Mutations in Promoter Region Enhanced PxαE27-Mediated β-Cypermethrin Resistance in Plutella xylostella

Insecticide resistance in insects often involves overexpression of carboxylesterase (CarE) genes. In the diamondback moth, Plutella xylostella, β-cypermethrin resistance is linked to elevated expressions of PxαE9, PxαE27, and PxαE14. To elucidate the underlying transcriptional regulation, we identified their core promoters and predicted, via in silico analysis (ALGGEN and JASPAR), that the transcription factor Dfd binds to PxαE27 and PxαE14. Experimental validation using dual-luciferase assays, site-directed mutagenesis, yeast one-hybrid assays, and RNA interference confirmed that Dfd enhances PxαE27 transcription by direct promoter binding. Furthermore, a two-nucleotide mutation in the PxαE27 promoter of a field-resistant population created a novel Dfd-binding site, further upregulating expression and increasing β-cypermethrin resistance. This study reveals how cis-regulatory mutations recruit specific transcription factors to elevate CarE expression and drive insecticide resistance, providing a mechanistic basis for potential resistance management strategies.