Fungal secondary metabolites play important roles not only in fungal ecology but also in humans living as beneficial medicine or harmful toxins. In filamentous fungi, bZIP-type transcription factors (TFs) are associated with the proteins involved in oxidative stress response and secondary metabolism. In this study, a connection between a bZIP TF and oxidative stress induction of secondary metabolism is uncovered in an opportunistic pathogen Aspergillus flavus, which produces carcinogenic and mutagenic aflatoxins. The bZIP transcription factor AflRsmA was identified by a homology research of A. flavus genome with the bZIP protein RsmA, involved in secondary metabolites production in Aspergillus nidulans. The AflrsmA deletion strain (∆AflrsmA) displayed less sensitivity to the oxidative reagents tert-Butyl hydroperoxide (tBOOH) in comparison with wild type (WT) and AflrsmA overexpression strain (AflrsmA OE ), while AflrsmA OE strain increased sensitivity to the oxidative reagents menadione sodium bisulfite (MSB) compared to WT and ∆AflrsmA strains. Without oxidative treatment, aflatoxin B 1 (AFB 1 ) production of ∆AflrsmA strains was consistent with that of WT, but AflrsmA OE strain produced more AFB 1 than WT; tBOOH and MSB treatment decreased AFB 1 production of ∆AflrsmA compared to WT. Besides, relative to WT, ∆AflrsmA strain decreased sclerotia, while AflrsmA OE strain increased sclerotia. The decrease of AFB 1 by ∆AflrsmA but increase of AFB 1 by AflrsmA OE was on corn. Our results suggest that AFB 1 biosynthesis is regulated by AflRsmA by oxidative stress pathways and provide insights into a possible function of AflRsmA in mediating AFB 1 biosynthesis response host defense in pathogen A. flavus.Key Contribution: Without oxidative treatment, deletion of AflrsmA has no impact on aflatoxin B 1 (AFB 1 ) biosynthesis in comparison with the wild type strain. However, the AflrsmA disruptant produces less AFB 1 on corn seeds and on YGT medium containing oxidative reagent menadione sodium bisulfite or tert-Butyl hydroperoxide.Toxins 2020, 12, 271 2 of 17 basic leucine zipper (bZIP) domain. The bZIP-type transcription factor as one of the largest families of dimerizing TFs is widely distributed in the all eukaryotes genomes. The first bZIP-type TF was discovered in humans over 30 years ago [2]. Then, it was found that bZIP-type TFs are involved in metabolism, development, cell cycle, reproduction, and programmed cell death [1]. In plants, the functions of bZIP-type TFs include abiotic stress response, metabolism, mediating pathogen defense, hormone signaling, and senescence [3]. bZIP-type TFs are involved in various biological processes in fungi (described below).In Saccharomyces cerevisiae, the first bZIP-type TF called Yap (yeast activator protein) was found [4], then a subset of YAP TFs (YAP1-YAP8) have been well defined [5]. Several bZIP proteins have been reported in filamentous fungi, such as Aspergillus spp. [6-11], Neurospora crassa [12], plant pathogens Fusarium graminearum [13], Magnaporthe oryzae [14], and ...

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 (ΔAflatfA) and AflatfA and AflatfB double deletion strain (ΔAflatfAΔAflatfB) produced more sclerotia, whereas the AflatfB deletion strain (ΔAflatfB) produced less sclerotia. After 4 °C preservation and incubation at 50 °C, conidia viability dramatically decreased in the ΔAflatfA and ΔAflatfAΔAflatfB but ΔAflatfB mutants, whereas conidia viability of the ΔAflatfAΔAflatfB strain was higher after storage at 4 °C than in AflatfA mutant. Conidia of ΔAflatfA, ΔAflatfB and ΔAflatfAΔAflatfB strains significantly increased in sensitivity to H2O2 in comparison with WT. Compared to WT, the mycelium of ΔAflatfA and ΔAflatfB strains were more sensitive to H2O2; conversely, the ΔAflatfAΔAflatfB strain showed less sensitivity to H2O2. ΔAflatfA and ΔAflatfAΔAflatfB strains displayed less sensitivity to the osmotic reagents NaCl, KCl and Sorbitol, in comparison with WT and ΔAflatfB strains. When on YES medium and hosts corn and peanut, ΔAflatfA and ΔAflatfAΔAflatfB strains produced less aflatoxin B1 (AFB1) than ΔAflatfB, and the AFB1 yield of ΔAflatfB was higher than that of WT. When WT and mutants were inoculated on corn and peanut, the ΔAflatfA and ΔAflatfAΔAflatfB but not ΔAflatfB 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 H2O2, which might result from the effect of AflatfA on the transcriptional level of AflatfB.

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