The MYB-like protein MylA contributes to conidiogenesis and conidial germination in Aspergillus nidulans.

Myeloblastosis (MYB)-like proteins are a family of highly conserved transcription factors in animals, plants, and fungi and are involved in the regulation of mRNA expression of genes. In this study, we identified and characterized one MYB-like protein in the model organism Aspergillus nidulans. We screened the mRNA levels of genes encoding MYB-like proteins containing two MYB repeats in conidia and found that the mRNA levels of four genes including flbD, cicD, and two uncharacterized genes, were high in conidia. To investigate the roles of two uncharacterized genes, AN4618 and AN10944, deletion mutants for each gene were generated. Our results revealed that AN4618 was required for fungal development. Therefore, we further investigated the role of AN4618, named as mylA, encoding the MYB-like protein containing two MYB repeats. Functional studies revealed that MylA was essential for normal fungal growth and development. Phenotypic and transcriptomic analyses demonstrated that deletion of mylA affected stress tolerance, cell wall integrity, and long-term viability in A. nidulans conidia. In addition, the germination rate of the mylA deletion mutant conidia was decreased compared with that of the wild-type conidia. Overall, this study suggests that MylA is critical for appropriate development, conidial maturation, dormancy, and germination in A. nidulans.

Phylogenomics analysis of velvet regulators in the fungal kingdom.

All life forms have evolved to respond appropriately to various environmental and internal cues. In the animal kingdom, the prototypical regulator class of such cellular responses is the Rel homology domain proteins including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Fungi, the close relatives of animals, have also evolved with their own NF-κB-like regulators called velvet family proteins to govern cellular and chemical development. Here, we conducted a detailed investigation of the taxonomic broad presence of velvet proteins. We observed that velvet proteins are widely distributed in the fungal kingdom. Moreover, we have identified and characterized 21 major velvet clades in fungi. We have further revealed that the highly conserved velvet domain is composed of three distinct motifs and acts as an evolutionarily independent domain, which can be shuffled with various functional domains. Such rearrangements of the velvet domain have resulted in the functional and type diversity of the present velvet regulators. Importantly, our in-deep analyses of the primary and 3D structures of the various velvet domains showed that the fungal velvet domains can be divided into two major clans: the VelB and the VosA clans. The 3D structure comparisons revealed a close similarity of the velvet domain with many other eukaryotic DNA-binding proteins, including those of the Rel, Runt, and signal transducer and activator of transcription families, sharing a common ß-sandwich fold. Altogether, this study improves our understanding of velvet regulators in the fungal kingdom.IMPORTANCEFungi are the relatives of animals in Opisthokonta and closely associated with human life by interactive ways such as pathogenicity, food, and secondary metabolites including beneficial ones like penicillin and harmful ones like the carcinogenic aflatoxins. Similar to animals, fungi have also evolved with NF-κB-like velvet family regulators. The velvet proteins constitute a large protein family of fungal transcription factors sharing a common velvet domain and play a key role in coordinating fungal secondary metabolism, developmental and differentiation processes. Our current understanding on velvet regulators is mostly from Ascomycota fungi; however, they remain largely unknown outside Ascomycota. Therefore, this study performed a taxonomic broad investigation of velvet proteins across the fungal kingdom and conducted a detailed analysis on velvet distribution, structure, diversity, and evolution. The results provide a holistic view of velvet regulatory system in the fungal kingdom.

The function of a conidia specific transcription factor CsgA in Aspergillus nidulans.

Aspergillus spp. mainly reproduce asexually via asexual spores called conidia. In this study, we identified CsgA, a conidia-specific Zn2Cys6 transcription factor containing the GAL4-like zinc-finger domain, and characterized the roles of CsgA in the model organism Aspergillus nidulans. In A. nidulans, the ΔcsgA strain produced abnormal conidiophores and exhibited increased conidial production. The deletion of csgA resulted in impaired production of sexual fruiting bodies (cleistothecia) and lower mutA expression levels. Overexpression of csgA led to decreased conidia production but increased cleistothecia production, suggesting that CsgA is essential for proper asexual and sexual development in A. nidulans. In conidia, the deletion of csgA resulted in increased trehalose content, higher spore viability, and increased tolerance to thermal and oxidative stresses. Transcriptomic analysis revealed that the loss of csgA affects the expression of genes related to conidia germination, DNA repair, and secondary metabolite biosynthesis. Further analysis revealed that the ΔcsgA strain exhibited delayed conidial germination and abnormal germ tube length. Additionally, the production of sterigmatocystin increased in the ΔcsgA conidia compared to that in the controls. Overall, these results suggest that CsgA is crucial for proper fungal development, spore viability, conidial germination, and sterigmatocystin production in A. nidulans.

Regulation of Conidiogenesis in Aspergillus flavus.

