Diclofop-methyl resistance conferred by LrGSTU181 overexpression in Lolium rigidum

Lolium rigidum significantly affects the global agricultural economy, with metabolic resistance becoming increasingly pronounced. Previous research indicated that the Australian L. rigidum population (WALR70) exhibits non-target-site resistance (NTSR) to diclofop-methyl. This study aims to investigate the resistance mechanisms. No point mutations or gene overexpression of the target-site results show the identified LrGSTU181 confers were detected. However, Glutathione S-transferase (GST) enzyme activities were significantly higher (by a factor of 2.8) in the resistant than the susceptible population (SVLR1), and the GST inhibitor (NBD-Cl) partially reversed the resistance to diclofop-methyl. RNA-seq and RT-qPCR identified a significantly upregulated GST23 gene (named LrGSTU181), and a UGT79 gene in WALR70 compared to SVLR1. LrGSTU181-transformed yeast cells exhibited enhanced tolerance to diclofop-methyl, while UGT79-transformed yeast did not, as compared to the empty vector control. LrGSTU181-transformed rice calli and seedlings exhibited moderate resistance to diclofop-methyl compared to the GFP-transformed control. Molecular docking indicates that the LrGSTU181 protein binds to diclofop acid with high affinity. This study functionally establishes, for the first time, that a tau class GST (LrGSTU181) mediates diclofop-methyl resistance in L. rigidum, expanding the known metabolic basis of NTSR in this species.