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Light is a key external signal factor that regulates asexual development, stress resistance, and secondary metabolism in fungi. In the presence of light, photoreceptors sense several light receptors and affect fungal life. In this study, we characterized the function of the blue light receptors LreA and LreB in Aspergillus flavus , a potent pathogenic and toxigenic fungus. lreA or lreB deletion increased the growth rate but decreased conidial production in the presence or absence of light. The Δ lreA -mutant strain and the Δ lreB -mutant strain produced abnormal conidiophores, suggesting that lreA and lreB were essential for proper conidiation in A. flavus . The absence of lreA or lreB slightly decreased the stress response tolerance against thermal and oxidative stresses. In kernel infection, the Δ lreA mutant strain and the Δ lreB mutant strain produced conidia and aflatoxin B1 that were less than those produced by the control strains. Therefore, LreA and LreB play key roles in the growth, asexual development, and pathogenicity of A. flavus .

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 .

Post-translational modifications of chromatin structure by histone acetyltransferase (HATs) play a pivotal role in the regulation of gene expression and diverse biological processes. However, the function of GNAT family HATs, especially Elp3, in the opportunistic human pathogenic fungus Aspergillus fumigatus is largely unknown. To investigate the roles of the GNAT family HATs Elp3 and GcnE in the A. fumigatus , we have generated and characterized individual null Δ elp3 and Δ gcnE mutants. The radial growth of fungal colonies was significantly decreased by the loss of elp3 or gcnE , and the number of asexual spores (conidia) in the Δ gcnE mutant was significantly reduced. Moreover, the mRNA levels of the key asexual development regulators were also significantly low in the Δ gcnE mutant compared to wild type (WT). Whereas both the Δ elp3 and Δ gcnE mutants were markedly impaired in the formation of adherent biofilms, the Δ gcnE mutant showed a complete loss of surface structure and of intercellular matrix. The Δ gcnE mutant responded differently to oxidative stressors and showed significant susceptibility to triazole antifungal agents. Furthermore, Elp3 and GcnE function oppositely in the production of secondary metabolites, and the Δ gcnE mutant showed attenuated virulence. In conclusion, Elp3 and GcnE are associated with diverse biological processes and can be potential targets for controlling the pathogenic fungus.

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.

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., (© 2021. The Microbiological Society of Korea.)

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 Δ dugB Δ dugC 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., (Copyright © 2021 American Society for Microbiology.)

The APSES family proteins are transcription factors (TFs) with a basic helix-loop-helix domain, known to regulate growth, development, secondary metabolism, and other biological processes in Aspergillus species. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus , five genes predicted to encode APSES TFs are present. Here, we report the characterization of one of these genes, called mbsA (Afu7g05620). The deletion (Δ) of mbsA resulted in significantly decreased hyphal growth and asexual sporulation (conidiation), and lowered mRNA levels of the key conidiation genes abaA , brlA, and wetA . Moreover, Δ mbsA resulted in reduced spore germination rates, elevated sensitivity toward Nikkomycin Z, and significantly lowered transcripts levels of genes associated with chitin synthesis. The mbsA deletion also resulted in significantly reduced levels of proteins and transcripts of genes associated with the SakA MAP kinase pathway. Importantly, the cell wall hydrophobicity and architecture of the Δ mbsA asexual spores (conidia) were altered, notably lacking the rodlet layer on the surface of the Δ mbsA conidium. Comparative transcriptomic analyses revealed that the Δ mbsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes, which was corroborated by elevated levels of GT production in the mutant. While the Δ mbsA mutant produced higher amount of GT, Δ mbsA strains showed reduced virulence in the murine model, likely due to the defective spore integrity. In summary, the putative APSES TF MbsA plays a multiple role in governing growth, development, spore wall architecture, GT production, and virulence, which may be associated with the attenuated SakA signaling pathway.

The APSES transcription factor (TF) in Aspergillus species is known to govern diverse cellular processes, including growth, development, and secondary metabolism. Here, we investigated functions of the rgdA gene (Afu3g13920) encoding a putative APSES TF in the opportunistic human-pathogenic fungus Aspergillus fumigatus The rgdA deletion resulted in significantly decreased hyphal growth and asexual sporulation. Consistently, transcript levels of the key asexual developmental regulators abaA , brlA , and wetA were decreased in the Δ rgdA mutant compared to those in the wild type (WT). Moreover, Δ rgdA resulted in reduced spore germination rates and elevated transcript levels of genes associated with conidium dormancy. The conidial cell wall hydrophobicity and architecture were changed, and levels of the RodA protein were decreased in the Δ rgdA mutant. Comparative transcriptomic analyses revealed that the Δ rgdA mutant showed higher mRNA levels of gliotoxin (GT)-biosynthetic genes and GT production. While the Δ rgdA mutant exhibited elevated production of GT, Δ rgdA strains showed reduced virulence in the mouse model. In addition, mRNA levels of genes associated with the cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway and the SakA mitogen-activated protein (MAP) kinase pathway were increased in the Δ rgdA mutant. In summary, RgdA plays multiple roles in governing growth, development, GT production, and virulence which may involve attenuation of PKA and SakA signaling. IMPORTANCE Immunocompromised patients are susceptible to infections with the opportunistic human-pathogenic fungus Aspergillus fumigatus This fungus causes systemic infections such as invasive aspergillosis (IA), which is one of the most life-threatening fungal diseases. To control this serious disease, it is critical to identify new antifungal drug targets. In fungi, the transcriptional regulatory proteins of the APSES family play crucial roles in controlling various biological processes, including mating, asexual sporulation and dimorphic growth, and virulence traits. This study found that a putative APSES transcription factor, RgdA, regulates normal growth, asexual development, conidium germination, spore wall architecture and hydrophobicity, toxin production, and virulence in A. fumigatus Better understanding the molecular mechanisms of RgdA in human-pathogenic fungi may reveal a novel antifungal target for future drug development., (Copyright © 2020 Jun et al.)

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 β-(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.

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.

