Multicopper oxidases (MCO) catalyze the biological oxidation of various aromatic substrates and have been identified in plants, insects, bacteria, and wood rotting fungi. In nature, they are involved in biodegradation of biopolymers such as lignin and humic compounds, but have also been tested for various industrial applications. In fungi, MCOs have been shown to play important roles during their life cycles, such as in fruiting body formation, pigment formation and pathogenicity. Coprophilous fungi, which grow on the dung of herbivores, appear to encode an unexpectedly high number of enzymes capable of at least partly degrading lignin. This study compared the MCO-coding capacity of the coprophilous filamentous ascomycetes Podospora anserina and Sordaria macrospora with closely related non-coprophilous members of the order Sordariales. An increase of MCO genes in coprophilic members of the Sordariales most probably occurred by gene duplication and horizontal gene transfer events.

The two β-carbonic anhydrases (CAs, EC 4.2.1.1) recently cloned and purified from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2, were investigated for their inhibition with a panel of 39 aromatic, heterocyclic, and aliphatic sulfonamides and one sulfamate, many of which are clinically used agents. CAS1 was efficiently inhibited by tosylamide, 3-fluorosulfanilamide, and 3-chlorosulfanilamide (KIs in the range of 43.2-79.6 nM), whereas acetazolamide, methazolamide, topiramate, ethoxzolamide, dorzolamide, and brinzolamide were medium potency inhibitors (KIs in the range of 360-445 nM). CAS2 was less sensitive to sulfonamide inhibitors. The best CAS2 inhibitors were 5-amino-1,3,4-thiadiazole-2-sulfonamide (the deacetylated acetazolamide precursor) and 4-hydroxymethyl-benzenesulfonamide, with KIs in the range of 48.1-92.5 nM. Acetazolamide, dorzolamide, ethoxzolamide, topiramate, sulpiride, indisulam, celecoxib, and sulthiame were medium potency CAS2 inhibitors (KIs of 143-857 nM). Many other sulfonamides showed affinities in the high micromolar range or were ineffective as CAS1/2 inhibitors. Small changes in the structure of the inhibitor led to important differences of the activity. As these enzymes may show applications for the removal of anthropically generated polluting gases, finding modulators of their activity may be crucial for designing environmental-friendly CO2 capture processes.

Marine Fungi are potent secondary metabolite producers. However, limited genetic information are available their biosynthetic gene clusters (BGCs) and their biotechnological applications. To overcome this lack of information, herein, we used next-generation sequencing methods for genome sequencing of two marine fungi, isolated from the German Wadden Sea, namely Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079. The assembled genome size of the marine isolate Calcarisporium sp. KF525 is about 36.8 Mb with 60 BGCs, while Pestalotiopsis sp. KF079 has a genome size of 47.5 Mb harboring 67 BGCs. Of all BGCs, 98% and 97% are novel clusters of Calcarisporium sp. and Pestalotiopsis sp., respectively. Only few of the BGCs were found to be expressed under laboratory conditions by RNA-seq analysis. The vast majority of all BGCs were found to be novel and unique for these two marine fungi. Along with a description of the identified gene clusters, we furthermore present important genomic features and life-style properties of these two fungi. The two novel fungal genomes provide a plethora of new BGCs, which may have biotechnological applications in the future, for example as novel drugs. The genomic characterizations will provide assistance in future genetics and genomic analyses of marine fungi.

Polar growth is a key characteristic of all filamentous fungi. It allows these eukaryotes to not only effectively explore organic matter but also interact within its own colony, mating partners, and hosts. Therefore, a detailed understanding of the dynamics in polar growth establishment and maintenance is crucial for several fields of fungal research. We developed a new marker protein, the actin-related protein 1 (Arp1) fused to red and green fluorescent proteins, which allows for the tracking of polar axis establishment and active hyphal growth in microscopy approaches. To exclude a probable redundancy with known polarity markers, we compared the localizations of the Spitzenkörper (SPK) and Arp1 using an FM4-64 staining approach. As we show in applications with the coprophilous fungus Sordaria macrospora and the hemibiotrophic plant pathogen Colletotrichum graminicola, the monitoring of Arp1 can be used for detailed studies of hyphal growth dynamics and ascospore germination, the interpretation of chemotropic growth processes, and the tracking of elongating penetration pegs into plant material. Since the Arp1 marker showed the same dynamics in both fungi tested, we believe this marker can be broadly applied in fungal research to study the manifold polar growth processes determining fungal life.

