AlphaFold3-guided single-amino acid substitution creates a dual-functional selection marker for herbicide resistance and genetic tagging in the cruciferous plant pathogen Colletotrichum higginsianum

Colletotrichum higginsianum, a plant pathogenic fungus threatening cruciferous crops, necessitates advanced genetic tools to study its pathogenesis and host interactions. However, selection markers available for fungal genome editing are often limited by scarcity and variable efficacy across species. Acetolactate synthase (ALS), a key enzyme in branched-chain amino acid (BCAA) biosynthesis, is the target of the herbicide chlorimuron ethyl (CE). Here, we utilized AlphaFold3-guided structural prediction to engineer ALS in C. higginsianum into a dual-functional endogenous selection marker. AlphaFold3-predicted ChALS structures revealed conserved catalytic regions and identified hydrophobic residues (V191, A200, F201) potentially critical for CE binding. Notably, introducing a single A200D substitution conferred the resistance of C. higginsianum to CE without impairing fungal growth, conidiation, or pathogenicity. Molecular docking simulations confirmed that the introduced aspartate side chain causes steric clashes that destabilize CE binding. Leveraging this mutation, we developed a homology-directed integration system enabling precise genetic tagging at the endogenous ChALS locus using the A200D allele for selection. Colletotrichum higginsianum strains expressing fluorescent proteins targeted to the nucleus, mitochondria, peroxisomes, or cytoplasm exhibited stable expression and unaltered infection dynamics in Chinese flowering cabbage (Brassica parachinensis). This work presents a dual-functional marker that addresses limitations in fungal genome editing tools. Moreover, the facility with which the A200D mutation confers resistance serves as a cautionary note on the potential evolutionary instability of single-target enzyme inhibitors in pathogen management.