Coordination of two regulators SscA and VosA in Aspergillus nidulans conidia.

Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in 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.

SscA is required for fungal development, aflatoxin production, and pathogenicity in Aspergillus flavus.

Fungal spores are specialized dormant cells that act as primary reproductive biological particles and exhibit strong viability under extremely harsh conditions. They contaminate a variety of crops and foods, causing severe health hazards to humans and animals. Previous studies demonstrated that a spore-specific transcription factor SscA plays pivotal roles in the conidiogenesis of the model organism Aspergillus nidulans. In this study, we investigated the biological and genetic functions of SscA in the aflatoxin-producing fungus A. flavus. Deletion of sscA showed reduced conidia formation, lost long-term viability, and exhibited more sensitivity to thermal, oxidative, and radiative stresses. The sscA-deficient strain showed increased aflatoxin B1 production in conidia as well as mycelia. Importantly, the absence of sscA affected fungal pathogenicity on crops. Further transcriptomic and phenotypic studies suggested that SscA coordinates conidial wall structures. Overall, SscA is important for conidial formation, maturation and dormancy, mycotoxin production, and pathogenicity in A. flavus.

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.

Pleiotropic functions of SscA on the asexual spore of the human pathogenic fungus Aspergillus fumigatus.

Asexual spores, called conidia, are key reproductive fungal particles that enable survival in harsh environmental conditions or host systems. The conidia can infect humans, animals, and plants to cause various fungal diseases. Transcription factors, including VosA, WetA, and SscA, have key roles in conidia formation and long-term survival in Aspergillus nidulans. Herein, we report the pleiotropic functions of SscA in the conidia of the human pathogen A. fumigatus. The deletion of sscA increased conidia formation despite decreased fungal growth. Absence of sscA impaired long-term survival and reduced spore resistance to various stresses, including heat, UV, and oxidation. Transcriptomic analyses showed that SscA involved the mRNA expression of cell wall organisation-related genes. Importantly, the sscA deletion mutant conidia contained an increased amount of ß-glucan and chitin compared to wild type conidia. In addition, conidial gliotoxin production was decreased in the sscA deletion strain. Overall, SscA has pleiotropic roles in conidia formation, maturation and dormancy and mycotoxin production in A. fumigatus.

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.

Regulators of the Asexual Life Cycle of Aspergillus nidulans.

The genus Aspergillus, one of the most abundant airborne fungi, is classified into hundreds of species that affect humans, animals, and plants. Among these, Aspergillus nidulans, as a key model organism, has been extensively studied to understand the mechanisms governing growth and development, physiology, and gene regulation in fungi. A. nidulans primarily reproduces by forming millions of asexual spores known as conidia. The asexual life cycle of A. nidulans can be simply divided into growth and asexual development (conidiation). After a certain period of vegetative growth, some vegetative cells (hyphae) develop into specialized asexual structures called conidiophores. Each A. nidulans conidiophore is composed of a foot cell, stalk, vesicle, metulae, phialides, and 12,000 conidia. This vegetative-to-developmental transition requires the activity of various regulators including FLB proteins, BrlA, and AbaA. Asymmetric repetitive mitotic cell division of phialides results in the formation of immature conidia. Subsequent conidial maturation requires multiple regulators such as WetA, VosA, and VelB. Matured conidia maintain cellular integrity and long-term viability against various stresses and desiccation. Under appropriate conditions, the resting conidia germinate and form new colonies, and this process is governed by a myriad of regulators, such as CreA and SocA. To date, a plethora of regulators for each asexual developmental stage have been identified and investigated. This review summarizes our current understanding of the regulators of conidial formation, maturation, dormancy, and germination in A. nidulans.

The novel spore-specific regulator SscA controls Aspergillus conidiogenesis.

