Functional characterization of the developmental genes asm2, asm3, and spt3 required for fruiting body formation in the filamentous ascomycete Sordaria macrospora.

The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

Improving enzyme production by solid-state cultivation in packed-bed bioreactors by changing bed porosity and airflow distribution.

Enzymes production by solid-state cultivation in packed-bed bioreactor needs to be improved by mathematical modeling and also by experimentation. In this work, a mixture of sugarcane bagasse and wheat bran was used for the growth of the fungus Myceliophthora thermophila I-1D3b, able to secrete endoglucanase and xylanase, enzymes of interest in the second-generation ethanol production. Bench and pilot-scale bioreactors were used for the experiments, while critical parameters as bed porosity and airflow distribution were evaluated. Results showed enzymes with higher activities for the most porous medium, even though the less substrate amount to be cultivated. For the pilot-scale bioreactor, only the most porous medium was evaluated using different airflow distribution techniques. Using an inner tube for air supply resulted in more homogeneous enzyme production, with higher activities. The results here presented will be helpful for the scale-up of this class of bioreactor into industrial apparatuses.

Growth kinetics of Myceliophthora thermophila M.7·7 in solid-state cultivation.

AIMS: This work aimed to estimate the growth of Myceliophthora thermophila M.7·7 in solid-state cultivation (SSC) through quantification of N-acetyl-d-glucosamine (NAG) and enzyme activity. METHODS AND RESULTS: The fungus was cultivated in sugarcane bagasse and wheat bran. A consistent statistical analysis was done to assess the reliability of experimental data. Logistic model equation was fitted to experimental data and growth parameters were estimated. The results showed strong influence of the sample size on NAG and a minimum recommended sample size was identified. Scanning electron microscopy (SEM) was used to identify the strategy of substrate colonization. Wheat bran was attacked firstly, while sugarcane bagasse was consumed after wheat bran depletion. The biomass growth was poorly estimated by secretion kinetics of α-amylase, endoglucanase, protease and xylanase, but enzyme kinetics were important for understanding substrate colonization. CONCLUSIONS: In conclusion, the NAG concentration was strongly affected by the sample size and sampling procedure. The strategy of fungal colonization on the substrates was well characterized through SEM analysis. The colonization strategy has direct influence on the kinetic parameters of the logistic model. Myceliophthora thermophila has a well-defined dynamic of enzyme secretion to degrade the substrate, although the kinetics of enzyme secretion has shown not adequate to characterize the kinetics of fungal growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides reliable growth kinetic parameters in the SSC of the cellulase producer fungus M. thermophila M.7·7, as well as a robust analysis on three indirect methods (NAG, enzymes and SEM) for estimation of fungal development.

The STRIPAK signaling complex regulates dephosphorylation of GUL1, an RNA-binding protein that shuttles on endosomes.

The striatin-interacting phosphatase and kinase (STRIPAK) multi-subunit signaling complex is highly conserved within eukaryotes. In fungi, STRIPAK controls multicellular development, morphogenesis, pathogenicity, and cell-cell recognition, while in humans, certain diseases are related to this signaling complex. To date, phosphorylation and dephosphorylation targets of STRIPAK are still widely unknown in microbial as well as animal systems. Here, we provide an extended global proteome and phosphoproteome study using the wild type as well as STRIPAK single and double deletion mutants (Δpro11, Δpro11Δpro22, Δpp2Ac1Δpro22) from the filamentous fungus Sordaria macrospora. Notably, in the deletion mutants, we identified the differential phosphorylation of 129 proteins, of which 70 phosphorylation sites were previously unknown. Included in the list of STRIPAK targets are eight proteins with RNA recognition motifs (RRMs) including GUL1. Knockout mutants and complemented transformants clearly show that GUL1 affects hyphal growth and sexual development. To assess the role of GUL1 phosphorylation on fungal development, we constructed phospho-mimetic and -deficient mutants of GUL1 residues. While S180 was dephosphorylated in a STRIPAK-dependent manner, S216, and S1343 served as non-regulated phosphorylation sites. While the S1343 mutants were indistinguishable from wild type, phospho-deficiency of S180 and S216 resulted in a drastic reduction in hyphal growth, and phospho-deficiency of S216 also affects sexual fertility. These results thus suggest that differential phosphorylation of GUL1 regulates developmental processes such as fruiting body maturation and hyphal morphogenesis. Moreover, genetic interaction studies provide strong evidence that GUL1 is not an integral subunit of STRIPAK. Finally, fluorescence microscopy revealed that GUL1 co-localizes with endosomal marker proteins and shuttles on endosomes. Here, we provide a new mechanistic model that explains how STRIPAK-dependent and -independent phosphorylation of GUL1 regulates sexual development and asexual growth.