The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis.

Carbon dioxide (CO(2)) is among the most important gases for all organisms. Its reversible interconversion to bicarbonate (HCO(3) (-)) reaches equilibrium spontaneously, but slowly, and can be accelerated by a ubiquitous group of enzymes called carbonic anhydrases (CAs). These enzymes are grouped by their distinct structural features into alpha-, beta-, gamma-, delta- and zeta-classes. While physiological functions of mammalian, prokaryotic, plant and algal CAs have been extensively studied over the past years, the role of beta-CAs in yeasts and the human pathogen Cryptococcus neoformans has been elucidated only recently, and the function of CAs in multicellular filamentous ascomycetes is mostly unknown. To assess the role of CAs in the development of filamentous ascomycetes, the function of three genes, cas1, cas2 and cas3 (carbonic anhydrase of Sordaria) encoding beta-class carbonic anhydrases was characterized in the filamentous ascomycetous fungus Sordaria macrospora. Fluorescence microscopy was used to determine the localization of GFP- and DsRED-tagged CAs. While CAS1 and CAS3 are cytoplasmic enzymes, CAS2 is localized to the mitochondria. To assess the function of the three isoenzymes, we generated knock-out strains for all three cas genes (Deltacas1, Deltacas2, and Deltacas3) as well as all combinations of double mutants. No effect on vegetative growth, fruiting-body and ascospore development was seen in the single mutant strains lacking cas1 or cas3, while single mutant Deltacas2 was affected in vegetative growth, fruiting-body development and ascospore germination, and the double mutant strain Deltacas1/2 was completely sterile. Defects caused by the lack of cas2 could be partially complemented by elevated CO(2) levels or overexpression of cas1, cas3, or a non-mitochondrial cas2 variant. The results suggest that CAs are required for sexual reproduction in filamentous ascomycetes and that the multiplicity of isoforms results in redundancy of specific and non-specific functions.

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30-90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in approximately 4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology.

The multiprotein Fab1p/PIKfyve-complex regulating the abundance of the phospholipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is highly conserved among eukaryotes. In yeast/mammals, it is composed of the phosphatidylinositol 3-phosphate 5-kinase Fab1p/PIKfyve, the PtdIns(3,5)P2 phosphatase Fig4p/Sac3 and the scaffolding subunit Vac14p/ArPIKfyve. The complex is located to vacuolar membranes in yeast and to endosomal membranes in mammals, where it controls the synthesis and turnover of PtdIns(3,5)P2. In this study, we analyzed the role and function of the Fab1p/PIKfyve-complex scaffold protein SmVAC14 in the filamentous ascomycete Sordaria macrospora (Sm). We generated the Smvac14 deletion strain ∆vac14 and performed phenotypic analysis of the mutant. Furthermore, we conducted fluorescence microscopic localization studies of fluorescently labeled SmVAC14 with vacuolar and late endosomal marker proteins. Our results revealed that SmVAC14 is important for maintaining vacuolar size and appearance as well as proper sexual development in S. macrospora. In addition, SmVAC14 plays an important role in starvation stress response. Accordingly, our results propose that the turnover of PtdIns(3,5)P2 is of great significance for developmental processes in filamentous fungi.

The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

Clonostachys rosea is a mycoparasitic fungus that can control several important plant diseases. Here, we report on the genome sequencing of C. rosea and a comparative genome analysis, in order to resolve the phylogenetic placement of C. rosea and to study the evolution of mycoparasitism as a fungal lifestyle. The genome of C. rosea is estimated to 58.3 Mb, and contains 14,268 predicted genes. A phylogenomic analysis shows that C. rosea clusters as sister taxon to plant pathogenic Fusarium species, with mycoparasitic/saprotrophic Trichoderma species in an ancestral position. A comparative analysis of gene family evolution reveals several distinct differences between the included mycoparasites. Clonostachys rosea contains significantly more ATP-binding cassette (ABC) transporters, polyketide synthases, cytochrome P450 monooxygenases, pectin lyases, glucose-methanol-choline oxidoreductases, and lytic polysaccharide monooxygenases compared with other fungi in the Hypocreales. Interestingly, the increase of ABC transporter gene number in C. rosea is associated with phylogenetic subgroups B (multidrug resistance proteins) and G (pleiotropic drug resistance transporters), whereas an increase in subgroup C (multidrug resistance-associated proteins) is evident in Trichoderma virens. In contrast with mycoparasitic Trichoderma species, C. rosea contains very few chitinases. Expression of six group B and group G ABC transporter genes was induced in C. rosea during exposure to the Fusarium mycotoxin zearalenone, the fungicide Boscalid or metabolites from the biocontrol bacterium Pseudomonas chlororaphis. The data suggest that tolerance toward secondary metabolites is a prominent feature in the biology of C. rosea.

