Genetically-linked simultaneous overexpression of multiple herbicide-metabolizing genes for broad-spectrum resistance in an agricultural weed Echinochloa phyllopogon

O_LIPrevious research unveiled that the overexpression of catalytically promiscuous CYP81A cytochrome P450s underlies the multiple-herbicide resistance (MHR) in a Californian population of Echinochloa phyllopogon. However, it does not fully accommodate the resistance to diverse herbicides in MHR E. phyllopogon although the genetic inheritance of MHR was suggested as under a single gene control. C_LIO_LIWe investigated the high-level resistance to diclofop-methyl in MHR E. phyllopogon. Detailed diclofop-methyl metabolism was analyzed, followed by gene expression study and functional characterization of P450 genes. The generality of the MHR mechanism was investigated using another MHR line. C_LIO_LIThe MHR line rapidly produced two distinct hydroxylated-diclofop-acid, only one of which was the major metabolite produced by CYP81A12/21. Gene expression study identified the genetically linked overexpression of a novel gene CYP709C69 with CYP81A12/21 in the MHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated-diclofop-acid in yeast. The activity was observed in some CYP709C in plants. Unlike the broad substrate-specificity in CYP81As, CYP709C69 showed narrow substrate-specificity. The overexpression of the CYP81A and CYP709C69 was also observed in another MHR line. C_LIO_LIThe present findings establish a novel concept that genetically-linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the profile of metabolic resistance in weeds. C_LI