Phosphoproteomic analysis of STRIPAK mutants identifies a conserved serine phosphorylation site in PAK kinase CLA4 to be important in fungal sexual development and polarized growth.

The highly conserved striatin-interacting phosphatases and kinases (STRIPAK) complex regulates phosphorylation/dephosphorylation of developmental proteins in eukaryotic microorganisms, animals and humans. To first identify potential targets of STRIPAK, we performed extensive isobaric tags for relative and absolute quantification-based proteomic and phosphoproteomic analyses in the filamentous fungus Sordaria macrospora. In total, we identified 4,193 proteins and 2,489 phosphoproteins, which are represented by 10,635 phosphopeptides. By comparing phosphorylation data from wild type and mutants, we identified 228 phosphoproteins to be regulated in all three STRIPAK mutants, thus representing potential targets of STRIPAK. To provide an exemplarily functional analysis of a STRIPAK-dependent phosphorylated protein, we selected CLA4, a member of the conserved p21-activated kinase family. Functional characterization of the ∆cla4 deletion strain showed that CLA4 controls sexual development and polarized growth. To determine the functional relevance of CLA4 phosphorylation and the impact of specific phosphorylation sites on development, we next generated phosphomimetic and -deficient variants of CLA4. This analysis identified (de)phosphorylation of a highly conserved serine (S685) residue in the catalytic domain of CLA4 as being important for fungal cellular development. Collectively, these analyses significantly contribute to the understanding of the mechanistic function of STRIPAK as a phosphatase and kinase signaling complex.

Derivatization of Natural Compound ß-Pinene Enhances Its In Vitro Antifungal Activity against Plant Pathogens.

BACKGROUND: The development of new antifungal agents has always been a hot research topic in pesticide development. In this study, a series of derivatives of natural compound ß-pinene were prepared, and the antifungal activities of these derivatives were evaluated. The purpose of this work is to develop some novel molecules as promising new fungicides. METHODS: Through a variety of chemical reactions, ß-pinene was transformed into a series of ß-pinene-based derivatives containing amide moieties and acylthiourea moieties. The antifungal activities of these derivatives against five plant pathogens including Colletotrichum gloeosporioides, Fusarium proliferatum, Alternaria kikuchiana, Phomopsis sp. and Phytophthora capsici were tested; preliminary structure-activity relationship was discussed. RESULTS: Some derivatives exhibited moderate or significant antifungal activity due to the fusion of the amide moiety or the acylthiourea moiety with the pinane skeleton. The structure-activity relationship analysis showed that the fluorine atom and the strong electron withdrawing nitro group, or trifluoromethyl group on the benzene ring of the derivatives had a significant effect on the improvement of the antifungal activity against Colletotrichum gloeosporioides, Fusarium proliferatum, Alternaria kikuchiana and Phomopsis sp. Meanwhile, the introduction of an ethyl group at the meta-position on the benzene ring of the derivatives could improve the antifungal activity against Phytophthora capsici. Compounds 4e, 4h, 4q, 4r exhibited broad-spectrum antifungal activity against the tested strains. Compound 4o had significant antifungal activity against Phytophthora capsici (IC50 = 0.18 µmol/L). These derivatives were expected to be used as precursor molecules for novel pesticide development in further research.

The Blue-Light Photoreceptor Sfwc-1 Gene Regulates the Phototropic Response and Fruiting-Body Development in the Homothallic Ascomycete Sordaria fimicola.

