College of Plant Protection, Nanjing Agricultural University, Nanjing, China.
Department of Entomology, University of Arizona, Tucson, Arizona, United States of America.
PLoS Pathog. 2020 Mar 19;16(3):e1008427. doi: 10.1371/journal.ppat.1008427. eCollection 2020 Mar.
Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Better understanding of the genetic basis of resistance is needed to more effectively monitor, manage, and counter pest resistance to Bt toxins. Here we used CRISPR/Cas9 gene editing to clarify the genetics of Bt resistance and the associated effects on susceptibility to other microbial insecticides in one of the world's most damaging pests, the cotton bollworm (Helicoverpa armigera). We discovered that CRISPR-mediated knockouts of ATP-binding cassette (ABC) transporter genes HaABCC2 and HaABCC3 together caused >15,000-fold resistance to Bt toxin Cry1Ac, whereas knocking out either HaABCC2 or HaABCC3 alone had little or no effect. Inheritance of resistance was autosomal and recessive. Bioassays of progeny from interstrain crosses revealed that one wild type allele of either HaABCC2 or HaABCC3 is sufficient to sustain substantial susceptibility to Cry1Ac. In contrast with previous results, susceptibility to two insecticides derived from bacteria other than Bt (abamectin and spinetoram), was not affected by knocking out HaABCC2, HaABCC3, or both. The results here provide the first evidence that either HaABCC2 or HaABCC3 protein is sufficient to confer substantial susceptibility to Cry1Ac. The functional redundancy of these two proteins in toxicity of Cry1Ac to H. armigera is expected to reduce the likelihood of field-evolved resistance relative to disruption of a toxic process where mutations affecting a single protein can confer resistance.
抗药性的进化降低了广泛应用于喷雾和转基因作物中的革兰氏阳性细菌苏云金芽孢杆菌（Bt）的杀虫蛋白的功效。更好地了解抗药性的遗传基础，有助于更有效地监测、管理和对抗Bt 毒素的害虫抗药性。在这里，我们使用 CRISPR/Cas9 基因编辑来阐明Bt 抗药性的遗传学基础，以及在世界上最具破坏性的害虫之一——棉铃虫（Helicoverpa armigera）中对其他微生物杀虫剂敏感性的相关影响。我们发现，CRISPR 介导的 ABC 转运蛋白基因 HaABCC2 和 HaABCC3 的敲除共同导致对 Bt 毒素 Cry1Ac 的抗性超过 15000 倍，而单独敲除 HaABCC2 或 HaABCC3 几乎没有或没有影响。抗性的遗传是常染色体隐性遗传。来自种间杂交的后代的生物测定显示，HaABCC2 或 HaABCC3 的一个野生型等位基因足以维持对 Cry1Ac 的高敏感性。与以前的结果相反，对两种源自除 Bt 以外的细菌的杀虫剂（阿维菌素和 spinetoram）的敏感性不受敲除 HaABCC2、HaABCC3 或两者的影响。这里的结果首次提供了证据，即 HaABCC2 或 HaABCC3 蛋白中的任何一种都足以赋予对 Cry1Ac 的高敏感性。这两种蛋白在 Cry1Ac 对 H. armigera 的毒性中的功能冗余预计会降低相对于破坏一个单一蛋白的突变可以赋予抗性的有毒过程的抗性进化的可能性。
