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Cap expansion in agaricoid mushroom species is an important event for sexual reproduction because meiosis occurs in basidia under the cap, and basidiospores can be released by opening the cap. However, molecular mechanisms underlying cap expansion in basidiomycetes remain poorly understood. We aimed to elucidate the molecular mechanisms of cap expansion in basidiomycetes by analyzing the unique cap-expansionless UV mutant #13 (exp2-1) in Coprinopsis cinerea. Linkage analysis and consequent genome sequence analysis revealed that the gene responsible for the mutant phenotypes encodes a putative transcription factor with two C2H2 zinc finger motifs. The mutant that was genome-edited to lack exp2 exhibited an expansionless phenotype. Some of the genes encoding cell wall degradation-related enzymes showed decreased expression during cap expansion and autolysis in the exp2 UV and genome-edited mutant. The exp2 gene is widely conserved in Agaricomycetes, suggesting that Exp2 homologs regulate fruiting body maturation in Agaricomycetes, especially cap expansion in Agaricoid-type mushroom-forming fungi. Therefore, exp2 homologs could be a target for mushroom breeding to maintain shape after harvest for some cultivating mushrooms, presenting a promising avenue for further research in breeding techniques., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing interests., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

The morphogenesis of sexual fruiting bodies of fungi is a complex process determined by a genetically encoded program. Fruiting bodies reached the highest complexity levels in the Agaricomycetes; yet, the underlying genetics is currently poorly known. In this work, we functionally characterized a highly conserved gene termed snb1 , whose expression level increases rapidly during fruiting body initiation. According to phylogenetic analyses, orthologs of snb1 are present in almost all agaricomycetes and may represent a novel conserved gene family that plays a substantial role in fruiting body development. We disrupted snb1 using CRISPR/Cas9 in the agaricomycete model organism Coprinopsis cinerea. snb1 deletion mutants formed unique, snowball-shaped, rudimentary fruiting bodies that could not differentiate caps, stipes, and lamellae. We took advantage of this phenotype to study fruiting body differentiation using RNA-Seq analyses. This revealed differentially regulated genes and gene families that, based on wild-type RNA-Seq data, were upregulated early during development and showed tissue-specific expression, suggesting a potential role in differentiation. Taken together, the novel gene family of snb1 and the differentially expressed genes in the snb1 mutants provide valuable insights into the complex mechanisms underlying developmental patterning in the Agaricomycetes., Importance: Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are complex multicellular structures, with a spatially and temporally integrated developmental program that is, however, currently poorly known. In this study, we present a novel, conserved gene family, Snowball (snb), termed after the unique, differentiation-less fruiting body morphology of snb1 knockout strains in the model mushroom Coprinopsis cinerea. snb is a gene of unknown function that is highly conserved among agaricomycetes and encodes a protein of unknown function. A comparative transcriptomic analysis of the early developmental stages of differentiated wild-type and non-differentiated mutant fruiting bodies revealed conserved differentially expressed genes which may be related to tissue differentiation and developmental patterning fruiting body development., Competing Interests: The authors declare no conflict of interest.

Hormographiella aspergillata is a basidiomycete exceptionally involved in invasive fungal infections (IFI). We report a case of H. aspergillata pulmonary infection in a 30-year-old female in a context of pancytopenia and relapsed of acute myeloid leukemia (AML). She presented with fever, thoracic pain, left pleural effusion and pneumonia, diagnosed on chest X-ray and CT-scan. Direct examination of a bronchoalveolar lavage (BAL) specimen performed on day (d) 10 was negative, while the culture was positive on d30. H. aspergillata was suspected, considering macroscopic and microscopic examination. Its identification was confirmed using Microflex® Bruker mass spectrometry and pan-fungal (PF)-PCR assay followed by DNA sequencing. After this initial diagnosis, the patient was monitored for 2.8 years. She was treated with liposomal amphotericin B and/or voriconazole until switching to isavuconazole on d298 due to side-effects. This antifungal treatment was maintained until d717 and then discontinued, the patient being considered as cured. Over this follow-up period, the patient was submitted to recurrent pulmonary sampling. Each time, cultures were negative, while PF - PCR assays and DNA sequencing confirmed the presence of H. aspergillata. The present case-report is the 32nd observation of H. aspergillata invasive infection showing that this IFI is still infrequent. Fifteen have occurred in patients with AML, which appears as the most frequent underlying disease favoring this IFI. Six recent case-reports in addition to ours highlight PF-PCR assays and DNA sequencing as relevant diagnostic tools that must be included in routine diagnosis and monitoring of IFI, specifically those due to rare basidiomycetes., Competing Interests: Declaration of competing interest The authors declare there is no conflict of interests., (Copyright © 2024 SFMM. Published by Elsevier Masson SAS. All rights reserved.)

