Asexual reproduction in filamentous fungi is a common, efficient, and fast differentiation process for producing large numbers of asexual spores (conidia), which can be distributed through the air to colonize new environments. The whole process is tightly controlled by specific regulatory proteins. Among the major regulators is the zinc cluster protein of SclB (sclerotia-like B), known to influence various aspects of asexual growth and secondary metabolism in Aspergillus nidulans as well as other filamentous fungi. Two different growth conditions of A. nidulans were compared to obtain a mechanistic overview of the role of SclB, mainly during the transition of the fungus from vegetative to asexual growth. Chromatin immunoprecipitation (IP) was coupled with next-generation sequencing (ChIP-seq) and combined with transcriptomic analyses (RNA-seq). SclB coordinates this developmental shift mainly by controlling the expression of genes encoding a few, however, prominent regulators of conidiation. These include the transcription factors BrlA, VelB, and SclB and the pheromone oxygenase PpoC. Association of SclB to promoter regions requires the newly identified putative SclB response element with a nine-base-pair DNA motif. Scl2 is the corresponding protein in the fungal plant pathogen Verticillium dahliae and partially complements the A. nidulans ΔsclB asexual deficiency. This supports a partial functional complementation of this regulator among different fungal species. In summary, SclB coordinates the transition from vegetative growth to asexual reproduction in A. nidulans through in vivo transcriptional control over genes coding for established players of conidia formation.IMPORTANCEFungi constantly adapt to environmental changes in their various habitats. Asexual spore formation allows for quickly leaving an unfriendly habitat through dispersal into the air. The asexual developmental program of fungi enables the production of a large number of spores in a short period of time and in an energetically efficient manner. The SclB transcription factor is a key regulator of asexual growth and secondary metabolism in numerous fungal species. The mechanism through which SclB orchestrates the transition of the filamentous fungus Aspergillus nidulans from vegetative to asexual growth was revealed. This regulator directly in vivo controls itself as well as the expression of master genes for the asexual program, such as brlA for transcriptional control or ppoC for pheromone production. This study enhances the molecular understanding of how fungal asexual differentiation is initiated and coordinated, which supports the development of better strategies to control fungal pathogens, improving human health, safety, and crop management.

Aspergillus fumigatus is a soil born fungus, which can induce severe aspergillosis in immunocompromised patients. In this study, we investigated the zinc cluster transcription factor EneA (enhancer of nscA expression A) of A. fumigatus, which is required for adaption to polyenes and for competition with soil living streptomycetes. In presence of polyenes, the polyketidesynthase encoding gene nscA, which is part of the neosartoricin/fumicycline gene cluster, is induced in dependency of EneA via NscR. In contrast, high levels of eneA transcripts lead to increased nscA expression in the absence of nscR, suggesting differing regulatory pathways for neosartoricin production depending on EneA. Moreover, overexpression of eneA leads to the induction of genes from nine different secondary metabolite clusters, comprising several of which are known to produce toxins, revealing EneA as a global regulator of secondary metabolism. We demonstrate that these metabolites reduce amphotericin B toxicity and inhibit the growth of streptomycetes. Finally, we show that polyenes and metabolites derived from Streptomycetes noursei promote increased neosartoricin production via EneA. These findings suggest that the EneA-dependent adaptation of A. fumigatus in its natural habitat may contribute to enhanced tolerance to antifungal agents, such as amphotericin B, and augmented resistance to the host’s immune defenses.

Development and secondary metabolism of the filamentous fungus Aspergillus nidulans are tightly controlled by concerted actions of several master regulator transcription factors (TFs). The connection between fungal development and cellular stress response programs is often elusive. Here we show that the zinc finger TF MsnA, which controls salt-stress response, is a novel major regulator of fungal development. A molecular circuit among MsnA and the velvet domain regulator VelB was discovered, which mutually fosters the actions of both regulatory proteins during development. Chromatin immunoprecipitation coupled with next generation sequencing (ChIP-seq) and gene expression studies have revealed that MsnA controls the expression of several genes encoding key transcriptional regulators of asexual as well as sexual development. The double mutant of msnA with velB showed that both genes share an additive genetic relationship, under normal and salt stress conditions, with each protein to control distinct phenotypical features. In addition, MsnA directly and indirectly affects the synthesis of specific secondary metabolites relevant for fungal defense against other organisms and growth, in addition to salt-stress responses. Moreover, the expression of genes encoding the epigenetic regulators VapA, VipC and LaeA are also directly controlled by MsnA. The VapA-VipC-VapB methyltransferase signal transduction complex promotes asexual differentiation, while the VeA-VelB-LaeA complex balances light response, development and the secondary metabolism of the fungus. MsnA is therefore placed at a novel prominent position of the central regulatory network, which coordinates stress responses with the developmental and metabolic fate of the fungus.

