Evolution of herbicide resistance through co-option of an ethylene-associated nitrilase

Adaptive evolution frequently proceeds through the co-option of pre-existing biological functions, yet the molecular mechanisms by which such co-option generates new adaptive traits over contemporary evolutionary timescales remain poorly understood. Herbicide-resistant weeds provide a powerful model for addressing this question because enhanced herbicide metabolism has repeatedly evolved within only a few decades. Here, we show that resistance to cyhalofop-butyl in Echinochloa crus-galli evolved through the co-option of EcNIT4A2, which encodes a nitrilase that normally functions in ethylene-associated cyanide detoxification. Genetic mapping, transcriptomics and transgenic analyses identified EcNIT4A2 as the major determinant of resistance. A D317G substitution enhanced its herbicide-detoxifying activity. Molecular dynamics simulations indicated that this substitution facilitates substrate access to the catalytic pocket. A promoter structural variant markedly increased EcNIT4A2 expression. Our findings reveal how coding and regulatory changes can jointly drive the co-option and optimization of a conserved metabolic function during rapid adaptation under intense anthropogenic selection.