IMPORTANCE: Filamentous fungi produce myriads of asexual spores, which are the main reproductive particles that act as infectious or allergenic agents. Although the serial of asexual sporogenesis is coordinated by various genetic regulators, there remain uncharacterized transcription factors in Aspergillus. To understand the underlying mechanism of spore formation, integrity, and viability, we have performed comparative transcriptomic analyses on three Aspergillus species and found a spore-specific transcription factor, SscA. SscA has a major role in conidial formation, maturation and dormancy, and germination in Aspergillus nidulans. Functional studies indicate that SscA coordinates conidial wall integrity, amino acid production, and secondary metabolism in A. nidulans conidia. Furthermore, the roles of SscA are conserved in other Aspergillus species. Our findings that the SscA has broad functions in Aspergillus conidia will help to understand the conidiogenesis of Aspergillus species.

The Forkhead Gene fkhB is Necessary for Proper Development in Aspergillus nidulans.

The forkhead domain genes are important for development and morphogenesis in fungi. Six forkhead genes fkhA-fkhF have been found in the genome of the model filamentous Ascomycete Aspergillus nidulans. To identify the fkh gene(s) associated with fungal development, we examined mRNA levels of these six genes and found that the level of fkhB and fkhD mRNA was significantly elevated during asexual development and in conidia. To investigate the roles of FkhB and FkhD, we generated fkhB and fkhD deletion mutants and complemented strains and investigated their phenotypes. The deletion of fkhB, but not fkhD, affected fungal growth and both sexual and asexual development. The fkhB deletion mutant exhibited decreased colony size with distinctly pigmented (reddish) asexual spores and a significantly lower number of conidia compared with these features in the wild type (WT), although the level of sterigmatocystin was unaffected by the absence of fkhB. Furthermore, the fkhB deletion mutant produced sexual fruiting bodies (cleistothecia) smaller than those of WT, implying that the fkhB gene is involved in both asexual and sexual development. In addition, fkhB deletion reduced fungal tolerance to heat stress and decreased trehalose accumulation in conidia. Overall, these results suggest that fkhB plays a key role in proper fungal growth, development, and conidial stress tolerance 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.

Unveiling the Functions of the VosA-VelB Target Gene vidD in Aspergillus nidulans.

The velvet regulators VosA and VelB are primarily involved in spore maturation and dormancy. Previous studies found that the VosA-VelB hetero-complex coordinates certain target genes that are related to fungal differentiation and conidial maturation in Aspergillus nidulans. Here, we characterized the VosA/VelB-inhibited developmental gene vidD in A. nidulans. Phenotypic analyses demonstrated that the vidD deleted mutant exhibited defect fungal growth, a reduced number of conidia, and delayed formation of sexual fruiting bodies. The deletion of vidD decreased the amount of conidial trehalose, increased the sensitivity against heat stress, and reduced the conidial viability. Moreover, the absence of vidD resulted in increased production of sterigmatocystin. Together, these results show that VidD is required for proper fungal growth, development, and sterigmatocystin production in A. nidulans.

Genetic Manipulation and Transformation Methods for Aspergillus spp.

Species of the genus Aspergillus have a variety of effects on humans and have been considered industrial cell factories due to their prominent ability for manufacturing several products such as heterologous proteins, secondary metabolites, and organic acids. Scientists are trying to improve fungal strains and re-design metabolic processes through advanced genetic manipulation techniques and gene delivery systems to enhance their industrial efficiency and utility. In this review, we describe the current status of the genetic manipulation techniques and transformation methods for species of the genus Aspergillus. The host strains, selective markers, and experimental materials required for the genetic manipulation and fungal transformation are described in detail. Furthermore, the advantages and disadvantages of these techniques are described.

HbxB Is a Key Regulator for Stress Response and ß-Glucan Biogenesis in Aspergillus nidulans.