Mushroom galectins are promising anticancer agents for their low IC50 values against cancer cells in vitro. In this study, two Coprinopsis cinerea galectins, CGL1 and CGL2, were heterologously expressed, and their biochemistry properties and anticancer effects were evaluated. The purified galectins were thermostable at neutral pH conditions. They both existed as tetramers and shared a high affinity towards lactose. CGL1 and CGL2 strongly inhibited the cell viability of many cancer cell lines, including three colorectal cancer cells, in a dose-dependent manner by inducing mitochondria-mediated caspase-dependent apoptosis. Furthermore, CGL1 exhibited higher apoptosis-inducing ability and cytotoxicity than CGL2. In vivo cell viability experiments based on two xenograft mouse models showed that CGL1 had a more substantial inhibitory effect than CGL2 on HCT116 tumor growth (p < 0.0001), whereas only CGL1 inhibited DLD1 tumor growth (p < 0.01). This is the first study to evaluate the anti-colorectal cancer effect of mushroom lectins in vivo, and our results showed that CGL1 is a potent agent for colorectal cancer treatment.

Multicellularity has been one of the most important innovations in the history of life. The role of gene regulatory changes in driving transitions to multicellularity is being increasingly recognized; however, factors influencing gene expression patterns are poorly known in many clades. Here, we compared the developmental transcriptomes of complex multicellular fruiting bodies of eight Agaricomycetes and Cryptococcus neoformans , a closely related human pathogen with a simple morphology. In-depth analysis in Pleurotus ostreatus revealed that allele-specific expression, natural antisense transcripts, and developmental gene expression, but not RNA editing or a 'developmental hourglass,' act in concert to shape its transcriptome during fruiting body development. We found that transcriptional patterns of genes strongly depend on their evolutionary ages. Young genes showed more developmental and allele-specific expression variation, possibly because of weaker evolutionary constraint, suggestive of nonadaptive expression variance in fruiting bodies. These results prompted us to define a set of conserved genes specifically regulated only during complex morphogenesis by excluding young genes and accounting for deeply conserved ones shared with species showing simple sexual development. Analysis of the resulting gene set revealed evolutionary and functional associations with complex multicellularity, which allowed us to speculate they are involved in complex multicellular morphogenesis of mushroom fruiting bodies., Competing Interests: ZM, MV, EG, JS, BK, TV, AG, BH, BB, LN No competing interests declared, (© 2022, Merényi et al.)

Frequently, laccases are triggered during fungal cocultivation for overexpression. The function of these activated laccases during coculture has not been clarified. Previously, we reported that Gongronella sp. w5 (w5) (Mucoromycota, Mucoromycetes) specifically triggered the laccase Lcc9 overexpression in Coprinopsis cinerea (Basidiomycota, Agaricomycetes). To systematically analyze the function of the overexpressed laccase during fungal interaction, C. cinerea mycelia before and after the initial Lcc9 overexpression were chosen for transcriptome analysis. Results showed that accompanied by specific utilization of fructose as carbohydrate substrate, oxidative stress derived from antagonistic compounds secreted by w5 appears to be a signal critical for laccase production in C. cinerea . A decrease in reactive oxygen species (ROS) in the C. cinerea wild-type strain followed the increase in laccase production, and then lcc9 transcription and laccase activity stopped. By comparison, increased H 2 O 2 content and mycelial ROS levels were observed during the entire cocultivation in lcc9 silenced C. cinerea strains. Moreover, lcc9 silencing slowed down the C. cinerea mycelial growth, affected hyphal morphology, and decreased the asexual sporulation in coculture. Our results showed that intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Lcc9 takes part in a defense strategy to eliminate oxidative stress during the interspecific interaction with w5. IMPORTANCE The overproduction of laccase during interspecific fungal interactions is well known. However, the exact role of the upregulated laccases remains underexplored. Based on comparative transcriptomic analysis of C. cinerea and gene silencing of laccase Lcc9, here we show that oxidative stress derived from antagonistic compounds secreted by Gongronella sp. w5 was a signal critical for laccase Lcc9 production in Coprinopsis cinerea. Intracellular ROS acted as signal molecules to stimulate defense responses by C. cinerea with the expression of oxidative stress response regulator Skn7 and various detoxification proteins. Ultimately, Lcc9 takes part in a defense strategy to eliminate oxidative stress and help cell growth and development during the interspecific interaction with Gongronella sp. w5. These findings deepened our understanding of fungal interactions in their natural population and communities.

