From Weed Evolution to Crop Design: A Computational Blueprint for a Novel, Synergistic Herbicide-Resistant Allele in Wheat

The escalating crisis of herbicide-resistant weeds threatens global wheat production. While key mutations are well-documented in weeds, the principles governing their interactions in wheat remain largely unknown. Here, we first developed a novel wheat germplasm carrying the acetolactate synthase (TaALS) Ser-627-Asn (S627N) mutation via ethyl methanesulfonate (EMS) mutagenesis. We then employed a computational design strategy to explore its synergy with the prevalent Trp-548-Leu (W548L) mutation—a combination not yet reported in nature. Integrated molecular dynamics (MD) simulations and free energy landscape analysis revealed that the in silico W548L/S627N double mutant triggers synergistic global destabilization of the herbicide–enzyme complex. Binding affinity progressively weakened from wild-type (−25.54 ± 2.05 kcal/mol) to the double mutant (−18.13 ± 2.76 kcal/mol), driven by a polarity inversion at the Arg-347 anchor. Comparative transcriptomic profiling of the S627N germplasm confirmed the absence of deleterious metabolic feedback in the branched-chain amino acid biosynthesis pathway. This work exemplifies a paradigm shift from mimicking natural variation to predictive crop design via multiplex gene editing.