ABSTRACT Velvet transcription factors are found throughout the fungal kingdom and share a common velvet domain with a fold similar to that of animal NF-κB. They act as homodimeric or heterodimeric master regulators of fungal secondary metabolism, development, and differentiation. A comparison of velvet domains from 4,999 fungal velvet proteins revealed a conserved overall architecture, including an N-terminal DNA-binding region of approximately 30 amino acids and a C-terminal dimerization region of about 100 amino acids. The dimerization region comprises α- and β-subunits separated by a linker region. A detailed analysis of the velvet domain in Aspergillus nidulans VelB identified glycine (glycine 240) and leucine (leucine 331) residues in the dimerization region as critical for interactions with velvet proteins. These core amino acid residues are conserved and also essential for the function of VeA or VosA, which corroborates their general importance in functional fungal velvet domains. Functional studies of VelB dimerization linkers suggest its tolerance for length shortening. These findings enhance our understanding of the working mechanisms of fungal velvet regulators.IMPORTANCEFungi, as relatives of animals within Opisthokonta, are closely connected to human life through interactions such as food, pathogenicity, and medicines. Similar to animals, fungi have developed NF-κB-like velvet family regulators to respond to various environmental and internal signals. Velvet regulators form homo- or heterodimers in implementing different functional roles. However, the molecular mechanism by which velvet proteins interact remains incompletely understood. In this study, we found a common architecture of fungal velvet domains and resolved the dimerization region using Aspergillus nidulans VelB as a paradigm. The growing understanding of the fungal velvet regulatory network may help to control fungi for pathogenic and industrial purposes and shed light on the general mechanisms shared with the animal NF-κB system in cellular responses to stimuli.

Colletotrichum graminicola, the maize anthracnose fungus, is known for its ability to invade above-ground tissues. This fungus forms two distinct asexual spore types in its life cycle, falcate conidia in acervuli on infected leaves and oval conidia in parenchyma cells in leaf and stem lesions. Our study reveals a previously unknown role for oval conidia in the infection of roots. We investigated whether root exudate from maize could redirect the growth of germlings generated by C. graminicola. Our results showed that only germlings derived from oval conidia were able to respond to root exudates, whereas those from falcate conidia did not. High-performance liquid chromatography/mass spectrometry (HPLC-MS) analyses combined with biological assays and genetic studies identified diterpenoids from maize as attractants perceived by the a-pheromone receptor CgSte3. We further explored the root-fungus interaction by analysing the germination of oval and falcate spores in soils of different composition. Oval conidia germinated rapidly under all conditions, whereas falcate conidia remained dormant and became highly vacuolated even in the presence of the host plant. Microscopic evaluation of root infection experiments showed that both conidia types attached to root material and formed penetration structures. However, only oval conidia enabled the pathogenic fungus to reach upper plant parts from infected roots. Our findings suggest a novel role for oval conidia in the infection of roots, highlighting the complexity of the anthracnose disease cycle.

Aspergillus nidulans is a common soil fungus, distributed worldwide. Adaptations in development and secondary metabolism of so far uncharacterized isolates from the Erbil province of Iraq were compared to each other and the commonly in laboratory work used strain A4 from Europe was used as a reference. Erb strains showed an increase in conidia formation during asexual development at 37°C when compared to A4. In contrast, fruiting body formation was strongly decreased during sexual development at 30°C or 37°C. The temperature had a strong impact on the synthesis of secondary metabolites. The amount of arugosin A, epi-/shamixanthone, and emericellin is increased in A4 compared to the Erb1 isolate during the various tested cultivation conditions. In contrast, the Erb1 isolate produced under these conditions increased amounts of sterigmatocystin. Deviations to the A4 in asperthecin production depends on the growth conditions e.g. changes in the incubation temperature. Increased conidiation of the Erb1 strain correlated with higher levels of austinol and dehydroaustinol production. These two metabolites were also more abundant in the Erb1 isolate during sexual development. Our data suggest that A. nidulans isolates from the Erbil province have adapted developmental and secondary metabolism patterns to local temperature conditions.

