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