Transformation of the basidiomycete, Schizophyllum commune

Summary Protoplasts of aSchizophyllum commune tryptophan auxotroph (trp1), deficient in indole-3-glycerol phosphate synthetase (IGPS), were transformed to trp⁺ with plasmid DNA containing the SchizophyllumTRP1 sequence. Efficiencies up to 30 transformants per microgram of plasmid DNA were obtained. Southern blots reveal that the transforming DNA is integrated in chromosomal DNA. The trp⁺ phenotype of transformants is stable in meiosis and mitosis. Transformants possess IGPS activity comparable to wild-type cells.     

Chimeric pheromone receptors in the basidiomycete Schizophyllum commune

The pheromone receptor system of the basidiomycete Schizophyllum commune is capable of ligand discrimination to confer mating specificity. The pheromone receptors of the B alpha locus were investigated for ligand discrimination in a strategy of domain swapping experiments. Several altered phenotypes of chimeric receptors have been found. These include constitutive pheromone receptors which need no ligand for activation of the downstream cascade of events. In addition, receptors still dependent on ligand were identified that had altered pheromone activation profiles, including promiscuous receptors that are activated by pheromones of all nine specificities, including the former self. In addition, highly discriminative receptors were created which are activated by only two of the eight non-self-specificities. The chimeric receptors identify the last third of the receptor as the determinant for B alpha 1 specificity, whereas B alpha 2 specificity resides in noncontiguous domains covering the first and middle parts of the receptor molecule.     

Heterokaryon formation in the basidiomycete Schizophyllum commune by electrofusion of protoplasts

Summary Conditions for high frequency electrofusion of protoplasts from the basidiomycete Schizophyllum commune are described. Visual inspection revealed up to 30% of the protoplasts engaged in fusion. Using complementing nutritional mutations, nearly 7% of the regenerated protoplasts could be recovered as heterokaryotic mycelia. The method is probably equally applicable to other basidiomycetes such as Agaricus bisporus, permitting the recovery of fusion products in the absence of selection markers.     

A bacteriolytic muramidase from the basidiomycete Schizophyllum commune

The basidiomycete Schizophyllum commune produces an extracellular bacteriolytic enzyme when grown on heat-killed cells of Bacillus subtilis as sole C, N and P source. The enzyme catalyses the dissolution of isolated B. subtilis cell walls at an optimum pH of 3.2-3.4, releasing muramyl reducing groups, which indicates that it is a muramidase. Although low levels of enzyme activity are present when the fungus is grown in the absence of bacteria, full enzyme production appears to be induced by bacterial cells and repressed by glucose. Whole bacteria are not lysed by the enzyme at pH 3.3, but are rendered osmotically fragile, and lyse when the pH is raised to 7 or higher. The muramidase is effective against several Gram-positive bacteria but did not lyse any of the Gram-negative species tested.     

Developmentally regulated proteases from the basidiomycete Schizophyllum commune

The basidiomycete Schizophyllum commune produces three chromatographically distinguishable proteases which are capable of attack on a variety of other enzymes from S. commune and other sources. These proteases, which are produced during a specific phase of the development cycle, exhibit typical enzyme kinetic patterns, are active in the neutral to weakly alkaline pH range and are inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, and ovomucoid. No pattern of specificity toward the test enzymes could be discerned. The proteases co-purify with the activity which causes the increase in cold lability of S. commune phosphoglucomutase reported previously. In addition, one of the protease enzymes could be purified to the point where it had no significant ability to release trichloroacetic acid products from denatured substrates at pH 3 or pH 7. When undenatured hemoglobin was used as a substrate, the purified protease releases a relatively large molecular weight nonheme peptide. Relatively large peptides are also formed after proteolysis of rabbit muscle phosphoglucomutase. These results suggest that the protease carries out only limited proteolysis.     

