Trichoderma harzianum is a filamentous ascomycete frequently applied as biocontrol agent in agriculture. While mycoparasitism and antagonism of Trichoderma spp. against fungal pathogens are well known, early fungal responses to the presence of a plant await broader investigation. Analyzing early stages of plant-fungus communication we show that T. harzianum B97 chemotropically responds to a plant extract and that both plant and fungus alter secondary metabolite secretion upon recognition. We developed a strategy for omics-analysis simulating conditions of early plant recognition eliciting a chemotropic response in the fungus and found 102 genes to be differentially regulated, including nitrate and nitrite reductases. Additionally, the previously uncharacterized Plant Communication Associated (PCA) gene cluster was strongly induced upon recognition of the plant, comprises a palindromic DNA motif and was essential for plant colonization. The PCA-cluster is only present in the Harzianum clade of Trichoderma and closely related to a homologous cluster in Metarhizium spp. Horizontal gene transfer (HGT) was detected for PCA-cluster genes by plants, while the cluster in T. harzianum is likely under balancing or positive selection. Hence, the PCA-cluster mediates early fungus-plant chemical communication and may be responsible for the high potential of T. harzianum and closely related species for biocontrol applications.

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.

Survival of multicellular organisms requires the coordinated interplay between networks regulating gene expression and controlled intracellular transport of respective regulators. Velvet domain proteins are fungal transcription factors, which form various heterodimers and play key roles in controlling early developmental decisions towards more either asexual or sexual differentiation. VeA is the central subunit of the trimeric velvet complex VelB-VeA-LaeA, which links transcriptional to epigenetic control for the coordination of fungal developmental programs to specific secondary metabolite synthesis. Nuclear localization of the VeA bridging factor is carefully controlled in fungi. In this work we demonstrate that VeA carries three nuclear localization signals NLS1, NLS2 and NLS3, which all contribute to nuclear import. We show that VeA has an additional nuclear export sequence (NES) which provides a shuttle function to allow the cell to relocate VeA to the cytoplasm. VeA is nuclear during vegetative growth, but has to be exported from the nucleus to allow and promote asexual development. In contrast, progression of the sexual pathway requires continuous nuclear VeA localization. Our work shows that an accurate nuclear import and export control of velvet proteins is further connected to specific stability control mechanism as prerequisites for fungal development and secondary metabolism. These results illustrate the various complex mutual dependencies of velvet regulatory proteins for coordinating fungal development and secondary metabolism.

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 soil microbiome comprises numerous filamentous fungi and bacteria that mutually react and challenge each other by the production of bioactive secondary metabolites. Herein, we show in liquid co-cultures that the presence of filamentous Streptomycetes producing antifungal glycopeptide antibiotics induces the production of the antibacterial and iron-chelating tropolones anhydrosepedonin (1) and antibiotic C (2) in the mold Aspergillus nidulans. Additionally, the biosynthesis of the related polyketide tripyrnidone (5) was induced, whose novel tricyclic scaffold we elucidated by NMR and HRESIMS data. The corresponding biosynthetic polyketide synthase-encoding gene cluster responsible for the production of these compounds was identified. The tropolones as well as tripyrnidone (5) are produced by genes that belong to the broad reservoir of the fungal genome for the synthesis of different secondary metabolites, which are usually silenced under standard laboratory conditions. These molecules might be part of the bacterium-fungus competition in the complex soil environment, with the bacterial glycopeptide antibiotic as specific environmental trigger for fungal induction of this cluster.

Saccharomyces cerevisiae is an industrially preferred cell factory for the heterologous production of proteins and chemicals. Here, we present the draft genome sequence of the laboratory strain Saccharomyces cerevisiae LW2591Y, which has been designed for robust and efficient assembly of multigene pathways.

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.

Aggregation of α-synuclein (αSyn) into proteinaceous deposits is a pathological hallmark of a range of neurodegenerative diseases including Parkinson’s disease (PD). Numerous lines of evidence indicate that the accumulation of toxic oligomeric and prefibrillar αSyn species may underpin the cellular toxicity and spread of pathology between cells. Therefore, aggregation of αSyn is considered a priority target for drug development, as aggregation inhibitors are expected to reduce αSyn toxicity and serve as therapeutic agents. Here, we used the budding yeast S. cerevisiae as a platform for the identification of short peptides that inhibit αSyn aggregation and toxicity. A library consisting of approximately one million peptide variants was utilized in two high-throughput screening approaches for isolation of library representatives that reduce αSyn-associated toxicity and aggregation. Seven peptides were isolated that were able to suppress specifically αSyn toxicity and aggregation in living cells. Expression of the peptides in yeast reduced the accumulation of αSyn-induced reactive oxygen species and increased cell viability. Next, the peptides were chemically synthesized and probed for their ability to modulate αSyn aggregation in vitro . Two synthetic peptides, K84s and K102s, of 25 and 19 amino acids, respectively, significantly inhibited αSyn oligomerization and aggregation at sub-stoichiometric molar ratios. Importantly, K84s reduced αSyn aggregation in human cells. These peptides represent promising αSyn aggregation antagonists for the development of future therapeutic interventions.

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.

The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae . Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V . dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V . dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta .

The in vivo identification of Hülle cells in cases of aspergillosis infections in animals and humans illustrates their biological relevance and suggests that they might be involved in pathogenicity. It is striking that aspergilli have developed and maintained a multinucleate nurse cell that is presumably energy-intensive to produce and is usually found only in higher eukaryotes. Our findings shed light on how the understudied Hülle cells might contribute to the success of aspergilli by acting not only as nurse cells under detrimental conditions (sexual development) but also as fungal backup stem cells with the capacity to produce genetically diverse spores in an accelerated manner, thereby substantially contributing to survival in response to predator attack or under otherwise severely destructive conditions. Our study solved the 90-year-old puzzle of Hülle cell germination and provides easy, reproducible methods that will facilitate future studies on biological and ecological roles of Hülle cells in aspergilli.

Monoterpenoids, such as the plant metabolite geraniol, are of high industrial relevance since they are important fragrance materials for perfumes, cosmetics, and household products. Chemical synthesis or extraction from plant material for industry purposes are complex, environmentally harmful or expensive and depend on seasonal variations. Heterologous microbial production offers a cost-efficient and sustainable alternative but suffers from low metabolic flux of the precursors and toxicity of the monoterpenoid to the cells. In this study, we evaluated two approaches to counteract both issues by compartmentalizing the biosynthetic enzymes for geraniol to the peroxisomes ofSaccharomyces cerevisiaeas production sites and by improving the geraniol tolerance of the yeast cells. The combination of both approaches led to an 80% increase in the geraniol titers. In the future, the inclusion of product tolerance and peroxisomal compartmentalization into the general chassis engineering toolbox for monoterpenoids or other host-damaging, industrially relevant metabolites may lead to an efficient, low-cost, and eco-friendly microbial production for industrial purposes.

Volume 78, no. 23, p. 8234–8244, [https://doi.org/10.1128/AEM.01808-12][1]. Page 8237: [Figure 2][2] should appear as shown below. ![Figure][3] Page 8237, column 1, paragraph 1, line 3: “AN7893, dbaA , dbaC , and dbaD genes” should read “AN7893, AN7895, dbaA , dbaC , and dbaD genes

E3 cullin-RING ubiquitin ligase (CRL) complexes recognize specific substrates and are activated by covalent modification with ubiquitin-like Nedd8. Deneddylation inactivates CRLs and allows Cand1/A to bind and exchange substrate recognition subunits. Human as well as most fungi possess a single gene for the receptor exchange factor Cand1, which is split and rearranged in aspergilli into two genes for separate proteins. Aspergillus nidulans CandA-N blocks the neddylation site, and CandA-C inhibits the interaction to the adaptor/substrate receptor subunits similar to the respective N-terminal and C-terminal parts of single Cand1. The pathogen Aspergillus fumigatus and related species express a CandA-C with a 190-aminoacid N-terminal extension domain encoded by an additional exon. This extension corresponds in most aspergilli, including A. nidulans, to a gene directly upstream of candA-C encoding a 20-kDa protein without human counterpart. This protein was named CandA-C1, because it is also required for the cellular deneddylation/neddylation cycle and can form a trimeric nuclear complex with CandA-C and CandA-N. CandA-C and CandA-N are required for asexual and sexual development and control a distinct secondary metabolism. CandA-C1 and the corresponding domain of A. fumigatus control spore germination, vegetative growth, and the repression of additional secondary metabolites. This suggests that the dissection of the conserved Cand1-encoding gene within the genome of aspergilli was possible because it allowed the integration of a fungus-specific protein required for growth into the CandA complex in two different gene set versions, which might provide an advantage in evolution. IMPORTANCE Aspergillus species are important for biotechnological applications, like the production of citric acid or antibacterial agents. Aspergilli can cause food contamination or invasive aspergillosis to immunocompromised humans or animals. Specific treatment is difficult due to limited drug targets and emerging resistances. The CandA complex regulates, as a receptor exchange factor, the activity and substrate variability of the ubiquitin labeling machinery for 26S proteasome-mediated protein degradation. Only Aspergillus species encode at least two proteins that form a CandA complex. This study shows that Aspergillus species had to integrate a third component into the CandA receptor exchange factor complex that is unique to aspergilli and required for vegetative growth, sexual reproduction, and activation of the ubiquitin labeling machinery. These features have interesting implications for the evolution of protein complexes and could make CandA-C1 an interesting candidate for target-specific drug design to control fungal growth without affecting the human ubiquitin-proteasome system.

[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.

The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.