Knockout of three aminopeptidase N genes does not affect susceptibility of Helicoverpa armigera larvae to Bacillus thuringiensis Cry1A and Cry2A toxins.

Jing Wang,Yi‐Hua Yang,Yi‐Dong Wu,Shao‐Yan Liu,Ya‐Yun Zuo,Hui Wang,Ling‐Li Li

doi:10.1111/1744-7917.12666

Jing Wang, Yi‐Hua Yang + Show 5 more

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https://doi.org/10.1111/1744-7917.12666

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Journal: Insect Science	Publication Date: Mar 14, 2019
Citations: 12	License type: CC BY 4.0

Affiliation: Nanjing Agricultural University

Abstract

Bacillus thuringiensis (Bt) insecticidal toxins have been globally utilized for control of agricultural insects through spraying or transgenic crops. Binding of Bt toxins to special receptors on midgut epithelial cells of target insects is a key step in the mode of action. Previous studies suggested aminopeptidase N1 (APN1) as a receptor or putative receptor in several lepidopteran insects including Helicoverpa armigera through evidence from RNA interefence‐based gene silencing approaches. In the current study we tested the role of APNs in the mode of action of Bt toxins using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated protein 9‐mediated gene knockout. Three APN genes (HaAPN1, HaAPN2 and HaAPN5) were individually knocked out in a susceptible strain (SCD) of H. armigera to establish three homozygous knockout strains. Qualitative in vitro binding studies indicated binding of Cry1Ac or Cry2Ab to midgut brush border membrane vesicles was not obviously affected by APN knockout. Bioassay results showed that none of the three knockouts had significant changes in susceptibility to Cry1A or Cry2A toxins when compared with the SCD strain. This suggests that the three HaAPN genes we tested may not be critical in the mode of action of Cry1A or Cry2A toxins in H. armigera.

Highlights

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces insecticidal crystal (Cry) proteins during sporulation (Schnepf et al, 1998)
Our study aimed to investigate the involvement of H. armigera aminopeptidase Ns (APNs) in mediating Cry toxin susceptibility through clustered regularly interspaced palindromic repeats (CRISPR)/Cas9-based reverse genetics approach
Genomic DNA of the G0 adults was prepared after oviposition, and sequencing results showed that 62.0% to 96.6% of the G0 individuals were edited for HaAPN1, HaAPN2 and HaAPN5

Summary

Introduction

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that produces insecticidal crystal (Cry) proteins during sporulation (Schnepf et al, 1998). The mechanism of action of Bt Cry proteins against susceptible lepidopterans is complex and consists of multiple steps The inclusion body is solubilized in the larval midgut to release protoxins, which are processed by gut proteases into active toxins. The toxins bind to specific receptors at the surface of midgut epithelia cells, subsequently resulting in membrane insertion, pore formation and cell lysis (de Maagd et al, 2001). There is general agreement on the above main steps of mode of action, the precise mechanism of toxinreceptor interactions and pore formation is still poorly understood

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