The velvet regulator VosA plays a pivotal role in asexual sporulation in the model filamentous fungus Aspergillus nidulans . In the present study, we characterize the roles of VosA in sexual spores (ascospores) in A . nidulans . During ascospore maturation, the deletion of vosA causes a rapid decrease in spore viability. The absence of vosA also results in a lack of trehalose biogenesis and decreased tolerance of ascospores to thermal and oxidative stresses. RNA-seq-based genome-wide expression analysis demonstrated that the loss of vosA leads to elevated expression of sterigmatocystin (ST) biosynthetic genes and a slight increase in ST production in ascospores. Moreover, the deletion of vosA causes upregulation of additional gene clusters associated with the biosynthesis of other secondary metabolites, including asperthecin, microperfuranone, and monodictyphenone. On the other hand, the lack of vosA results in the downregulation of various genes involved in primary metabolism. In addition, vosA deletion alters mRNA levels of genes associated with the cell wall integrity and trehalose biosynthesis. Overall, these results demonstrate that the velvet regulator VosA plays a key role in the maturation and the cellular and metabolic integrity of sexual spores in A. nidulans .

The regulator of G-protein signaling (RGS) proteins play an important role in upstream control of heterotrimeric G-protein signaling pathways. In the genome of the human opportunistic pathogenic fungus Aspergillus fumigatus , six RGS protein-encoding genes are present. To characterize the rgsA gene predicted to encode a protein with an RGS domain, we generated an rgsA null mutant and observed the phenotypes of the mutant. The deletion (Δ) of rgsA resulted in increased radial growth and enhanced asexual sporulation in both solid and liquid culture conditions. Accordingly, transcripts levels of the key asexual developmental regulators abaA , brlA, and wetA are elevated in the Δ rgsA mutant. Moreover, Δ rgsA resulted in elevated spore germination rates in the absence of a carbon source. The activity of cAMP-dependent protein kinase A (PKA) and mRNA levels of genes encoding PKA signaling elements are elevated by Δ rgsA . In addition, mRNA levels of genes associated with stress-response signaling increased with the lack of rgsA , and the Δ rgsA spores showed enhanced tolerance against oxidative stressors. Comparative transcriptomic analyses revealed that the Δ rgsA mutant showed higher mRNA levels of gliotoxin (GT) biosynthetic genes. Accordingly, the rgsA null mutant exhibited increased production of GT and elevated virulence in the mouse. Conversely, the majority of genes encoding glucan degrading enzymes were down-regulated by Δ rgsA, and endoglucanase activities were reduced. In summary, RgsA plays multiple roles, governing growth, development, stress responses, virulence, and external polymer degradation-likely by attenuating PKA signaling.

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 C 2 H 2 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 β-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.

Tremellomycetes , a fungal class in the subphylum Agaricomycotina , contain well-known opportunistic and emerging human pathogens. The azole drug fluconazole, used in the treatment of diseases caused by some species of Tremellomycetes , inhibits cytochrome P450 monooxygenase CYP51, an enzyme that converts lanosterol into an essential component of the fungal cell membrane ergosterol. Studies indicate that mutations and over-expression of CYP51 in species of Tremellomycetes are one of the reasons for fluconazole resistance. Moreover, the novel drug, VT-1129, that is in the pipeline is reported to exert its effect by binding and inhibiting CYP51. Despite the importance of CYPs, the CYP repertoire in species of Tremellomycetes has not been reported to date. This study intends to address this research gap. Comprehensive genome-wide CYP analysis revealed the presence of 203 CYPs (excluding 16 pseudo-CYPs) in 23 species of Tremellomycetes that can be grouped into 38 CYP families and 72 CYP subfamilies. Twenty-three CYP families are new and three CYP families (CYP5139, CYP51 and CYP61) were conserved across 23 species of Tremellomycetes . Pathogenic cryptococcal species have 50% fewer CYP genes than non-pathogenic species. The results of this study will serve as reference for future annotation and characterization of CYPs in species of Tremellomycetes .

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.

Asexual sporulation is fundamental to the ecology and lifestyle of filamentous fungi and can facilitate both plant and human infection. In Aspergillus , the production of asexual spores is primarily governed by the BrlA→AbaA→WetA regulatory cascade. The final step in this cascade is controlled by the WetA protein and governs not only the morphological differentiation of spores but also the production and deposition of diverse metabolites into spores. While WetA is conserved across the genus Aspergillus , the structure and degree of conservation of the wetA gene regulatory network (GRN) remain largely unknown. We carried out comparative transcriptome analyses of comparisons between wetA null mutant and wild-type asexual spores in three representative species spanning the diversity of the genus Aspergillus : A. nidulans , A. flavus , and A. fumigatus We discovered that WetA regulates asexual sporulation in all three species via a negative-feedback loop that represses BrlA, the cascade's first step. Furthermore, data from chromatin immunoprecipitation sequencing (ChIP-seq) experiments in A. nidulans asexual spores suggest that WetA is a DNA-binding protein that interacts with a novel regulatory motif. Several global regulators known to bridge spore production and the production of secondary metabolites show species-specific regulatory patterns in our data. These results suggest that the BrlA→AbaA→WetA cascade's regulatory role in cellular and chemical asexual spore development is functionally conserved but that the wetA -associated GRN has diverged during Aspergillus evolution. IMPORTANCE The formation of resilient spores is a key factor contributing to the survival and fitness of many microorganisms, including fungi. In the fungal genus Aspergillus , spore formation is controlled by a complex gene regulatory network that also impacts a variety of other processes, including secondary metabolism. To gain mechanistic insights into how fungal spore formation is controlled across Aspergillus , we dissected the gene regulatory network downstream of a major regulator of spore maturation (WetA) in three species that span the diversity of the genus: the genetic model A. nidulans , the human pathogen A. fumigatus , and the aflatoxin producer A. flavus Our data show that WetA regulates asexual sporulation in all three species via a negative-feedback loop and likely binds a novel regulatory element that we term the WetA response element (WRE). These results shed light on how gene regulatory networks in microorganisms control important biological processes and evolve across diverse species., (Copyright © 2018 Wu et al.)

Bridging cellular reproduction and survival is essential for all life forms. Aspergillus fungi primarily reproduce by forming asexual spores called conidia, whose formation and maturation is governed by the central genetic regulatory circuit BrlA→AbaA→WetA. Here, we report that WetA is a multi-functional regulator that couples spore differentiation and survival, and governs proper chemical development in Aspergillus flavus. The deletion of wetA results in the formation of conidia with defective cell walls and no intra-cellular trehalose, leading to reduced stress tolerance, a rapid loss of viability, and disintegration of spores. WetA is also required for normal vegetative growth, hyphal branching, and production of aflatoxins. Targeted and genome-wide expression analyses reveal that WetA exerts feedback control of brlA and that 5,700 genes show altered mRNA levels in the mutant conidia. Functional category analyses of differentially expressed genes in ΔwetA RNA-seq data indicate that WetA contributes to spore integrity and maturity by properly regulating the metabolic pathways of trehalose, chitin, α-(1,3)-glucan, β-(1,3)-glucan, melanin, hydrophobins, and secondary metabolism more generally. Moreover, 160 genes predicted to encode transcription factors are differentially expressed by the absence of wetA, suggesting that WetA may play a global regulatory role in conidial development. Collectively, we present a comprehensive model for developmental control that bridges spore differentiation and survival in A. flavus.