Sordaria fimicola, a coprophilous ascomycete, is a homothallic fungus that can undergo sexual differentiation with cellular and morphological changes followed by multicellular tissue development to complete its sexual cycle. In this study, we identified and characterized the blue-light photoreceptor gene in S. fimicola The S. fimicola white collar-1 photoreceptor (SfWC-1) contains light-oxygen-voltage-sensing (LOV), Per-Arnt-Sim (PAS), and other conserved domains and is homologous to the WC-1 blue-light photoreceptor of Neurospora crassa The LOV domain of Sfwc-1 was deleted by homologous recombination using Agrobacterium-mediated protoplast transformation. The Sfwc-1(Δlov) mutant showed normal vegetative growth but produced less carotenoid pigment under illumination. The mutant showed delayed and less-pronounced fruiting-body formation, was defective in phototropism of the perithecial beaks, and lacked the fruiting-body zonation pattern compared with the wild type under the illumination condition. Gene expression analyses supported the light-induced functions of the Sfwc-1 gene in the physiology and developmental process of perithecial formation in S. fimicola Moreover, green fluorescent protein (GFP)-tagged SfWC-1 fluorescence signals were transiently strong upon light induction and prominently located inside the nuclei of living hyphae. Our studies focused on the putative blue-light photoreceptor in a model ascomycete and contribute to a better understanding of the photoregulatory functions and networks mediated by the evolutionarily conserved blue-light photoreceptors across diverse fungal phyla.IMPORTANCESordaria sp. has been a model for study of fruiting-body differentiation in fungi. Several environmental factors, including light, affect cellular and morphological changes during multicellular tissue development. Here, we created a light-oxygen-voltage-sensing (LOV) domain-deleted Sfwc-1 mutant to study blue-light photoresponses in Sordaria fimicola Phototropism and rhythmic zonation of perithecia were defective in the Sfwc-1(Δlov) mutant. Moreover, fruiting-body development in the mutant was reduced and also significantly delayed. Gene expression analysis and subcellular localization study further revealed the light-induced differential gene expression and cellular responses upon light stimulation in S. fimicola.

Surveying of acid-tolerant thermophilic lignocellulolytic fungi in Vietnam reveals surprisingly high genetic diversity.

Thermophilic fungi can represent a rich source of industrially relevant enzymes. Here, 105 fungal strains capable of growing at 50 °C and pH 2.0 were isolated from compost and decaying plant matter. Maximum growth temperatures of the strains were in the range 50 °C to 60 °C. Sequencing of the internal transcribed spacer (ITS) regions indicated that 78 fungi belonged to 12 species of Ascomycota and 3 species of Zygomycota, while no fungus of Basidiomycota was detected. The remaining 27 strains could not be reliably assigned to any known species. Phylogenetically, they belonged to the genus Thielavia, but they represented 23 highly divergent genetic groups different from each other and from the closest known species by 12 to 152 nucleotides in the ITS region. Fungal secretomes of all 105 strains produced during growth on untreated rice straw were studied for lignocellulolytic activity at different pH and temperatures. The endoglucanase and xylanase activities differed substantially between the different species and strains, but in general, the enzymes produced by the novel Thielavia spp. strains exhibited both higher thermal stability and tolerance to acidic conditions. The study highlights the vast potential of an untapped diversity of thermophilic fungi in the tropics.

A novel STRIPAK complex component mediates hyphal fusion and fruiting-body development in filamentous fungi.