Most plant pathogenic microorganisms have evolved to infect distinct host tissues. The maize anthracnose fungus, Colletotrichum graminicola , is known for its ability to invade above-ground tissues with asexual falcate conidia. Besides, C. graminicola generates a second asexual spore type, oval conidia. We analyzed in this study specific adaptations making oval or falcate conidia prone for maize root infection, providing evidence that only oval conidia show characteristics of a root pathogen. This includes the ability to germinate in soil and to grow chemotropically toward root-secreted molecules. HPLC/MS analyses combined with biological assays revealed that diterpenoids like dihydrotanshinone I are most likely responsible for the chemical attraction of C. graminicola . A genetics approach identified the a-pheromone receptor CgSte3 as being responsible for the perception of diterpenoids by the fungal pathogen. In conclusion, the understanding of the maize anthracnose disease has to be expanded to include an elaborated root infection cycle by oval conidia.### Competing Interest StatementThe authors have declared no competing interest.

Zinc cluster transcription factors (Zcfs) encoding zcf genes are exclusive for fungi and required for multiple cellular processes including metabolism and development. Genome-wide screening of 228 zinc cluster transcription factor encoding genes by overexpression in Aspergillus fumigatus revealed 11 genes which provided increased tolerance to the broadly applied azole voriconazole or to the polyene amphotericin B or to both. These include four oxidative stress and drug resistance genes (odrA-D) genes encoding factors, which provide broad cellular stress protection. Thereby, the corresponding fungal OdrA/Mdu2- and AtrR/OdrD-dependent genetic networks are interconnected. OdrA/Mdu2 activates atrR/odrD transcription by direct binding to the promoter, whereas AtrR/OdrD functions as repressor of odrA/mdu2 expression. odrA/mdu2 overexpression provides combined resistance to amphotericin B, voriconazole, itraconazole, and reactive oxygen species generated by menadione. OdrA/Mdu2-mediated itraconazole resistance is evoked by direct regulation of the transporter encoding gene mdr1. Oxidative stress-inducing substances like amphotericin B and menadione promote OdrA/Mdu2 accumulation in the nucleus to regulate stress response genes like mdr1 and the putative glutathione-S-transferase encoding gene gstD. The expression levels and external stress conditions fostering nuclear accumulation of OdrA/Mdu2 determine the regulation of the target genes. Hence, OdrA/Mdu2 provides a combined adaptation strategy for survival in nature or within a potential host, where this fungus represents the most common agent for human mold pneumonia worldwide. The OdrA/Mdu2 controlled genetic network highlights the tight connection between oxidative stress response and antifungal drug adaption to secure A. fumigatus survival in various hostile environments.IMPORTANCEAn overexpression screen of 228 zinc cluster transcription factor encoding genes of A. fumigatus revealed 11 genes conferring increased tolerance to antifungal drugs. Out of these, four oxidative stress and drug tolerance transcription factor encoding odr genes increased tolerance to oxidative stress and antifungal drugs when overexpressed. This supports a correlation between oxidative stress response and antifungal drug tolerance in A. fumigatus. OdrA/Mdu2 is required for the cross-tolerance between azoles, polyenes, and oxidative stress and activates genes for detoxification. Under oxidative stress conditions or when overexpressed, OdrA/Mdu2 accumulates in the nucleus and activates detoxifying genes by direct binding at their promoters, as we describe with the mdr1 gene encoding an itraconazole specific efflux pump. Finally, this work gives new insights about drug and stress resistance in the opportunistic pathogenic fungus A. fumigatus.