Aspergillus fumigatus reproduces and infects host by forming a high number of small asexual spores (conidia). The velvet proteins are global transcriptional regulators governing the complex process of conidiogenesis in this fungus. Here, to further understand the velvet-mediated regulation, we carried out comparative proteomic analyses of conidia of wild type (WT) and three velvet mutants (ΔveA, ΔvelB and ΔvosA). Cluster analysis of 184 protein spots showing at least 1.5-fold differential accumulation between WT and mutants reveal the clustering of WT- ΔveA and ΔvelB-ΔvosA. Among 43 proteins identified by Nano-LC-ESI-MS/MS, 23 including several heat shock proteins showed more than two-fold reduction in both the ∆velB and ∆vosA conidia. On the contrary, three proteins exhibited more than five-fold increase in ∆veA only, including the putative RNA polymerase II degradation factor DefA. The deletion of defA resulted in a reduced number of conidia and restricted colony growth. In addition, the defA deletion mutant conidia showed hypersensitivity against the DNA damaging agents NQO and MMS, while the ΔveA mutant conidia were more resistant against to NQO. Taken together, we propose that VeA controls protein level of DefA in conidia, which are dormant and equipped with multiple layers of protection against environmental cues., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Asexual development (conidiation) in the filamentous fungus Aspergillus nidulans is governed by orchestrated gene expression. The three key negative regulators of conidiation SfgA, VosA, and NsdD act at different control point in the developmental genetic cascade. Here, we have revealed that NsdD is a key repressor affecting the quantity of asexual spores in Aspergillus. Moreover, nullifying both nsdD and vosA results in abundant formation of the development specific structure conidiophores even at 12 h of liquid culture, and near constitutive activation of conidiation, indicating that acquisition of developmental competence involves the removal of negative regulation exerted by both NsdD and VosA. NsdD's role in repressing conidiation is conserved in other aspergilli, as deleting nsdD causes enhanced and precocious activation of conidiation in Aspergillus fumigatus or Aspergillus flavus. In vivo NsdD-DNA interaction analyses identify three NsdD binding regions in the promoter of the essential activator of conidiation brlA, indicating a direct repressive role of NsdD in conidiation. Importantly, loss of flbC or flbD encoding upstream activators of brlA in the absence of nsdD results in delayed activation of brlA, suggesting distinct positive roles of FlbC and FlbD in conidiation. A genetic model depicting regulation of conidiation in A. nidulans is presented.

Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host/ecological niche can influence shaping the P450 content of an organism. The present study initiates our understanding of P450 family patterns in basidiomycete biotrophic plant pathogens.

Orchestration of cellular growth and development occurs during the life cycle of Aspergillus nidulans. A multi-copy genetic screen intended to unveil novel regulators of development identified the AN6578 locus predicted to encode a protein with the WOPR domain, which is a broadly present fungi-specific DNA-binding motif. Multi-copy of AN6578 disrupted the normal life cycle of the fungus leading to enhanced proliferation of vegetative cells, whereas the deletion resulted in hyper-active sexual fruiting with reduced asexual development (conidiation), thus named as osaA (Orchestrator of Sex and Asex). Further genetic studies indicate that OsaA balances development mainly by repressing sexual development downstream of the velvet regulator VeA. The absence of osaA is sufficient to suppress the veA1 allele leading to the sporulation levels comparable to veA+ wild type (WT). Genome-wide transcriptomic analyses of WT, veA1, and ΔosaA veA1 strains by RNA-Seq further corroborate that OsaA functions in repressing sexual development downstream of VeA. However, OsaA also plays additional roles in controlling development, as the ΔosaA veA1 mutant exhibits precocious and enhanced formation of Hülle cells compared to WT. The OsaA orthologue of Aspergillus flavus is able to complement the osaA null phenotype in A. nidulans, suggesting a conserved role of this group of WOPR domain proteins. In summary, OsaA is an upstream orchestrator of morphological and chemical development in Aspergillus that functions downstream of VeA.

The opportunistic human pathogenic fungus Aspergillus fumigatus primarily reproduces by forming a large number of asexual spores (conidia). Sequential activation of the central regulators BrlA, AbaA and WetA is necessary for the fungus to undergo asexual development. In this study, to address the presumed roles of these key developmental regulators during proliferation of the fungus, we analyzed and compared the proteomes of vegetative cells of wild type (WT) and individual mutant strains. Approximately 1300 protein spots were detectable from 2-D electrophoresis gels. Among these, 13 proteins exhibiting significantly altered accumulation levels were further identified by ESI-MS/MS. Markedly, we found that the GliM and GliT proteins associated with gliotoxin (GT) biosynthesis and self-protection of the fungus from GT were significantly down-regulated in the ΔabaA and ΔbrlA mutants. Moreover, mRNA levels of other GT biosynthetic genes including gliM, gliP, gliT, and gliZ were significantly reduced in both mutant strains, and no and low levels of GT were detectable in the ΔbrlA and ΔabaA mutant strains, respectively. As GliT is required for the protection of the fungus from GT, growth of the ΔbrlA mutant with reduced levels of GliT was severely impaired by exogenous GT. Our studies demonstrate that AbaA and BrlA positively regulate expression of the GT biosynthetic gene cluster in actively growing vegetative cells, and likely bridge morphological and chemical development during the life-cycle of A. fumigatus., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Beta-glucans are a heterologous group of fibrous glucose polymers that are a major constituent of cell walls in Ascomycetes and Basidiomycetes fungi. Synthesis of β (1,3)- and (1,6)-glucans is coordinated with fungal cell growth and development, thus, is under tight genetic regulation. Here, we report that β-glucan synthesis in both asexual and sexual spores is turned off by the NF-kB like fungal regulators VosA and VelB in Aspergillus nidulans. Our genetic and genomic analyses have revealed that both VosA and VelB are necessary for proper down-regulation of cell wall biosynthetic genes including those associated with β-glucan synthesis in both types of spores. The deletion of vosA or velB results in elevated accumulation of β-glucan in asexual spores. Double mutant analyses indicate that VosA and VelB play an inter-dependent role in repressing β-glucan synthesis in asexual spores. In vivo chromatin immuno-precipitation analysis shows that both VelB and VosA bind to the promoter region of the β-glucan synthase gene fksA in asexual spores. Similarly, VosA is required for proper repression of β-glucan synthesis in sexual spores. In summary, the VosA-VelB hetero-complex is a key regulatory unit tightly controlling proper levels of β-glucan synthesis in asexual and sexual spores.