Coprinopsis cinerea has seven homologs of the Aspergillus nidulans transcription factor NsdD. Of these, CcNsdD1 and CcNsdD2 from C. cinerea show the best identities of 62 and 50% to A. nidulans NsdD, respectively. After 4 days of constant darkness cultivation, CcnsdD2 , but not CcnsdD1 , was upregulated on the first day of light/dark cultivation to induce fruiting bodies, and overexpression of CcnsdD2 , but not CcnsdD1 , produced more fruiting bodies under a light/dark rhythm. Although single knockdown of CcnsdD2 did not affect fruiting body production due to upregulation of its homolog CcnsdD1 , the double-knockdown CcNsdD1/NsdD2-RNAi transformant showed defects in fruiting body formation under a light/dark rhythm. Knockdown of CcnsdD1 / nsdD2 led to the differentiation of primary hyphal knots into sclerotia rather than secondary hyphal knots under a light/dark rhythm, similar to the differentiation of primary hyphal knots into sclerotia of the wild-type strain under darkness. The CcNsdD2-overexpressing transformant produced more primary hyphal knots, secondary hyphal knots, and fruiting bodies under a light/dark rhythm but only more primary hyphal knots and sclerotia under darkness. RNA-seq revealed that some genes reported previously to be involved in formation of hyphal knots and primordia, cyclopropane-fatty-acyl-phospholipid synthases cfs1-3 , galectins cgl1-3 , and hydrophobins hyd1-3 were downregulated in the CcNsdD1/NsdD2-RNAi transformant compared to the mock transformant. ChIP-seq and electrophoretic mobility shift assay demonstrated that CcNsdD2 bound to promoter regulatory sequences containing a GATC motif in cfs1 , cfs2 , cgl1 , and hyd1 . A molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions is proposed. IMPORTANCE The model mushroom Coprinopsis cinerea exhibits remarkable photomorphogenesis during fruiting body development. This study reports that the C. cinerea transcription factor CcNsdD2 promotes primary hyphal knot formation by upregulating cfs1 , cfs2 , cgl1 , and hyd1 . Although the induction of CcnsdD2 is not under direct control of light and photoreceptors, the CcNsdD2-mediated developmental fates of the primary hyphal knots depend on the following light/dark rhythm cultivation or dark cultivation after full growth of mycelia in the constant dark cultivation. This study provides new insight into the molecular mechanism by which CcNsdD2 regulates the developmental fate of C. cinerea under dark or light conditions. In addition, the result that overexpression of CcnsdD2 induced more secondary hyphal knots, primordia, and fruiting bodies under light/dark rhythm cultivation conditions has potential applied value in the edible mushroom industry.

The homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The chitin deacetylase CDA3 from C. cinerea deacetylated chitin-oligosaccharides with dp ≥ 2. Since CDA3 firstly removed the intermediate acetyl group of (GlcNAc) 4 , it was an endo-acting deacetylase. Different from previously reported deacetylation modes, CDA3 deacetylated chitinbiose at either the reducing end or the nonreducing end; CDA3 deacetylated chitintriose at any subsite including the end and the intermediate; CDA3 further removed acetyl groups at any subsite, the intermediate, nonreducing and reducing end of chitintetraose after removal of the first intermediate acetyl group. 3D structural analysis showed that CDA3 has aromatic amino acids distributing at both the +1 and -1 subsites of the catalytic site, which may be responsible for its distinctive deacetylation mode. Furthermore, CDA3 was active on crystalline chitin, its deacetylation activity increased with the DA decreases of chitinous substrates and showed a higher activity towards the cell wall of the basal stipe with the higher molar ratio of GlcN/GlcNAc than that of the apical stipe with the lower molar ratio of GlcN/GlcNAc. CDA3 with distinctive deacetylation mode and activity indicates its function during the maturation of the fruiting bodies of C. cinerea and a potential for preparation of mushroom chitosan for application in the food, cosmetics, and pharmaceutical industries., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