The conserved eight-subunit COP9 signalosome (CSN) is required for multicellular fungal development. The CSN deneddylase cooperates with the Cand1 exchange factor to control replacements of E3 ubiquitin cullin RING ligase receptors, providing specificity to eukaryotic protein degradation. Aspergillus nidulans CSN assembles through a heptameric pre-CSN, which is activated by integration of the catalytic CsnE deneddylase. Combined genetic and biochemical approaches provided the assembly choreography within a eukaryotic cell for native fungal CSN. Interactomes of functional GFP-Csn subunit fusions in pre-CSN deficient fungal strains were compared by affinity purifications and mass spectrometry. Two distinct heterotrimeric CSN subcomplexes were identified as pre-CSN assembly intermediates. CsnA-C-H and CsnD-F-G form independently of CsnB, which connects the heterotrimers to a heptamer and enables subsequent integration of CsnE to form the enzymatically active CSN complex. Surveillance mechanisms control accurate Csn subunit amounts and correct cellular localization for sequential assembly since deprivation of Csn subunits changes the abundance and location of remaining Csn subunits.

Fungal Hülle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hülle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hülle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones accumulate emodins and are delayed in maturation and growth of fruiting bodies. Emodin represses fruiting body and resting structure formation in other fungi. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. Our findings reveal a novel role of Hülle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

Fungal Hülle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hülle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hülle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones are delayed in maturation and growth of fruiting bodies. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. These findings reveal a novel role of Hülle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

Background Aspergillus spp. comprises a very diverse group of lower eukaryotes with a high relevance for industrial applications and clinical implications. These multinucleate species are often cultured for many generations in the laboratory, which can unknowingly propagate hidden genetic mutations. To assess the likelihood of such events, we studied the genome stability of aspergilli by using a combination of mutation accumulation (MA) lines and whole genome sequencing. Results We sequenced the whole genomes of 30 asexual and 10 sexual MA lines of three Aspergillus species ( A. flavus , A. fumigatus and A. nidulans ) and estimated that each MA line accumulated mutations for over 4000 mitoses during asexual cycles. We estimated mutation rates of 4.2 × 10 −11 ( A. flavus ), 1.1 × 10 −11 ( A. fumigatus ) and 4.1 × 10 −11 ( A. nidulans ) per site per mitosis, suggesting that the genomes are very robust. Unexpectedly, we found a very high rate of GC → TA transversions only in A. flavus . In parallel, 30 asexual lines of the non-homologous end-joining (NHEJ) mutants of the three species were also allowed to accumulate mutations for the same number of mitoses. Sequencing of these NHEJ MA lines gave an estimated mutation rate of 5.1 × 10 −11 ( A. flavus ), 2.2 × 10 −11 ( A. fumigatus ) and 4.5 × 10 −11 ( A. nidulans ) per base per mitosis, which is slightly higher than in the wild-type strains and some ~ 5–6 times lower than in the yeasts. Additionally, in A. nidulans , we found a NHEJ-dependent interference of the sexual cycle that is independent of the accumulation of mutations. Conclusions We present for the first time direct counts of the mutation rate of filamentous fungal species and find that Aspergillus genomes are very robust. Deletion of the NHEJ machinery results in a slight increase in the mutation rate, but at a rate we suggest is still safe to use for biotechnology purposes. Unexpectedly, we found GC→TA transversions predominated only in the species A. flavus , which could be generated by the hepatocarcinogen secondary metabolite aflatoxin. Lastly, a strong effect of the NHEJ mutation in self-crossing was observed and an increase in the mutations of the asexual lines was quantified.

Additional file 10: Table S6. SRA accessions.

Most of the metal transporters in Aspergillus fumigatus are yet uncharacterized. Their role in fungal metabolism and virulence remains unclear. This paper describes the novel PIB-type cation ATPase PcaA, which links metal homeostasis and heavy metal tolerance in the opportunistic human pathogen A. fumigatus. The protein possesses conserved ATPase motif and shares 51% amino acid sequence identity with the Saccharomyces cerevisiae cadmium exporter Pca1p. A pcaA deletion, an overexpression and a gfp-pcaA complementation strain of A. fumigatus were constructed and their heavy metal susceptibilities were studied. The pcaA knock out strain showed drastically decreased cadmium tolerance, however, its growth was not affected by the exposure to high concentrations of copper, iron, zinc, or silver ions. Although the lack of PcaA had no effect on copper adaption, we demonstrated that not only cadmium but also copper ions are able to induce the transcription of pcaA in A. fumigatus wild type Af293. Similarly, cadmium and copper ions could induce the copper exporting ATPase crpA. These data imply a general response on the transcriptomic level to heavy metals in A. fumigatus through the induction of detoxification systems. Confocal microscopy of the gfp-pcaA complementation strain expressing functional GFP-PcaA supports the predicted membrane localization of PcaA. The GFP-PcaA fusion protein is located in the plasma membrane of A. fumigatus in the presence of cadmium ions. Virulence assays support a function of PcaA for virulence of A. fumigatus in the Galleria mellonella wax moth larvae model, which might be linked to the elimination of reactive oxygen species.

