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.

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.

Decolorization of the anthraquinone dye Cibacron Blue 3G-A with immobilized Coprinus cinereus in fluidized bed bioreactor.

Coprinus cinereus, which was able to decolorize the anthraquinone dye Cibacron Blue 3G-A (CB) enzymatically, was used as a biocatalyst for the decolorization of synthetic solutions containing this reactive dye. Coprinus cinereus was immobilized in both calcium alginate and polyacrylamide gels, and was used for the decolorization of CB from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when Coprinus cinereus was entrapped in calcium alginate beads, and was of about 3.84 mg g(-1) h(-1) with a 50% conversion time (t1/2) of about 2.60 h. Moreover, immobilized fungal biomass in calcium alginate continuously decolorized CB even after 7 repeated experiments without significant loss of activity, while polyacrylamide-immobilized fungal biomass retained only 67% of its original activity. The effects of some physicochemical parameters such as temperature, pH and dye concentration on decolorization performance of isolated fungal strain were also investigated.

Meiotic cytogenetics in Coprinus cinereus.

The basidiomycete fungus Coprinus cinereus has naturally synchronous meiosis and is amenable to analysis using an array of well-developed genetic and molecular tools. In this chapter, we explain in detail the two methods most commonly employed for C. cinereus, staining of intact gill segments and chromosome spreads, with an example of the application of each. We describe iron-hematoxylin staining of intact gill segments for the brightfield examination of meiotic progression, and the use of surface spreads and fluorescence in situ hybridization (FISH) to investigate meiotic chromosome pairing. Gill segments can alternatively be stained with DAPI for the determination of meiotic stage, or propidium iodide for the quantitation of nuclear DNA content, and the chromosome fixation and spreading techniques used for FISH are also suitable for immunolocalization studies of chromosomal proteins.

Optimization of the functional expression of Coprinus cinereus peroxidase in Pichia pastoris by varying the host and promoter.

Peroxidase from Coprinus cinereus (CiP) has attracted attention for its high specific activity and broad substrate spectrum compared with other peroxidases. In this study, the functional expression of this peroxidase was successfully achieved in the methylotrophic yeast Pichia pastoris. The expression level of CiP was increased by varying the microbial hosts and the expression promoters. Since a signal sequence, such as the alpha mating factor of Saccharomyces cerevisiae, was placed preceding the cDNA of the CiP coding gene, expressed recombinant CiP (rCiP) was secreted into the culture broth. The Mut+ Pichia pastoris host showed a 3-fold higher peroxidase activity, as well as 2-fold higher growth rate, compared with the Muts Pichia pastoris host. Furthermore, the AOX1 promoter facilitated a 5-fold higher expression of rCiP than did the GAP promoter.

Inheritance of DNA methylation in Coprinus cinereus.

We examined the inheritance of 5-methylcytosine residues at a centromere-linked locus in the basidiomycete Coprinus cinereus. Although methylated and unmethylated tracts were inherited both mitotically and meiotically the lengths of these tracts were variable. This variation was not confined to any one phase of the life cycle of the organism, and it usually involved the simultaneous de novo methylation of at least four HpaII-MspI sites. We also found that the higher levels of methylation at this locus were transmitted through meiosis, regardless of the level of methylation of the homologous chromosome.

A new meiotic endonuclease from Coprinus meiocytes.

Two different types of Coprinus meiotic nuclease have been previously reported by the authors which are believed to be involved in meiotic chromosome recombination [1,2]. A third meiotic endonuclease was purified from the cap tissues of the basidiocarp of Coprinus cinereus. The enzyme is a 60 kDa molecule composed of a monopolypeptide as revealed by SDS-PAGE and FPLC-Sephacryl S-300 gel filtration. The enzyme belongs to a type of endonuclease which can preferentially digest single-stranded DNA and requires divalent cations as a co-factor, most commonly Mg2+ ions. In the presence of this co-factor, the enzyme converts the supercoiled plasmid DNA (form I) to both the relaxed form (form II) and the linear form (form III). Ca2+ ions can also function as a co-factor, though, in this case, not only is form I plasmid converted to form II, but a few ladder bands between form I and form II are also produced. The Ca2+ ion effect as a cofactor can be prevented with ATP. Immunohistochemical observation shows that the enzyme is distributed in the surface of the gills, which contain the meiotic tissues. These characteristics clearly differ from those of the meiotic nucleases reported previously.

Production and characterization of radiation-sensitive meiotic mutants of Coprinus cinereus.

