Unraveling the Gene Regulatory Networks of the Global Regulators VeA and LaeA in Aspergillus nidulans.

In the filamentous fungus Aspergillus nidulans, the velvet family protein VeA and the global regulator of secondary metabolism LaeA govern development and secondary metabolism mostly by acting as the VelB/VeA/LaeA heterotrimeric complex. While functions of these highly conserved controllers have been well studied, the genome-wide regulatory networks governing cellular and chemical development remain to be uncovered. Here, by integrating transcriptomic analyses, protein-DNA interactions, and the known A. nidulans gene/protein interaction data, we have unraveled the gene regulatory networks governed by VeA and LaeA. Within the networks, VeA and LaeA directly control the expression of numerous genes involved in asexual/sexual development and primary/secondary metabolism in A. nidulans. Totals of 3,190 and 1,834 potential direct target genes of VeA and LaeA were identified, respectively, including several important developmental and metabolic regulators such as flbA·B·C, velB·C, areA, mpkB, and hogA. Moreover, by analyzing over 8,800 ChIP-seq peaks, we have revealed the predicted common consensus sequences 5'-TGATTGGCTG-3' and 5'-TCACGTGAC-3' that VeA and LaeA might bind to interchangeably. These findings further expand the biochemical and genomic studies of the VelB/VeA/LaeA complex functionality in the gene regulation. In summary, this study unveils genes that are under the regulation of VeA and LaeA, proposes the VeA- and LaeA-mediated gene regulatory networks, and demonstrates their genome-wide developmental and metabolic regulations in A. nidulans. IMPORTANCE Fungal development and metabolism are genetically programmed events involving specialized cellular differentiation, cellular communication, and temporal and spatial regulation of gene expression. In genus Aspergillus, the global regulators VeA and LaeA govern developmental and metabolic processes by affecting the expression of downstream genes, including multiple transcription factors and signaling elements. Due to their vital roles in overall biology, functions of VeA and LaeA have been extensively studied, but there still has been a lack of knowledge about their genome-wide regulatory networks. In this study, employing the model fungus A. nidulans, we have identified direct targets of VeA and LaeA and their gene regulatory networks by integrating transcriptome, protein-DNA interaction, and protein-protein interaction analyses. Our results demonstrate the genome-wide regulatory mechanisms of these global regulators, thereby advancing the knowledge of fungal biology and genetics.

The Putative C2H2 Transcription Factor VadH Governs Development, Osmotic Stress Response, and Sterigmatocystin Production in Aspergillus nidulans.

The VosA-VelB hetero-dimeric complex plays a pivotal role in regulating development and secondary metabolism in Aspergillus nidulans. In this work, we characterize a new VosA/VelB-activated gene called vadH, which is predicted to encode a 457-amino acid length protein containing four adjacent C2H2 zinc-finger domains. Mutational inactivation of vosA or velB led to reduced mRNA levels of vadH throughout the lifecycle, suggesting that VosA and VelB have a positive regulatory effect on the expression of vadH. The deletion of vadH resulted in decreased asexual development (conidiation) but elevated production of sexual fruiting bodies (cleistothecia), indicating that VadH balances asexual and sexual development in A. nidulans. Moreover, the vadH deletion mutant exhibited elevated susceptibility to hyperosmotic stress compared to wild type and showed elevated production of the mycotoxin sterigmatocystin (ST). Genome-wide expression analyses employing RNA-Seq have revealed that VadH is likely involved in regulating more genes and biological pathways in the developmental stages than those in the vegetative growth stage. The brlA, abaA, and wetA genes of the central regulatory pathway for conidiation are downregulated significantly in the vadH null mutant during asexual development. VadH also participates in regulating the genes, mat2, ppgA and lsdA, etc., related to sexual development, and some of the genes in the ST biosynthetic gene cluster. In summary, VadH is a putative transcription factor with four C2H2 finger domains and is involved in regulating asexual/sexual development, osmotic stress response, and ST production in A. nidulans.

The velvet-activated putative C6 transcription factor VadZ regulates development and sterigmatocystin production in Aspergillus nidulans.

The NF-ƙB-type VosA-VelB velvet complex acts as a global regulator governing development and metabolism in fungi. One of the VosA-VelB-activated developmental (VAD) genes called vadZ is predicted to encode a 557-amino acid protein containing a highly conserved GAL4-type Zn(II)2Cys6 (or C6 zinc) binuclear cluster DNA-binding domain in Aspergillus nidulans. In this report, we characterize the function of the vadZ gene in controlling development and sterigmatocystin (ST) production in A. nidulans. To verify VosA-VelB mediated activation of vadZ, we checked relative mRNA levels of vadZ in wild-type (WT), ΔvosA, and ΔvelB mutant strains during vegetative, asexual, and sexual development phases. At the beginning of asexual development, the absence of vosA led to a 66.2-fold lowered vadZ mRNA levels, whereas ΔvelB resulted in a 3.6-fold decrease in vadZ mRNA levels. The deletion of vadZ resulted in significantly restricted colony growth coupled with reduced asexual development, but increased formation of sexual fruiting bodies called cleistothecia. In addition, nullifying vadZ caused elevated mRNA levels of the two key sexual developmental activators esdC and nsdD throughout the lifecycle. Moreover, the ΔvadZ mutant showed elevated production of ST and enhanced mRNA levels of ST biosynthetic genes. In summary, the putative C6 transcription factor VadZ promotes asexual development and suppresses the sexual development and the ST production in A. nidulans.

