The developmental biology underlying the morphogenesis of mushrooms remains poorly understood despite the essential role of fungi in the terrestrial environment and global carbon cycle. The mushroom ...Coprinopsis cinerea is a leading model system for the molecular and cellular basis of fungal morphogenesis. The dikaryotic vegetative hyphae of this fungus grow by tip growth with clamp cell formation, conjugate nuclear division, septation, subapical peg formation, and fusion of the clamp cell to the peg. Studying these processes provides many opportunities to gain insights into fungal cell morphogenesis. Here, we report the dynamics of five septins, as well as the regulators CcCla4, CcSpa2, and F-actin, visualized by tagging with fluorescent proteins, EGFP, PA-GFP or mCherry, in the growing dikaryotic vegetative hyphae. We also observed the nuclei using tagged Sumo proteins and histone H1. The five septins colocalized at the hyphal tip in the shape of a dome with a hole (DwH). CcSpa2-EGFP signals were observed in the hole, while CcCla4 signals were observed as the fluctuating dome at the hyphal tip. Before septation, CcCla4-EGFP was also occasionally recruited transiently around the future septum site. Fluorescent protein-tagged septins and F-actin together formed a contractile ring at the septum site. These distinct specialized growth machineries at different sites of dikaryotic vegetative hyphae provide a foundation to explore the differentiation program of various types of cells required for fruiting body formation.
•Pex1 disruption causes defects in lignin biodegradation in Pleurotus ostreatus.•It causes defects in fruiting in P. ostreatus and Coprinopsis cinerea.•It affects hyphal growth on certain media in P. ...ostreatus but not C. cinerea.
Peroxisomes are well-known organelles that are present in most eukaryotic organisms. Mutant phenotypes caused by the malfunction of peroxisomes have been shown in many fungi. However, these have never been investigated in Agaricomycetes, which include white-rot fungi that degrade wood lignin in nature almost exclusively and play an important role in the global carbon cycle. Based on the results of a forward genetics study to identify mutations causing defects in the ligninolytic activity of the white-rot Agaricomycete Pleurotus ostreatus, we report phenotypes of pex1 disruptants in P. ostreatus, which are defective in two major features of white-rot Agaricomycetes: lignin biodegradation and mushroom formation. Pex1 disruption was also shown to cause defects in the hyphal growth of P. ostreatus on certain sawdust and minimum media. We also demonstrated that pex1 is essential for fruiting initiation in the non-wood decaying Agaricomycete Coprinopsis cinerea. However, unlike P. ostreatus, significant defects in hyphal growth on the aforementioned agar medium were not observed in C. cinerea. This result, together with previous C. cinerea genetic studies, suggests that the regulation mechanisms for the utilization of carbon sources are altered during the evolution of Agaricomycetes or Agaricales.
The homobasidiomycete Coprinus cinereus exhibits remarkable photomorphogenesis during fruiting-body development. Under proper light conditions, fruiting-body primordia proceed to the maturation phase ...in which basidia in the pileus undergo meiosis, producing sexual spores, followed by stipe elongation and pileus expansion for efficient dispersal of the spores. In the continuous darkness, however, the primordia do not proceed to the maturation phase but are etiolated: the pileus and stipe tissues at the upper part of the primordium remain rudimentary and the basal part of the primordium elongates, producing "dark stipe." In this study we genetically analyzed five strains that produce dark stipes even if light conditions promoting the maturation are given and then characterized one of them, Uar801 (dst1-1). The dst1 gene was cloned as a DNA fragment that rescues the dst1-1 mutation. Dst1 is predicted to be a protein of 1175 amino acids that contains two PAS domains, a coiled-coil structure, and a putative, glutamine-rich, transcriptional activation domain (AD). One of the PAS domains exhibits significant similarity to the LOV domains of known blue-light receptors, suggesting that Dst1 is a blue-light receptor of C. cinereus. The dst1-1 mutation is predicted to truncate the putative AD in the C-terminal region.
•The eln8-1 mutant fails to elongate the stipe in Coprinopsis cinerea.•Five of six septin genes are highly transcribed during wild-type stipe elongation.•The eln8-1 mutant has defects in expression ...of both Cc.cdc3 and Cc.cdc10.•EGFP-Cc.Cdc3 assembles into abundant thin filaments in the stipe cell cortex.
We have identified and characterized a Coprinopsis cinerea mutant defective in stipe elongation during fruiting body development. In the wild-type, stipe cells elongate at the maturation stage of fruiting, resulting in very slender cells. In the mutant, the stipe cells fail to elongate, but become rather globular at the maturation stage. We found that the mutant phenotype is rescued by a gene encoding a homolog of Saccharomyces cerevisiae CDC3 septin, Cc.Cdc3. The C. cinerea genome includes 6 septin genes, 5 of which, including Cc.cdc3, are highly transcribed during stipe elongation in the wild type. In the mutant, the level of Cc.cdc3 transcription in the stipe cells remains the same as that in the mycelium, and the level of Cc.cdc10 transcription is approximately 100 times lower than that in the wild-type stipe cells. No increase in transcription of Cc.cdc3 in the mutant may be due to the fact that the Cc.cdc3 gene has a 4-base pair insertion in its promoter and/or that the promoter region is methylated in the mutant. Overexpressed EGFP-Cc.Cdc3 fusion protein rescues the stipe elongation in the transformants, localizes to the cell cortex and assembles into abundant thin filaments in the elongating stipe cells. In contrast, in vegetative hyphae, EGFP-Cc.Cdc3 is localized to the hyphal tips of the apical cells of hyphae. Cellular defects in the mutant, combined with the localization of EGFP-Cc.Cdc3, suggest that septin filaments in the cell cortex provide the localized rigidity to the plasma membrane and allow cells to elongate cylindrically.