Homeobox transcription factors are conserved in eukaryotes and act as multi-functional transcription factors in filamentous fungi. Previously, it was demonstrated that HbxB governs fungal development and spore viability in Aspergillus nidulans. Here, the role of HbxB in A. nidulans was further characterized. RNA-sequencing revealed that HbxB affects the transcriptomic levels of genes associated with trehalose biosynthesis and response to thermal, oxidative, and radiation stresses in asexual spores called conidia. A phenotypic analysis found that hbxB deletion mutant conidia were more sensitive to ultraviolet stress. The loss of hbxB increased the mRNA expression of genes associated with ß-glucan degradation and decreased the amount of ß-glucan in conidia. In addition, hbxB deletion affected the expression of the sterigmatocystin gene cluster and the amount of sterigmatocystin. Overall, these results indicated that HbxB is a key transcription factor regulating trehalose biosynthesis, stress tolerance, ß-glucan degradation, and sterigmatocystin production in A. nidulans conidia.

Transcriptomic, Protein-DNA Interaction, and Metabolomic Studies of VosA, VelB, and WetA in Aspergillus nidulans Asexual Spores.

In filamentous fungi, asexual development involves cellular differentiation and metabolic remodeling leading to the formation of intact asexual spores. The development of asexual spores (conidia) in Aspergillus is precisely coordinated by multiple transcription factors (TFs), including VosA, VelB, and WetA. Notably, these three TFs are essential for the structural and metabolic integrity, i.e., proper maturation, of conidia in the model fungus Aspergillus nidulans To gain mechanistic insight into the complex regulatory and interdependent roles of these TFs in asexual sporogenesis, we carried out multi-omics studies on the transcriptome, protein-DNA interactions, and primary and secondary metabolism employing A. nidulans conidia. RNA sequencing and chromatin immunoprecipitation sequencing analyses have revealed that the three TFs directly or indirectly regulate the expression of genes associated with heterotrimeric G-protein signal transduction, mitogen-activated protein (MAP) kinases, spore wall formation and structural integrity, asexual development, and primary/secondary metabolism. In addition, metabolomics analyses of wild-type and individual mutant conidia indicate that these three TFs regulate a diverse array of primary metabolites, including those in the tricarboxylic acid (TCA) cycle, certain amino acids, and trehalose, and secondary metabolites such as sterigmatocystin, emericellamide, austinol, and dehydroaustinol. In summary, WetA, VosA, and VelB play interdependent, overlapping, and distinct roles in governing morphological development and primary/secondary metabolic remodeling in Aspergillus conidia, leading to the production of vital conidia suitable for fungal proliferation and dissemination.IMPORTANCE Filamentous fungi produce a vast number of asexual spores that act as efficient propagules. Due to their infectious and/or allergenic nature, fungal spores affect our daily life. Aspergillus species produce asexual spores called conidia; their formation involves morphological development and metabolic changes, and the associated regulatory systems are coordinated by multiple transcription factors (TFs). To understand the underlying global regulatory programs and cellular outcomes associated with conidium formation, genomic and metabolomic analyses were performed in the model fungus Aspergillus nidulans Our results show that the fungus-specific WetA/VosA/VelB TFs govern the coordination of morphological and chemical developments during sporogenesis. The results of this study provide insights into the interdependent, overlapping, or distinct genetic regulatory networks necessary to produce intact asexual spores. The findings are relevant for other Aspergillus species such as the major human pathogen Aspergillus fumigatus and the aflatoxin producer Aspergillus flavus.

Genome Wide Analysis Reveals the Role of VadA in Stress Response, Germination, and Sterigmatocystin Production in Aspergillus nidulans Conidia.

In the Aspergillus species, conidia are asexual spores that are infectious particles responsible for propagation. Conidia contain various mycotoxins that can have detrimental effects in humans. Previous study demonstrated that VadA is required for fungal development and spore viability in the model fungus Aspergillus nidulans. In the present study, vadA transcriptomic analysis revealed that VadA affects the mRNA expression of a variety of genes in A. nidulans conidia. The genes that were primarily affected in conidia were associated with trehalose biosynthesis, cell-wall integrity, stress response, and secondary metabolism. Genetic changes caused by deletion of vadA were related to phenotypes of the vadA deletion mutant conidia. The deletion of vadA resulted in increased conidial sensitivity against ultraviolet stress and induced germ tube formation in the presence and absence of glucose. In addition, most genes in the secondary metabolism gene clusters of sterigmatocystin, asperfuranone, monodictyphenone, and asperthecin were upregulated in the mutant conidia with vadA deletion. The deletion of vadA led to an increase in the amount of sterigmatocystin in the conidia, suggesting that VadA is essential for the repression of sterigmatocystin production in conidia. These results suggest that VadA coordinates conidia maturation, stress response, and secondary metabolism in A. nidulans conidia.

