Multiscalar electrical spiking in Schizophyllum commune.

Growing colonies of the split-gill fungus Schizophyllum commune show action potential-like spikes of extracellular electrical potential. We analysed several days of electrical activity recording of the fungus and discovered three families of oscillatory patterns. Very slow activity at a scale of hours, slow activity at a scale of 10 min and very fast activity at scale of half-minute. We simulated the spiking behaviour using FitzHugh-Nagume model, uncovered mechanisms of spike shaping. We speculated that spikes of electrical potential might be associated with transportation of nutrients and metabolites.

The anaerobic survival mechanism of Schizophyllum commune 20R-7-F01, isolated from deep sediment 2 km below the seafloor.

Fungi dominated the eukaryotic group in the anaerobic sedimentary environment below the ocean floor where they play an essential ecological role. However, the adaptive mechanism of fungi to these anaerobic environments is still unclear. Here, we reported the anaerobic adaptive mechanism of Schizophyllum commune 20R-7-F01, isolated from deep coal-bearing sediment down to ~2 km below the seafloor, through biochemical, metabolomic and transcriptome analyses. The fungus grows well, but the morphology changes obviously and the fruit body develops incompletely under complete hypoxia. Compared with aerobic conditions, the fungus has enhanced branched-chain amino acid biosynthesis and ethanol fermentation under anaerobic conditions, and genes related to these metabolisms have been significantly up-regulated. Additionally, the fungus shows novel strategies for synthesizing ethanol by utilizing both glycolysis and ethanol fermentation pathways. These findings suggest that the subseafloor fungi may adopt multiple mechanisms to cope with lack of oxygen.

Schizophyllum commune induces IL-17-mediated neutrophilic airway inflammation in OVA-induced asthma model mice.

Schizophyllum commune is a ubiquitous basidiomycetous fungus typically found across the world, which has been detected in indoor and outdoor air. Some studies indicated that sensitization to S. commune is correlated with asthma severity in patients. Patients with chronic severe or acute fatal asthma have neutrophil-dominant airway inflammation. We hypothesized that S. commune can exacerbate asthma. To test this hypothesis, we evaluated the direct immunomodulatory activities of S. commune in allergic airway inflammation induced by non-fungal sensitization. Ovalbumin (OVA)-induced asthma model mice were generated using wild-type (WT) and Il-17a-/-Il-17f-/- mice that were intratracheally exposed to S. commune, then immune responses in the lungs were assessed after 24 h. Intratracheal administration of S. commune in OVA-induced asthma model mice enhanced neutrophilic airway inflammation, increased the mRNA expression of CXCL1 and CXCL2 in the lungs, and provoked IL-17A, and IL-17F production in BAL fluid. In addition, neutrophilic airway inflammation was significantly inhibited in Il-17a-/-Il-17f-/- mice compared with those found in WT mice. We demonstrated that S. commune induces neutrophilic airway inflammation in OVA-induced asthma model mice, and IL-17A and IL-17F had central roles in this activity. As S. commune inhabits the general environment, including indoor and outdoor air, our results suggested that S. commune is a causative agent of asthma exacerbation. This study has provided clues regarding the mechanisms behind fungi and asthma exacerbation.

Nuclear arms races: Experimental evolution for mating success in the mushroom-forming fungus Schizophyllum commune.

When many gametes compete to fertilize a limited number of compatible gametes, sexual selection will favour traits that increase competitive success during mating. In animals and plants, sperm and pollen competition have yielded many interesting adaptations for improved mating success. In fungi, similar processes have not been shown directly yet. We test the hypothesis that sexual selection can increase competitive fitness during mating, using experimental evolution in the mushroom-forming fungus Schizophyllum commune (Basidiomycota). Mating in mushroom fungi occurs by donation of nuclei to a mycelium. These fertilizing 'male' nuclei migrate through the receiving 'female' mycelium. In our setup, an evolving population of nuclei was serially mated with a non-evolving female mycelium for 20 sexual generations. From the twelve tested evolved lines, four had increased and one had decreased fitness relative to an unevolved competitor. Even though only two of those five remained significant after correcting for multiple comparisons, for all five lines we found a correlation between the efficiency with which the female mycelium is accessed and fitness, providing additional circumstantial evidence for fitness change in those five lines. In two lines, fitness change was also accompanied by increased spore production. The one line with net reduced competitive fitness had increased spore production, but reduced fertilisation efficiency. We did not find trade-offs between male reproductive success and other fitness components. We compare these findings with examples of sperm and pollen competition and show that many similarities between these systems and nuclear competition in mushrooms exist.

