Glutathione Transferases Are Linked to Penoxsulam Resistance in Echinochloa crus-galli (L.) P. Beauv.

Increasing evidence indicates that nontarget-site resistance (NTSR) plays a crucial role in herbicide stress tolerance, though its mechanisms remain largely unexplored. We identified a penoxsulam-resistant Echinochloa crus-galli (R-JS) that exhibits a 13.8-fold increase in resistance without any target-site resistance mutations. Glutathione transferase (GST) inhibitor reduced resistance, whereas GST activity increased under penoxsulam stress. RNA-seq revealed the upregulation of detoxification genes, including GSTS, CYP450s, GTs, ABC transporters, and AKRs. Among the six overexpressed GSTs, GST4 exhibited both consistent upregulation in R-JS and specific induction by penoxsulam in RT-qPCR analysis. Molecular docking analyses demonstrated that GSTF1 and GST4 exhibited the highest affinity for penoxsulam, and GST4 overexpression enhanced yeast resistance to penoxsulam. Site-directed mutagenesis and HPLC-based metabolic assays demonstrated the impact of Lys-80, Leu-84, Gly-85, and Glu-91 on penoxsulam metabolism. This study advances our understanding of GST4-mediated NTSR mechanisms underlying penoxsulam resistance in the R-JS population and provides valuable insights for E. crus-galli management.