Asexual development (conidiation) of the filamentous fungus Aspergillus nidulans occurs via balanced activities of multiple positive and negative regulators. For instance, FluG (+) and SfgA (-) govern upstream regulation of the developmental switch, and BrlA (+) and VosA (-) control the progression and completion of conidiation. To identify negative regulators of conidiation downstream of FluG-SfgA, we carried out multicopy genetic screens using sfgA deletion strains. After visually screening >100,000 colonies, we isolated 61 transformants exhibiting reduced conidiation. Responsible genes were identified as AN3152 (nsdD), AN7507, AN2009, AN1652, AN5833, and AN9141. Importantly, nsdD, a key activator of sexual reproduction, was present in 10 independent transformants. Furthermore, deletion, overexpression, and double-mutant analyses of individual genes have led to the conclusion that, of the six genes, only nsdD functions in the FluG-activated conidiation pathway. The deletion of nsdD bypassed the need for fluG and flbA∼flbE, but not brlA or abaA, in conidiation, and partially restored production of the mycotoxin sterigmatocystin (ST) in the ΔfluG, ΔflbA, and ΔflbB mutants, suggesting that NsdD is positioned between FLBs and BrlA in A. nidulans. Nullifying nsdD caused formation of conidiophores in liquid submerged cultures, where wild-type strains do not develop. Moreover, the removal of both nsdD and vosA resulted in even more abundant development of conidiophores in liquid submerged cultures and high-level accumulation of brlA messenger (m)RNA even at 16 hr of vegetative growth. Collectively, NsdD is a key negative regulator of conidiation and likely exerts its repressive role via downregulating brlA.

Fungal development and secondary metabolism is intimately associated via activities of the fungi-specific velvet family proteins including VeA, VosA, VelB and VelC. Among these, VelC has not been characterized in Aspergillus nidulans. In this study, we characterize the role of VelC in asexual and sexual development in A. nidulans. The velC mRNA specifically accumulates during the early phase of sexual development. The deletion of velC leads to increased number of conidia and reduced production of sexual fruiting bodies (cleistothecia). In the velC deletion mutant, mRNA levels of the brlA, abaA, wetA and vosA genes that control sequential activation of asexual sporulation increase. Overexpression of velC causes increased formation of cleistothecia. These results suggest that VelC functions as a positive regulator of sexual development. VelC is one of the five proteins that physically interact with VosA in yeast two-hybrid and GST pull down analyses. The ΔvelC ΔvosA double mutant produced fewer cleistothecia and behaved similar to the ΔvosA mutant, suggesting that VosA is epistatic to VelC in sexual development, and that VelC might mediate control of sex through interacting with VosA at specific life stages for sexual fruiting.

Growth, development, virulence and secondary metabolism in fungi are governed by heterotrimeric G proteins (G proteins). A Gβ-like protein called Gib2 has been shown to function as an atypical Gβ in Gpa1-cAMP signaling in Cryptococcus neoformans. We found that the previously reported CpcB (cross pathway control B) protein is the ortholog of Gib2 in Aspergillus nidulans and Aspergillus fumigatus. In this report, we further characterize the roles of CpcB in governing growth, development and toxigenesis in the two aspergilli. The deletion of cpcB results in severely impaired cellular growth, delayed spore germination, and defective asexual sporulation (conidiation) in both aspergilli. Moreover, CpcB is necessary for proper expression of the key developmental activator brlA during initiation and progression of conidiation in A. nidulans and A. fumigatus. Somewhat in accordance with the previous study, the absence of cpcB results in the formation of fewer, but not micro-, cleistothecia in A. nidulans in the presence of wild type veA, an essential activator of sexual development. However, the cpcB deletion mutant cleistothecia contain no ascospores, validating that CpcB is required for progression and completion of sexual fruiting including ascosporogenesis. Furthermore, unlike the canonical GβSfaD, CpcB is not needed for the biosynthesis of the mycotoxin sterigmatocystin (ST) as the cpcB null mutant produced reduced amount of ST with unaltered STC gene expression. However, in A. fumigatus, the deletion of cpcB results in the blockage of gliotoxin (GT) production. Further genetic analyses in A. nidulans indicate that CpcB may play a central role in vegetative growth, which might be independent of FadA- and GanB-mediated signaling. A speculative model summarizing the roles of CpcB in conjunction with SfaD in A. nidulans is presented.

FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus., Biological Significance: Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion mutant and 8 proteins differentially secreted in wild type and the flbA mutant. Based on proteomic analyses, we further examined the role of FlbA and found that FlbA down-regulates gliT expression and SOD activity. Our results proposed that FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and exogenous gliotoxin in A. fumigatus., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Fungal development and secondary metabolism is intimately associated via activities of the fungi-specific velvet family proteins. Here we characterize the four velvet regulators in the opportunistic human pathogen Aspergillus fumigatus. The deletion of AfuvosA, AfuveA and AfuvelB causes hyperactive asexual development (conidiation) and precocious and elevated accumulation of AfubrlA during developmental progression. Moreover, the absence of AfuvosA, AfuveA or AfuvelB results in the abundant formation of conidiophores and highly increased AfubrlA mRNA accumulation in liquid submerged culture, suggesting that they act as repressors of conidiation. The deletion of AfuvosA or AfuvelB causes a reduction in conidial trehalose amount, long-term spore viability, conidial tolerance to oxidative and UV stresses, and accelerated and elevated conidial germination regardless of the presence or absence of an external carbon source, suggesting an interdependent role of them in many aspects of fungal biology. Genetic studies suggest that AfuAbaA activates AfuvosA and AfuvelB expression during the mid to late phase of conidiation. Finally, the AfuveA null mutation can be fully complemented by Aspergillus nidulans VeA, which can physically interact with AfuVelB and AfuLaeA in vivo. A model depicting the similar yet different roles of the velvet regulators governing conidiation and sporogenesis in A. fumigatus is presented., (© 2012 Blackwell Publishing Ltd.)