The engineered monomeric version of the lancelet Branchiostoma lanceolatum fluorescent protein, mNeonGreen (mNG), has several positive characteristics, such as a very bright fluorescence, high photostability and fast maturation. These features make it a good candidate for the utilization as fluorescent tool for cell biology and biochemical applications in filamentous fungi. We report the generation of plasmids for the expression of the heterologous mNG gene under the control of an inducible and a constitutive promoter in the filamentous ascomycete Sordaria macrospora and display a stable expression of mNG in the cytoplasm. To demonstrate its usefulness for labeling of organelles, the peroxisomal targeting sequence serine-lysine-leucine (SKL) was fused to mNG. Expression of this tagged version led to protein import of mNG into peroxisomes and their bright fluorescence in life cell imaging.

CAS3 is a newly cloned cytosolic β-class carbonic anhydrase (CA, EC 4.2.1.1) from the filamentous ascomycete Sordaria macrospora. This enzyme has a high catalytic activity for the physiological CO2 hydration reaction and herein, we report the inhibition profile of CAS3 with anions and small molecules. The most effective CAS3 anions/small molecule inhibitors were diethyl-dithiocarbamate, sulfamide, sulfamate, phenyl boronic and phenyl arsonic acids, with KIs in the range of 0.89 mM-97 µM. Anions such as iodide, the pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrogensulfide, stannate, selenate, tellurate, tetraborate, perrhenate, perruthenate, selenocyanide and trithiocarbonate were low millimolar CAS3 inhibitors. The light halides, sulfate, hydrogensulfite, peroxydisulfate, diphosphate, divanadate, perchlorate, tetrafluoroborate, fluorosulfonate and iminodisulfonate did not significantly inhibit this enzyme. These data may be useful for developing antifungals based on CA inhibition, considering the fact that many of the inhibitors reported here may be used as lead molecules and, by incorporating the appropriate organic scaffolds, potent nanomolar inhibitors could be developed.

A new β-class carbonic anhydrase was cloned and purified from the filamentous ascomycete Sordaria macrospora, CAS3. This enzyme has a higher catalytic activity compared to the other two such enzymes from this fungus, CAS1 and CAS2, which were reported earlier, with the following kinetic parameters: kcat of (7.9 ± 0.2) × 105 s-1, and kcat/Km of (9.5 ± 0.12) × 107 M-1∙s-1. An inhibition study with a panel of sulfonamides and one sulfamate was also performed. The most effective CAS3 inhibitors were benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfanilamide, methanilamide, and benzene-1,3-disulfonamide, with KIs in the range of 54-95 nM. CAS3 generally shows a higher affinity for this class of inhibitors compared to CAS1 and CAS2. As S. macrospora is a model organism for the study of fruiting body development in fungi, these data may be useful for developing antifungal compounds based on CA inhibition.

Members of the striatin family and their highly conserved interacting protein phocein/Mob3 are key components in the regulation of cell differentiation in multicellular eukaryotes. The striatin homologue PRO11 of the filamentous ascomycete Sordaria macrospora has a crucial role in fruiting body development. Here, we functionally characterized the phocein/Mob3 orthologue SmMOB3 of S. macrospora. We isolated the gene and showed that both, pro11 and Smmob3 are expressed during early and late developmental stages. Deletion of Smmob3 resulted in a sexually sterile strain, similar to the previously characterized pro11 mutant. Fusion assays revealed that ∆Smmob3 was unable to undergo self-fusion and fusion with the pro11 strain. The essential function of the SmMOB3 N-terminus containing the conserved mob domain was demonstrated by complementation analysis of the sterile S. macrospora ∆Smmob3 strain. Downregulation of either pro11 in ∆Smmob3, or Smmob3 in pro11 mutants by means of RNA interference (RNAi) resulted in synthetic sexual defects, demonstrating for the first time the importance of a putative PRO11/SmMOB3 complex in fruiting body development.

