were predicted from S. exigua transcriptomes. Subsequent analyses indicated that SeCHY2 was widely expressed in different developmental stages and larval tissues by RT-PCR and its expression knockdown by RNAi caused high mortality along with immunosuppression. However, a large amount of dsRNA was required to efficiently kill late instars of S. exigua because of high RNase activity in their midgut lumen. To minimize dsRNA degradation, bacterial expression and formulation of dsRNA were performed in HT115 Escherichia coli using L4440 expression vector. dsRNA (300 bp) specific to SeCHY2 overexpressed in E. coli was toxic to S. exigua larvae after oral administration. To enhance dsRNA release from E. coli, bacterial cells were sonicated before oral administration. RNAi efficiency of sonicated bacteria was significantly increased, causing higher larval mortality at oral administration. Moreover, targeting young larvae possessing weak RNase activity in the midgut lumen significantly enhanced RNAi efficiency and subsequent insecticidal activity against S. exigua.

Highlights

RNA interference (RNAi) regulates gene expression at post-transcriptional level by reducing the half-life of specific mRNA or interfering with translational efficiency [1]
DsRNA insecticide using transgenic crops expressing double-stranded RNA (dsRNA) support dsRNA-based insect pest control strategy [4,5]
CHY was selected as a target gene to construct dsRNA to dsRNA insecticide control S. exigua because inhibitor assay against different digestive enzymes indicated that CHY-specific inhibitor highly intimidated survival of S. exigua larvae

Summary

Introduction

RNA interference (RNAi) regulates gene expression at post-transcriptional level by reducing the half-life of specific mRNA or interfering with translational efficiency [1]. DsRNA insecticide using transgenic crops expressing dsRNA support dsRNA-based insect pest control strategy [4,5]. To this end, dsRNA strategy has been considered for controlling S. exigua. A developmental gene, has been tested as RNAi target through dsRNA strategy, causing significant mortality [11]. To interfere with cell-cell interaction, a β-subunit of integrin has been knockeddown by dsRNA, resulting in significant mortality of S. exigua [2]. These results support that it is feasible to use dsRNA to control S. exigua. To improve insecticidal efficiency, better target gene and efficient dsRNA delivery method are needed

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Results

Conclusion