Hydrophobin gene deletion and environmental growth conditions impact mechanical properties of mycelium by affecting the density of the material.

Filamentous fungi colonize substrates by forming a mycelium. This network of hyphae can be used as a bio-based material. Here, we assessed the impact of environmental growth conditions and deletion of the hydrophobin gene sc3 on material properties of the mycelium of the mushroom forming fungus Schizophyllum commune. Thermogravimetric analysis showed that Δsc3 mycelium retained more water with increasing temperature when compared to the wild type. The Young's modulus (E) of the mycelium ranged between 438 and 913 MPa when the wild type strain was grown in the dark or in the light at low or high CO2 levels. This was accompanied by a maximum tensile strength (σ) of 5.1-9.6 MPa. In contrast, E and σ of the Δsc3 strain were 3-4- fold higher with values of 1237-2727 MPa and 15.6-40.4 MPa, respectively. These values correlated with mycelium density, while no differences in chemical composition of the mycelia were observed as shown by ATR-FTIR. Together, genetic modification and environmental growth conditions impact mechanical properties of the mycelium by affecting the density of the mycelium. As a result, mechanical properties of wild type mycelium were similar to those of natural materials, while those of Δsc3 were more similar to thermoplastics.

Fungal keratitis due to Schizophyllum commune: an emerging pathogenic fungus.

Fungal keratitis due to Schizophyllum commune is very rare. In this study, we report the clinical and microbiological profile of five patients with fungal keratitis due to S. commune. Direct microscopic examination of corneal scrapings from all five patients showed septate branching hyaline fungal filaments. Similarly, in all five patients Sabouraud dextrose agar (SDA) plates inoculated with corneal scrapings showed white, cottony colonies on the second day of incubation. Lactophenol cotton blue stained wet preparation of 7-day-old colonies on SDA revealed clamp connections and no spores. The fungus was identified by its characteristic clamp connections, fan-shaped bracket fruiting body with pinkish-grey longitudinally split-radiating gills. The phenotypic identification of one of the five isolates further conformed by ITS sequencing. Treatment outcome was available for two of the five patients; in these two patients, the keratitis resolved with topical natamycin.

The transcriptional regulator c2h2 accelerates mushroom formation in Agaricus bisporus.

The Cys2His2 zinc finger protein gene c2h2 of Schizophyllum commune is involved in mushroom formation. Its inactivation results in a strain that is arrested at the stage of aggregate formation. In this study, the c2h2 orthologue of Agaricus bisporus was over-expressed in this white button mushroom forming basidiomycete using Agrobacterium-mediated transformation. Morphology, cap expansion rate, and total number and biomass of mushrooms were not affected by over-expression of c2h2. However, yield per day of the c2h2 over-expression strains peaked 1 day earlier. These data and expression analysis indicate that C2H2 impacts timing of mushroom formation at an early stage of development, making its encoding gene a target for breeding of commercial mushroom strains.

The blue light receptor complex WC-1/2 of Schizophyllum commune is involved in mushroom formation and protection against phototoxicity.

Blue light is necessary for initiation of mushroom formation in Schizophyllum commune. The genome of this basidiomycete contains homologues of the blue light receptor genes wc-1 and wc-2 of Neurospora crassa. Here, it is shown that inactivation of either or both of these genes in S. commune results in a blind phenotype. Mushroom formation was abolished in dikaryons and they formed symmetrical instead of asymmetrical colonies. Development was restored in a temperature dependent way in a Δwc-2Δwc-2 strain by introducing a construct encompassing the wc-2 gene under control of the promoter of the heat shock gene hsp3. A genome-wide expression analysis showed that the transcription factor genes c2h2 and hom1 as well as many hydrophobin genes are downregulated in light-grown colonies of the Δwc-2Δwc-2 mutant when compared with the wild-type dikaryon. Inactivation of wc-1 and/or wc-2 also resulted in sensitivity of the mycelium to intense light. Monokaryotic mutant strains only survived exposure to 6500 lux of light by growing into the agar. Expression analysis indicates that the photosensitivity of the Δwc-1 and Δwc-2 strains is due to lower levels of photolyase and ferrochelatase.

Identification of fungal proteases potentially suitable for environmentally friendly cleaning-in-place in the dairy industry.