The STRIPAK complex is involved in growth, cell fusion, development and signaling pathways, and thus malfunctions in the human STRIPAK complex often result in severe neuronal diseases and cancer. Despite the high degree of general conservation throughout the complex, several STRIPAK complex-associated small coiled-coil proteins of animals and yeasts are not conserved across species. As there are no data for filamentous ascomycetes, we addressed this through affinity purification with HA-tagged striatin ortholog PRO11 in Sordaria macrospora. Combining the method with liquid chromatography-mass spectrometry, we were able to co-purify STRIPAK complex interactor 1 (SCI1), the first STRIPAK-associated small coiled-coil protein in filamentous ascomycetes. Using yeast two-hybrid experiments, we identified SCI1 protein regions required for SCI1-PRO11 interaction, dimerization of SCI1 and interaction with other STRIPAK components. Further, both proteins PRO11 and SCI1 co-localize with the nuclear basket protein SmPOM152 at the nuclear envelope. Expression of the gene sci1 occurs during early developmental stages of S. macrospora, and the protein SCI1 in combination with PRO11 is required for cell fusion, vegetative growth and sexual development. The results of the present study will help to understand the underlying molecular mechanisms of STRIPAK signaling and function in cellular development and diseases in higher eukaryotes.

A Hippo Pathway-Related GCK Controls Both Sexual and Vegetative Developmental Processes in the Fungus Sordaria macrospora.

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.

Molecular characterization of two alkylresorcylic acid synthases from Sordariomycetes fungi.

Two putative type III polyketide synthase genes (PKS) were identified from Sordariomycetes fungi. These two type III PKS genes from Sordaria macrospora (SmPKS) and Chaetomium thermophilum (CtPKS), shared 59.8% sequence identity. Both, full-length and truncated versions of type III PKSs were successfully cloned and overexpressed in a bacterial host, Escherichia Coli BL21 (DE3) using a N-terminus hexa-histidine tag. The full-length and the truncated construct of PKSs showed similar activity profiles, suggesting that additional amino acid residues at the C-terminal of both SmPKS and CtPKS may not be involved in catalytic functions. We demonstrate that these two recombinant polyketide synthases could efficiently synthesize tri- and tetraketide pyrones, resorcinols and resorcylic acids using various acyl-CoAs (C4-C20) as starter units. The truncated S. macrospora polyketide synthases (TrSmPKS) showed a maximum of 7.0 × 104 s-1 M-1 catalytic efficiency towards stearoyl-CoA.Whereas, truncated C. thermophilum polyketide synthases (TrCtPKS) preferred the long-chain acyl-CoA starter arachidoyl-CoA, to produce pentaketide and hexaketide resorcinols with a high catalytic efficiency of 6.2 × 104 s-1 M-1. Homology model and substrate docking analyses suggest a shorter distance between sulfur of catalytic Cys152 and thioester carbonyl group of arachidoyl-CoA as well as stronger imidazolium-thiolate ion pair distance in TrCtPKS between catalytic Cys152-His309 compared to TrSmPKS- arachidoyl CoA complex. Enhanced binding interactions of CtPKS residues forming intermolecular contacts at the active site could be attributed to its high specificity towards arachidoyl-CoA. This study reports the functional characterization of two fungal type III polyketide synthases, SmPKS and CtPKS with high catalytic efficiency from S. macrospora and C. thermophilum respectively. Furthermore, the results suggested that the both SmPKS and CtPKS could be attractive targets for protein engineering to discern the unique substrate specificity and catalytic efficiency.

The DNA-methyltransferase inhibitor 5-aza-2-deoxycytidine affects Humicola grisea enzyme activities and the glucose-mediated gene repression.

Humicola grisea var. thermoidea (Hgvt) is a thermophilic ascomycete that produces lignocellulolytic enzymes and it is proposed for the conversion of agricultural residues into useful byproducts. Drugs that inhibit the DNA methyltransferases (DNMTs) activity are employed in epigenetic studies but nothing is known about a possible effect on the production of fungal enzymes. We evaluated the effect of 5-aza-2'-deoxycytidine (5-Aza; a chemical inhibitor of DNMTs activity) on the secreted enzyme activity and on the transcription of cellulase and xylanase genes from Hgvt grown in agricultural residues and in glucose. Upon cultivation on wheat bran (WB), the drug provoked an increase in the xylanase activity at 96 h. When Hgvt was grown in glucose (GLU), a repressor of Hgvt glycosyl hydrolase genes, 5-Aza led to increased transcript accumulation for the cellobiohydrolases and for the xyn2 xylanase genes. In WB, 5-Aza enhanced the expression of the transcription factor CreA gene. Growth on WB or GLU, in presence of 5-Aza, led to a significant increase in transcripts of the pH-response regulator PacC gene. To our knowledge, this is the first report on the effect of a DNMT inhibitor in the production of fungal plant cell wall degradation enzymes.

