Summary

The use of herbicides in agricultural fields has driven the evolution of weeds for resistance, causing a grave threat to the current agriculture. One big mystery of weed resistance involves multiple-herbicide resistance (MHR) concomitant to enhanced herbicide metabolism. Previous research unveiled that the overexpression of catalytically promiscuous cytochrome P450s underlies the metabolism-based cross-resistance in multiple species. However, the concept of activation of promiscuous enzymes does not fully accommodate the resistance to diverse herbicides in MHR Echinochloa phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control. Here, we show that the high-level resistance to diclofop-methyl in E. phyllopogon is caused by the simultaneous overexpression of CYP81A12/21, the previously identified promiscuous P450s, and a novel P450 CYP709C69. We found that the MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81As. RNA-seq followed by real-time PCR in the crossed progeny of MHR and sensitive lines identified several P450 genes whose overexpressions were associated with MHR. Gene functional characterization revealed that only CYP709C69 conferred diclofop-methyl resistance in rice calli and produced another hydroxylated-diclofop-acid in yeast, reinforcing the relatively low activity of CYP81As to diclofop-methyl. Plants transformed with CYP709C69 had unchanged sensitivity to 46 herbicides except for clomazone, where transgenic plants became more susceptible. The present findings establish a novel concept that simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds.