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Schizophyllum commune is a mushroom-forming fungus notable for its distinctive fruiting bodies with split gills. It is used as a model organism to study mushroom development, lignocellulose degradation and mating type loci. It is a hypervariable species with considerable genetic and phenotypic diversity between the strains. In this study, we systematically phenotyped 16 dikaryotic strains for aspects of mushroom development and 18 monokaryotic strains for lignocellulose degradation. There was considerable heterogeneity among the strains regarding these phenotypes. The majority of the strains developed mushrooms with varying morphologies, although some strains only grew vegetatively under the tested conditions. Growth on various carbon sources showed strain-specific profiles. The genomes of seven monokaryotic strains were sequenced and analyzed together with six previously published genome sequences. Moreover, the related species Schizophyllum fasciatum was sequenced. Although there was considerable genetic variation between the genome assemblies, the genes related to mushroom formation and lignocellulose degradation were well conserved. These sequenced genomes, in combination with the high phenotypic diversity, will provide a solid basis for functional genomics analyses of the strains of S. commune., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2023. Springer Nature Limited.)

Wood-decaying fungi of the class Agaricomycetes (phylum Basidiomycota) are saprotrophs that break down lignocellulose and play an important role in nutrient recycling. They secrete a wide range of extracellular plant cell wall degrading enzymes that break down cellulose, hemicellulose, and lignin, the main building blocks of plant biomass. Although the production of these enzymes is regulated mainly at the transcriptional level, no activating regulators have been identified in any wood-decaying fungus in the class Agaricomycetes. We studied the regulation of cellulase expression in the wood-decaying fungus Schizophyllum commune . Comparative genomics and transcriptomics on two wild isolates revealed a Zn 2 Cys 6 -type transcription factor gene ( roc1 ) that was highly upregulated during growth on cellulose, compared to glucose. It is only conserved in the class Agaricomycetes. A roc1 knockout strain showed an inability to grow on medium with cellulose as sole carbon source, and growth on cellobiose and xylan (other components of wood) was inhibited. Growth on non-wood-related carbon sources was not inhibited. Cellulase gene expression and enzyme activity were reduced in the Δ roc1 strain. ChIP-Seq identified 1474 binding sites of the Roc1 transcription factor. Promoters of genes involved in lignocellulose degradation were enriched with these binding sites, especially those of LPMO (lytic polysaccharide monooxygenase) CAZymes, indicating that Roc1 directly regulates these genes. A conserved motif was identified as the binding site of Roc1, which was confirmed by a functional promoter analysis. Together, Roc1 is a key regulator of cellulose degradation and the first identified in wood-decaying fungi in the phylum Basidiomycota. IMPORTANCE Wood-degrading fungi in the phylum Basidiomycota play a crucial role in nutrient recycling by breaking down all components of wood. Fungi have evolved transcriptional networks that regulate expression of wood-degrading enzymes, allowing them to prioritize one nutrient source over another. However, to date all these transcription factors have been identified in the phylum Ascomycota, which is only distantly related to the phylum Basidiomycota. Here, we identified the transcription factor Roc1 as a key regulator of cellulose degradation in the mushroom-forming and wood-degrading fungus Schizophyllum commune . Roc1 is highly conserved in the phylum Basidiomycota. Using comparative genomics, transcriptomics, ChIP-Seq and promoter analysis we have identified direct targets of Roc1, as well as other aspects of the transcriptional response to cellulose.

Efficient gene deletion methods are essential for the high-throughput study of gene function. Compared to most ascomycete model systems, gene deletion is more laborious in mushroom-forming basidiomycetes due to the relatively low incidence of homologous recombination (HR) and relatively high incidence of non-homologous end-joining (NHEJ). Here, we describe the use of pre-assembled Cas9-sgRNA ribonucleoproteins (RNPs) to efficiently delete the homeodomain transcription factor gene hom2 in the mushroom-forming basidiomycete Schizophyllum commune by replacing it with a selectable marker. All components (Cas9 protein, sgRNA, and repair template with selectable marker) were supplied to wild type protoplasts by PEG-mediated transformation, abolishing the need to optimize the expression of cas9 and sgRNAs. A Δku80 background further increased the efficiency of gene deletion. A repair template with homology arms of 250 bp was sufficient to efficiently induce homologous recombination. This is the first report of the use of pre-assembled Cas9 RNPs in a mushroom-forming basidiomycete and this approach may also improve the genetic accessibility of non-model species.

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 CO 2 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.