The ancestral levels of transcription and the evolution of sexual phenotypes in filamentous fungi.

Changes in gene expression have been hypothesized to play an important role in the evolution of divergent morphologies. To test this hypothesis in a model system, we examined differences in fruiting body morphology of five filamentous fungi in the Sordariomycetes, culturing them in a common garden environment and profiling genome-wide gene expression at five developmental stages. We reconstructed ancestral gene expression phenotypes, identifying genes with the largest evolved increases in gene expression across development. Conducting knockouts and performing phenotypic analysis in two divergent species typically demonstrated altered fruiting body development in the species that had evolved increased expression. Our evolutionary approach to finding relevant genes proved far more efficient than other gene deletion studies targeting whole genomes or gene families. Combining gene expression measurements with knockout phenotypes facilitated the refinement of Bayesian networks of the genes underlying fruiting body development, regulation of which is one of the least understood processes of multicellular development.

Production of chitinase from thermophilic Humicola grisea and its application in production of bioactive chitooligosaccharides.

A novel thermophilic chitinase producing strain Humicola grisea ITCC 10,360.16 was isolated from soil of semi-arid desert region of Rajasthan. Maximum enzyme production (116±3.45Ul-1) was achieved in submerged fermentation. Nutritional requirement for maximum production of chitinase under submerged condition was optimized using response surface methodology. Among the eight nutritional elements studied, chitin, colloidal chitin, KCl and yeast-extract were identified as the most critical variables for chitinase production by Plackett-Burman design first. Further optimization of these variables was done by four-factor central composite design. The model came out to be significant and statistical analysis of results showed that an appropriate ratio of chitin and colloidal chitin had resulted into enhancement in enzyme production levels. Optimum concentration of the variables for enhanced chitinase production were 7.49, 4.91, 0.19 and 5.50 (gl-1) for chitin, colloidal chitin, KCl and yeast extract, respectively. 1.43 fold enhancement in chitinase titres was attained in shake flasks, when the variables were used at their optimum levels. Thin layer chromatography revealed that enzyme can effectively hydrolyze colloidal chitin to produce chitooligosaccharides. Chitinase production from H. grisea and optimization of economic production medium heighten the employment of enzyme for large scale production of bioactive chitooligosaccharides.

RNA Editing During Sexual Development Occurs in Distantly Related Filamentous Ascomycetes.

RNA editing is a post-transcriptional process that modifies RNA molecules leading to transcript sequences that differ from their template DNA. A-to-I editing was found to be widely distributed in nuclear transcripts of metazoa, but was detected in fungi only recently in a study of the filamentous ascomycete Fusarium graminearum that revealed extensive A-to-I editing of mRNAs in sexual structures (fruiting bodies). Here, we searched for putative RNA editing events in RNA-seq data from Sordaria macrospora and Pyronema confluens, two distantly related filamentous ascomycetes, and in data from the Taphrinomycete Schizosaccharomyces pombe. Like F. graminearum, S. macrospora is a member of the Sordariomycetes, whereas P. confluens belongs to the early-diverging group of Pezizomycetes. We found extensive A-to-I editing in RNA-seq data from sexual mycelium from both filamentous ascomycetes, but not in vegetative structures. A-to-I editing was not detected in different stages of meiosis of S. pombe. A comparison of A-to-I editing in S. macrospora with F. graminearum and P. confluens, respectively, revealed little conservation of individual editing sites. An analysis of RNA-seq data from two sterile developmental mutants of S. macrospora showed that A-to-I editing is strongly reduced in these strains. Sequencing of cDNA fragments containing more than one editing site from P. confluens showed that at the beginning of sexual development, transcripts were incompletely edited or unedited, whereas in later stages transcripts were more extensively edited. Taken together, these data suggest that A-to-I RNA editing is an evolutionary conserved feature during fruiting body development in filamentous ascomycetes.