Heterotrimeric G proteins (G proteins) govern growth, development, and secondary metabolism in various fungi. Here, we characterized ricA, which encodes a putative GDP/GTP exchange factor for G proteins in the model fungus Aspergillus nidulans and the opportunistic human pathogen Aspergillus fumigatus. In both species, ricA mRNA accumulates during vegetative growth and early developmental phases, but it is not present in spores. The deletion of ricA results in severely impaired colony growth and the total (for A. nidulans) or near (for A. fumigatus) absence of asexual sporulation (conidiation). The overexpression (OE) of the A. fumigatus ricA gene (AfricA) restores growth and conidiation in the ΔAnricA mutant to some extent, indicating partial conservation of RicA function in Aspergillus. A series of double mutant analyses revealed that the removal of RgsA (an RGS protein of the GanB Gα subunit), but not sfgA, flbA, rgsB, or rgsC, restored vegetative growth and conidiation in ΔAnricA. Furthermore, we found that RicA can physically interact with GanB in yeast and in vitro. Moreover, the presence of two copies or OE of pkaA suppresses the profound defects caused by ΔAnricA, indicating that RicA-mediated growth and developmental signaling is primarily through GanB and PkaA in A. nidulans. Despite the lack of conidiation, brlA and vosA mRNAs accumulated to normal levels in the ΔricA mutant. In addition, mutants overexpressing fluG or brlA (OEfluG or OEbrlA) failed to restore development in the ΔAnricA mutant. These findings suggest that the commencement of asexual development requires unknown RicA-mediated signaling input in A. nidulans.

Carbon-starving Aspergillus nidulans cultures produce high activities of versatile hydrolytic enzymes and, among these, ChiB endochitinase and EngA β-1,3-endoglucanase showed significant antifungal activity against various fungal species. Double deletion of engA and chiB diminished the antifungal activity of the fermentation broths and increased conidiogenesis and long-term viability of A. nidulans, but decreased the growth rate on culture media containing weak carbon sources. Production of ChiB and EngA can influence fungal communities either directly due to their antifungal properties or indirectly through their effects on vegetative growth. Our data suggest saprophytic fungi as promising future candidates to develop novel biocontrol technologies.

Understanding in vivo protein-protein interactions is critical to dissect precise functions of the regulatory proteins of fungal secondary metabolites. As many fungi differentially produce a diverse array of secondary metabolites during their lifecycle, it is important to understand the cell-type specific regulation of secondary metabolism. However, due to the difficulty of sample preparation of biologically active proteins in fungal spores, protein-protein interaction studies have been generally restricted. While some outstanding studies revealed protein-protein interactions of selected regulators, including the velvet proteins in vegetative cells, a detailed protocol for investigating the protein-protein interactions in the fungal spores has not yet been reported. Here, we describe a working protocol for the purification and identification of interacting protein partners of the spores of Aspergillus nidulans employing the VelB protein as an example.

The multifunctional regulator VelB physically interacts with other velvet regulators and the resulting complexes govern development and secondary metabolism in the filamentous fungus Aspergillus nidulans. Here, we further characterize VelB's role in governing asexual development and conidiogenesis in A. nidulans. In asexual spore formation, velB deletion strains show reduced number of conidia, and decreased and delayed mRNA accumulation of the key asexual regulatory genes brlA, abaA, and vosA. Overexpression of velB induces a two-fold increase of asexual spore production compared to wild type. Furthermore, the velB deletion mutant exhibits increased conidial germination rates in the presence of glucose, and rapid germination of conidia in the absence of external carbon sources. In vivo immuno-pull-down analyses reveal that VelB primarily interacts with VosA in both asexual and sexual spores, and VelB and VosA play an inter-dependent role in spore viability, focal trehalose biogenesis and control of conidial germination. Genetic and in vitro studies reveal that AbaA positively regulates velB and vosA mRNA expression during sporogenesis, and directly binds to the promoters of velB and vosA. In summary, VelB acts as a positive regulator of asexual development and regulates spore maturation, focal trehalose biogenesis and germination by interacting with VosA in A. nidulans.

The β-lactam producing filamentous fungus Penicillium chrysogenum secretes a 6.25 kDa small molecular mass antifungal protein, PAF, which has a highly stable, compact 3D structure and is effective against a wide spectrum of plant and zoo pathogenic fungi. Its precise physiological functions and mode of action need to be elucidated before considering possible biomedical, agricultural or food technological applications. According to some more recent experimental data, PAF plays an important role in the fine-tuning of conidiogenesis in Penicillium chrysogenum. PAF triggers apoptotic cell death in sensitive fungi, and cell death signaling may be transmitted through two-component systems, heterotrimeric G protein coupled signal transduction and regulatory networks as well as via alteration of the Ca(2+) -homeostasis of the cells. Possible biotechnological applications of PAF are also outlined in the review., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The opportunistic human pathogen Aspergillus fumigatus produces a massive number of asexual spores (conidia) as the primary means of dispersal, survival, genome protection and infection of hosts. In this report, we investigate the functions of two developmental regulators, AfuAbaA and AfuWetA, in A. fumigatus. The AfuabaA gene is predicted to encode an ATTS/TEA DNA-binding domain protein and is activated by AfuBrlA during the middle stage of A. fumigatus asexual development (conidiation). The deletion of AfuabaA results in the formation of aberrant conidiophores exhibiting reiterated cylinder-like terminal cells lacking spores. Furthermore, the absence of AfuabaA causes delayed autolysis and cell death, whereas the overexpression of AfuabaA accelerates these processes, indicating an additional role for AfuAbaA. The AfuwetA gene is sequentially activated by AfuAbaA in the late phase of conidiation. The deletion of AfuwetA causes the formation of defective spore walls and a lack of trehalose biogenesis, leading to a rapid loss of spore viability and reduced tolerance to various stresses. This is the first report to demonstrate that WetA is essential for trehalose biogenesis in conidia. Moreover, the absence of AfuwetA causes delayed germ-tube formation and reduced hyphal branching, suggesting a role of AfuWetA in the early phase of fungal growth. A genetic model depicting the regulation of conidiation in A. fumigatus is proposed.

