The exp2 gene, which encodes a protein with two zinc finger domains, regulates cap expansion and autolysis in Coprinopsis cinerea.

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.

Live-cell imaging of septins and cell polarity proteins in the growing dikaryotic vegetative hypha of the model mushroom Coprinopsis cinerea.

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.

Blue light exposure and nutrient conditions influence the expression of genes involved in simultaneous hyphal knot formation in Coprinopsis cinerea.

Light and nutrients are crucial environmental factors influencing fungal sexual reproduction. Blue light induces simultaneous hyphal knot formation in Coprinopsis cinerea mycelia grown on low-glucose media but not in mycelia grown on high-glucose media. Many hyphal knots are visible in the arc near the edge of the colony one day after 15 min of blue light stimulation. These findings collectively suggest that blue light accelerates hyphal knot induction in nutrient-limited conditions. Transcriptome analysis revealed that gene expression after light exposure is divided into at least two major stages. In the first stage, genes coding for fasciclin (fas1), cyclopropane-fatty-acyl-phospholipid synthases (cfs1 and cfs2), and putative lipid exporter (nod1) are highly expressed after 1 h of light exposure in the mycelial region where the hyphal knot will be developed. These genes are upregulated by blue light and not influenced by glucose condition and mating. These results suggest that although some of the genes are critical for induction of the hyphal knots, they are not sufficient for hyphal knot development. In the second gene expression stage, genes encoding galectins (cgl1-3), farnesyl cysteine-carboxyl methyltransferases, mating pheromone-containing protein, nucleus protein (ich1), and laccase (lcc1) are specifically upregulated at 10-16 h after blue light exposure when the mycelia are cultivated on low-glucose media. These genes might be involved in the architecture of hyphal knots or signal transduction for further fruiting body development. These results contribute to the understanding of the effect of environmental factors on sexual reproduction in basidiomycetous fungi.

Effects of pex1 disruption on wood lignin biodegradation, fruiting development and the utilization of carbon sources in the white-rot Agaricomycete Pleurotus ostreatus and non-wood decaying Coprinopsis 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 Coprinopsis cinerea Tup1 homologue Cag1 is required for gill formation during fruiting body morphogenesis.

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.

Differential interference contrast imaging using a pair of twisted nematic cells.

Nematic liquid crystal behaves like an optically uniaxial crystal whose optical axis coincides with the direction of molecular orientation. When an electric field is applied, a lateral shear of incident light is induced, depending on the angle of molecular inclination. While this may degrade the image quality for display applications, the precise electrical tunability of the lateral shear distance is desirable for differential interference contrast (DIC) imaging. In this Letter, a pair of twisted nematic (TN) cells is used for DIC imaging instead of the normal DIC prisms, and the unique optical properties of the TN cell are investigated for DIC imaging applications.

Strand-Specific RNA-Seq Analyses of Fruiting Body Development in Coprinopsis cinerea.

The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.

Determination of birefringence and slow axis distribution using an interferometric measurement system with liquid crystal phase shifter.

It is known that liquid crystal (LC) cells are useful as compact and easy-to-handle phase shifters that are readily coupled into the optics of standard microscope systems. Here, a uniformly aligned molecular LC phase shifter is introduced into a polarization microscope to attain a birefringence imaging system, using the phase-shift interferometric technique. Since the birefringence can be determined accurately only when the optical axis of the sample is parallel or perpendicular to the slow axis (variable axis) of the LC phase shifter, an improved data analysis method is proposed for determining the birefringence independently of the direction; a simple method of determining the slow axis distribution is also demonstrated. Measurements of the birefringence and slow axis distribution properties of a potato starch particle are demonstrated to confirm the novel determination method.

The Coprinopsis cinerea septin Cc.Cdc3 is involved in stipe cell elongation.

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.

Intact structure of EGAM1 homeoproteins and basic amino acid residues in the common homeodomain of EGAM1 and EGAM1C contribute to their nuclear localization in mouse embryonic stem cells.

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.

Insights into evolution of multicellular fungi from the assembled chromosomes of the mushroom Coprinopsis cinerea (Coprinus cinereus).