Box-Behnken analysis and storage of spray-dried collagenolytic proteases from Myceliophthora thermophila submerged bioprocess.

Enzymes do not have long-term storage stability in soluble forms, thus drying methods could minimize the loss of enzymatic activity, the spray dryer removes water under high temperatures and little time. The aims of this study were to improve the stability of enzymatic extract from Myceliophthora thermophila for potential applications in industry and to evaluate the best conditions to remove the water by spray drying technique. The parameters were tested according to Box-Behnken and evaluated by analysis of variance (ANOVA), all the parameters measured were found to influence the final enzyme activity and spray drying process yield ranged from 38.65 to 63.75%. Enzyme powders showed increased storage stability than extract and maintained about 100% of collagenolytic activity after 180 days of storage at 30°C. The results showed that the microbial enzymes maintained activity during the spray drying process and were stable during long-term storage; these are promising characteristics for industrial applications.

Nuclear dynamics during ascospore germination in Sordaria macrospora.

The ascomycete Sordaria macrospora has a long history as a model organism for studying fungal sexual development. Starting from an ascospore, sexual fruiting bodies (perithecia) develop within seven days and discharge new ascospores. Sexual development has been studied in detail, revealing genes required for perithecium formation and ascospore germination. However, the germination process per se has not yet been examined. Here I analyze nuclear dynamics during ascospore germination using a fluorescently labeled histone. Live-cell imaging revealed that nuclei are transported into germination vesicles that form on one side of the spore. Polar growth is established from these vesicles.

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable ß-1,4-xylanase with exo- and endo-activity.

A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC), and characterized biochemically. TtXynA showed optimal xylanolytic activity at pH 5.5 and at 85 °C, and retained more than 90% of its activity at a broad pH range (4.5-10). The enzyme is highly thermostable with a half-life of 23.1 days at 65 °C, and active in the presence of several metal ions. Circular dichroism spectra strongly suggest the enzyme gains secondary structures when temperature increases. TtXynA displayed higher substrate affinity and higher catalytic efficiency towards beechwood xylan than towards birchwood xylan, oat-spelt xylan, and CMC. According to its final hydrolysis products, TtXynA displays endo-/exo-activity, yielded xylobiose, an unknown oligosaccharide containing about five residues of xylose and a small amount of xylose on beechwood xylan. Finally, this report represents the description of the first fungal hyperthermophilic xylanase which is produced by T. terrestris Co3Bag1. Since TtXynA displays relevant biochemical properties, it may be a suitable candidate for biotechnological applications carried out at high temperatures, like the enzymatic pretreatment of plant biomass for the production of bioethanol.

New insights from an old mutant: SPADIX4 governs fruiting body development but not hyphal fusion in Sordaria macrospora.

During the sexual life cycle of filamentous fungi, multicellular fruiting bodies are generated for the dispersal of spores. The filamentous ascomycete Sordaria macrospora has a long history as a model system for studying fruiting body formation, and two collections of sterile mutants have been generated. However, for most of these mutants, the underlying genetic defect remains unknown. Here, we investigated the mutant spadix (spd) that was generated by X-ray mutagenesis in the 1950s and terminates sexual development after the formation of pre-fruiting bodies (protoperithecia). We sequenced the spd genome and found a 22 kb deletion affecting four genes, which we termed spd1-4. Generation of deletion strains revealed that only spd4 is required for fruiting body formation. Although sterility in S. macrospora is often coupled with a vegetative hyphal fusion defect, Δspd4 was still capable of fusion. This feature distinguishes SPD4 from many other regulators of sexual development. Remarkably, GFP-tagged SPD4 accumulated in the nuclei of vegetative hyphae and fruiting body initials, the ascogonial coils, but not in sterile tissue from the developing protoperithecium. Our results point to SPD4 as a specific determinant of fruiting body formation. Research on SPD4 will, therefore, contribute to understanding cellular reprogramming during initiation of sexual development in fungi.

Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora.

In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.