Characterization of the genome of the basidiomycete Schizophyllum commune

DNA of Schizophyllum commune was isolated both from mycelial cells and from protoplasts. Nuclear DNA was isolated after solubilization of the mitochondria with the detergent Nonidet. The G + C content of the nuclear DNA was 57%, calculated from its buoyant density (1.7165 g/ml) and from the Tm (77.4 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). The buoyant density of the ribosomal cistrons was 1.707 g/ml. DNA isolated from purified mitochondria had a very low G + C content: 22% (rho = 1.6845 g/ml, Tm = 61.8 degrees C in 15 mM NaCl/1.5 mM trisodium citrate). Analysis of CsCl profiles and melting patterns suggested that mitochondrial DNA contains interspersed (A + T)-rich sequences. From reassociation analysis of sheared nuclear DNA the genome size of S. commune was determined to be 22.8 . 10(9) daltons. A small amount of DNA (0.5 . 10(9) daltons) bound to hydroxyapatite at zero time Cot. 7% of the genome (1.6 . 10(9) daltons) represented repetitive DNA.     

Action spectrum for fruiting in the basidiomycete Schizophyllum commune

An action spectrum for fruit body formation was determined in the range 280–723 nm for a dikaryon of Schizophyllum commune Fr. Action maxima occurred at 280 and 340–360 nm (main peak), and there were minor peaks at 437 and 467 nm. The quantum effectiveness at 360 nm was ca seven-fold compared to that of 437 nm light. Wavelengths longer than 500 nm were ineffective. Light also induced formation of brown pigment in the area producing fruit bodies. Wavelengths ranging from 260 to 300 nm injured aerial hyphae at the border of the colony. The possibility that a flavin or a pteridine may be the photoreceptor is discussed.     

Regulation of the pattern of protein synthesis in Schizophyllum commune by the incompatibility genes

The pattern of protein synthesis in various coisogenic mycelial types of Schizophyllum commune, viz. monokaryon, dikaryon, and homokaryons carrying primary mutations in the A and the B factors, was studied by two-dimensional gel electrophoresis. After pulse-labeling with ³⁵S-methionine, approximately 650 of 710 proteins analyzed were common to all mycelial types. Coisogenic monokaryons differed by only 2%, whereas the largest difference was found between these monokaryons and the dikaryon derived from them (6.6 and 7.7%). The majority of these differences fell into two about equally sized categories, i.e., proteins which were either specifically absent (“switched-off” proteins) or present (“switched-on” proteins) in the dikaryon. “Switched-on” proteins were on the average larger and slightly more acidic than “switched-off” proteins. The double factor mutant which best mimicked the dikaryon in morphology also best resembled the dikaryon in types of proteins synthesized. Unexpected, however, was the large overlap in proteins apparently controlled by each of the two incompatibility factors individually, despite the distinct morphological sequences directed by each of them.     

Cell wall metabolism during fruiting of the basidiomycete Schizophyllum commune

During the development of fruit bodies of the basidiomycete Schizophyllum commune, the alkali-insoluble (R glucan) and alkali-soluble (S glucan) cell wall fractions are synthesized during the entire course of morphogenesis. The water soluble glucan (WSG) is not synthesized after an early stage. There is also a relative increase in the proportion of S glucan during development which appears related to a change in the proportion of the components synthesized. Data are also presented to show that several fruiting mutants also have specific cell wall differences, and that there is a significant contribution to cell wall structure by genes which do not cause a macroscopically observable change in phenotype.     

