SummaryPopulations of Polypogon fugax have developed resistance to many acetyl‐CoA carboxylase (ACCase)‐inhibiting herbicides. This resistance threats the effectiveness and sustainability of herbicide use. In our previous research, a field P. fugax population exhibited GST‐based metabolic resistance to the widely used ACCase‐inhibiting herbicide quizalofop‐p‐ethyl. Here, in this current study, we identified and characterized two GST genes (named as PfGSTF2 and PfGSTF58) that showed higher expression levels in the resistant than the susceptible population. Transgenic rice calli overexpressing PfGSTF2, but not PfGSTF58, became resistant to quizalofop‐p‐ethyl and haloxyfop‐R‐methyl. This reflects similar cross‐resistance pattern to what was observed in the resistant P. fugax population. Transgenic rice seedlings overexpressing PfGSTF2 also exhibited resistance to quizalofop‐p‐ethyl. In contrast, CRISPR/Cas9 knockout of the orthologue gene in rice seedlings increased their sensitivity to quizalofop‐p‐ethyl. LC–MS analysis of in vitro herbicide metabolism by Escherichia coli‐expressed recombinant PfGSTF2 revealed that quizalofop (but not haloxyfop) was detoxified at the ether bond, generating the GSH‐quizalofop conjugate and a propanoic acid derivative with greatly reduced herbicidal activity. Equally, these two metabolites accumulated at higher levels in the resistant population than the susceptible population. In addition, both recombinant PfGSTF2 and PfGSTF58 can attenuate cytotoxicity by reactive oxygen species (ROS), suggesting a role in plant defence against ROS generated by herbicides. Furthermore, the GST inhibitor (NBD‐Cl) reversed resistance in the resistant population, and PfGSTF2 (but not PfGSTF58) responded to NBD‐Cl inhibition. All these suggest that PfGSTF2 plays a significant role in the evolution of quizalofop resistance through enhanced herbicide metabolism in P. fugax.

中文翻译：

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PfGSTF2 通过 GSH 偶联赋予 Polypogon fugax 对 quizalofop‐p-ethyl 的抗性


摘要Polypogon fugax 种群已经对许多乙酰辅酶 A 羧化酶 （ACCase） 抑制除草剂产生了耐药性。这种抗药性威胁到除草剂使用的有效性和可持续性。在我们之前的研究中，田间 P. fugax 种群对广泛使用的 ACCase 抑制除草剂 quizalofop‐p-ethyl 表现出基于 GST 的代谢抗性。在这里，在目前的这项研究中，我们鉴定并表征了两个 GST 基因 （命名为 PfGSTF2 和 PfGSTF58），它们在耐药人群中的表达水平高于易感人群。过表达 PfGSTF2 但不表达 PfGSTF58 的转基因水稻愈伤组织对 quizalofop-p-ethyl 和 haloxyfop-R-methyl 产生抗性。这反映了与在抗性 P. fugax 种群中观察到的相似的交叉抗性模式。过表达 PfGSTF2 的转基因水稻幼苗也表现出对 quizalofop-p-ethyl 的抗性。相比之下，水稻幼苗中直系同源基因的 CRISPR/Cas9 敲除增加了它们对 quizalofop-p-ethyl 的敏感性。大肠杆菌表达的重组 PfGSTF2 对体外除草剂代谢的 LC-MS 分析表明，喹喹嘉（但不是 haloxyfop）在醚键处解毒，生成 GSH-喹喹喹咪咪偶联物和除草活性大大降低的丙酸衍生物。同样，这两种代谢物在耐药人群中的积累水平高于易感人群。此外，重组 PfGSTF2 和 PfGSTF58 都可以减轻活性氧 （ROS） 的细胞毒性，表明在植物防御除草剂产生的 ROS 中发挥作用。此外，GST 抑制剂 （NBD-Cl） 逆转了耐药人群的耐药性，并且 PfGSTF2 （而不是 PfGSTF58） 对 NBD-Cl 抑制有反应。 所有这些都表明，PfGSTF2 通过增强 P. fugax 中的除草剂代谢，在 quizalofop 抗性的进化中发挥重要作用。