Transcriptionally linked simultaneous overexpression of P450 genes for broad-spectrum herbicide resistance.

Broad-spectrum herbicide resistance (BSHR), often linked to weeds with metabolism-based herbicide resistance, poses a threat to food production. Past studies have revealed that overexpression of catalytically-promiscuous enzymes explains BSHR in some weeds; however, the mechanism of BSHR expression remains poorly understood. Here, we investigated the molecular basis of high-level resistance to diclofop-methyl in BSHR late watergrass (Echinochloa phyllopogon) found in the US, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced two distinct hydroxylated-diclofop-acids, only one of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent RT-qPCR-based segregation screening identified the transcriptionally-linked overexpression of a gene, CYP709C69, with CYP81A12/21 in the BSHR line. The gene conferred diclofop-methyl resistance in plants and produced another hydroxylated-diclofop-acid in yeast (Saccharomyces cerevisiae). Unlike CYP81A12/21, CYP709C69 showed no other herbicide-metabolizing function except for a presumed clomazone-activating function. The overexpression of the three herbicide-metabolizing genes was also identified in another BSHR late watergrass in Japan, suggesting a convergence of BSHR evolution at the molecular level. Synteny analysis of the P450 genes implied that they are located at mutually independent loci, which supports the idea that a single trans-element regulates the three genes. We propose that transcriptionally-linked simultaneous overexpression of herbicide-metabolizing genes enhances and broadens the metabolic resistance in weeds. The convergence of the complex mechanism in BSHR late watergrass from two countries suggests that BSHR evolved through co-opting a conserved gene-regulatory system in late watergrass.