We have isolated four gamma-ray-sensitive mutants of the basidiomycete Coprinus cinereus. When homozygous, two of these (rad 3-1 and rad 9-1) produce fruiting bodies with very few viable basidiospores, the products of meiosis in this organism. A less radiation-sensitive allele of RAD 3, rad 3-2, causes no apparent meiotic defect in homozygous strains. Quantitative measurements of oidial survival of rad 3-1; rad 9-1 double mutants compared to the single mutants indicated that rad 3-1 and rad 9-1 mutants are defective in the same DNA repair pathway. In the few viable basidiospores that are produced by these two strains, essentially normal levels of meiotic recombination can be detected. None of the mutants exhibits increased sensitivity to UV radiation. Cytological examination of meiotic chromosomes from mutant and wild-type fruiting bodies showed that rad 3-1 homozygous strains fail to condense and pair homologous chromosomes during prophase I. Although rad 9-1 strains are successful at chromosome pairing, meiosis is usually not completed in these mutants.

Production of a lignocellulolytic enzyme system for simultaneous bio-delignification and saccharification of corn stover employing co-culture of fungi.

Aiming at improving the efficiency of transferring corn stover into sugars, an efficient lignocellulolytic enzyme system was developed and investigated by co-cultivation of the Coprinus comatus with Trichoderma reesei in a single bioreactor. The results showed that the lignocellulolytic enzyme activities of the co-culture exceeded that of the monoculture, suggesting synergistic interaction between two fungi. The highest laccase activity from the co-culture was 2.6-fold increase over that of the C. comatus monoculture and reached a peak 3days earlier. The maximum delignification obtained was 66.5% and about 82% of the original polysaccharides were converted into fermentable sugars by simultaneous bio-delignification and saccharification process. Correlation analysis showed that sugar yields were directly proportional to the lignin degradation. Our results suggested that co-fungi cultivation was a valuable technique for corn stover bioconversion, which could produce high efficiency of lignocellulolytic enzyme system as a cheaper alternative to commercial enzymes for industrial utilization.

Morphometric analysis of cell size patterning involved in gravitropic curvature of the stipe of Coprinus cinereus.

During gravitropic bending of the stipe of Coprinus cinereus the majority of elongation occurred in the apical region of the lower surface of the stipe, although some elongation was seen throughout the stipe. The final rate of elongation was similar at both the upper and lower stipe surfaces but the lower surface achieved this rate first (close to the reaction time 25 min), whilst the upper surface of the stipe only attained its final elongation rate after a period of acceleration of 150 min. Detailed morphometric analysis of cell size patterning in transverse sections revealed no significant differences in cross sectional area, spatial or proportional distribution of different cell types between the upper and lower regions of the gravitropic bend. Measurements of longitudinal cell size revealed significant differences in compartment size between the lower and upper region. Hyphal compartments of lower regions of the bend were on average four to five times longer than those of the upper region.

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.

Proliferating cell nuclear antigen (PCNA) interacts with a meiosis-specific RecA homologues, Lim15/Dmc1, but does not stimulate its strand transfer activity.

PCNA is a multi-functional protein that is involved in various nuclear events. Here we show that PCNA participates in events occurring during early meiotic prophase. Analysis of protein-protein interactions using surface plasmon resonance indicates that Coprinus cinereus PCNA (CoPCNA) specifically interacts with a meiotic specific RecA-like factor, C. cinereus Lim15/Dmc1 (CoLim15) in vitro. The binding efficiency increases with addition of Mg(2+) ions, while ATP inhibits the interaction. Co-immunoprecipitation experiments indicate that the CoLim15 protein interacts with the CoPCNA protein in vitro and in the cell extracts. Despite the interaction between these two factors, no enhancement of CoLim15-dependent strand transfer activity by CoPCNA was found in vitro. We propose that the interaction between Lim15/Dmc1 and PCNA mediates the recombination-associated DNA synthesis during meiosis.

Replication-dependent early meiotic requirement for Spo11 and Rad50.

