Aspergillus flavus and its primary secondary metabolite, aflatoxin B1, pose a persistent threat to global food security and public health, highlighting the need to identify novel molecular targets for the development of highly specific fungicides. In this study, the transcription factor Aflrpn4 was investigated by constructing gene deletion and complementation strains to elucidate its regulatory mechanisms in controlling the growth, development, and pathogenicity of
A. flavus. Phenotypic analysis revealed that, compared with the wild-type and complemented strains, loss of
Aflrpn4 severely restricted radial colony growth, reduced conidial yield, and caused structural defects in conidiophores. Furthermore, AFB1 content was reduced by 52% compared with the wild-type. In storage simulation assays using peanut and maize kernels, the
ΔAflrpn4 strain exhibited significantly compromised colonization capacity, reduced biomass, and lower AFB1 accumulation. Under aflatoxin-inducing YES culture conditions, deletion of
Aflrpn4 was associated with significant downregulation of key pathway-specific regulatory and structural genes, including
aflR,
aflS, and
aflP. Furthermore, under osmotic stress induced by 1.2 M NaCl and KCl, the growth inhibition rates of the
ΔAflrpn4 strain reached 70% and 59%, respectively, and cell membrane integrity was severely compromised. Loss of
Aflrpn4 also disrupted intracellular redox homeostasis, characterized by a significant decrease in superoxide dismutase activity, compensatory increases in catalase and peroxidase activities, and substantial accumulation of reactive oxygen species. Collectively, these findings demonstrate that Aflrpn4 acts as a pivotal regulator coordinating vegetative growth, asexual development, stress adaptation, and aflatoxin biosynthesis in
A. flavus. Consequently, Aflrpn4 represents a promising molecular target for developing targeted interventions to control
A. flavus and aflatoxin contamination during grain storage.
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