Characterization of 260 Isolates of Aspergillus Section Flavi Obtained from Sesame Seeds in Punjab, Pakistan.

Sesame Sesamum indicum L. is a major oil-based seed crop that has been widely cultivated and consumed in Pakistan. Unfortunately, sesame is highly prone to Aspergillus fungal growth in the field, and under inappropriate storage conditions can become contaminated with aflatoxins, the most potent carcinogen found in nature. Here, we have isolated a high number of Aspergillus isolates from sesame seeds in fresh and stored conditions obtained from rainfed and irrigated zones of Punjab, Pakistan, and characterized them for aflatoxigenic potentials. Using morphological identification techniques, 260 isolates were grouped as potential Aspergillus section Flavi, with 126 and 134 originating from the rainfed and irrigated zones, respectively. Out of 260 in total, 188 isolates were confirmed to produce aflatoxins. There were no significant differences in potential aflatoxigenic isolates with respect to the rainfed and irrigated zones. However, the number of potential aflatoxigenic isolates was significantly higher (p < 0.05) in stored samples than that of those from fresh sesame seeds in the rainfed and irrigated zone. Whole genome sequencing and comparative analyses of 12 select isolates have revealed that one of the A. flavus isolates, which produced very low aflatoxins (AFP10), has an elevated missense variant rate, numerous high impact mutations, and a 600 base pair deletion in the norB gene. In summary, our study provides insights into aflatoxigenic potential and the associated genetic diversity of indigenous Aspergillus section Flavi isolates and potential management strategies for reducing aflatoxin contamination levels in a major crop consumed in Punjab, Pakistan.

Upstream Regulation of Development and Secondary Metabolism in Aspergillus Species.

In filamentous fungal Aspergillus species, growth, development, and secondary metabolism are genetically programmed biological processes, which require precise coordination of diverse signaling elements, transcription factors (TFs), upstream and downstream regulators, and biosynthetic genes. For the last few decades, regulatory roles of these controllers in asexual/sexual development and primary/secondary metabolism of Aspergillus species have been extensively studied. Among a wide spectrum of regulators, a handful of global regulators govern upstream regulation of development and metabolism by directly and/or indirectly affecting the expression of various genes including TFs. In this review, with the model fungus Aspergillus nidulans as the central figure, we summarize the most well-studied main upstream regulators and their regulatory roles. Specifically, we present key functions of heterotrimeric G proteins and G protein-coupled receptors in signal transduction), the velvet family proteins governing development and metabolism, LaeA as a global regulator of secondary metabolism, and NsdD, a key GATA-type TF, affecting development and secondary metabolism and provide a snapshot of overall upstream regulatory processes underlying growth, development, and metabolism in Aspergillus fungi.

AtfA-Independent Adaptation to the Toxic Heavy Metal Cadmium in Aspergillus nidulans.

Cadmium is an exceptionally toxic industrial and environmental pollutant classified as a human carcinogen. In order to provide insight into how we can keep our environment safe from cadmium contamination and prevent the accumulation of it in the food chain, we aim to elucidate how Aspergillus nidulans, one of the most abundant fungi in soil, survives and handles cadmium stress. As AtfA is the main transcription factor governing stress responses in A. nidulans, we examined genome-wide expression responses of wild-type and the atfA null mutant exposed to CdCl2. Both strains showed up-regulation of the crpA Cu2+/Cd2+ pump gene and AN7729 predicted to encode a putative bis(glutathionato)-cadmium transporter, and transcriptional changes associated with elevated intracellular Cys availability leading to the efficient adaptation to Cd2+. Although the deletion of atfA did not alter the cadmium tolerance of the fungus, the cadmium stress response of the mutant differed from that of a reference strain. Promoter and transcriptional analyses of the "Phospho-relay response regulator" genes suggest that the AtfA-dependent regulation of these genes can be relevant in this phenomenon. We concluded that the regulatory network of A. nidulans has a high flexibility allowing the fungus to adapt efficiently to stress both in the presence and absence of this important transcription factor.

The putative sensor histidine kinase VadJ coordinates development and sterigmatocystin production in Aspergillus nidulans.