This study reports that a high concentration of the endo-β-1,3-glucanase ENG (200 μg ml -1 ) induced heat-inactivated stipe wall extension of Coprinopsis cinerea , whereas a high concentration of the extracellular β-glucosidase BGL2 (1,000 μg ml -1 ) did not; however, in combination, low concentrations of ENG (25 μg ml -1 ) and BGL2 (260 μg ml -1 ) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, β-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60 min; the stipe cell wall extension induced by a high concentration of β-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the β-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of β-1,6 branched β-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the β-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases. IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis cinerea In this study, we explored that β-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of β-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, β-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of β-1,3-glucanases is necessary for the cell wall remodeling and the addition of new β-glucans during stipe elongation growth., (Copyright © 2019 American Society for Microbiology.)

We previously reported that β-glucosidase BGL1 at low concentration (15 μg mL -1 ) from Coprinopsis cinerea exhibited hydrolytic activity only toward laminarioligosaccharides but not toward cellooligosaccharides and gentiobiose. This study shows that BGL1 at high concentration (200 μg mL -1 ) also hydrolyzed cellobiose and gentiobiose, which accounted for only 0.83 and 2.05% of its activity toward laminaribiose, respectively. Interestingly, BGL1 at low concentration (1.5 μg mL -1 ) showed transglycosylation but BGL1 at high concentration (200 μg mL -1 ) did not. BGL1 utilizes only laminarioligosaccharides but not glucose, gentiobiose, and cellobiose to synthesize the higher oligosaccharides. BGL1 transferred one glucosyl residue from substrate laminarioligosaccharide to another laminarioligosaccharide as an acceptor in a β(1 → 3) or β(1 → 6) fashion to produce higher laminarioligosaccharides or 3-O-β-d-gentiobiosyl-d-laminarioligosaccharides. The BGL1-digested laminaritriose exhibited approximately 90% enhancement in the anti-oxidant activity compared to that of untreated laminaritriose, implying a potential application of BGL1-based transglycosylation for the production of high value-added rare oligosaccharides.

The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N -acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea , ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N -acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro ; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension. IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by β-1,6-branched β-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and β-1,3-glucans under turgor pressure in vivo ., (Copyright © 2019 American Society for Microbiology.)

The glycoside hydrolase (GH) 16 family of Coprinopsis cinerea includes 15 members distributed in four subgroups (A1, A2, B and D) by phylogenetic analysis. The expression patterns match well with the requirement of wall-softening in the germination of basidiospores, hyphal growth and branching, primordium formation, stipe elongation, pileus expansion and autolysis. Remarkably, expression levels of different GH16 members varied with different morphogenetic events. Like orthologs of Aspergillus fumigatus GH16 glucanases (ENG2-5), which were expressed in the dormant conidia and conidiogenesis, and essential for segregation of conidia, some members such as ENG in the subgroup A1 in C. cinerea were also predominantly expressed in dormant basidiospores, primordia and maturing pilei during basidiosporogenesis. In contrast, other members in subgroup A2, subgroup B or D were dominantly expressed in the germinating basidiospores, the growing mycelia, and the elongating stipes. We did not find the members of the GH81 or GH55 family in C. cinerea genome, which was different from A. fumigatus. However, C. cinerea contains an extra three subgroups (A2, B and D) compared with A. fumigatus. These extra subgroups of GH16 family members may function as those endo-β-1,3-glucanases belonging to other GH families in the development and growth of C. cinerea., (© FEMS 2019.)