VeA is the founding member of the velvet superfamily of fungal regulatory proteins. This protein is involved in light response and coordinates sexual reproduction and secondary metabolism in Aspergillus nidulans. In the dark, VeA bridges VelB and LaeA to form the VelB-VeA-LaeA (velvet) complex. The VeA-like protein VelB is another developmental regulator, and LaeA has been known as global regulator of secondary metabolism. In this study, we show that VelB forms a second light-regulated developmental complex together with VosA, another member of the velvet family, which represses asexual development. LaeA plays a key role, not only in secondary metabolism, but also in directing formation of the VelB-VosA and VelB-VeA-LaeA complexes. LaeA controls VeA modification and protein levels and possesses additional developmental functions. The laeA null mutant results in constitutive sexual differentiation, indicating that LaeA plays a pivotal role in inhibiting sexual development in response to light. Moreover, the absence of LaeA results in the formation of significantly smaller fruiting bodies. This is due to the lack of a specific globose cell type (Hülle cells), which nurse the young fruiting body during development. This suggests that LaeA controls Hülle cells. In summary, LaeA plays a dynamic role in fungal morphological and chemical development, and it controls expression, interactions, and modification of the velvet regulators., Competing Interests: The authors have declared that no competing interests exist.

Several upstream developmental activators control asexual development (conidiation) in Aspergillus. In this study, we characterize one of such activators called flbE in Aspergillus fumigatus and Aspergillus nidulans. The predicted FlbE protein is composed of 222 and 201 aa in A. fumigatus and A. nidulans, respectively. While flbE is transiently expressed during early phase of growth in A. nidulans, it is somewhat constitutively expressed during the lifecycle of A. fumigatus. The deletion of flbE causes reduced conidiation and delayed expression of brlA and vosA in both species. Moreover, FlbE is necessary for salt-induced development in liquid submerged culture in A. fumigatus. The A. nidulans flbE null mutation is fully complemented by A. fumigatus flbE, indicating a functional conservancy of FlbE in Aspergillus. Both the deletion and overexpression of flbE in A. nidulans result in developmental defects, enhanced autolysis, precocious cell death, and delayed expression of brlA/vosA, suggesting that balanced activity of FlbE is crucial for proper growth and development. Importantly, the N-terminal portion of FlbE exhibits the trans-activation ability in yeast, whereas the C-terminal half negatively affects its activity. Site-directed mutagenesis of certain conserved N-terminal amino acids abolishes the ability of trans-activation, overexpression-induced autolysis, and complementing the null mutation. Finally, overexpression of flbD, but not flbB or flbC, restores conidiation in A. nidulans ΔflbE, generally supporting the current genetic model for developmental regulation., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The opportunistic human pathogen Aspergillus fumigatus reproduces asexually by forming a massive number of mitospores called conidia. In this study, we characterize the upstream developmental regulator A. fumigatus flbB (AfuflbB). Northern blotting and cDNA analyses reveal that AfuflbB produces two transcripts predicted to encode two basic leucine zipper domain (bZIP) polypeptides, AfuFlbBβ (420 amino acids [aa]) and AfuFlbBα (390 aa). The deletion of AfuflbB results in delayed/reduced sporulation, precocious cell death, the lack of conidiophore development in liquid submerged culture, altered expression of AfubrlA and AfuabaA, and blocked production of gliotoxin. While introduction of the wild-type (WT) AfuflbB allele fully complemented these defects, disruption of the ATG start codon for either one of the AfuFlbB polypeptides leads to a partial complementation, indicating the need of both polypeptides for WT levels of asexual development and gliotoxin biogenesis. Consistent with this, Aspergillus nidulans flbB(+) encoding one polypeptide (426 aa) partially complements the AfuflbB null mutation. The presence of 0.6 M KCl in liquid submerged culture suppresses the defects caused by the lack of one, but not both, of the AfuFlbB polypeptides, suggesting a genetic prerequisite for AfuFlbB in A. fumigatus development. Finally, Northern blot analyses reveal that both AfuflbB and AfuflbE are necessary for expression of AfuflbD, suggesting that FlbD functions downstream of FlbB/FlbE in aspergilli.

Asexual development (conidiation) in Aspergillus is governed by multiple regulators. Here, we characterize the upstream developmental activator FlbC in Aspergillus nidulans. flbC mRNA is detectable throughout the life cycle, at relatively high levels during vegetative growth, early asexual and late sexual developmental phases. The deletion of flbC causes a delay/reduction in conidiation, brlA and vosA expression, and conidial germination. While overexpression of flbC (OEflbC) does not elaborate conidiophores, it inhibits hyphal growth and activates expression of brlA, abaA and vosA, but not wetA. FlbC is conserved in filamentous Ascomycetes containing two C(2) H(2) zinc fingers at the C-terminus and a putative activation domain at the N-terminus. FlbC localizes in the nuclei of both hyphae and developmental cells. Localization and expression of FlbC are not affected by the absence of FlbB or FlbE, and vice versa. Importantly, overexpression of flbC causes growth inhibition and activation of abaA and vosA in the absence of brlA and abaA respectively. In vitro DNA-binding assay reveals that FlbC binds to the brlA, abaA and vosA, but not the wetA, promoters. In summary, FlbC is a putative nuclear transcription factor necessary for proper activation of conidiation, and its balanced activity is crucial for governing growth and development in A. nidulans., (© 2010 Blackwell Publishing Ltd.)

Phosphatidylcholines (PCs) are a class of major cell membrane phospholipids that participate in many physiological processes. Three genes, choA, choB and choC, have been proposed to function in the endogenous biosynthesis of PC in Aspergillus nidulans. In this study, we characterize the choC gene encoding a putative highly conserved phospholipid methyltransferase. The previously reported choC3 mutant allele results from a mutation leading to the E177K amino acid substitution. The transcript of choC accumulates at high levels during vegetative growth and early asexual developmental phases. The deletion of choC causes severe impairment of vegetative growth, swelling of hyphal tips and the lack of both asexual and sexual development, suggesting the requirement of ChoC and PC in growth and development. Noticeably, supplementation of the mutant with the penultimate precursor of PC N, N-dimethylaminoethanol leads to full recovery of vegetative growth, but incomplete progression of asexual and sexual development, implying differential roles of PC and its intermediates in fungal growth and development. Importantly, while the choC deletion mutant shows reduced vegetative growth and precocious cell death until day 4, it regains hyphal proliferation and cell viability from day 5, indicating the presence of an alternative route for cellular membrane function in A. nidulans.

