Aspergillus flavus produces carcinogenic and mutagenic aflatoxins, which cause economic losses and risk of food safety by contaminating grains, food and feed. In this study, we characterized two bZIP transcription factors, AflatfA and AflatfB, and their genetic interaction. Compared to the wild type (WT),
AflatfA deletion and
AflatfA and
AflatfB double deletion both caused retarded vegetative growth of mycelia. Relative to WT, the
AflatfA deletion strain (Δ
AflatfA) and
AflatfA and
AflatfB double deletion strain (Δ
AflatfAΔ
AflatfB) produced more sclerotia, whereas the
AflatfB deletion strain (Δ
AflatfB) produced less sclerotia. After 4 °C preservation and incubation at 50 °C, conidia viability dramatically decreased in the Δ
AflatfA and Δ
AflatfAΔ
AflatfB but Δ
AflatfB mutants, whereas conidia viability of the Δ
AflatfAΔ
AflatfB strain was higher after storage at 4 °C than in
AflatfA mutant. Conidia of Δ
AflatfA, Δ
AflatfB and Δ
AflatfAΔ
AflatfB strains significantly increased in sensitivity to H
2O
2 in comparison with WT. Compared to WT, the mycelium of Δ
AflatfA and Δ
AflatfB strains were more sensitive to H
2O
2; conversely, the Δ
AflatfAΔ
AflatfB strain showed less sensitivity to H
2O
2. Δ
AflatfA and Δ
AflatfAΔ
AflatfB strains displayed less sensitivity to the osmotic reagents NaCl, KCl and Sorbitol, in comparison with WT and Δ
AflatfB strains. When on YES medium and hosts corn and peanut, Δ
AflatfA and Δ
AflatfAΔ
AflatfB strains produced less aflatoxin B1 (AFB1) than Δ
AflatfB, and the AFB1 yield of Δ
AflatfB was higher than that of WT. When WT and mutants were inoculated on corn and peanut, the Δ
AflatfA and Δ
AflatfAΔ
AflatfB but not Δ
AflatfB mutants produced less conidia than did WT. Taken together, this study reveals that AflatfA controls more cellular processes, and the function of AflatfA is stronger than that of AflatfB when of the same process is regulated, except the response to H
2O
2, which might result from the effect of AflatfA on the transcriptional level of AflatfB.
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