The mushroom Coprinopsis cinerea is a classic experimental model for multicellular development in fungi because it grows on defined media, completes its life cycle in 2 weeks, produces some 10(8) synchronized meiocytes, and can be manipulated at all stages in development by mutation and transformation. The 37-megabase genome of C. cinerea was sequenced and assembled into 13 chromosomes. Meiotic recombination rates vary greatly along the chromosomes, and retrotransposons are absent in large regions of the genome with low levels of meiotic recombination. Single-copy genes with identifiable orthologs in other basidiomycetes are predominant in low-recombination regions of the chromosome. In contrast, paralogous multicopy genes are found in the highly recombining regions, including a large family of protein kinases (FunK1) unique to multicellular fungi. Analyses of P450 and hydrophobin gene families confirmed that local gene duplications drive the expansions of paralogous copies and the expansions occur in independent lineages of Agaricomycotina fungi. Gene-expression patterns from microarrays were used to dissect the transcriptional program of dikaryon formation (mating). Several members of the FunK1 kinase family are differentially regulated during sexual morphogenesis, and coordinate regulation of adjacent duplications is rare. The genomes of C. cinerea and Laccaria bicolor, a symbiotic basidiomycete, share extensive regions of synteny. The largest syntenic blocks occur in regions with low meiotic recombination rates, no transposable elements, and tight gene spacing, where orthologous single-copy genes are overrepresented. The chromosome assembly of C. cinerea is an essential resource in understanding the evolution of multicellularity in the fungi.

The dst2 gene essential for photomorphogenesis of Coprinopsis cinerea encodes a protein with a putative FAD-binding-4 domain.

The fruiting-body primordium of Coprinopsis cinerea exhibits remarkable photomorphogenesis. Under a 12-h light/12-h dark regime, the primordium proceeds to the fruiting-body maturation phase in which the primordium successively undergoes basidiospore formation, stipe elongation and pileus expansion, resulting in the mature fruiting-body. In continuous darkness, however, the primordium never proceeds to the maturation phase: the pileus and stipe tissues at the upper part of the primordium remain rudimentary while the basal part of the primordium elongates, producing the etiolated "dark stipe" phenotype. In our previous studies, blind mutants, which produce dark stipes under light conditions that promote fruiting-body maturation in the wild-type, have been isolated, and two genes, dst1 and dst2, responsible for the mutant phenotype have been identified. In this study we show that the dst2-1 mutant exhibits a blind phenotype during asexual spore production in addition to that in fruiting-body photomorphogenesis. We also reveal that dst2 is predicted to encode a protein with a putative flavin adenine dinucleotide (FAD)-binding-4 domain. The two blind phenotypes, together with the existence of an FAD-binding domain in Dst2, suggest that Dst2 may play a role in perceiving blue light.

Identification and characterisation of structural maintenance of chromosome 1 (smc1) mutants of Coprinopsis cinerea.

A Coprinopsis cinerea homokaryotic fruiting strain was mutagenised, identifying a mutant that exhibited a hyphal growth temperature sensitive defect and hyphal knot development defect at an early fruiting stage, even at the hyphal growth permissive temperature. Microscopic observation suggested that the mutant nuclei exhibited defects in the metaphase to anaphase transition at the restrictive temperature. The gene in which the mutation occurred was cloned, sequenced and determined to be homologous to smc1. Sequence analyses of the mutant revealed deletion of 28 base pairs in the 19th intron of the Cc.smc1 gene, resulting in complete failure of splicing of that intron and in insertion of 14 amino acids in the C-terminal region of the Cc.Smc1 protein. We isolated eight hyphal growth revertants and identified four intragenic suppressors. All were the result of amino acid substitutions in the C-terminal region. Three of the suppressors caused reversion of the arrest in an early fruiting stage. One of the suppressors exhibited cold sensitivity and failed to suppress the fruiting defect, suggesting that flexibility of a lobe in the C-terminal region is important for proper function of Cc.Smc1.

The exp1 gene essential for pileus expansion and autolysis of the inky cap mushroom Coprinopsis cinerea (Coprinus cinereus) encodes an HMG protein.

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 3h 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 3h 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.

The dst1 gene involved in mushroom photomorphogenesis of Coprinus cinereus encodes a putative photoreceptor for blue light.

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.

A linkage map of the basidiomycete Coprinus cinereus based on random amplified polymorphic DNAs and restriction fragment length polymorphisms.

A genetic linkage map of the basidiomycete Coprinus cinereus was constructed on the basis of the segregation of 219 RAPD markers, 28 RFLP markers and the A and B mating-type loci among 40 random basidiospore progeny from a single cross between a wild-type homokaryon, KF(3)#2, and an AmutBmut strain, #326. Thirteen linkage groups covering a total of 1346cM were identified and correlated to the 13 chromosomes of this fungus by hybridization of RFLP and RAPD marker probes to CHEF blots. These probes also revealed chromosome length polymorphisms (CLP), which could be associated with haplotype plots of the progeny. The average kb/cM ratio in this cross was approximately 27.9kb/cM. The AmutBmut strain undergoes sexual development without mating, because of mutations in both A and B mating-type loci, and has been used to identify mutations affecting developmental processes such as dikaryosis, fruit body morphogenesis, and meiosis. The markers in the map, especially the RAPD ones, would facilitate mapping of genes responsible for such mutations induced in the AmutBmut strain.