The roles of the Gbetagamma subunits of the opportunistic human pathogen Aspergillus fumigatus were investigated. The predicted AfuSfaD (Gbeta) protein consists of 353 amino acids and shows 94-98% similarity with other Aspergillus Gbeta subunits. AfuGpgA consists of 90 amino acids showing 95-98% identity with other fungal G-protein gamma subunits. The deletion (Delta) of AfusfaD or AfugpgA resulted in severe impairment in vegetative growth, conidial germination and conidial trehalose breakdown. While the total number of conidia produced by DeltaAfusfaD and DeltaAfugpgA strains on solid medium was only about 1% of wild type, the growth-adjusted conidiation levels were twofold higher than those of wild type. Enhanced formation of conidiophores and elevated AfubrlA mRNA levels were observable in DeltaAfusfaD or DeltaAfugpgA strains in liquid submerged culture. Moreover, overexpression of AfusfaD or AfugpgA caused reduced levels of submerged culture conidiation, indicating that Gbetagamma is involved in negative regulation of conidiation. Gliotoxin and other metabolites were not detected in the chloroform extracts of DeltaAfusfaD and DeltaAfugpgA culture filtrates. Northern blot analyses revealed that, while AfulaeA mRNA levels unchanged, accumulation of gliZ mRNA was delayed by DeltaAfusfaD or DeltaAfugpgA. A model summarizing the roles of AfusfaD and AfugpgA in A. fumigatus is presented.

Aims: Elucidation of the regulation of ChiB production in Aspergillus nidulans., Methods and Results: Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures., Conclusions: The endochitinase ChiB plays an important role in autolysis of A. nidulans, and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling., Significance and Impact of the Study: Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.

Autolysis is a natural event that occurs in most filamentous fungi. Such self-degradation of fungal cells becomes a predominant phenomenon in the absence of the regulator of G protein signaling FlbA in Aspergillus nidulans. Among a number of potential hydrolytic enzymes in the A. nidulans genome, the secreted endochitinase ChiB was shown to play a major role in autolysis. In this report, we investigate the roles of ChiB in fungal autolysis and cell death processes through genetic, biochemical, and cellular analyses using a set of critical mutants. Determination of mycelial mass revealed that, while the flbA deletion (DeltaflbA) mutant autolyzed completely after a 3-day incubation, the DeltaflbA DeltachiB double mutant escaped from hyphal disintegration. These results indicate that ChiB is necessary for the DeltaflbA-induced autolysis. However, importantly, both DeltaflbA and DeltaflbA DeltachiB strains displayed dramatically reduced cell viability compared to the wild type. These imply that ChiB is dispensable for cell death and that autolysis and cell death are separate processes. Liquid chromatography-tandem mass spectrometry analyses of the proteins that accumulate at high levels in the DeltaflbA and DeltaflbA DeltachiB mutants identify chitinase (ChiB), dipeptidyl peptidase V (DppV), O-glycosyl compound hydrolase, beta-N-acetylhexosaminidase (NagA), and myo-inositol-1-phosphate synthase (InoB). Functional characterization of these four genes reveals that the deletion of nagA results in reduced cell death. A working model bridging G protein signaling and players in autolysis/cell death is proposed.

Differentiation and secondary metabolism are correlated processes in fungi that respond to light. In Aspergillus nidulans, light inhibits sexual reproduction as well as secondary metabolism. We identified the heterotrimeric velvet complex VelB/VeA/LaeA connecting light-responding developmental regulation and control of secondary metabolism. VeA, which is primarily expressed in the dark, physically interacts with VelB, which is expressed during sexual development. VeA bridges VelB to the nuclear master regulator of secondary metabolism, LaeA. Deletion of either velB or veA results in defects in both sexual fruiting-body formation and the production of secondary metabolites.

Candida magnoliae, an osmotolerant and erythritol producing yeast, prefers D-fructose to D-glucose as carbon sources. For the investigation of the fructophilic characteristics with respect to sugar transportation, a sequential extraction method using various detergents and ultracentrifugation was developed to isolate cellular membrane proteins in C. magnoliae. Immunoblot analysis with the Pma1 antibody and twodimensional electrophoresis analysis coupled with MS showed that the fraction II was enriched with membrane proteins. Eighteen proteins out of 36 spots were identified as membrane or membrane-associated proteins involved in sugar uptake, stress response, carbon metabolism, and so on. Among them, three proteins were significantly upregulated under the fructose supplying conditions. The hexose transporter was highly homologous to Ght6p in Schizosaccharomyces pombe, which was known as a predominant transporter for the fructose uptake of S. pombe because it exhibited higher affinity to D-fructose than D-glucose. The physicochemical properties of the ATP-binding cassette transporter and inorganic transporter explained their direct or indirect associations with the fructophilic behavior of C. magnoliae. The identification and characterization of membrane proteins involved in sugar uptake might contribute to the elucidation of the selective utilization of fructose to glucose by C. magnoliae at a molecular level.

The fungal colony is a complex multicellular unit consisting of various cell types and functions. Asexual spore formation (conidiation) is integrated through sensory and regulatory elements into the general morphogenetic plan, in which the activation of the transcription factor BrlA is the first determining step. A number of early regulatory elements acting upstream of BrlA (fluG and flbA-E) have been identified, but their functional relations remain to be further investigated. In this report we describe FlbB as a putative basic-zipper-type transcription factor restricted to filamentous fungi. FlbB accumulates at the hyphal apex during early vegetative growth but is later found in apical nuclei, suggesting that an activating modification triggers nuclear import. Moreover, proper temporal and quantitative expression of FlbB is a prerequisite for brlA transcription, and misscheduled overexpression inhibits conidiation. We also present evidence that FlbB activation results in the production of a second diffusible signal, acting downstream from the FluG factor, to induce conidiation.