Intercellular distribution of nutrients and coordination of responses to internal and external cues via endogenous signaling molecules are hallmarks of multicellular organisms. Vegetative mycelia of multicellular fungi are syncytial networks of interconnected hyphae resulting from hyphal tip growth, branching, and fusion. Such mycelia can reach considerable dimensions and, thus, different parts can be exposed to quite different environmental conditions. Our knowledge about the mechanisms by which fungal mycelia can adjust nutrient gradients or coordinate their defense response to fungivores is scarce, in part due to limitations in technologies currently available for examining different parts of a mycelium over longer time periods at the microscopic level. Here, we combined a tailor-made microfluidic platform with time-lapse fluorescence microscopy to visualize the dynamic response of the vegetative mycelium of a basidiomycete to two different stimuli. The microfluidic platform allows simultaneous monitoring at both the colony and single-hypha level. We followed the dynamics of the distribution of a locally administered nutrient analog and the defense response to spatially confined predation by a fungivorous nematode. Although both responses of the mycelium were constrained locally, we observed long-distance propagation for both the nutrient analog and defense response in a subset of hyphae. This propagation along hyphae occurred in both acropetal and basipetal directions and, intriguingly, the direction was found to alternate every 3 hr in an individual hypha. These results suggest that multicellular fungi have, as of yet, undescribed mechanisms to coordinate the distribution of nutrients and their behavioral response upon attack by fungivores., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

When sucrose was used as the carbon source, the Basidiomycete Coprinopsis cinerea showed poor growth and low laccase activity in pure culture, but greatly enhanced the level of laccase activity (>1800 U/L) during coculture with the Mucoromycete Gongronella sp. w5. As a result, the mechanism of laccase overproduction in coculture was investigated by starting from clarifying the function of sucrose. Results demonstrated that Gongronella sp. w5 in the coculture system hydrolyzed sucrose to glucose and fructose by an intracellular invertase. Fructose rather than glucose was supplied by Gongronella sp. w5  as the readily available carbon source for C. cinerea, and contributed to an alteration of its growth behavior and a basal laccase secretion of 110.6 ± 3.3 U/L. On the other hand, separating Gongronella sp. w5 of C. cinerea by transfer into dialysis tubes yielded the same level of laccase activity as without separation, indicating that enhanced laccase production probably resulted from the metabolites in the fermentation broth. Further investigation showed that the ethyl acetate-extracted metabolites generated by Gongronella sp. w5 induced C. cinerea laccase production. One of the laccase-inducing compounds namely p-hydroxybenzoic acid (HBA) was purified and identified from the extract. When using HBA as the inducer and fructose as the carbon source in monoculture, C. cinerea observed similar high laccase activity to that in coculture, and zymograms revealed the same expression of laccase Lcc9 as the main and Lcc1 and Lcc5 as the minor enzymes. Overall, our experiments verified that Gongronella sp. w5 elevates Coprinopsis cinerea laccase production by carbon source syntrophism and secondary metabolite induction.

Fruiting bodies that exhibit strong autolysis of Coprinopsis cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications.

Objectives: The aim of the study was to obtain information about the enzymatic properties of aryl-alcohol oxidase from the plant saprophytic basidiomycete Coprinopsis cinerea (rCcAAO), which is classified into the auxiliary activities family 3 subfamily 2 (AA3&#95;2)., Results: The gene encoding AAO from the plant saprophytic basidiomycete Coprinopsis cinerea (CcAAO) was cloned, and the recombinant CcAAO (rCcAAO) was heterologously expressed in the methylotrophic yeast Pichia pastoris. The purified rCcAAO showed significant activity not only against trans,trans-2,4-hexadien-1-ol but also against a broad range of aromatic alcohols including aromatic compounds that were reported to be poor substrates for known AAOs. Moreover, site-directed mutagenesis analysis demonstrated that mutants with substitutions from leucine to phenylalanine and tryptophan at position 416 exhibited decreases of activity for aromatic alcohols but still maintained the activity for trans,trans-2,4-hexadien-1-ol., Conclusions: Leucine 416 in CcAAO contributes to the broad substrate specificity against various aromatic alcohols, which is useful for the production of hydrogen peroxide using this enzyme.

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions.

