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 United States, which cannot be solely explained by the overexpression of promiscuous cytochrome P450 monooxygenases CYP81A12/21. The BSHR late watergrass line rapidly produced 2 distinct hydroxylated diclofop acids, only 1 of which was the major metabolite produced by CYP81A12/21. RNA-seq and subsequent reverse transcription quantitative PCR (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 3 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 3 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 2 countries suggests that BSHR evolved through co-opting a conserved gene regulatory system in late watergrass.

CYP81A P450s are involved in concomitant cross-resistance to acetolactate synthase and acetyl-CoA carboxylase herbicides in Echinochloa phyllopogon.

Californian populations of Echinochloa phyllopogon have evolved multiple-herbicide resistance (MHR), posing a threat to rice production in California. Previously, we identified two CYP81A cytochrome P450 genes whose overexpression is associated with resistance to acetolactate synthase (ALS) inhibitors from two chemical groups. Resistance mechanisms to other herbicides remain unknown. We analyzed the sensitivity of an MHR line to acetyl-CoA carboxylase (ACCase) inhibitors from three chemical groups, followed by an analysis of herbicide metabolism and segregation of resistance of the progenies in sensitive (S) and MHR lines. ACCase herbicide metabolizing function was investigated in the two previously identified P450s. MHR plants exhibited resistance to all the ACCase inhibitors by enhanced herbicide metabolism. Resistance to the ACCase inhibitors segregated in a 3 : 1 ratio in the F2 generation and completely co-segregated with ALS inhibitor resistance in F6 lines. Expression of the respective P450 genes conferred resistance to the three herbicides in rice, which is in line with the detection of hydroxylated herbicide metabolites in vivo in transformed yeast. CYP81As are super P450s that metabolize multiple herbicides from five chemical classes, and concurrent overexpression of the P450s induces metabolism-based resistance to the three ACCase inhibitors in MHR E. phyllopogon, as it does to ALS inhibitors.