The VosA-VelB heterocomplex governs expression of several genes associated with fungal development and secondary metabolism. In this study, we have investigated the functions of one of the VosA-VelB-activated developmental genes vadJ in development and production of the mycotoxin sterigmatocystin in the model fungus Aspergillus nidulans. The vadJ gene is predicted to encode a 957-amino acid length protein containing a highly conserved sensor histidine kinase domain. The deletion of vosA or velB resulted in decreased mRNA levels of vadJ throughout the life cycle, suggesting that VosA and VelB are necessary for proper expression of vadJ. Nullifying vadJ led to highly restricted colony growth, lowered formation of asexual spores, and about two-fold reduction in conidial viability. Conversely, the deletion of vadJ resulted in elevated production of sexual fruiting bodies and sterigmatocystin. These suggest that VadJ is necessary for proper coordination of asexual and sexual development, and sterigmatocystin production. In accordance with this idea, the deletion of vadJ led to elevated mRNA levels of the two key sexual developmental activators esdC and nsdD. In summary, the putative sensor histidine kinase VadJ represses sexual development and sterigmatocystin production, but activates asexual development in A. nidulans.

The DUG Pathway Governs Degradation of Intracellular Glutathione in Aspergillus nidulans.

Glutathione (GSH) is an abundant tripeptide that plays a crucial role in shielding cellular macromolecules from various reactive oxygen and nitrogen species in fungi. Understanding GSH metabolism is of vital importance for deciphering redox regulation in these microorganisms. In the present study, to better understand the GSH metabolism in filamentous fungi, we investigated functions of the dugB and dugC genes in the model fungus Aspergillus nidulans These genes are orthologues of dug2 and dug3, which are involved in cytosolic GSH degradation in Saccharomyces cerevisiae The deletion of dugB, dugC, or both resulted in a moderate increase in the GSH content in mycelia grown on glucose, reduced conidium production, and disturbed sexual development. In agreement with these observations, transcriptome data showed that genes encoding mitogen-activated protein (MAP) kinase pathway elements (e.g., steC, sskB, hogA, and mkkA) or regulatory proteins of conidiogenesis and sexual differentiation (e.g., flbA, flbC, flbE, nosA, rosA, nsdC, and nsdD) were downregulated in the ΔdugB ΔdugC mutant. Deletion of dugB and/or dugC slowed the depletion of GSH pools during carbon starvation. It also reduced accumulation of reactive oxygen species and decreased autolytic cell wall degradation and enzyme secretion but increased sterigmatocystin formation. Transcriptome data demonstrated that enzyme secretions-in contrast to mycotoxin production-were controlled at the posttranscriptional level. We suggest that GSH connects starvation and redox regulation to each other: cells utilize GSH as a stored carbon source during starvation. The reduction of GSH content alters the redox state, activating regulatory pathways responsible for carbon starvation stress responses.IMPORTANCE Glutathione (GSH) is a widely distributed tripeptide in both eukaryotes and prokaryotes. Owing to its very low redox potential, antioxidative character, and high intracellular concentration, GSH profoundly shapes the redox status of cells. Our observations suggest that GSH metabolism and/or the redox status of cells plays a determinative role in several important aspects of fungal life, including oxidative stress defense, protein secretion, and secondary metabolite production (including mycotoxin formation), as well as sexual and asexual differentiations. We demonstrated that even a slightly elevated GSH level can substantially disturb the homeostasis of fungi. This information could be important for development of new GSH-producing strains or for any biotechnologically relevant processes where the GSH content, antioxidant capacity, or oxidative stress tolerance of a fungal strain is manipulated.

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.

Revised Transcriptome-Based Gene Annotation for Aspergillus flavus Strain NRRL 3357.

Aspergillus flavus is an agriculturally and medically important filamentous fungus that produces mycotoxins, including aflatoxins, which are potent carcinogens. Here, we generated short- and long-read transcript sequence data from the growth of A. flavus strain NRRL 3357 under both typical and stress conditions to produce a new annotation of its genome.

Characterization of the velvet regulators in Aspergillus flavus.

Fungal development and secondary metabolism are closely associated via the activities of the fungal NK-kB-type velvet regulators that are highly conserved in filamentous fungi. Here, we investigated the roles of the velvet genes in the aflatoxigenic fungus Aspergillus flavus. Distinct from other Aspergillus species, the A. flavus genome contains five velvet genes, veA, velB, velC, velD, and vosA. The deletion of velD blocks the production of aflatoxin B1, but does not affect the formation of sclerotia. Expression analyses revealed that vosA and velB mRNAs accumulated at high levels during the late phase of asexual development and in conidia. The absence of vosA or velB decreased the content of conidial trehalose and the tolerance of conidia to the thermal and UV stresses. In addition, double mutant analyses demonstrated that VosA and VelB play an inter-dependent role in trehalose biosynthesis and conidial stress tolerance. Together with the findings of previous studies, the results of the present study suggest that the velvet regulators play the conserved and vital role in sporogenesis, conidial trehalose biogenesis, stress tolerance, and aflatoxin biosynthesis in A. flavus.