Cheng Yang, Chen Yu-Xin, Gao Jia-Nan, Chen Jie, Huang Jia, Qiao Xiaomu
Ministry of Agriculture Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Institute of Insect Sciences, Zhejiang University, Hangzhou 310058, China.
Collaborative Innovation Center of Green Pesticide, National Joint Local Engineering Laboratory of Biopesticide Preparation, School of Forestry and Biotechnology, Zhejiang A&F University, Hangzhou 311300, China.
J Agric Food Chem. 2025 May 21;73(20):12176-12183. doi: 10.1021/acs.jafc.5c01017. Epub 2025 May 12.
The evolution of resistance to neonicotinoid insecticides threatens global agriculture. To elucidate its molecular basis, we employed as a model system to investigate resistance-associated mutations in the β1 subunit of nicotinic acetylcholine receptors (nAChRs). Using a CRISPR/Cas9-mediated allele replacement, we generated homozygous knock-in mutants (V62I, V101I, R81E, and A60T,R81E) without apparent fitness costs. Toxicity bioassays revealed that these mutations confer varying resistance levels, with the R81E mutation exhibiting over 225-fold resistance to thiamethoxam, clothianidin, and dinotefuran. A heteropentameric α1β1 nAChR model, generated using an AI-based protein-ligand prediction (Chai-1), showed that imidacloprid binds at the orthosteric site, where R81 forms a critical electrostatic interaction. Residues A60, V62, and V101, positioned further from the binding site, showed spatial distances correlated with their resistance ratios. These findings provide genetic and structural insights into neonicotinoid resistance mechanisms, offering a foundation for the design of next-generation insecticides and resistance management strategies.
对新烟碱类杀虫剂的抗性进化威胁着全球农业。为了阐明其分子基础，我们以烟碱型乙酰胆碱受体（nAChRs）的β1亚基为模型系统，研究与抗性相关的突变。利用CRISPR/Cas9介导的等位基因替换，我们生成了无明显适合度代价的纯合敲入突变体（V62I、V101I、R81E和A60T，R81E）。毒性生物测定表明，这些突变赋予了不同的抗性水平，其中R81E突变对噻虫嗪、氯噻啉和呋虫胺表现出超过225倍的抗性。使用基于人工智能的蛋白质-配体预测（Chai-1）生成的异源五聚体α1β1 nAChR模型表明，吡虫啉结合在正构位点，其中R81形成关键的静电相互作用。距离结合位点较远的A60、V62和V101残基显示出与其抗性比率相关的空间距离。这些发现为新烟碱类抗性机制提供了遗传和结构方面的见解，为下一代杀虫剂的设计和抗性管理策略奠定了基础。
