H3K27me3 is vital for fungal development and secondary metabolite gene silencing, and substitutes for the loss of H3K9me3 in the plant pathogen Fusarium proliferatum.

Facultative heterochromatin marked by histone H3 lysine 27 trimethylation (H3K27me3) is an important regulatory layer involved in secondary metabolite (SM) gene silencing and crucial for fungal development in the genus Fusarium. While this histone mark is essential in some (e.g., the rice pathogen Fusarium fujikuroi), it appears dispensable in other fusaria. Here, we show that deletion of FpKMT6 is detrimental but not lethal in the plant pathogen Fusarium proliferatum, a member of the Fusarium fujikuroi species complex (FFSC). Loss of FpKmt6 results in aberrant growth, and expression of a large set of previously H3K27me3-silenced genes is accompanied by increased H3K27 acetylation (H3K27ac) and an altered H3K36me3 pattern. Next, H3K9me3 patterns are affected in Δfpkmt6, indicating crosstalk between both heterochromatic marks that became even more obvious in a strain deleted for FpKMT1 encoding the H3K9-specific histone methyltransferase. In Δfpkmt1, all H3K9me3 marks present in the wild-type strain are replaced by H3K27me3, a finding that may explain the subtle phenotype of the Δfpkmt1 strain which stands in marked contrast to other filamentous fungi. A large proportion of SM-encoding genes is allocated with H3K27me3 in the wild-type strain and loss of H3K27me3 results in elevated expression of 49% of them. Interestingly, genes involved in the biosynthesis of the phytohormones gibberellins (GA) are among the most upregulated genes in Δfpkmt6. Although several FFSC members harbor GA biosynthetic genes, its production is largely restricted to F. fujikuroi, possibly outlining the distinct lifestyles of these notorious plant pathogens. We show that H3K27me3 is involved in GA gene silencing in F. proliferatum and at least one additional FFSC member, and thus, may serve as a regulatory layer for gene silencing under non-favoring conditions.

The KdmB-EcoA-RpdA-SntB chromatin complex binds regulatory genes and coordinates fungal development with mycotoxin synthesis.

Chromatin complexes control a vast number of epigenetic developmental processes. Filamentous fungi present an important clade of microbes with poor understanding of underlying epigenetic mechanisms. Here, we describe a chromatin binding complex in the fungus Aspergillus nidulans composing of a H3K4 histone demethylase KdmB, a cohesin acetyltransferase (EcoA), a histone deacetylase (RpdA) and a histone reader/E3 ligase protein (SntB). In vitro and in vivo evidence demonstrate that this KERS complex is assembled from the EcoA-KdmB and SntB-RpdA heterodimers. KdmB and SntB play opposing roles in regulating the cellular levels and stability of EcoA, as KdmB prevents SntB-mediated degradation of EcoA. The KERS complex is recruited to transcription initiation start sites at active core promoters exerting promoter-specific transcriptional effects. Interestingly, deletion of any one of the KERS subunits results in a common negative effect on morphogenesis and production of secondary metabolites, molecules important for niche securement in filamentous fungi. Consequently, the entire mycotoxin sterigmatocystin gene cluster is downregulated and asexual development is reduced in the four KERS mutants. The elucidation of the recruitment of epigenetic regulators to chromatin via the KERS complex provides the first mechanistic, chromatin-based understanding of how development is connected with small molecule synthesis in fungi.

Soil fertility determines whether ectomycorrhizal fungi accelerate or decelerate decomposition in a temperate forest.

Ectomycorrhizal (ECM) fungi can both accelerate and decelerate decomposition of organic matter in forest soils, but a mechanistic understanding of this differential influence is limited. Here, we tested how ECM fungi affect decomposition along a natural fertility gradient in a temperate forest of European beech. Trees were girdled to reduce belowground carbon supply to the soil. Girdling shifted soil fungal community composition and decreased hyphal biomass production and soil CO2 efflux, indicating a reduced ECM fungal activity. Girdling also affected decomposition processes, but the effects depended on fertility. Our results indicate that ECM fungi decelerate decomposition under conditions of low fertility while under conditions of high fertility ECM fungi and their host roots have an accelerating effect. We conclude that both acceleration and deceleration of decomposition of organic matter by ECM fungi can occur within a forest, with soil fertility determining the direction and magnitude of these effects. We suggest a positive feedback between fertility, stand productivity and soil carbon and nitrogen dynamics that is mediated to a large extent by ECM fungi.

Polaramycin B, and not physical interaction, is the signal that rewires fungal metabolism in the Streptomyces-Aspergillus interaction.

Co-culturing the bacterium Streptomyces rapamycinicus and the ascomycete Aspergillus nidulans has previously been shown to trigger the production of orsellinic acid (ORS) and its derivates in the fungal cells. Based on these studies it was assumed that direct physical contact is a prerequisite for the metabolic reaction that involves a fungal amino acid starvation response and activating chromatin modifications at the biosynthetic gene cluster (BGC). Here we show that not physical contact, but a guanidine containing macrolide, named polaramycin B, triggers the response. The substance is produced constitutively by the bacterium and above a certain concentration, provokes the production of ORS. In addition, several other secondary metabolites were induced by polaramycin B. Our genome-wide transcriptome analysis showed that polaramycin B treatment causes downregulation of fungal genes necessary for membrane stability, general metabolism and growth. A compensatory genetic response can be observed in the fungus that included upregulation of BGCs and genes necessary for ribosome biogenesis, translation and membrane stability. Our work discovered a novel chemical communication, in which the antifungal bacterial metabolite polaramycin B leads to the production of antibacterial defence chemicals and to the upregulation of genes necessary to compensate for the cellular damage caused by polaramycin B.

RimO (SrrB) is required for carbon starvation signaling and production of secondary metabolites in Aspergillus nidulans.

Depending on the prevailing environmental, developmental and nutritional conditions, fungi activate biosynthetic gene clusters (BGCs) to produce condition-specific secondary metabolites (SMs). For activation, global chromatin-based de-repression must be integrated with pathway-specific induction signals. Here we describe a new global regulator needed to activate starvation-induced SMs. In our transcriptome dataset, we found locus AN7572 strongly transcribed solely under conditions of starvation-induced SM production. The predicted AN7572 protein is most similar to the stress and nutritional regulator Rim15 of Saccharomyces cerevisiae, and to STK-12 of Neurospora crassa. Based on this similarity and on stress and nutritional response phenotypes of A. nidulans knock-out and overexpression strains, AN7572 is designated rimO. In relation to SM production, we found that RimO is required for the activation of starvation-induced BGCs, including the sterigmatocystin (ST) gene cluster. Here, RimO regulates the pathway-specific transcription factor AflR both at the transcriptional and post-translational level. At the transcriptional level, RimO mediates aflR induction following carbon starvation and at the post-translational level, RimO is required for nuclear accumulation of the AflR protein. Genome-wide transcriptional profiling showed that cells lacking rimO fail to adapt to carbon starvation that, in the wild type, leads to down-regulation of genes involved in basic metabolism, membrane biogenesis and growth. Consistently, strains overexpressing rimO are more resistant to oxidative and osmotic stress, largely insensitive to glucose repression and strongly overproduce several SMs. Our data indicate that RimO is a positive regulator within the SM and stress response network, but this requires nutrient depletion that triggers both, rimO gene transcription and activation of the RimO protein.

Soil fertility relates to fungal-mediated decomposition and organic matter turnover in a temperate mountain forest.

Fungi are known to exert a significant influence over soil organic matter (SOM) turnover, however understanding of the effects of fungal community structure on SOM dynamics and its consequences for ecosystem fertility is fragmentary. Here we studied soil fungal guilds and SOM decomposition processes along a fertility gradient in a temperate mountain beech forest. High-throughput sequencing was used to investigate fungal communities. Carbon and nitrogen stocks, enzymatic activity and microbial respiration were measured. While ectomycorrhizal fungal abundance was not related to fertility, saprotrophic ascomycetes showed higher relative abundances under more fertile conditions. The activity of oxidising enzymes and respiration rates in mineral soil were related positively to fertility and saprotrophic fungi. In addition, organic layer carbon and nitrogen stocks were lower on the more fertile plots, although tree biomass and litter input were higher. Together, the results indicated a faster SOM turnover at the fertile end of the gradient. We suggest that there is a positive feedback mechanism between SOM turnover and fertility that is mediated by soil fungi to a significant extent. By underlining the importance of fungi for soil fertility and plant growth, these findings furthermore emphasise the dependency of carbon cycling on fungal communities below ground.

High Fungal Diversity but Low Seasonal Dynamics and Ectomycorrhizal Abundance in a Mountain Beech Forest.

Forests on steep slopes constitute a significant proportion of European mountain areas and are important as production and protection forests. This study describes the soil fungal community structure in a European beech-dominated mountain forest stands in the Northern Calcareous Alps and investigates how it is determined by season and soil properties. Samples were collected at high spatial resolution in an area of ca. 100 m × 700 m in May (spring) and August (summer). Illumina MiSeq high-throughput sequencing of the ITS2-region revealed distinct patterns for the soil fungal communities. In contrast to other studies from temperate European beech forest stands, Ascomycota dominated the highly diverse fungal community, while ectomycorrhizal fungi were of lower abundance. Russulaceae, which are often among the dominant ectomycorrhizal fungi associated with European beech, were absent from all samples. Potentially plant pathogenic fungi were more prevalent than previously reported. Only subtle seasonal differences were found between fungal communities in spring and summer. Especially, dominant saprotrophic taxa were largely unaffected by season, while slightly stronger effects were observed for ectomycorrhizal fungi. Soil characteristics like pH and organic carbon content, on the other hand, strongly shaped abundant taxa among the saprotrophic fungal community.

A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning.

Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillus nidulans that uses enzymatically disabled "dead" Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway. Graphical abstract KEYPOINTS: • An inducible RNA-guided transcriptional regulator based on VPR-dCas9 was established in Aspergillus nidulans. • Genome-wide nucleosome positioning maps were created that facilitate sgRNA positioning. • The system was successfully applied to activate genes within two silent biosynthetic gene clusters.

KdmB, a Jumonji Histone H3 Demethylase, Regulates Genome-Wide H3K4 Trimethylation and Is Required for Normal Induction of Secondary Metabolism in Aspergillus nidulans.

Histone posttranslational modifications (HPTMs) are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB) in which the corresponding genes-usually physically linked in co-regulated clusters-are silenced under optimal physiological conditions (nutrient-rich) but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS), genome-wide chromatin immunoprecipitation (ChIP-seq) and transcriptional network analysis (RNA-seq) that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3) are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism) and nutrient-limiting (secondary metabolism) conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production.

Assessment of Cu applications in two contrasting soils-effects on soil microbial activity and the fungal community structure.

Copper (Cu)-based fungicides have been used in viticulture to prevent downy mildew since the end of the 19th century, and are still used today to reduce fungal diseases. Consequently, Cu has built up in many vineyard soils, and it is still unclear how this affects soil functioning. The present study aimed to assess the short and medium-term effects of Cu contamination on the soil fungal community. Two contrasting agricultural soils, an acidic sandy loam and an alkaline silt loam, were used for an eco-toxicological greenhouse pot experiment. The soils were spiked with a Cu-based fungicide in seven concentrations (0-5000 mg Cu kg-1 soil) and alfalfa was grown in the pots for 3 months. Sampling was conducted at the beginning and at the end of the study period to test Cu toxicity effects on total microbial biomass, basal respiration and enzyme activities. Fungal abundance was analysed by ergosterol at both samplings, and for the second sampling, fungal community structure was evaluated via ITS amplicon sequences. Soil microbial biomass C as well as microbial respiration rate decreased with increasing Cu concentrations, with EC50 ranging from 76 to 187 mg EDTA-extractable Cu kg-1 soil. Oxidative enzymes showed a trend of increasing activity at the first sampling, but a decline in peroxidase activity was observed for the second sampling. We found remarkable Cu-induced changes in fungal community abundance (EC50 ranging from 9.2 to 94 mg EDTA-extractable Cu kg-1 soil) and composition, but not in diversity. A large number of diverse fungi were able to thrive under elevated Cu concentrations, though within the order of Hypocreales several species declined. A remarkable Cu-induced change in the community composition was found, which depended on the soil properties and, hence, on Cu availability.

KdmA, a histone H3 demethylase with bipartite function, differentially regulates primary and secondary metabolism in Aspergillus nidulans.

Aspergillus nidulans kdmA encodes a member of the KDM4 family of jumonji histone demethylase proteins, highly similar to metazoan orthologues both within functional domains and in domain architecture. This family of proteins exhibits demethylase activity towards lysines 9 and 36 of histone H3 and plays a prominent role in gene expression and chromosome structure in many species. Mass spectrometry mapping of A. nidulans histones revealed that around 3% of bulk histone H3 carried trimethylated H3K9 (H3K9me3) but more than 90% of histones carried either H3K36me2 or H3K36me3. KdmA functions as H3K36me3 demethylase and has roles in transcriptional regulation. Genetic manipulation of KdmA levels is tolerated without obvious effect in most conditions, but strong phenotypes are evident under various conditions of stress. Transcriptome analysis revealed that - in submerged early and late cultures - between 25% and 30% of the genome is under KdmA influence respectively. Transcriptional imbalance in the kdmA deletion mutant may contribute to the lethal phenotype observed upon exposure of mutant cells to low-density visible light on solid medium. Although KdmA acts as transcriptional co-repressor of primary metabolism genes, it is required for full expression of several genes involved in biosynthesis of secondary metabolites.

Incorporation of the histone variant H2A.Z counteracts gene silencing mediated by H3K27 trimethylation in Fusarium fujikuroi.

BACKGROUND: Fusarium fujikuroi is a pathogen of rice causing diverse disease symptoms such as 'bakanae' or stunting, most likely due to the production of various natural products (NPs) during infection. Fusaria have the genetic potential to synthesize a plethora of these compounds with often diverse bioactivity. The capability to synthesize NPs exceeds the number of those being produced by far, implying a gene regulatory network decisive to induce production. One such regulatory layer is the chromatin structure and chromatin-based modifications associated with it. One prominent example is the exchange of histones against histone variants such as the H2A variant H2A.Z. Though H2A.Z already is well studied in several model organisms, its regulatory functions are not well understood. Here, we used F. fujikuroi as a model to explore the role of the prominent histone variant FfH2A.Z in gene expression within euchromatin and facultative heterochromatin. RESULTS: Through the combination of diverse '-omics' methods, we show the global distribution of FfH2A.Z and analyze putative crosstalks between the histone variant and two prominent histone marks, i.e., H3K4me3 and H3K27me3, important for active gene transcription and silencing, respectively. We demonstrate that, if FfH2A.Z is positioned at the + 1-nucleosome, it poises chromatin for gene transcription, also within facultative heterochromatin. Lastly, functional characterization of FfH2A.Z overexpression and depletion mutants revealed that FfH2A.Z is important for wild type-like fungal development and secondary metabolism. CONCLUSION: In this study, we show that the histone variant FfH2A.Z is a mark of positive gene transcription and acts independently of the chromatin state most likely through the stabilization of the + 1-nucleosome. Furthermore, we demonstrate that FfH2A.Z depletion does not influence the establishment of both H3K27me3 and H3K4me3, thus indicating no crosstalk between FfH2A.Z and both histone marks. These results highlight the manifold functions of the histone variant FfH2A.Z in the phytopathogen F. fujikuroi, which are distinct regarding gene transcription and crosstalk with the two prominent histone marks H3K27me3 and H3K4me3, as proposed for other model organisms.

Genome analysis of Cephalotrichum gorgonifer and identification of the biosynthetic pathway for rasfonin, an inhibitor of KRAS dependent cancer.

BACKGROUND: Fungi are important sources for bioactive compounds that find their applications in many important sectors like in the pharma-, food- or agricultural industries. In an environmental monitoring project for fungi involved in soil nitrogen cycling we also isolated Cephalotrichum gorgonifer (strain NG_p51). In the course of strain characterisation work we found that this strain is able to naturally produce high amounts of rasfonin, a polyketide inducing autophagy, apoptosis, necroptosis in human cell lines and showing anti-tumor activity in KRAS-dependent cancer cells. RESULTS: In order to elucidate the biosynthetic pathway of rasfonin, the strain was genome sequenced, annotated, submitted to transcriptome analysis and genetic transformation was established. Biosynthetic gene cluster (BGC) prediction revealed the existence of 22 BGCs of which the majority was not expressed under our experimental conditions. In silico prediction revealed two BGCs with a suite of enzymes possibly involved in rasfonin biosynthesis. Experimental verification by gene-knock out of the key enzyme genes showed that one of the predicted BGCs is indeed responsible for rasfonin biosynthesis. CONCLUSIONS: This study identified a biosynthetic gene cluster containing a key-gene responsible for rasfonin production. Additionally, molecular tools were established for the non-model fungus Cephalotrichum gorgonifer which allows strain engineering and heterologous expression of the BGC for high rasfonin producing strains and the biosynthesis of rasfonin derivates for diverse applications.

Transcriptome analysis of nitrate assimilation in Aspergillus nidulans reveals connections to nitric oxide metabolism.

Nitrate is a dominant form of inorganic nitrogen (N) in soils and can be efficiently assimilated by bacteria, fungi and plants. We studied here the transcriptome of the short-term nitrate response using assimilating and non-assimilating strains of the model ascomycete Aspergillus nidulans. Among the 72 genes positively responding to nitrate, only 18 genes carry binding sites for the pathway-specific activator NirA. Forty-five genes were repressed by nitrate metabolism. Because nirA(-) strains are N-starved at nitrate induction conditions, we also compared the nitrate transcriptome with N-deprived conditions and found a partial overlap of differentially regulated genes between these conditions. Nitric oxide (NO)-metabolizing flavohaemoglobins were found to be co-regulated with nitrate assimilatory genes. Subsequent molecular characterization revealed that the strongly inducible FhbA is required for full activity of nitrate and nitrite reductase enzymes. The co-regulation of NO-detoxifying and nitrate/nitrite assimilating systems may represent a conserved mechanism, which serves to neutralize nitrosative stress imposed by an external NO source in saprophytic and pathogenic fungi. Our analysis using membrane-permeable NO donors suggests that signalling for NirA activation only indirectly depends on the nitrate transporters NrtA (CrnA) and NrtB (CrnB).

Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans.

Fungal secondary metabolites are important bioactive compounds but the conditions leading to expression of most of the putative secondary metabolism (SM) genes predicted by fungal genomics are unknown. Here we describe a novel mechanism involved in SM-gene regulation based on the finding that, in Aspergillus nidulans, mutants lacking components involved in heterochromatin formation show de-repression of genes involved in biosynthesis of sterigmatocystin (ST), penicillin and terrequinone A. During the active growth phase, the silent ST gene cluster is marked by histone H3 lysine 9 trimethylation and contains high levels of the heterochromatin protein-1 (HepA). Upon growth arrest and activation of SM, HepA and trimethylated H3K9 levels decrease concomitantly with increasing levels of acetylated histone H3. SM-specific chromatin modifications are restricted to genes located inside the ST cluster, and constitutive heterochromatic marks persist at loci immediately outside the cluster. LaeA, a global activator of SM clusters in fungi, counteracts the establishment of heterochromatic marks. Thus, one level of regulation of the A. nidulans ST cluster employs epigenetic control by H3K9 methylation and HepA binding to establish a repressive chromatin structure and LaeA is involved in reversal of this heterochromatic signature inside the cluster, but not in that of flanking genes.

A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions.

Traditional chromatin analysis methods only test one locus at the time or use different templates for each locus, making a standardized analysis of large genomic regions or many co-regulated genes at different loci a difficult task. On the other hand, genome-wide high-resolution mapping of chromatin accessibility employing massive parallel sequencing platforms generates an extensive data set laborious to analyse and is a cost-intensive method, only applicable to the analysis of a limited set of biological samples. To close this gap between the traditional and the high-throughput procedures we have developed a method in which a condition-specific, genome-wide chromatin fragment library is produced and then used for locus-specific DNA fragment analysis. To validate the method, we used, as a test locus, the well-studied promoter of the divergently transcribed niiA and niaD genes coding for nitrate assimilation enzymes in Aspergillus. Additionally, we have used the condition-specific libraries to study nucleosomal positioning at two different loci, the promoters of the general nitrogen regulator areA and the regulator of secondary metabolism, aflR.

Polyphasic Approach Utilized for the Identification of Two New Toxigenic Members of Penicillium Section Exilicaulis, P. krskae and P. silybi spp. nov.

Two new species, Penicillium krskae (isolated from the air as a lab contaminant in Tulln (Austria, EU)) and Penicillium silybi (isolated as an endophyte from asymptomatic milk thistle (Silybum marianum) stems from Josephine County (Oregon, USA)) are described. The new taxa are well supported by phenotypic (especially conidial ornamentation under SEM, production of red exudate and red pigments), physiological (growth at 37 °C, response to cycloheximide and CREA), chemotaxonomic (production of specific extrolites), and multilocus phylogenetic analysis using RNA-polymerase II second largest subunit (RPB2), partial tubulin (benA), and calmodulin (CaM). Both new taxa are resolved within the section Exilicaulis in series Restricta and show phylogenetic affiliation to P. restrictum sensu stricto. They produce a large spectrum of toxic anthraquinoid pigments, namely, monomeric anthraquinones related to emodic and chloremodic acids and other interesting bioactive extrolites (i.e., endocrocin, paxilline, pestalotin, and 7-hydroxypestalotin). Of note, two bianthraquinones (i.e., skyrin and oxyskyrin) were detected in a culture extract of P. silybi. Two new chloroemodic acid derivatives (2-chloro-isorhodoptilometrin and 2-chloro-desmethyldermoquinone) isolated from the exudate of P. krskae ex-type culture were analyzed by nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS).

Nuclear export of the transcription factor NirA is a regulatory checkpoint for nitrate induction in Aspergillus nidulans.

NirA, the specific transcription factor of the nitrate assimilation pathway of Aspergillus nidulans, accumulates in the nucleus upon induction by nitrate. NirA interacts with the nuclear export factor KapK, which bridges an interaction with a protein of the nucleoporin-like family (NplA). Nitrate induction disrupts the NirA-KapK interaction in vivo, whereas KapK associates with NirA when this protein is exported from the nucleus. A KpaK leptomycin-sensitive mutation leads to inducer-independent NirA nuclear accumulation in the presence of the drug. However, this does not lead to constitutive expression of the genes controlled by NirA. A nirA(c)1 mutation leads to constitutive nuclear localization and activity, remodeling of chromatin, and in vivo binding to a NirA upstream activation sequence. The nirA(c)1 mutation maps in the nuclear export signal (NES) of the NirA protein. The NirA-KapK interaction is nearly abolished in NirA(c)1 and NirA proteins mutated in canonical leucine residues in the NirA NES. The latter do not result in constitutively active NirA protein, which implies that nuclear retention is necessary but not sufficient for NirA activity. The results are consistent with a model in which activation of NirA by nitrate disrupts the interaction of NirA with the NplA/KapK nuclear export complex, thus resulting in nuclear retention, leading to AreA-facilitated DNA binding of the NirA protein and subsequent chromatin remodeling and transcriptional activation.

Draft Genome Sequences of Three Strains of Verticillium nonalfalfae Exhibiting Different Levels of Aggressiveness on Ailanthus altissima.

Verticillium nonalfalfae, a soilborne vascular fungus, shows promise for biocontrol of highly invasive Ailanthus altissima strains. This announcement provides draft genome sequences of the aggressive isolate G1/5 (wild-type strain), the highly aggressive isolate Vert56 (improved strain), and the mildly aggressive isolate I3/2, all obtained from symptomatic A. altissima trees in Austria.

The G-protein Coupled Receptor GPR8 Regulates Secondary Metabolism in Trichoderma reesei.

Changing environmental conditions are of utmost importance for regulation of secondary metabolism in fungi. Different environmental cues including the carbon source, light and the presence of a mating partner can lead to altered production of compounds. Thereby, the heterotrimeric G-protein pathway is of major importance for sensing and adjustment of gene regulation. Regulation of secondary metabolism is crucial in the biotechnological workhorse Trichoderma reesei for knowledge-based adjustment in industrial fermentations, but also with respect to the potential use as a host for heterologous compound production. We investigated the function of the class VII G-protein coupled receptor (GPCR) gene gpr8 that is localized in the vicinity of the SOR cluster, which is responsible for biosynthesis of sorbicillinoids. GPR8 positively impacts regulation of the genes in this cluster in darkness. Accordingly, abundance of trichodimerol and dihydrotrichotetronine as well as other secondary metabolites is decreased in the deletion mutant. Transcriptome analysis moreover showed the major role of GPR8 being exerted in darkness with a considerable influence on regulation of secondary metabolism. Genes regulated in Δgpr8 overlap with those regulated directly or indirectly by the transcription factor YPR2, especially concerning genes related to secondary metabolism. The predicted FAD/FMN containing dehydrogenase gene sor7, one of the positive targets of the cascade triggered by GPR8, has a positive effect on secondary metabolite production, but also cellulase gene expression. Hence SOR7 has some overlapping, but also additional functions compared to GPR8. The G-protein coupled receptor GPR8 exerts a light dependent impact on secondary metabolism, which is in part mediated by the transcription factor YPR2 and the function of SOR7. Hence, T. reesei may apply GPR8 to adjust production of secondary metabolites and hence chemical communication to signals from the environment.