Aspergillus flavus bZIP-Type Transcription Factors as Promising Novel Targets for Future Aflatoxin Control Strategies

The bZIP type transcription factors (bZIPs) are global regulators governing vegetative growth, development, stress defense and secondary metabolism, including mycotoxin production in filamentous fungi. In this work, we constructed and phenotypically characterized gene deletion and complementation mutants of some bZIPs, including Afap1, AflatfA, LziP, AflatfB and bZIP6 in Aspergillus flavus. Environmental and fungicide stress responses, as well as aflatoxin production of the mutants in both surface cultures and infected maize kernels, were studied. Phenotypic characterization of the mutants revealed that Afap1 and AflatfA were involved in the oxidative (H₂O₂, menadione, tert-butyl hydroperoxide), cell wall integrity (Congo Red) and heavy metal (CdCl₂) stress responses of A. flavus. In addition, the Afap1 and AflatfA gene deletions decreased the diamide and prothioconazole tolerances of the fungus, respectively. The ΔLziP strain showed increased growth in the presence of diamide, while reduced colony diameters were observed after exposure to CdCl₂ and fludioxonil. The ΔAflatfB gene deletion mutant was also sensitive to tBOOH, while azoxystrobin and prothioconazole fungicides significantly inhibited the growth of ΔbZIP6. When aflatoxin (AFB1) production was measured in surface cultures, decreased AFB1 levels were detected only in the ΔAflatfA gene deletion mutant strain. However, in corn kernel infection assays, the ΔAfap1, ΔAflatfA, and ΔAflatfB mutants were characterized by significantly reduced aflatoxin production, while the deletion of bZIP6 almost completely abolished AFB1 biosynthesis. Our results suggest that Afap1 and AflatfA appear to be promising targets for the development of new antifungal agents, as their inhibition may increase the sensitivity of A. flavus to environmental stress, simultaneously reducing the aflatoxin production of the fungus and the use of azoles (AflatfA) in the antifungal protection of maize. In addition, bZIP6 may also be considered as an attractive target for further studies when aiming to eliminate aflatoxin production and minimize the use of azoxystrobin and prothioconazole in maize crop protection.