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.

Velvet activated McrA plays a key role in cellular and metabolic development in Aspergillus nidulans.

McrA is a key transcription factor that functions as a global repressor of fungal secondary metabolism in Aspergillus species. Here, we report that mcrA is one of the VosA-VelB target genes and McrA governs the cellular and metabolic development in Aspergillus nidulans. The deletion of mcrA resulted in a reduced number of conidia and decreased mRNA levels of brlA, the key asexual developmental activator. In addition, the absence of mcrA led to a loss of long-term viability of asexual spores (conidia), which is likely associated with the lack of conidial trehalose and increased ß-(1,3)-glucan levels in conidia. In supporting its repressive role, the mcrA deletion mutant conidia contain more amounts of sterigmatocystin and an unknown metabolite than the wild type conidia. While overexpression of mcrA caused the fluffy-autolytic phenotype coupled with accelerated cell death, deletion of mcrA did not fully suppress the developmental defects caused by the lack of the regulator of G-protein signaling protein FlbA. On the contrary to the cellular development, sterigmatocystin production was restored in the ΔflbA ΔmcrA double mutant, and overexpression of mcrA completely blocked the production of sterigmatocystin. Overall, McrA plays a multiple role in governing growth, development, spore viability, and secondary metabolism in A. nidulans.

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.

Conserved Roles of MonA in Fungal Growth and Development in Aspergillus Species.

MonA is a subunit of a guanine nucleotide exchange factor that is important for vacuole passing and autophagy processes in eukaryotes. In this study, we characterized the function of MonA, an orthologue of Saccharomyces cerevisiae Mon1, in the model fungus Aspergillus nidulans and a toxigenic fungus A. flavus. In A. nidulans, the absence of AnimonA led to decreased fungal growth, reduced asexual reproduction, and defective cleistothecia production. In addition, AnimonA deletion mutants exhibited decreased spore viability, had reduced trehalose contents in conidia, and were sensitive to thermal stress. In A. flavus, deletion of AflmonA caused decreased fungal growth and defective production of asexual spores and sclerotia structures. Moreover, the absence of monA affected vacuole morphology in both species. Taken together, these results indicate that MonA plays conserved roles in controlling fungal growth, development and vacuole morphology in A. nidulans and A. flavus.

The velvet repressed vidA gene plays a key role in governing development in Aspergillus nidulans.

Fungal development is regulated by a variety of transcription factors in Aspergillus nidulans. Previous studies demonstrated that the NF-κB type velvet transcription factors regulate certain target genes that govern fungal differentiation and cellular metabolism. In this study, we characterize one of the VosA/VelB-inhibited developmental genes called vidA, which is predicted to encode a 581-amino acid protein with a C2H2 zinc finger domain at the C-terminus. Levels of vidA mRNA are high during the early and middle phases of asexual development and decrease during the late phase of asexual development and asexual spore (conidium) formation. Deletion of either vosA or velB results in increased vidA mRNA accumulation in conidia, suggesting that vidA transcript accumulation in conidia is repressed by VosA and VelB. Phenotypic analysis demonstrated that deletion of vidA causes decreased colony growth, reduced production of asexual spores, and abnormal formation of sexual fruiting bodies. In addition, the vidA deletion mutant conidia contain more trehalose and ß-glucan than wild type. Overall, these results suggest that VidA is a putative transcription factor that plays a key role in governing proper fungal growth, asexual and sexual development, and conidia formation in A. nidulans.