Multiple Mutations in the β1 Subunit of the Nicotinic Acetylcholine Receptor Confer Resistance to Neonicotinoids

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.