Overexpression of the cytochrome P450 gene CYP99A3 likely mediates mesosulfuron-methyl resistance in Lolium multiflorum from China.

Lolium multiflorum L. (Italian ryegrass) is a noxious weed causing severe yield losses in wheat production globally, and its rapid expansion across major wheat-producing regions in China poses an escalating agricultural threat. Recently, field-recommended doses of the acetolactate synthase (ALS) inhibitor mesosulfuron-methyl have failed to effectively control certain L. multiflorum populations. This study evaluated the resistance level and elucidated the underlying mechanisms in a suspected mesosulfuron-methyl-resistant L. multiflorum population (ZMHF-R) collected from China. Whole-plant bioassays demonstrated that the ZMHF-R population exhibited a high level of resistance (43.34-fold) to mesosulfuron-methyl. Replicate sequencing of the ALS gene revealed no known target-site mutations, thereby excluding target-site mutation-mediated resistance. Pretreatment with the cytochrome P450 inhibitors piperonyl butoxide (PBO) and malathion significantly synergized mesosulfuron-methyl toxicity in ZMHF-R, implicating P450 monooxygenases in the resistance phenotype. Comparative RNA-seq and RT-qPCR validation identified the constitutive overexpression of a specific P450 gene, LmCYP99A3, in the R population. Molecular docking simulations predicted favorable binding and potential metabolic activity of the LmCYP99A3 protein toward mesosulfuron-methyl. Furthermore, transgenic rice calli heterologously overexpressing LmCYP99A3 exhibited a distinct resistance phenotype against the herbicide. Taken together, this study functionally validates that LmCYP99A3 mediates mesosulfuron-methyl resistance in L. multiflorum, expanding our understanding of the metabolic basis of NTSR in this global weed species.