Spo11 and the Rad50-Mre11 complex have been indirectly implicated in processes associated with DNA replication. These proteins also have been shown to have early meiotic roles essential for the formation of a programmed DNA double-strand break known in Saccharomyces cerevisiae to initiate meiotic recombination. In both S. cerevisiae and the basidiomycete Coprinus cinereus, spo11 and rad50 mutants are defective in chromosome synapsis during meiosis. Here we demonstrate that a partial restoration of synapsis occurs in C. cinereus spo11 and rad50 mutants if premeiotic DNA replication is prevented. Double mutants were constructed with spo11-1 or rad50-4 and another mutant, spo22-1, which does not undergo premeiotic DNA replication. In both cases, we observed an increase in the percentage of nuclei containing synaptonemal complex (SC) structures, with concomitant decreases in the percentage of nuclei containing axial elements (AE) only or no structures. Both types of double mutants demonstrated significant increases in the average numbers of AE and SC, although SC-containing nuclei did not on average contain more AE than did nuclei showing no synapsis. Our results show that Spo11-induced recombination is not absolutely required for synapsis in C. cinereus, and that the early meiotic role of both Spo11 and Rad50 in SC formation partially depends on premeiotic S phase. This dependency likely reflects either a requirement for these proteins imposed by the premeiotic replication process itself or a requirement for these proteins in synapsis when a sister chromatid (the outcome of DNA replication) is present.

Coordinated cell elongation alone drives tropic bending in stems of the mushroom fruit body of Coprinus cinereus.

During tropic bending in the stem of the mushroom fruit body of Coprinus cinereus the majority of extension occurred in the upper 20-30% of the stem. By attaching inert markers to the stem, it was shown that the outer flank of the bend initially has a faster rate of extension, although the inner flank matches this growth rate later in the response. Thus bending results from differential enhancement of growth rate rather than sustained differences. Large voids, up to 85 micrometers in diameter, observed in tropically bent stems showed no significant difference in number between inner and outer flanks but are implicated in bending because of their absence from unbent stems. Such voids may prevent the propagation of cracks through the stem tissue during bending. Creases at the external and lumen surfaces were also peculiar to bent stems and could represent constrictions caused by localized accumulation of stresses. Cell morphometric analysis of transverse sections of both flanks of the bend revealed no significant differences in hyphal diameter, distribution, or populations of cell types, but cells of the outer flank were four to five times longer than those of the inner. Thus, tropic bending requires only an increase in length of pre-existing inflated hyphae in the outer flank tissue.

Purification and characterization of an endonuclease from fruiting caps of basidiomycete Coprinus cinereus.

An endonuclease was purified from the cap tissues of basidiocarp of Coprinus cinereus collected at early meiotic prophase. It has an optimal activity at pH 7.0 and 37 degrees C. It is a cationic enzyme with a molecular mass of 22 kDa by gel filtration, and contains a 12-kDa and a 14-kDa peptide as revealed by SDS gel electrophoresis and Western blot analysis. An antiserum was produced in rabbits against the purified Coprinus endonuclease. The specificity of this antiserum was demonstrated in a dot-blot analysis and, more critically, in an immunoinhibition of endonuclease activity. The Coprinus endonuclease requires Mg2+ and/or Ca2+ as co-factors. Ca2+ is more efficient than Mg2+ while the effect of combining both co-factors is the highest. The Coprinus endonuclease has a substrate preference for single-strand and supercoiled DNA. It gives only single-strand nicks on supercoiled DNA at low enzyme concentration and limited time of incubation. At high enzyme concentration and/or long incubation time, double-strand fragmentation occurred. As is discussed, this endonuclease is believed to be involved in the early phase of meiotic recombination.

Meiosis in Coprinus VII. The prekaryogamy S-phase and the postkaryogamy DNA replication in C. lagopus.

The kinetics of incorporation of 32P into DNA have unequivocally shown that the premeiotic S-phase in Coprinus lagopus occurs before the onset of karyogamy. It takes 8 h under the control conditions (25 degrees C with a 16 h light-8 h dark regime) but only 6 h under the arrest-release conditions. An important discovery in this study is that the initiation of premeiotic DNA replication is subject to an arrest by restrictive conditions (35 degrees C under a continuous light regime) whereas that of the mitotic replication is not. Once initiated, meiotic DNA replication can continue even under the restrictive conditions. Incorporation of 32P into DNA at pachytene is quite extensive. These replications are considered to be repair replications.

A new meiotic protein factor which enhances activity of meiotic DNA polymerase from Coprinus cinereus.

Meiotic cells of Coprinus contain a protein that can be identified by its ability to enhance activity of the meiotic DNA polymerase reported previously (8). Its activity is found only during the prophase stages in meiotic cells, and is accompanied by the meiotic polymerase. The protein, which was purified to near homogeneity, is a single polypeptide with a molecular mass of 30k, and shows no activities of nuclease, ATPase, or DNA-binding protein. The protein could enhance the meiotic polymerase activity by at least 5 fold. The other Coprinus polymerases were not influenced by the protein. The protein could increase Vmax value of the meiotic polymerase, but not the Km. The significance of this protein to meiotic DNA synthesis is considered in relation to other biochemical properties of meiotic cells.