Fourteen fungi were screened for ability to produce proteases with activity on milk protein. The proteases produced were assessed on a lab-scale in terms of their potential suitability for cleaning-in-place (CIP) in the dairy industry. Cleaning performance was assessed by determining the ability of the enzymes to remove an industrial-like milk fouling deposit from stainless steel. Based on the results observed, the extracellular protease activity produced by Schizophyllum commune was selected as most suitable for potential CIP application. A CIP procedure involving a sodium carbonate rinse followed by enzymatic cleaning with this fungal enzyme activity was developed. Satisfactory cleaning, judged by quantification of residual organic matter and protein on the stainless steel surface after cleaning, was achieved using the developed CIP procedure at 40°C. This CIP procedure, based on biodegradable enzymes working at low temperature is more environmentally favourable than conventional CIP methods using caustic based cleaning solutions at 70-80°C. Potential environmental benefits of the developed enzymatic CIP procedure include reduced energy consumption, decreased chemical usage and a reduced requirement for pH neutralisation of the resultant waste prior to release.

The mycelial response of the white-rot fungus, Schizophyllum commune to the biocontrol agent, Trichoderma viride.

In this study, agar plate interaction between Schizophyllum commune and Trichoderma viride was investigated to characterise the physiological responses occurring during interspecific mycelial combat. The metabolite profiles and morphological changes in both fungi paired on agar were studied relative to the modulation of phenoloxidase activity in S. commune. The calcium ionophore A23187 was incorporated in self-paired cultures of S. commune to explore possible involvement of calcium influx in the response of S. commune to T. viride. The levels of lipid peroxides and protein carbonyls in the confronted mycelia of S. commune were also measured. Contact with T. viride induced pigmentation and cell wall hydrolysis in S. commune with concomitant increase in phenoloxidase activity, rise in the levels of oxidative stress indicators and increased levels of phenolic compounds, antioxidant γ-amino butyric acid, and pyridoxine and osmo-protective sugar alcohols. Calcium ionophore mimicked the pigmentation in the T. viride-confronted mycelia of S. commune, implicating calcium influx in the response to T. viride. The changes in S. commune are indicative of targeted responses to osmotic and oxidative stresses and phenoloxidase-mediated detoxification of noxious compounds in the contact interface with T. viride, which may confer resistance in natural environments.

Quantitative proteomic analysis of the response of the wood-rot fungus, Schizophyllum commune, to the biocontrol fungus, Trichoderma viride.

AIMS: Investigation of changes in the protein profile of the wood-rot fungus, Schizophyllum commune, when paired against the biocontrol fungus, Trichoderma viride, for 48 h. METHODS AND RESULTS: Variations in protein profile resulting from contact with T. viride were assessed by spot separation using 2 dimensional protein gel electrophoresis followed by MALDI-TOF-TOF MS/MS protein identification. Contact with T. viride elicited a systematic response in S. commune, characterized by marked increases in proteins involved for transcription and translation (61%) and cell wall/hyphal biogenesis and stabilization (17%), whereas metabolism-associated proteins decreased in amounts (64%). Trichoderma viride, however, exhibited typical mycoparasitic behaviour with increases in the amounts of proteins involved in proteolysis and carbohydrate metabolism. CONCLUSIONS: The protein profile of S. commune confronted by T. viride indicates the up-regulation of mechanisms specifically targeted at the mycoparasitic machinery of T. viride, particularly cell wall lysis and antibiosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The proteomic responses observed in S. commune may occur in natural environments, providing an insight to the mechanism involved in conferring resistance to mycoparasitic attack. This study, therefore, warrants further investigation for the targeted design of more robust biocontrol agents.

Cytoplasmic continuity revisited: closure of septa of the filamentous fungus Schizophyllum commune in response to environmental conditions.

BACKGROUND: Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50% and 90% of the cases, respectively. Interestingly, only 24 and 37% of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature. CONCLUSIONS/SIGNIFICANCE: Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of a higher fungus is not continuous per se.

Phleomycin increases transformation efficiency and promotes single integrations in Schizophyllum commune.