Mushrooms are the most conspicuous fungal structures. Transcription factors (TFs) Bri1 and Hom1 of the model fungus Schizophyllum commune are involved in late stages of mushroom development, while Wc-2, Hom2, and Fst4 function early in development. Here, it is shown that Bri1 and Hom1 also stimulate vegetative growth, while biomass formation is repressed by Wc-2, Hom2, and Fst4. The Δbri1Δbri1 and the Δhom1Δhom1 strains formed up to 0.6 fold less biomass when compared to wild-type, while Δwc-2Δwc-2, Δhom2Δhom2, and Δfst4Δfst4 strains formed up to 2.8 fold more biomass. Inactivation of TF gene tea1, which was downregulated in the Δwc-2Δwc-2, Δhom2Δhom2, and Δfst4Δfst4 strains, resulted in a strain that was severely affected in mushroom development and that produced 1.3 fold more biomass than the wild-type. In contrast, introducing a constitutive active version of hom2 that had 4 predicted phosphorylation motifs eliminated resulted in radial growth inhibition and prompt fructification in both Δhom2 and wild-type strains, even in sterile monokaryons. Together, it is concluded that TFs involved in mushroom formation also modulate vegetative growth. Among these TFs is the homeodomain protein Hom2, being the first time that this class of regulatory proteins is implicated in repression of vegetative growth in a eukaryote.

Production of commercially interesting sesquiterpenes was previously examined in plants and microorganisms such as Escherichia coli and Saccharomyces cerevisiae. We here investigate the potential of the mushroom Schizophyllum commune for the production of sesquiterpenes. Genomic analysis of S. commune revealed that the mevalonate pathway required for the synthesis of the farnesyl diphosphate substrate for sesquiterpene production is operational. Introduction of a valencene synthase gene resulted in production of the sesquiterpene (+)-valencene, both in mycelium and in fruiting bodies. Levels of (+)-valencene in culture media of strains containing a mutated RGS regulatory protein gene (thn) were increased fourfold compared to those in wild-type transformants. Up to 16 mg L(-1) (+)-valencene was produced in these strains. In addition, the amount of (+)-valencene containing n-dodecane recovered from the culture medium increased sixfold to sevenfold in the thn mutant strains due to the absence of schizophyllan.

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., (© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.)

Alg3 of Saccharomyces cerevisiae catalyzes the mannosyl transfer from Man-P-Dol to Man(5)GlcNAc(2)-PP-Dol resulting in the formation of Man(6)GlcNAc(2)-PP-Dol, which is then further processed to the final precursor oligosaccharide Glc(3)Man(9)GlcNAc(2) for N-glycosylation of proteins. Here, we identified the alg3 gene of the mushroom-forming fungus Schizophyllum commune by homology search. Its function was confirmed by the complementation of the Δalg3 strain of S. cerevisiae. Inactivation of alg3 in S. commune resulted in the production of predominantly Man(3)GlcNAc(2) protein-linked N-glycans. No impact on growth nor a developmental phenotype due to the deletion was observed. This provides a first step toward engineering of a homogeneous, humanized N-glycosylation pattern for the production of therapeutic glycoproteins in mushrooms.

Mushrooms represent the most conspicuous structures of fungi. Their development is being studied in the model basidiomycete Schizophyllum commune. The genome of S. commune contains 472 genes encoding predicted transcription factors. Of these, fst3 and fst4 were shown to inhibit and induce mushroom development respectively. Here, we inactivated five additional transcription factor genes. This resulted in absence of mushroom development (in the case of deletion of bri1 and hom2), in arrested development at the stage of aggregate formation (in the case of c2h2) and in the formation of more but smaller mushrooms (in the case of hom1 and gat1). Moreover, strains in which hom2 and bri1 were inactivated formed symmetrical colonies instead of irregular colonies like the wild type. A genome-wide expression analysis identified several gene classes that were differentially expressed in the strains in which either hom2 or fst4 was inactivated. Among the genes that were downregulated in these strains were c2h2 and hom1. Based on these results, a regulatory model of mushroom development in S. commune is proposed. This model most likely also applies to other mushroom-forming fungi and will serve as a basis to understand mushroom formation in nature and to enable and improve commercial mushroom production., (© 2011 Blackwell Publishing Ltd.)

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.

Schizophyllum commune is the only mushroom-forming fungus in which targeted gene deletions by homologous recombination have been reported. However, these deletions occur with a low frequency. To overcome this, the ku80 gene of S. commune was deleted. This gene is involved in the nonhomologous end-joining system for DNA repair. The Deltaku80 strain was not affected in growth and development. However, the transformation efficiency was reduced up to 100-fold. This was accompanied by a strong increase in the relative number of transformants with a homologous integration of a knockout construct. Genes sc15, jmj3 and pri2 were deleted in the Deltaku80 strain. In total, seven out of 10 transformants showed a gene deletion. This frequency will facilitate a systematic analysis of gene function in S. commune.

