Regulation of signal transduction pathways is crucial for the maintenance of cellular homeostasis and organismal development in fungi. Transcription factors are key elements of this regulatory network. The basic-region leucine zipper (bZIP) domain of the bZIP-type transcription factors is responsible for DNA binding while their leucine zipper structural motifs are suitable for dimerization with each other facilitiating the formation of homodimeric or heterodimeric bZIP proteins. This review highlights recent knowledge on the function of fungal orthologs of the Schizosaccharomyces pombe Atf1, Aspergillus nidulans AtfA, and Fusarium verticillioides FvAtfA, bZIP-type transcription factors with a special focus on pathogenic species. We demonstrate that fungal Atf1-AtfA-FvAtfA orthologs play an important role in vegetative growth, sexual and asexual development, stress response, secondary metabolite production, and virulence both in human pathogens, including Aspergillus fumigatus, Mucor circinelloides, Penicillium marneffei, and Cryptococcus neoformans and plant pathogens, like Fusarium ssp., Magnaporthe oryzae, Claviceps purpurea, Botrytis cinerea, and Verticillium dahliae. Key points: • Atf1 orthologs play crucial role in the growth and development of fungi. • Atf1 orthologs orchestrate environmental stress response of fungi. • Secondary metabolite production and virulence are coordinated by Atf1 orthologs.

Transcription factors (TFs) with the basic leucin zipper domain are key elements of the stress response pathways in filamentous fungi. In this study, we functionally characterized the two bZIP type TFs AtfA and AtfB by deletion (Δ) and overexpression (OE) of their encoding genes in all combination: ΔatfA, ΔatfB, ΔatfAΔatfB, ΔatfAatfBOE, ΔatfBatfAOE, atfAOE, atfBOE and atfAOEatfBOE in Aspergillus nidulans. Based on our previous studies, ΔatfA increased the sensitivity of the fungus to oxidative stress mediated by menadione sodium bisulfite (MSB) and tert-butylhydroperoxide (tBOOH), while ΔatfB was not sensitive to any oxidative stress generating agents, namely MSB, tBOOH and diamide at all. Contrarily, the ΔatfB mutant was sensitive to NaCl, but tolerant to sorbitol. Overexpression of atfB was able to compensate the MSB sensitivity of the ΔatfA mutant. Heavy metal stress elicited by CdCl₂ reduced diameter of the atfBOE and atfAOEatfBOE mutant colonies to about 50% of control colony, while the cell wall stress generating agent CongoRed increased the tolerance of the ΔatfA mutant. When we tested the heat stress sensitivity of the asexual spores (conidiospores) of the mutants, we found that conidiospores of ΔatfAatfBOE and ΔatfBatfAOE showed nearly 100% tolerance to heat stress. Asexual development was negatively affected by ΔatfA, while atfAOE and atfAOE coupled with ΔatfB increased the number of conidiospores of the fungus approximately 150% compared to the control. Overexpression of atfB led to a 25% reduction in the number of conidiospores, but increased levels of abaA mRNA and size of conidiospores. Sexual fruiting body (cleistothecium) formation was diminished in the ΔatfA and the ΔatfAΔatfB mutants, while relatively elevated in the ΔatfB and the ΔatfBatfAOE mutants. Production of the mycotoxin sterigmatocystin (ST) was decreased to undetectable levels in the ΔatfA mutant, yet ST production was restored in the ΔatfAΔatfB mutant, suggesting that ΔatfB can suppress ST production defect caused by ΔatfA. Levels of ST were also significantly decreased in the ΔatfAatfBOE, ΔatfBatfAOE and atfAOEatfBOE mutants.