Phleomycin is mutagenic by introducing double-strand breaks in DNA. The ble gene of Streptoalloteychus hindustanus, which confers resistance to this substance, is widely used as a selection marker for transformation. Schizophyllum commune grows on 25 microg of phleomycin ml(-1) after introduction of a resistance cassette based on the ble gene. However, we here report that growth of resistant colonies on this concentration of phleomycin resulted in aberrant colony morphologies. Apparently, phleomycin was mutagenic despite acquired resistance. Therefore, a new selection system was developed based on resistance to the antibiotic nourseothricin. However, the transformation efficiency was tenfold lower than that obtained with phleomycin as a selection agent. This low transformation efficiency could be rescued by addition of a nonselective concentration of phleomycin during protoplast regeneration. This was accompanied by a higher incidence of single-copy integrations and with an increase of expression of key genes involved in double-strand break repair. Taken together, we conclude that the effect of a nonselective concentration of phleomycin strongly resembles the effect of restriction enzyme-mediated integration (REMI) but, unlike REMI, it does not depend on the presence of a target restriction site.

Purification and characterization of chitinase A of Streptomyces cyaneus SP-27: an enzyme participates in protoplast formation from Schizophyllum commune mycelia.

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia. alpha-1,3-Glucanase and chitinase I, which were isolated from the filtrate, did not form the protoplast by itself while a mixture of them showed protoplast-forming activity. Streptomyces cyaneus SP-27 was isolated based on the productivity of chitinase. The culture filtrate of S. cyaneus SP-27 did not form S. commune protoplasts, but addition of it to alpha-1,3-glucanase of B. circulans KA-304 brought about protoplast-forming activity. Chitinase A isolated from the S. cyaneus SP-27 culture filtrate was more effective than chitinase I of B. circulans KA-304 for the protoplast formation in combination with alpha-1,3-glucanase. The N-terminal amino acid sequence of chitinase A (MW 29,000) has a sequential similarity to those of several Streptomycete family 19 chitinases. Chitinase A adsorbed to chitinous substrate and inhibited the growth of Trichoderma reesei mycelia. Anomer analysis of the reaction products also suggested that the enzyme is a family 19 chitinase.

A macroarray expression analysis of novel cDNAs vital for growth initiation and primary metabolism during development of Heterobasidion parviporum conidiospores.

The pathogen Heterobasidion parviporum causes significant losses to forest industries in Europe and North America. The fungus is spread by basidiospores on fresh stumps where it differentiates into a specialized infection hyphae to colonize its host. This differentiation is driven by recognition and its strategic success lies in its ability to do this rapidly and efficiently. To investigate gene expression pattern during the spore germination stages, mRNA of germinated and ungerminated conidiospores of H. parviporum harvested at distinct developmental time points (18, 36, 72 and 120 h) corresponding to periods of isotropic/germ tube emergence, polarized apical, early and late mycelial lateral branching growth stages was hybridized to macroarrays containing 338 cDNAs from H. parviporum. The results of the statistical analysis identified a total of 24, 39, 38 and 30 genes that were differentially upregulated at 18, 36, 72 and 120 h, respectively, relative to time 0. The number of the downregulated genes was 4, 6, 8 and 13 genes respectively. During isotropic, polarized and mycelial growth stages, majority of the differentially expressed genes belonged to functional categories metabolism (21-32%) and protein formation (21-30%). Real-time polymerase chain reaction (real-time-PCR) data essentially confirmed the macroarray analyses. The real-time-PCR result showed that transcript levels of genes involved in glucose metabolism (phosphoglucomutase), amino acid metabolism (arginase, delta-1-pyrroline-5-carboxylate reductase, sulfur metabolism-negative regulator, imidazoleglycerol phosphate dehydratase) and protein synthesis (40S ribosomal protein S15) were significantly increased during polarized growth (36 h) stage but decreased at early and late stages of mycelial growth (72-120 h). An understanding of the various molecular and physiological processes during the development of H. parviporum spores is an important step towards the goal of identifying novel antifungal strategies.

Cloning and expression of an alpha-1,3-glucanase gene from Bacillus circulans KA-304: the enzyme participates in protoplast formation of Schizophyllum commune.

A culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation of Schizophyllum commune has an activity to form protoplasts from S. commune mycelia, and a combination of alpha-1,3-glucanase and chitinase I, which were isolated from the filtrate, brings about the protoplast-forming activity. The gene of alpha-1,3-glucanase was cloned from B. circulans KA-304. It consists of 3,879 nucleotides, which encodes 1,293 amino acids including a putative signal peptide (31 amino acid residues), and the molecular weight of alpha-1,3-glucanase without the putative signal peptide was calculated to be 132,184. The deduced amino acid sequence of alpha-1,3-glucanase of B. circulans KA-304 showed approximately 80% similarity to that of mutanase (alpha-1,3-glucanase) of Bacillus sp. RM1, but no significant similarity to those of fungal mutanases. The recombinant alpha-1,3-glucanase was expressed in Escherichia coli Rosetta-gami B (DE 3), and significant alpha-1,3-glucanase activity was detected in the cell-free extract of the organism treated with isopropyl-beta-D-thiogalactopyranoside. The recombinant alpha-1,3-glucanase showed protoplast-forming activity when the enzyme was combined with chitinase I.

