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

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.

Target site–based resistance to penoxsulam in late watergrass (Echinochloa phyllopogon) from China

Late watergrass [Echinochloa phyllopogon (Stapf) Koso-Pol.] is one of the most persistent weeds in rice fields and shows resistance to some acetolactate synthase (ALS)-inhibiting herbicides, such as penoxsulam. Previous studies of E. phyllopogon’s herbicide resistance have focused on non–target site resistance mechanisms. In this study, E. phyllopogon populations from Heilong Jiang Province, China, that were possibly resistant to penoxsulam were used to identify the target site–based mechanisms of resistance. Population HSRH-520 showed a 25.4-fold higher resistance to penoxsulam than the sensitive population, HSRH-538. HSRH-520 was resistant to other ALS inhibitors, with resistance indexes ranging from 17.1 to 166. Target-gene sequence analysis revealed two different ALS genes in E. phyllopogon; a Pro-197-Ser substitution occurred in the ALS-2 gene of HSRH-520. In vitro activity assays revealed that the penoxsulam concentrations required to inhibit 50% of the ALS activity were 13.7 times higher in HSRH-520 than in HSRH-538. Molecular-docking tests showed that the Pro-197-Ser mutation reduced the binding affinity between ALS and ALS inhibitors belonging to the triazolopyrimidine, sulfonylaminocarbonyltriazolinone, and sulfonylurea families, and there were almost no effects on binding affinity when the ALS inhibitors were of the pyrimidinylthiobenzoate and imidazolinone families. Overall, the results indicated and verified that the Pro-197-Ser mutation leads to increased ALS activity by reducing the binding affinity of the inhibitor and ALS. This is the first report on the Pro-197-Ser mutation in the complete ALS gene of E. phyllopogon and will aid future research of target site–based resistance mechanisms of E. phyllopogon to ALS inhibitors.

Ocorrência e importância da resistência a herbicidas causada por incremento de metabolização para o manejo de plantas daninhas

A resistência a herbicidas causada por mecanismos não relacionados ao local de ação (NRLA), principalmente causada pelo incremento de metabolização, é altamente problemática devido à ocorrência de biótipos com resistência múltipla, e é um novo desafio para o manejo de plantas daninhas e utilização de herbicidas. Recentemente, enzimas associadas à degradação de xenobióticos, especialmente citocromo P450, GSTs e transportadores ABC, têm sido associadas à resistência a herbicidas em várias plantas daninhas. Os objetivos desta revisão são descrever o conhecimento atual da regulação gênica associada à resistência a herbicidas causada pelo incremento de metabolização e analisar as principais implicações deste problema para definir estratégias adequadas de manejo com o uso de herbicidas. A resistência múltipla a herbicidas causada pelo incremento da degradação ocorre em Lolium rigidum, Alopecurus myosuroides, Echinochloa phyllopogon e outras várias espécies. O nível de resistência a herbicidas causada pelo incremento de metabolização é afetado por fatores ambientais em vários casos, o que dificulta o diagnóstico da resistência e facilita sua distribuição. O manejo moderno de plantas daninhas deve considerar as características de degradação do herbicida, uma vez que a simples rotação de mecanismo de ação de herbicidas pode não ser suficiente para prevenir a ocorrência da resistência a estes produtos. A utilização de misturas sinergísticas de herbicidas e outros inibidores enzimáticos pode contribuir para prevenir a evolução e distribuição da NRLA em plantas daninhas.