Aspergillus flavus is a representative fungal species in the Aspergillus section Flavi and has been used as a model system to gain insights into fungal development and toxin production. A. flavus has several adverse effects on humans, including the production of the most carcinogenic mycotoxin aflatoxins and causing aspergillosis in immune-compromised patients. In addition, A. flavus infection of crops results in economic losses due to yield loss and aflatoxin contamination. A. flavus is a saprophytic fungus that disperses in the ecosystem mainly by producing asexual spores (conidia), which also provide long-term survival in the harsh environmental conditions. Conidia are composed of the rodlet layer, cell wall, and melanin and are produced from an asexual specialized structure called the conidiophore. The production of conidiophores is tightly regulated by various regulators, including the central regulatory cascade composed of BrlA-AbaA-WetA, the fungi-specific velvet regulators, upstream regulators, and developmental repressors. In this review, we summarize the findings of a series of recent studies related to asexual development in A. flavus and provide insights for a better understanding of other fungal species in the section Flavi.

Homeobox proteins are essential for fungal differentiation and secondary metabolism in Aspergillus nidulans.

The homeobox domain-containing transcription factors play an important role in the growth, development, and secondary metabolism in fungi and other eukaryotes. In this study, we characterized the roles of the genes coding for homeobox-type proteins in the model organism Aspergillus nidulans. To examine their roles in A. nidulans, the deletion mutant strains for each gene coding for homeobox-type protein were generated, and their phenotypes were examined. Phenotypic analyses revealed that two homeobox proteins, HbxA and HbxB, were required for conidia production. Deletion of hbxA caused abnormal conidiophore production, decreased the number of conidia in both light and dark conditions, and decreased the size of cleistothecia structures. Overexpressing hbxA enhanced the production of asexual spores and formation of conidiophore under the liquid submerged conditions. The hbxB deletion mutant strains exhibited decreased asexual spore production but increased cleistothecia production. The absence of hbxB decreased the trehalose content in asexual spores and increased their sensitivity against thermal and oxidative stresses. The ΔhbxA strains produced more sterigmatocystin, which was decreased in the ΔhbxB strain. Overall, our results show that HbxA and HbxB play crucial roles in the differentiation and secondary metabolism of the fungus A. nidulans.

The role of the VosA-repressed dnjA gene in development and metabolism in Aspergillus species.

The DnaJ family of proteins (or J-proteins) are molecular chaperones that govern protein folding, degradation, and translocation in many organisms. Although J-proteins play key roles in eukaryotic and prokaryotic biology, the role of J-proteins in Aspergillus species is currently unknown. In this study, we characterized the dnjA gene, which encodes a putative DnaJ protein, in two Aspergillus species: Aspergillus nidulans and Aspergillus flavus. Expression of the dnjA gene is inhibited by the velvet regulator VosA, which plays a pivotal role in spore survival and metabolism in Aspergillus. The deletion of dnjA decreased the number of asexual spores (conidia), produced abnormal conidiophores, and reduced sexual fruiting bodies (cleistothecia) or sclerotia. In addition, the absence of dnjA caused increased sterigmatocystin or aflatoxin production in A. nidulans and A. flavus, respectively. These results suggest that DnjA plays a conserved role in asexual and sexual development and mycotoxin production in Aspergillus species. However, DnjA also plays a species-specific role; AniDnjA but not AflDnjA, affects conidial viability, trehalose contents, and thermal tolerance of conidia. In plant virulence assay, the infection ability of the ΔAfldnjA mutant decreased in the kernels, suggesting that DnjA plays a crucial role in the pathogenicity of A. flavus. Taken together, these results demonstrate that DnjA is multifunctional in Aspergillus species; it is involved in diverse biological processes, including fungal differentiation and secondary metabolism.

Characterizing the role of Zn cluster family transcription factor ZcfA in governing development in two Aspergillus species.

Filamentous fungi reproduce asexually or sexually, and the processes of asexual and sexual development are tightly regulated by a variety of transcription factors. In this study, we characterized a Zn2Cys6 transcription factor in two Aspergillus species, A. nidulans (AN5859) and A. flavus (AFLA_046870). AN5859 encodes a Zn2Cys6 transcription factor, called ZcfA. In A. nidulans, ΔzcfA mutants exhibit decreased fungal growth, a reduction in cleistothecia production, and increased asexual reproduction. Overexpression of zcfA results in increased conidial production, suggesting that ZcfA is required for proper asexual and sexual development in A. nidulans. In conidia, deletion of zcfA causes decreased trehalose levels and decreased spore viability but increased thermal sensitivity. In A. flavus, the deletion of the zcfA homolog AFLA_046870 causes increased conidial production but decreased sclerotia production; these effects are similar to those of zcfA deletion in A. nidulans development. Overall, these results demonstrate that ZcfA is essential for maintaining a balance between asexual and sexual development and that some roles of ZcfA are conserved in Aspergillus spp.