A versatile contribution of both aminopeptidases N and ABC transporters to Bt Cry1Ac toxicity in the diamondback moth

Dan Sun,Le Guo,Liuhong Zhu,Youjun Zhang,Shaoli Wang,Alejandra Bravo,Qingjun Wu,Neil Crickmore,Zhaojiang Guo,Xuguo Zhou,Mario Soberón

doi:10.1186/s12915-022-01226-1

Abstract

BackgroundBiopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial.ResultsWe have examined the contribution of two paralogous ABC transporters and two aminopeptidases N to Bt Cry1Ac toxicity in the diamondback moth, Plutella xylostella, using CRISPR/Cas9 to generate a series of homozygous polygenic knockout strains. A double-gene knockout strain, in which the two paralogous ABC transporters ABCC2 and ABCC3 were deleted, exhibited 4482-fold resistance to Cry1A toxin, significantly greater than that previously reported for single-gene knockouts and confirming the mutual functional redundancy of these ABC transporters in acting as toxin receptors in P. xylostella. A double-gene knockout strain in which APN1 and APN3a were deleted exhibited 1425-fold resistance to Cry1Ac toxin, providing the most direct evidence to date for these APN proteins acting as Cry1Ac toxin receptors, while also indicating their functional redundancy. Genetic crosses of the two double-gene knockouts yielded a hybrid strain in which all four receptor genes were deleted and this resulted in a > 34,000-fold resistance, indicating that while both types of receptor need to be present for the toxin to be fully effective, there is a level of functional redundancy between them. The highly resistant quadruple knockout strain was less fit than wild-type moths, but no fitness cost was detected in the double knockout strains.ConclusionOur results provide direct evidence that APN1 and APN3a are important for Cry1Ac toxicity. They support our overarching hypothesis of a versatile mode of action of Bt toxins, which can compensate for the absence of individual receptors, and are consistent with an interplay among diverse midgut receptors in the toxins’ mechanism of action in a super pest.

Highlights

Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness
The best established Bt resistance mechanism in insects is via alterations of these midgut receptors such as cadherin (CAD), aminopeptidases-N (APNs), alkaline phosphatases (ALPs), and ABC transporters, that interrupt toxin binding [15, 16]
Construction of double knockout strains by CRISPR/Cas9 Based on the fact that the PxABCC2 and PxABCC3 receptor genes are adjacent on the P. xylostella genome, as are the PxAPN1 and PxAPN3a receptor genes, a dual single guide RNA (sgRNA) CRISPR/Cas9 approach was used to knockout each pair of genes

Summary

Introduction

Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. The best established Bt resistance mechanism in insects is via alterations of these midgut receptors such as cadherin (CAD), aminopeptidases-N (APNs), alkaline phosphatases (ALPs), and ABC transporters, that interrupt toxin binding [15, 16]. Among these receptors, structural mutations or expression alterations of ABC transporters (especially ABCC2) have been widely reported to be associated with high levels of resistance in lepidopteran insects [17,18,19,20,21,22,23,24,25,26,27,28]. Previous studies suggested that the interactions of ABC transporters with cadherin could enhance cell toxicity, and co-operation between these two receptors was demonstrated by in vitro ectopic expression in different cell lines [36,37,38,39]

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