Genetic Knockouts Indicate That the ABCC2 Protein in the Bollworm Helicoverpa zea Is Not a Major Receptor for the Cry1Ac Insecticidal Protein.

Omaththage P Perera,Gadi V P Reddy,Juan Luis Jurat-Fuentes,Heba Abdelgaffar,Nathan S Little

doi:10.3390/genes12101522

Abstract

Members of the insect ATP binding cassette transporter subfamily C2 (ABCC2) in several moth species are known as receptors for the Cry1Ac insecticidal protein from Bacillus thuringiensis (Bt). Mutations that abolish the functional domains of ABCC2 are known to cause resistance to Cry1Ac, although the reported levels of resistance vary widely depending on insect species. In this study, the function of the ABCC2 gene as a putative Cry1Ac receptor in Helicoverpa zea, a major pest of over 300 crops, was evaluated using CRISPR/Cas9 to progressively eliminate different functional ABCC2 domains. Results from bioassays with edited insect lines support that mutations in ABCC2 were associated with Cry1Ac resistance ratios (RR) ranging from 7.3- to 39.8-fold. No significant differences in susceptibility to Cry1Ac were detected between H. zea with partial or complete ABCC2 knockout, although the highest levels of tolerance were observed when knocking out half of ABCC2. Based on >500–1000-fold RRs reported in similar studies for closely related moth species, the low RRs observed in H. zea knockouts support that ABCC2 is not a major Cry1Ac receptor in this insect.

Highlights

The cry1Ac gene was first commercialized in the USA in 1996 as a plant incorporated protectant (PIP) in cotton [1], and since has been commercialized or authorized as PIP in corn (1997), eggplant in Bangladesh (2013) and the Philippines (2021), rice in China (2009) and the USA (2018), and soybean in several countries [2]
The ATP binding cassette transporter subfamily C2 (ABCC2) gene of H. zea consisted of 25 exons from the putative promoter to the polyadenylation site, spanning nucleotides 97,034 to 110,370 of scaffold 4812
Only the first 24 exons coded for the ABCC2 transporter polypeptide

Summary

Introduction

The main threat to the sustainable use of Cry1Ac as a PIP is the development of resistance in target pests. Practical resistance to Cry1Ac as a PIP in cotton has been reported in populations of bollworm (Helicoverpa zea) in the USA [3], and the pink bollworm (Pectinophora gossypiella) in India [4]. In P. gossypiella, this resistance was associated with alternative splicing which generated aberrant transcripts of cadherin, a receptor for Cry1Ac in that insect [5]. This cadherin that lacks a functional receptor is associated with reduced Cry1Ac binding and resistance [6]. The mechanistic description of resistance in H. zea remains elusive, mutations in a novel cadherin gene [7]

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