The asexual spore is one of the most crucial factors contributing to the fecundity and fitness of filamentous fungi. Although the developmental activator FluG was shown to be necessary for activation of asexual sporulation (conidiation) and production of the carcinogenic mycotoxin sterigmatocystin (ST) in the model filamentous fungus Aspergillus nidulans, the molecular mechanisms underlying the developmental switch have remained elusive. In this study, we report that the FluG-mediated conidiation in A. nidulans occurs via derepression. Suppressor analyses of fluG led to the identification of the sfgA gene encoding a novel protein with the Gal4-type Zn(II)2Cys6 binuclear cluster DNA-binding motif at the N terminus. Deletion (delta) and 31 other loss-of-function sfgA mutations bypassed the need for fluG in conidiation and production of ST. Moreover, both delta sfgA and delta sfgA delta fluG mutations resulted in identical phenotypes in growth, conidiation, and ST production, indicating that the primary role of FluG is to remove repressive effects imposed by SfgA. In accordance with the proposed regulatory role of SfgA, overexpression of sfgA inhibited conidiation and delayed/reduced expression of conidiation- and ST-specific genes. Genetic analyses demonstrated that SfgA functions downstream of FluG but upstream of transcriptional activators (FlbD, FlbC, FlbB, and BrlA) necessary for normal conidiation.

Filamentous fungal genomes contain two distantly related cyclic AMP-dependent protein kinase A catalytic subunits (PKAs), but only one PKA is found to play a principal role. In Aspergillus nidulans, PkaA is the primary PKA that positively functions in vegetative growth and spore germination but negatively controls asexual sporulation and production of the mycotoxin sterigmatocystin. In this report, we present the identification and characterization of pkaB, encoding the secondary PKA in A. nidulans. Although deletion of pkaB alone does not cause any apparent phenotypic changes, the absence of both pkaB and pkaA is lethal, indicating that PkaB and PkaA are essential for viability of A. nidulans. Overexpression of pkaB enhances hyphal proliferation and rescues the growth defects caused by DeltapkaA, indicating that PkaB plays a role in vegetative growth signaling. However, unlike DeltapkaA, deletion of pkaB does not suppress the fluffy-autolytic phenotype resulting from DeltaflbA. While upregulation of pkaB rescues the defects of spore germination resulting from DeltapkaA in the presence of glucose, overexpression of pkaB delays spore germination. Furthermore, upregulation of pkaB completely abolishes spore germination on medium lacking a carbon source. In addition, upregulation of pkaB enhances the level of submerged sporulation caused by DeltapkaA and reduces hyphal tolerance to oxidative stress. In conclusion, PkaB is the secondary PKA that has a synthetic lethal interaction with PkaA, and it plays an overlapping role in vegetative growth and spore germination in the presence of glucose but an opposite role in regulating asexual sporulation, germination in the absence of a carbon source, and oxidative stress responses in A. nidulans.

The filamentous fungus Aspergillus nidulans possesses both asexual and sexual reproductive cycles. Sexual fruiting bodies (cleistothecia) can be formed in both homothallic (self) and heterothallic (outcross) conditions. In this study, we characterized two genes, gprA and gprB, that are predicted to encode putative G protein-coupled receptors (GPCRs) similar to fungal pheromone receptors. Deletion (Delta) of gprA or gprB resulted in the production of a few small cleistothecia carrying a reduced number of ascospores, whereas DeltagprADeltagprB eliminated fruiting body formation in homothallic conditions. However, nullifying gprA and/or gprB did not affect vegetative growth, asexual sporulation, Hülle cell formation or even cleistothecia formation in outcross, indicating that GprA and GprB are specifically required for self-fertilization. The gprA and gprB genes encode two transcripts and, for both genes, larger transcripts are detectable during vegetative growth and asexual development whereas smaller transcripts accumulate during sexual development. Upregulation of nsdD encoding a key sexual developmental activator resulted in the production of barren cleistothecia in the DeltagprADeltagprB mutant, suggesting that NsdD can partially rescue the developmental defects caused by deletion of GPCRs and that GprA/B-mediated signalling may activate other genes necessary for maturation of cleistothecia and ascosporogenesis. Deletion of gprA and/or gprB suppressed growth defects caused by DeltagprD, implying that GprA/B function downstream of GprD-mediated negative control of sexual development.

Regulators of G-protein signalling play a crucial role in controlling the degree of heterotrimeric G-protein signalling. In addition to the previously studied flbA, we have identified three genes (rgsA, rgsB and rgsC) encoding putative RGS proteins in the genome of Aspergillus nidulans. Characterization of the rgsA gene revealed that RgsA downregulates pigment production and conidial germination, but stimulates asexual sporulation (conidiation). Deletion of rgsA (DeltargsA) resulted in reduced colony size with increased aerial hyphae, elevated accumulation of brown pigments as well as enhanced tolerance of conidia and vegetative hyphae against oxidative and thermal stress. Moreover, DeltargsA resulted in conidial germination in the absence of a carbon source. Deletion of both flbA and rgsA resulted in an additive phenotype, suggesting that the G-protein pathways controlled by FlbA and RgsA are different. Morphological and metabolic alterations caused by DeltargsA were suppressed by deletion of ganB encoding a Galpha subunit, indicating that the primary role of RgsA is to control negatively GanB-mediated signalling. Overexpression of rgsA caused inappropriate conidiation in liquid submerged culture, supporting the idea that GanB signalling represses conidiation. Our findings define a second and specific RGS-Galpha pair in A. nidulans, which may govern upstream regulation of fungal cellular responses to environmental changes.

G protein-coupled receptors (GPCRs) are key components of heterotrimeric G protein-mediated signalling pathways that detect environmental signals and confer rapid cellular responses. To broaden our understanding of signalling mechanisms in the filamentous fungus Aspergillus nidulans, intensive analyses of the Aspergillus nidulans genome have been carried out and nine genes (gprA approximately gprI) that are predicted to encode seven transmembrane spanning GPCRs have been identified. Six of nine putative GPCRs have been disrupted and the gprD gene was found to play a central role in coordinating hyphal growth and sexual development. Deletion of gprD (Delta gprD) causes extremely restricted hyphal growth, delayed conidial germination and uncontrolled activation of sexual development resulting in a small colony covered by sexual fruiting bodies. Genetic studies indicate that GprD may not signal through the FadA (G alpha)-protein kinase A (PKA) pathway. Elimination of sexual development rescues both growth and developmental abnormalities caused by Delta gprD, suggesting that the primary role of GprD is to negatively regulate sexual development. This is supported by the fact that environmental conditions inhibiting sexual development suppress growth defects of the Delta gprD mutant. We propose that the GprD-mediated signalling cascade negatively regulates sexual development, which is required for proper proliferation of A. nidulans.