[This corrects the article DOI: 10.1371/journal.pgen.1007511.].

The NF-κB-like velvet domain protein VosA (viability of spores) binds to more than 1,500 promoter sequences in the filamentous fungus Aspergillus nidulans. VosA inhibits premature induction of the developmental activator gene brlA, which promotes asexual spore formation in response to environmental cues as light. VosA represses a novel genetic network controlled by the sclB gene. SclB function is antagonistic to VosA, because it induces the expression of early activator genes of asexual differentiation as flbC and flbD as well as brlA. The SclB controlled network promotes asexual development and spore viability, but is independent of the fungal light control. SclB interactions with the RcoA transcriptional repressor subunit suggest additional inhibitory functions on transcription. SclB links asexual spore formation to the synthesis of secondary metabolites including emericellamides, austinol as well as dehydroaustinol and activates the oxidative stress response of the fungus. The fungal VosA-SclB regulatory system of transcription includes a VosA control of the sclB promoter, common and opposite VosA and SclB control functions of fungal development and several additional regulatory genes. The relationship between VosA and SclB illustrates the presence of a convoluted surveillance apparatus of transcriptional control, which is required for accurate fungal development and the linkage to the appropriate secondary metabolism.

Antifungal resistance is an emerging problem and one of the reasons for treatment failure of invasive aspergillosis (IA). Voriconazole has become a standard therapeutic for the treatment of this often fatal infection. We studied the differentially expressed proteins as a response of Aspergillus fumigatus to voriconazole by employing the two-dimensional difference gel electrophoresis (DIGE) technique. Due to addition of drug, a total of 135 differentially synthesized proteins were identified by MALDI-TOF/TOF-mass spectrometry. In particular, the level of proteins involved in the general stress response and cell detoxification increased prominently. In contrast, cell metabolism and energy proteins were down-regulated, which suggests the cellular effort to maintain balance in energy utilization while trying to combat the cellular stress exerted by the drug. We detected several so-far uncharacterized proteins which may play a role in stress response and drug metabolism and which could be future targets for antifungal treatment. A mutant strain, which is deleted in the cross-pathway control gene cpcA, was treated with voriconazole to investigate the contribution of the general control of amino acid biosynthesis to drug resistance. We compared the mutant strain’s protein expression profile with the wild-type strain. The absence of CpcA led to an increased resistance to voriconazole and a reduced activation of some general stress response proteins, while the transcript level of the triazole target gene erg11A (cyp51A) remained unchanged. In contrast, the sensitivity of strain ΔcpcA to terbinafine and amphotericin B was slightly increased. These findings imply a role of CpcA in the cellular stress response to azole drugs at the post transcriptional level.

SUMMARY The incidence of azole resistance in Aspergillus species has increased over the past years, most importantly for Aspergillus fumigatus . This is partially attributable to the global spread of only a few resistance alleles through the environment. Secondary resistance is a significant clinical concern, as invasive aspergillosis with drug-susceptible strains is already difficult to treat, and exclusion of azole-based antifungals from prophylaxis or first-line treatment of invasive aspergillosis in high-risk patients would dramatically limit drug choices, thus increasing mortality rates for immunocompromised patients. Management options for invasive aspergillosis caused by azole-resistant A. fumigatus strains were recently reevaluated by an international expert panel, which concluded that drug resistance testing of cultured isolates is highly indicated when antifungal therapy is intended. In geographical regions with a high environmental prevalence of azole-resistant strains, initial therapy should be guided by such analyses. More environmental and clinical screening studies are therefore needed to generate the local epidemiologic data if such measures are to be implemented on a sound basis. Here we propose a first workflow for evaluating isolates from screening studies, and we compile the MIC values correlating with individual amino acid substitutions in the products of cyp51 genes for interpretation of DNA sequencing data, especially in the absence of cultured isolates.