Inteins are internal protein domains found inside the coding region of different proteins. They can autocatalytically self-excise from their host protein and ligate the protein flanks, called exteins, with a peptide bond via a post-translational process called protein cis-splicing. In contrast, protein trans-splicing involves inteins split into an N- and a C-terminal domain. Both domains are synthesized as two separate components and each joined to an extein; the intein domains can reassemble and link the joined exteins into one functional protein. In this study, we introduced three split sites into the PRP8 mini-intein of Penicillium chrysogenum and demonstrated for the first time trans-splicing of a fungal PRP8 intein. Two of the sites introduced allowed splicing to occur in trans while the third was not functional.

Proximity-dependent biotin identification (BioID) has emerged as a powerful methodology to identify proteins co-localizing with a given bait protein in vivo. The approach has been established in animal cells, plants and yeast but not yet in filamentous fungi. BioID relies on promiscuous biotin ligases fused to bait proteins to covalently label neighboring proteins with biotin. Biotinylated proteins are specifically enriched through biotin affinity capture from denatured cell lysates and subsequently identified and quantified with liquid chromatography-mass spectrometry (LC-MS). In contrast to many other affinity capture approaches for studying protein-protein interactions, BioID does not rely on physical protein-protein binding within native cell lysates. This feature allows the identification of protein proximities of weak or transient and dynamic nature. Here, we demonstrate the application of BioID for the fungal model organism Sordaria macrospora (Sm) using the example of the STRIPAK complex interactor 1 (SCI1) of the well-characterized striatin-interacting phosphatase and kinase (SmSTRIPAK) complex as proof of concept. For the establishment of BioID in S. macrospora, a codon-optimized TurboID biotin ligase was fused to SCI1. Biotin capture of the known SmSTRIPAK components PRO11, SmMOB3, PRO22 and SmPP2Ac1 demonstrates the successful BioID application in S. macrospora. BioID proximity labeling approaches will provide a powerful proteomics tool for fungal biologists.

Microbodies, including peroxisomes, glyoxysomes and Woronin bodies, are ubiquitous dynamic organelles that play important roles in fungal development. The ATP-dependent chaperone and protease family Lon that maintain protein quality control within the organelle significantly regulate the functionality of microbodies. The filamentous ascomycete Sordaria macrospora is a model organism for studying fruiting-body development. The genome of S. macrospora encodes one Lon protease with the C-terminal peroxisomal targeting signal (PTS1) serine-arginine-leucine (SRL) for import into microbodies. Here, we investigated the function of the protease SmLON2 in sexual development and during growth under stress conditions. Localization studies revealed a predominant localization of SmLON2 in glyoxysomes. This localization depends on PTS1, since a variant without the C-terminal SRL motif was localized in the cytoplasm. A ΔSmlon2 mutant displayed a massive production of aerial hyphae, and produced a reduced number of fruiting bodies and ascospores. In addition, the growth of the ΔSmlon2 mutant was completely blocked under mild oxidative stress conditions. Most of the defects could be complemented with both variants of SmLON2, with and without PTS1, suggesting a dual function of SmLON2, not only in microbody, but also in cytosolic protein quality control.

The supramolecular striatin-interacting phosphatases and kinases (STRIPAK) complex is conserved from yeast to human, and regulates a variety of key biological processes. In animals, this complex consists of the scaffold protein striatin, the protein phosphatase 2A, and kinases, such as germinal center kinase (GCK) III and GCKIV family members, as well as other associated proteins. The STRIPAK complex was identified as a negative regulator of the Hippo pathway, a large eukaryotic signaling network with a core composed of a GCK and a nuclear Dbf2-related kinase. The signaling architecture of the Hippo core resembles the fungal septation initiation network (SIN) that regulates cytokinesis in fission yeast as well as septation in filamentous fungi. In the filamentous model fungus Sordaria macrospora, core components of the STRIPAK complex have been functionally described and the striatin homolog PRO11 has been shown to interact with the GCK SmKIN3. However, the exact role of SmKIN3 in fungal development has not yet been fully elucidated. Here, we provide comprehensive genetic and functional analysis of SmKIN3 from S. macrospora Using deletion mutants and site-directed mutagenesis, along with phenotypic and phylogenetic analysis, we provide compelling evidence that SmKIN3 is involved in fruiting body formation, hyphal fusion, and septation. Strains carrying the ATP-binding mutant SmKIN3K39R, as well as a double-deletion strain lacking SmKIN3 and the core STRIPAK subunit PRO11, also revealed severe developmental defects. Collectively, this study suggests that SmKIN3 links both the SIN and STRIPAK complex, thereby regulating multiple key cellular processes.