| Summary: | Aflatoxin is a carcinogenic secondary metabolite that poses a serious threat to human and animal health. Some C<sub>2</sub>H<sub>2</sub> transcription factors are associated with fungal growth and secondary metabolic regulation. In this study, we characterized the role of <i>AflZKS3</i>, a putative C<sub>2</sub>H<sub>2</sub> transcription factor based on genome annotation, in the growth and aflatoxin biosynthesis of <i>A. flavus</i> and explored its possible mechanisms of action. Surprisingly, the protein was found to be located in the cytoplasm, and gene deletion in <i>A. flavus</i> resulted in defective growth and conidia formation, as well as increased sensitivity to the fluorescent brightener Calcofluor white, Congo red, NaCl, and sorbitol stress. Notably, the biosynthesis of aflatoxin B<sub>1</sub> was completely inhibited in the Δ<i>AflZKS3</i> deletion strain, and its ability to infect peanut and corn seeds was also reduced. RNA sequencing showed that differentially expressed genes in the Δ<i>AflZKS3</i> strain compared with the control and complementation strains were mainly associated with growth, aflatoxin biosynthesis, and oxidative stress. Thus, <i>AflZKS3</i> likely contributes to growth, cell development, and aflatoxin synthesis in <i>A. flavus</i>. These findings lay the foundation for a deeper understanding of the roles of C<sub>2</sub>H<sub>2</sub> transcription factors in <i>A. flavus</i> and provide a potential biocontrol target for preventing aflatoxin contamination.
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