Extracellular proteins secreted by the basidiomycete Schizophyllum commune in response to carbon source

The secretion of 1,4-beta-D-glucanases by the basidiomycete Schizophyllum commune in response to cellulose or cellobiose has been studied. The proteins were labeled with 35S, and the secretion of enzymes was measured by beta-glucosidase and carboxymethyl cellulase activities and by immunoprecipitation with specific antibodies. The antigen proteins used were a beta-glucosidase (Mr, 93,000), an avicelase (avicelase II; Mr, 64,000), and a carboxymethyl cellulose (carboxymethyl cellulase I; Mr 41,000). The beta-glucosidase was initially secreted as an Mr 110,000 form, which was followed later by lower-molecular-weight (88,000 to 93,000) forms. The avicelase II, which accounted for about 50% of the secreted labeled protein, had an Mr of 64,000. Secretion of the related avicelase I (Mr 61,000) followed later. The carboxymethyl cellulose I was secreted in two molecular weight forms, Mr 44,000 and 41,000. The evidence is consistent with the idea that three genes account for the secreted glucanase activities. Other species result from different glycosylation or proteolytic cleavage processing, which may occur during or after secretion. The beta-glucosidase secretion appears to be regulated differently than that of avicelase II or carboxymethyl cellulase I; the latter two were regulated coordinately under the conditions used in this work. No common immune determinants between the three antigens were observed.     

Dikaryons of the basidiomycete fungus Schizophyllum commune: evolution in long-term culture

The impact of ploidy on adaptation is a central issue in evolutionary biology. While many eukaryotic organisms exist as diploids, with two sets of gametic genomes residing in the same nucleus, most basidiomycete fungi exist as dikaryons in which the two genomes exist in separate nuclei that are physically paired and that divide in a coordinated manner during hyphal extension. To determine if haploid monokaryotic and dikaryotic mycelia adapt to novel environments under natural selection, we serially transferred replicate populations of each ploidy state on minimal medium for 18 months (approximately 13,000 generations). Dikaryotic mycelia responded to selection with increases in growth rate, while haploid monokaryotic mycelia did not. To determine if the haploid components of the dikaryon adapt reciprocally to one another's presence over time, we recovered the intact haploid components of dikaryotic mycelia at different time points (without meiosis) and mated them with nuclei of different evolutionary histories. We found evidence for coadaptation between nuclei in one dikaryotic line, in which a dominant deleterious mutation in one nucleus was followed by a compensatory mutation in the other nucleus; the mutant nuclei that evolved together had the best overall fitness. In other lines, nuclei had equal or higher fitness when paired with nuclei of other histories, indicating a heterozygote advantage. To determine if genetic exchange occurs between the two nuclei of a dikaryon, we developed a 24-locus genotyping system based on single nucleotide polymorphisms to monitor somatic exchange. We observed genetic exchange and recombination between the nuclei of several different dikaryons, resulting in genotypic variation in these mitotic cell lineages.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.     

Commitment to fruiting in synchronously developing cultures of the basidiomycete Schizophyllum commune

Summary 1. A simple technique is described for obtaining large numbers of localized and synchronized fruit bodies of Schizophyllum commune by lowering the carbon dioxide level in mated cultures. 2. The fruiting process is unaffected by a continuous supply of glucose (0.4–2%). However, synchrony and commitment are lost when the glucose concentration is raised to 4%. 3. Five per cent carbon dioxide inhibits the further development of fruit bodies at all stages except the expansion of the already formed basidiocarp. 4. Fruit bodies can develop at 30°C when a CO₂ trapping agent is employed, contrary to previous reports. 5. A possible relationship between the rate of vegetative growth and the initiation of fruiting is discussed.     

An efficient gene deletion procedure for the mushroom-forming basidiomycete Schizophyllum commune

Gene deletion in Schizophyllum commune is hampered by a low incidence of homologous integration. As a consequence, extensive screening is required to identify a transformant with the desired genotype. To alleviate this and to facilitate the assembly of deletion plasmids, vector pDelcas was constructed. This construct has a set of restriction sites, which allows for directional cloning of the flanking sequences at both sides of a nourseothricin resistance cassette. Moreover, it contains a phleomycin resistance cassette elsewhere in the plasmid, which is used to screen for transformants with an ectopic integration of the pDelcas derivative. The use of pDelcas derivatives in combination with an improved PCR screening protocol permitted the efficient identification of S. commune deletion strains. This procedure may also function in other basidiomycetes.