Mushroom-forming basidiomycetes colonize large areas in nature. Their hyphae are compartmentalized by perforated septa, which are usually covered by a septal pore cap (SPC). Here, we describe, for the first time, the composition and function of SPCs using the model system Schizophyllum commune. The SPC of S. commune was shown to consist of a proteinaceous matrix covered by a lipid membrane. The matrix was demonstrated to define the ultrastructure of the SPC and to consist of two main proteins, Spc14 and Spc33. Gene spc14 encodes a protein of 86 amino acids, which lacks known domain, signal or localization sequences. Gene spc33 encodes a 239 and a 340 amino acid variant. Both forms contain a predicted signal anchor that targets them to the ER. Immuno-localization showed the presence of Spc33 in the SPC but not in ER. From this and previous reports it is concluded that the SPC is derived from this organelle. Inactivation of spc33 resulted in loss of SPCs and the inability to close septa. The latter may well explain why vegetative growth and mushroom formation were severely reduced in strains in which spc33 was inactivated.

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 microg ml(-1)) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (beta(1-3),beta(1-6)-glucan) and beta(1-3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.

N-linked glycans of Schizophyllum commune consist of Man(5-9)GlcNAc(2) structures. Lack of further glycan maturation is explained by the absence of genes encoding such functions in this and other homobasidiomycetes. N-linked glycans in vegetative mycelium and fruiting bodies of S. commune are mainly Man(7)GlcNAc(2) and Man(5)GlcNAc(2), respectively, suggesting more efficient mannose trimming in the mushroom.

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 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.

Hydrophobins fulfill a wide spectrum of functions in fungal growth and development. These proteins self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. Hydrophobins are divided into two classes based on their hydropathy patterns and solubility. We show here that the properties of the class II hydrophobins HFBI and HFBII of Trichoderma reesei differ from those of the class I hydrophobin SC3 of Schizophyllum commune. In contrast to SC3, self-assembly of HFBI and HFBII at the water-air interface was neither accompanied by a change in secondary structure nor by a change in ultrastructure. Moreover, maximal lowering of the water surface tension was obtained instantly or took several minutes in the case of HFBII and HFBI, respectively. In contrast, it took several hours in the case of SC3. Oil emulsions prepared with HFBI and SC3 were more stable than those of HFBII, and HFBI and SC3 also interacted more strongly with the hydrophobic Teflon surface making it wettable. Yet, the HFBI coating did not resist treatment with hot detergent, while that of SC3 remained unaffected. Interaction of all the hydrophobins with Teflon was accompanied with a change in the circular dichroism spectra, indicating the formation of an alpha-helical structure. HFBI and HFBII did not affect self-assembly of the class I hydrophobin SC3 of S. commune and vice versa. However, precipitation of SC3 was reduced by the class II hydrophobins, indicating interaction between the assemblies of both classes of hydrophobins.

Disruption of genes by homologous recombination occurs at a low frequency in the basidiomycete Schizophyllum commune. For instance, the SC3 and SC15 genes were inactivated at frequencies of 1 and 5%, respectively. As an alternative to disruption, we used gene silencing through the introduction of a hairpin construct. The SC15 gene, which encodes an abundantly secreted structural protein, was silenced at a frequency of 80% in monokaryons of S. commune after introduction of a hairpin construct of the gene. Silencing also occurred in dikaryons in which one of the partners was not a silenced strain. The silencing mechanism resembles RNAi in other filamentous fungi and is a powerful tool for the functional analysis of genes expressed in monokaryons or dikaryons.

Fluorescent DNA and peptide nucleic acid (PNA) probes were used for in situ hybridisations in colonies of Schizophyllum commune and Aspergillus niger. DNA probes for 18S rRNA did not diffuse through the cell wall after mild chemical fixation. After permeabilising the cell wall with lysing enzymes or slow freezing and embedding, hybridisation was still poor and not reproducible. In contrast, PNA probes did diffuse through the cell wall after mild chemical fixation and reproducible fluorescent signals were obtained. The rRNA signal was most intense in the apical compartment of hyphae of S. commune. Within this compartment, the signal was lower at the extreme apex. Apparently, ribosomes are unevenly distributed in hyphae. In S. commune, the mRNA of the SC3 gene was also detected with a PNA probe. The ratio between 18S rRNA and SC3 mRNA signals were variable between hyphae and their compartments. This is the first report of using PNA probes for in situ hybridisation of mRNA in fungi. The method provides a powerful tool to study gene expression.

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.