The pileus (cap) of the fruiting body in homobasidiomycete fungi bears the hymenium, a layer of cells that includes the basidia where nuclear fusion, meiosis and sporulation occur. Coprinopsis ...cinerea is a model system for studying fruiting body development. The hymenium of C. cinerea forms at the surface of the gills in the pileus. In a previous study, we identified a mutation called cap-growthless1-1 (cag1-1) that blocks gill formation, which yields primordia that never mature. In this study, we found that the cag1 gene encodes a homologue of Saccharomyces cerevisiae Tup1. The C. cinerea genome contains another Tup1 homologue gene called Cc.tupA Reciprocal tagging of Cag1 and Cc.TupA with green and red fluorescent proteins revealed that the relative ratios of the amounts of the two Tup1 paralogues varied among tissues. Compared with Cc.TupA, Cag1 was preferentially expressed in the gill trama tissue cells, suggesting that the function of Cag1 is required for gill trama tissue differentiation and maintenance. Yeast two-hybrid analysis and co-localisation of Cag1 and Cc.TupA suggested that Cag1 interacts with Cc.TupA in the nuclei of certain cells.
The homobasidiomycete
Coprinopsis cinerea is a member of the fungi known as inky cap mushrooms, and its fruiting-body pileus autolyzes soon after completion of the development. During the last 3
h of ...the development, the pileus exhibits umbrella-like expansion: the pileal tissue is cracked at the base of each gill and then each gill tissue is split to form a V-shape, as seen in a cross section. We identified two
C. cinerea mutants defective in both pileus expansion and autolysis. The defects in both mutants are due to recessive mutations in a single gene, designated
exp1. The
exp1 gene is predicted to encode an HMG1/2-like protein with two HMG domains. The transcription of
exp1 is strongly induced in the pileus 3
h before pileus expansion. This result, together with the fact that the
exp1 mutations cause a specific developmental phenotype, suggest that Exp1 is a novel, transcriptional regulator controlling the final phase of fruiting-body morphogenesis.
Recently, we identified the structurally related homeoproteins EGAM1, EGAM1N, and EGAM1C in both preimplantation mouse embryos and mouse embryonic stem (ES) cells. These EGAM1 homeoproteins act as ...positive or negative regulators of differentiation and cell growth in mouse ES cells, such that these proteins are considered transcriptional regulators. In this study, we investigated their nuclear localization and identified the amino acid residues crucial for the nuclear translocation of EGAM1 and EGAM1C. When expressed exogenously in pluripotent ES cells and somatic NIH3T3 cells, all EGAM1 homeoproteins localized to the nucleus. Analysis using the web-based tool PSORTII predicted a potential nuclear localization signal (NLS) motif, RKDLIRSWFITQRHR, in the homeodomain shared by EGAM1 and EGAM1C. The introduction of mutations, such as mutations from K or R, both basic amino acid residues, to A, in this potential NLS resulted in significant impairment of the nuclear localization of both EGAM1 and EGAM1C. In contrast, GFP fusion proteins of all the full-length EGAM1 homeoproteins failed to localize to the nucleus. These results, when taken together, suggest that basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C and the intact structures of the EGAM1 homeoproteins contribute, at least in part, to the nuclear localization of these proteins in mouse ES cells.
Genomic DNA encoding the Pleurotus ostreatus LccK laccase was fused with the Coprinopsis cinerea β-tubulin promoter and terminator, and introduced into a C. cinerea strain. Linkage analysis, native ...PAGE separations, substrate specificity investigations and expression profiling indicated that C. cinerea transformants secrete P. ostreatus LccK, suggesting that the introns of the lccK gene are correctly spliced and the signal peptide for secretion is functional in C. cinerea. Transformants constitutively expressing laccase may be useful for the degradation of aromatic compounds.
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.
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•A novel cap-expansionless mutant, (exp2–1), was isolated from Coprinopsis cinerea.•The exp2 gene encodes a putative transcription factor that has two C2H2 zinc finger motifs.•The exp2 gene was edited by CRISPR/Cas9.•The Exp2 protein regulates expression of cell wall-degrading enzymes.
A dominant mutation of the elongationless2 (eln2) gene of the mushroom Coprinus cinereus (eln2-1) affects pattern formation in the development of fruit body primordia, causing dumpy primordia which ...culminate in mature fruit bodies with short stipes. Phenotypic analyses revealed disturbance of tissue organization and cell morphogenesis in the primordial shaft and suggested that the defects in the primordial shaft interfere with differentiation of the stipe tissue in the mutant primordia. The eln2 locus was assigned to chromosome XII and the eln2-1 mutant gene was cloned from a chromosome XII-specific cosmid library as a genomic fragment that brings about the dominant mutant phenotype. The eln2 gene encodes a novel type of microsomal cytochrome P450 enzyme (CYP502) and is constitutively expressed. The eln2-1 mutation is a 4-bp deletion in the 3′-terminal region of the gene, leading to truncation of C-terminal 18 amino acids. We suggest that the truncation affects the activity of the CYP502 enzyme.
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