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

Anthracnose caused by Colletotrichum gloeosporioides 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 C. gloeosporioides significantly reduced fluazinam sensitivity of C. gloeosporioides. Notably, the MFS gene CgAflT was characterized as a fluazinam resistance-related gene. Molecular docking confirmed a high-affinity interaction between fluazinam and CgAflT through two hydrogen bonds. Additionally, CgAflT regulates melanin synthesis, stress responses, and virulence in C. gloeosporioides. These findings provide a novel strategy for discovering non-target-site resistance-related genes and demonstrate that CgAflT is involved in fluazinam resistance and pathogenicity of C. gloeosporioides, laying a foundation for further analysis of fluazinam resistance mechanisms and the development of new fungicide targets.