Yeast protein sequence-based homology search for glutathione (GSH) metabolic enzymes and GSH transporters demonstrated that Aspergillus nidulans has a robust GSH uptake and metabolic system with several paralogous genes. In wet laboratory experiments, two key genes of GSH metabolism, gcsA, and glrA, encoding γ-l-glutamyl-l-cysteine synthetase and glutathione reductase, respectively, were deleted. The gene gcsA was essential, and the ΔgcsA mutant required GSH supplementation at considerably higher concentration than the Saccharomyces cerevisiae gsh1 mutant (8–10 mmol l⁻¹ vs. 0.5 μmol l⁻¹). In addition to some functions known previously, both genes were important in the germination of conidiospores, and both gene deletion strains required the addition of extra GSH to reach wild-type germination rates in liquid cultures. Nevertheless, the supplementation of cultures with 10 mmol l⁻¹ GSH was toxic for the control and ΔglrA strains especially during vegetative growth, which should be considered in future development of high GSH-producer fungal strains. Importantly, the ΔglrA strain was characterized by increased sensitivity toward a wide spectrum of osmotic, cell wall integrity and antimycotic stress conditions in addition to previously reported temperature and oxidative stress sensitivities. These novel phenotypes underline the distinguished functions of GSH and GSH metabolic enzymes in the stress responses of fungi.

The bZIP transcription factors (TFs) govern regulation of development, secondary metabolism, and various stress responses in filamentous fungi. In this work, we carried out genome-wide expression studies employing Illumina RNAseq to understand the roles of the two bZIP transcription factors AtfA and AtfB in Aspergillus nidulans. Comparative analyses of transcriptomes of control, ΔatfA, ΔatfB, and ΔatfAΔatfB mutant strains were performed. Dependence of a gene on AtfA (AtfB) was decided by its differential downregulation both between the reference and ΔatfA (ΔatfB) strains and between the ΔatfB (ΔatfA) and the ΔatfAΔatfB strains in vegetatively grown cells (mycelia) and asexual spores (conidia) of menadione sodium bisulfite (MSB)-treated or untreated cultures. As AtfA is the primary bZIP TF governing stress-response in A. nidulans, the number of differentially expressed genes for ΔatfA was significantly higher than for ΔatfB in both mycelial and conidial samples, and most of the AtfB-dependent genes showed AtfA dependence, too. Moreover, the low number of genes depending on AtfB but not on AtfA can be a consequence of ΔatfA leading to downregulation of atfB expression. Conidial samples showed much higher abundance of atfA and atfB mRNAs and more AtfA- and AtfB-affected genes than mycelial samples. In the presence of MSB, the number of AtfB- (but not of AtfA-) affected genes decreased markedly, which was accompanied with decreased mRNA levels of atfB in MSB-treated mycelial (reference strain) and conidial (ΔatfA mutant) samples. In mycelia, the overlap between the AtfA-dependent genes in MSB-treated and in untreated samples was low, demonstrating that distinct genes can be under AtfA control under different conditions. Carbohydrate metabolism genes were enriched in the set of AtfA-dependent genes. Among them, AtfA-dependence of glycolytic genes in conidial samples was the most notable. Levels of transcripts of certain secondary metabolitic gene clusters, such as the Emericellamide cluster, also showed AtfA-dependent regulation. Genes encoding catalase and histidine-containing phosphotransfer proteins showed AtfA-dependence under all experimental conditions. There were 23 AtfB-dependent genes that did not depend on AtfA under any of our experimental conditions. These included a putative α-glucosidase (agdB), a putative α-amylase, calA, which is involved in early conidial germination, and an alternative oxidase. In summary, in A. nidulans there is a complex interaction between the two bZIP transcription factors, where AtfA plays the primary regulatory role.

PAF, which is produced by the filamentous fungus Pencicillium chrysogenum, is a small antifungal protein, triggering ROS-mediated apoptotic cell death in Aspergillus nidulans. In this work, we provide information on the function of PAF in the host P. chrysogenum considering that carbon-starving cultures of the Δpaf mutant strain showed significantly reduced apoptosis rates in comparison to the wild-type (wt) strain. Moreover, the addition of PAF to the Δpaf strain resulted in a twofold increase in the apoptosis rate. PAF was also involved in the regulation of the autophagy machinery of this fungus, since several Saccharomyces cerevisiae autophagy-related ortholog genes, e.g. those of atg7, atg22 and tipA, were repressed in the deletion strain. This phenomenon was accompanied by the absence of autophagosomes in the Δpaf strain, even in old hyphae.