As one of the most abundant renewable resources, rice straw is an attractive lignocellulosic material for animal feeding or for the production of biochemical. An appropriate pre-treatment technique is essential for converting rice straw to rich fodder or biofuel. Based on previous work, Coprinopsis cinerea can grow on rice straw medium and therefore it is useful for the treatment of rice straw. However, little is known regarding its degradation systems and nutrition values. In this study, we firstly found that C. cinerea could grow rapidly on rice straw without any additives by the production of a series of enzymes (laccase, cellulase, and xylanase) and that the microstructure and contents of rice straw changed significantly after being treated by C. cinerea. We propose that a possible underlying mechanism exists in the degradation. Moreover, C. cinerea has a high nutrition value (23.5% crude protein and 22.2% total amino acids). Hence, fermented rice straw with mycelium could be a good animal feedstuff resource instead of expensive forage. The direct usage of C. cinerea treatment is expected to be a practical, cost-effective, and environmental-friendly approach for enhancing the nutritive value and digestibility of rice straw.

Chitinase ChiEn1 did not hydrolyze insoluble chitin but showed hydrolysis and transglycosylation activities toward chitin-oligosaccharides. Interestingly, the addition of ChiEn1 increased the amount of reducing sugars released from chitin powder by endochitinase ChiIII by 105.0%, and among the released reducing sugars the amount of (GlcNAc) 2 was increased by 149.5%, whereas the amount of GlcNAc was decreased by 10.3%. The percentage of GlcNAc in the products of chitin powder with the combined ChiIII and ChiEn1 was close to that in the products of chitin-oligosaccharides with ChiEn1, rather than that with ChiIII. These results indicate that chitin polymers are first degraded into chitin oligosaccharides by ChiIII and the latter are further degraded to monomers and dimers by ChiEn1, and the synergistic action of ChiEn1 and ChiIII is involved in the efficient degradation of chitin in cell walls during pileus autolysis. The structure modeling explores the molecular base of ChiEn1 action.

A putative class III endochitinase (ChiIII) was reported previously to be expressed dominantly in fruiting bodies of Coprinopsis cinerea, and its expression levels increased with the maturation of the fruiting bodies. This paper further reports that ChiIII is a novel chitinase with exo- and endoactivities. When the substrate was (GlcNAc)3-5, ChiIII exhibited exoactivity, releasing GlcNAc processively from the reducing end of (GlcNAc)3-5; when the substrate was (GlcNAc)6-7, the activity of ChiIII shifted to an endoacting enzyme, randomly splitting chitin oligosaccharides to various shorter oligosaccharides. This shift in the mode of action of ChiIII may be related to its stronger hydrolytic capacity to degrade chitin in fungal cell walls. The predicted structure of ChiIII shows that it lacks the α+β domain insertion; however, its substrate binding cleft seems to be deeper than that of common endochitinases but shallower and more open than that of common exochitinases, which may be related to its exo- and endohydrolytic activities.

A β-glycoside hydrolase was isolated from cell walls material in Coprinopsis cinerea elongating stipes. By analysis of SDS-PAGE, MALDI-TOF/TOF MS and substrate specificity, this enzyme was characterized as an extracellular β-glucosidase which is a trimer consisting of three homosubunits. β-Glucosidase did not degrade β-glucans with modified ends, whereas it hydrolyzed various β-glucans with free ends and related oligosaccharides with β-1,3-, β-1,4- or β-1,6-linkages. Although this β-glucosidase possesses glycosyltransferase activity on laminarioligosaccharides, it did not transfer glucose residues from laminaritriose to β-glucan in stipe cell walls to produce larger β-glucan molecules; instead, it caused a decrease in the molecular size of stipe wall β-glucan by removing glucose. Relatively, the molecular size of wall β-glucans in the elongating apical stipe was less than that found in the non-elongating basal stipes, and this β-glucosidase was more highly expressed in the elongating apical stipe than in non-elongating basal regions. Therefore, we propose that β-glucosidase functions by trimming or cutting the β-glucan side chains on the β-1,3-glucan backbone to prevent them from forming longer branches, keeping the wall plastic to promote diffuse wall growth., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Autolysis of Coprinopsis cinerea fruiting bodies affects its commercial value. In this study, a mutant of C. cinerea that exhibits pileus expansion without pileus autolysis was obtained using ultraviolet mutagenesis. This suggests that pileus expansion and pileus autolysis involve different enzymes or proteins. Among the detected hydrolytic enzymes, only β-1,3-glucanase activity increased with expansion and autolysis of pilei in the wild-type strain, but the increase was abolished in the mutant. This suggests that β-1,3-glucanases plays a major role in the autolysis. Although there are 43 possible β-1,3-glucoside hydrolases genes, only 4 known genes, which have products that are thought to act synergistically to degrade the β-1,3-glucan backbone of cell walls during fruiting body autolysis, and an unreported gene were upregulated during pileus expansion and autolysis in the wild-type stain but were suppressed in the mutant. This suggests that expression of these β-1,3-glucanases is potentially controlled by a single regulatory mechanism.

Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs) have been extensively studied in mammals, whereas fungus CaMKs still remain largely uncharacterized. We previously obtained CaMK homolog in Coprinopsis cinerea, designated CoPK12, and revealed its unique catalytic properties in comparison with the mammalian CaMKs. To further clarify the regulatory mechanisms of CoPK12, we investigated post-translational modification and subcellular localization of CoPK12 in this study. In C. cinerea, full-length CoPK12 (65 kDa) was fractionated in the membrane fraction, while the catalytically active fragment (46 kDa) of CoPK12 was solely detected in the soluble fraction by differential centrifugation. Expressed CoPK12-GFP was localized on the cytoplasmic and vacuolar membranes as visualized by green fluorescence in yeast cells. In vitro N-myristoylation assay revealed that CoPK12 is N-myristoylated at Gly-2 in the N-terminal position. Furthermore, calmodulin could bind not only to CaM-binding domain but also to the N-terminal myristoyl moiety of CoPK12. These results, taken together, suggest that the cellular localization and function of CoPK12 are regulated by protein N-myristoylation and limited proteolysis., (© The Authors 2014. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

The fungal fruiting body or mushroom is a multicellular structure essential for sexual reproduction. It is composed of dikaryotic cells that contain one haploid nucleus from each mating partner sharing the same cytoplasm without undergoing nuclear fusion. In the mushroom, the pileus bears the hymenium, a layer of cells that includes the specialized basidia in which nuclear fusion, meiosis, and sporulation occur. Coprinopsis cinerea is a well-known model fungus used to study developmental processes associated with the formation of the fruiting body. Here we describe that knocking down the expression of Atr1 and Chk1, two kinases shown to be involved in the response to DNA damage in a number of eukaryotic organisms, dramatically impairs the ability to develop fruiting bodies in C. cinerea, as well as other developmental decisions such as sclerotia formation. These developmental defects correlated with the impairment in silenced strains to sustain an appropriated dikaryotic cell cycle. Dikaryotic cells in which chk1 or atr1 genes were silenced displayed a higher level of asynchronous mitosis and as a consequence aberrant cells carrying an unbalanced dose of nuclei. Since fruiting body initiation is dependent on the balanced mating-type regulator doses present in the dikaryon, we believe that the observed developmental defects were a consequence of the impaired cell cycle in the dikaryon. Our results suggest a connection between the DNA damage response cascade, cell cycle regulation, and developmental processes in this fungus.

Sections Lanatuli and Atramentarii of the genus Coprinopsis contain some of the best known and most important agaric species, including C. cinerea and C. lagopus, yet a critical, phylogeny-based assessment of the species limits has not been carried out. Taxa have been characterized chiefly on the basis of morphological characters, which however show little discriminatory power and/or considerable overlap between several species pairs. We used ITS and LSU sequence data of 29 described taxa in Coprinopsis sections Lanatuli and Atramentarii to infer species limits and the correspondence between morphological characters and species lineages, as well as to examine the phylogenetic affinities of sections Lanatuli and Atramentarii. Our analyses recovered three large clades, implying a paraphyly for section Lanatuli. Based on morphology and clade structure, we estimate ca. 38 species in the two sections, including several potentially new taxa, three of which are described herein. Coprinopsis pachyderma, C. lagopus var. vacillans, C. acuminata, C. spelaiophila, Coprinus citrinovelatus and Cop. brunneistrangulatus were found to be synonymous with other, earlier described species. Congruent with previous mating studies, our analyses recovered multiple, morphologically indistinguishable lineages within C. lagopus, which included C. lagopus var. vacillans, an ephemeral, developmental variant. Morphological traits supporting the inferred clade structure are discussed. Three new taxa (C. fusispora, C. babosiae, C. villosa), and one new combination (C. mitraespora) are proposed. [ABSTRACT FROM AUTHOR]