ALS target-site mutations and overexpression synergistically enhance mesosulfuron resistance in Lolium perenne

The invasive weed Lolium perenne (perennial ryegrass) poses a considerable threat to winter wheat crops in China and is hardly controlled by acetolactate synthase (ALS) inhibitors after years of application. In this study, four L. perenne populations from different locations in Henan were collected to evaluate the mechanisms of target site resistance to mesosulfuron-methyl. The resistant index (RI) of these four populations ranged from 122.73 to 149.26 compared to the susceptible population. ALS gene sequencing revealed three specific target site mutations: Pro-197-Thr, Asp-376-Glu, and Trp-574-Leu. Among these, Trp-574-Leu has not been previously reported in L. perenne. Three simultaneous double mutations were found in the same individual: Pro-197-Thr and Asp-376-Glu (Group I); Pro-197-Thr and Trp-574-Leu (Group II); and Asp-376-Glu and Trp-574-Leu (Group III). The gene expression levels of the double mutant groups were significantly higher than those of the susceptible plants. The I₅₀ values obtained from the ALS enzyme assays showed that all three double mutant groups demonstrated a 136.75 to 149-fold increase compared to the enzyme activity of susceptible plants. Molecular docking analysis of mutant ALS proteins with mesosulfuron-methyl indicated that the mutant proteins had a reduced binding affinity to the herbicide. This reduced affinity was due to the disruption of hydrogen bonds and other key interactions, which contributed to increased herbicide resistance. Double target site mutations in a single ALS gene are a crucial mechanism for conferring high levels of mesosulfuron-methyl resistance in L. perenne.