High-Throughput Identification of Fluazinam Resistance-Related Genes Based on a Yeast Screening System and Functional Characterization of <i>CgAflT</i> in <i>Colletotrichum gloeosporioides</i>

Anthracnose caused by <i>Colletotrichum gloeosporioides</i> inflicts severe economic losses and is controllable with fluazinam, yet the pathogen's resistance mechanism to this fungicide remains elusive. Here, we first applied yeast-based high-throughput screening combined with sequencing to a cDNA library of a fluazinam-resistant mutant and sequencing, identifying potential fluazinam resistance-related genes. Overexpression of the candidate genes in <i>C. gloeosporioides</i> significantly reduced fluazinam sensitivity of <i>C. gloeosporioides</i>. Notably, the MFS gene <i>CgAflT</i> was characterized as a fluazinam resistance-related gene. Molecular docking confirmed a high-affinity interaction between fluazinam and CgAflT through two hydrogen bonds. Additionally, <i>CgAflT</i> regulates melanin synthesis, stress responses, and virulence in <i>C. gloeosporioides</i>. These findings provide a novel strategy for discovering non-target-site resistance-related genes and demonstrate that <i>CgAflT</i> is involved in fluazinam resistance and pathogenicity of <i>C. gloeosporioides</i>, laying a foundation for further analysis of fluazinam resistance mechanisms and the development of new fungicide targets.