PfCYP94C179 endows resistance to fenoxaprop‐ P ‐ethyl in Polypogon fugax : insights into its herbicide detoxification function and transcription regulation

SUMMARY Cytochrome P450s (P450s) play a central role in phase I detoxification in plants; however, the specific enzymes and the underlying transcriptional networks driving metabolic herbicide resistance in arable weeds remain poorly understood. In this study, we characterized the molecular basis of metabolic resistance to fenoxaprop‐ P ‐ethyl (FPE), an acetyl‐CoA carboxylase (ACCase) inhibitor, in the globally invasive weed Polypogon fugax . We identified PfCYP94C179 , a member of the CYP94 family, as a primary resistance determinant that is constitutively overexpressed across multiple metabolic‐FPE‐resistant populations. Heterologous expression in yeast demonstrated that PfCYP94C179 metabolizes FPE via O ‐deethylation and subsequent cleavage, a catalytic process dependent on the conserved active site residue Val‐289. Overexpression (OE) of PfCYP94C179 in rice ( Oryza sativa L.) conferred more than fourfold FPE resistance, whereas silencing of the gene in resistant P. fugax via virus‐induced gene silencing significantly increased herbicide susceptibility. By excluding cis ‐regulatory mutations as the cause of OE, we identified the WRKY family member PfWRKY71 as a key trans ‐acting regulator. Yeast one‐hybrid, transient expression, and electrophoretic mobility shift assays demonstrated that PfWRKY71 directly binds to the PfCYP94C179 promoter to activate transcription. Gain‐ and loss‐of‐function experiments established PfWRKY71 as a positive regulator sufficient to enhance FPE tolerance in rice and essential for the maintenance of PfCYP94C179 expression and FPE resistance in P. fugax . Collectively, our findings reveal a WRKY‐P450 regulatory module that underpins metabolic herbicide resistance, providing critical mechanistic insights into the evolution of metabolic herbicide resistance and offering potential molecular targets for sustainable weed management.