Hydrophobins: proteins with potential.

Hydrophobins are self-assembling proteins of fungal origin. Their ability to self-assemble into an amphipathic membrane is of interest for many different applications, ranging from medical and technical coatings to the production of proteinaceous glue and cosmetics. Assembled hydrophobins can modify surface characteristics, thus controling the binding properties of the surface; for example, enzymes can be actively and non-covalently immobilized on electrode surfaces and medical coatings can be improved for biocompatibility. Over the past few years research on hydrophobins has contributed to a better understanding of the self-assembly process and is generating more handles to control and manipulate the process. This knowledge could have an immediate effect on production levels, which are not yet adequate, and provide the boost needed for hydrophobins to reach their full potential.

Ectopic expression of a constitutively active Cdc42 small GTPase alters the morphology of haploid and dikaryotic hyphae in the filamentous homobasidiomycete Schizophyllum commune.

Cloning of the Cdc42 gene from Schizophyllum commune enabled investigation of the role of ScCdc42 in the regulation of vegetative growth and sexual reproduction in this fungus, which has a well-characterized hyphal cell structure, cytoskeleton, and mating system. Ectopic expression of the constitutively active Sccdc42(G12V) or Sccdc42(Q61L) alleles from native or inducible ScCel1 promoters in haploid hyphae had dramatic effects on hyphal morphology, cytoskeletal structure, and Cdc42 localization. For transformants with constitutively active Sccdc42, polar tip growth of apical cells in the leading hyphae was normal but polar tip growth in side branches was altered, implying different regulation of polarity establishment in the two groups of apical cells. Branch emergence at exceptional sites and isotropic growth of cells near the septum indicated that ScCdc42 regulates branch site selection and subsequent hyphal development. Poor dikaryotization along with irregular clamp connections in mates expressing Sccdc42(G12V) or Sccdc42(Q61L) suggested that Cdc42 also contributes to efficient mating in S. commune.

The SC15 protein of Schizophyllum commune mediates formation of aerial hyphae and attachment in the absence of the SC3 hydrophobin.

Disruption of the SC3 gene in the basidiomycete Schizophyllum commune affected not only formation of aerial hyphae but also attachment to hydrophobic surfaces. However, these processes were not completely abolished, indicating involvement of other molecules. We here show that the SC15 protein mediates formation of aerial hyphae and attachment in the absence of SC3. SC15 is a secreted protein of 191 aa with a hydrophilic N-terminal half and a highly hydrophobic C-terminal half. It is not a hydrophobin as it lacks the eight conserved cysteine residues found in these proteins. Besides being secreted into the medium, SC15 was localized in the cell wall and the mucilage that binds aerial hyphae together. In a strain in which the SC15 gene was deleted (DeltaSC15) formation of aerial hyphae and attachment were not affected. However, these processes were almost completely abolished when the SC15 gene was deleted in the DeltaSC3 background. The absence of aerial hyphae in the DeltaSC3DeltaSC15 strain can be explained by the inability of the strain to lower the water surface tension and to make aerial hyphae hydrophobic.

Crossing the boundary between the Balpha and Bbeta mating-type loci in Schizophyllum commune.

In this study, genes of the Schizophyllum commune Balpha and Bbeta mating-type loci are shown to be within a few kilobases of each other. The region between the nearest Balpha and Bbeta genes contains many short direct repeats. Predicted amino acid sequences and activity spectra of three pheromones encoded in the Balpha3 mating-type specificity are presented along with a re-evaluation of pheromone activity of many previously reported S. commune lipopeptide pheromones. This analysis showed that S. commune pheromones belong to five subtypes. Several pheromones activate both a Bbeta receptor and a Balpha receptor, a phenomenon previously unrecognized. Clues from mating tests and DNA hybridization led to the cloning of bar8, the gene encoding the Balpha8 pheromone receptor, Bar8. Bar8 is similar in sequence to Bbr1, the Bbeta1 pheromone receptor, and functionally identical to it. These data begin to elucidate the enigmatic recombination patterns previously encountered at the B mating-type complex.