In order to confirm the presence of putative glucoamylase gene in Tricholoma matsutake genome, the genomic DNA was prepared from T. matsutake NBRC30773 strain and was used as template to clone the glucoamylases gene (TmGlu1). We obtained the nucleotide sequence of TmGlu1 and its franking region. The coding region (from ATG to stop codon) is 2,186 bp. The locations of exons and introns were determined from the nucleotide sequences of 3’- and 5’-RACE PCR and RT- PCR products. On the other hand, to investigate the relationship between composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime reverse transcription PCR and measurement of glucoamylase enzyme activity. It was found that enzyme activity of glucoamylase was very low in different medium. Expression of glucoamylases gene appeared to not be affected by different carbon source.

In the bipolar basidiomycete Pholiota nameko, a pair of homeodomain protein genes located at the A mating-type locus regulates mating compatibility. In the present study, we used a DNA-mediated transformation system in P. nameko to investigate the homeodomain proteins that control the clamp formation. When a single homeodomain protein gene (A3- hox1 or A3-hox2) from the A3 monokaryon strain was introduced into the A4 monokaryon strain, the transformants produced many pseudo-clamps but very few clamps. When two homeodomain protein genes (A3-hox1 and A3-hox2) were transformed either separately or together into the A4 monokaryon, the ratio of clamps to the clamp-like cells in the transformants was significantly increased to approximately 50%. We, therefore, concluded that the gene dosage of homeodomain protein genes is important for clamp formation. When the sip promoter was connected to the coding region of A3-hox1 and A3-hox2 and the fused fragments were introduced into NGW19-6 (A4), the transformants achieved more than 85% clamp formation and exhibited two nuclei per cell, similar to the dikaryon (NGW12 -163 × NGW19-6). The results of real-time RT-PCR confirmed that sip promoter activity is greater than that of the native promoter of homeodomain protein genes in P. nameko. So, we concluded that nearly 100% clamp formation requires high expression levels of homeodomain protein genes and that altered expression of the A mating-type genes alone is sufficient to drive true clamp formation.

In the past studies of Lyophlium shimeji, it was reported that the quantity of sufficient starch used as a carbon source was able to supply the factor that allows successful fruit-body formation without raising osmotic pressure in the medium. Glucoamylase are exo Glucosyl hydrolase, which catalyze the release glucose from the nonreducing ends of amylose, amylopectin, and other polysaccharides. Glucoamylase genes are found in many prokaryotic and eukaryotic microbes that use starch as a carbon source. It was believed to be important in the utilization of starch by the basidiomycetous fungus. Glucoamylase activity in the medium increased markedly during fruit-body formation. So study of the characteristic of glucoamylase in Pholiota nameko will provide the basis for P .nameko fruit body formation. In this research, in order to confirm the presence of glucoamylase gene in P. nameko genome, the genomic DNA was prepared from P. nameko NGW19-6 strain and was used as template to amplify the glucoamylases gene (PnGlu1). To prepare genomic DNA from the P. nameko NGW19-6 strain, the mycelium was grown on 10 ml of PD liquid medium (potato 200 g/l ,Glucose 20 g/l) prepared with tap water in a 100 ml Erlenmeyer flask and at 25°C for 7 days. Genomic DNA fragment encoding the glucoamylase protein (PnGlu1) were amplified by PCR with degenerate primer F15-GP2-AF/F15-GP2-BR. The primer pair was designed based on the amino acid sequences GLGEPKF and FDLWEEI, respectively, which are conserved in the glucoamylase protein of Laccaria bicolor. This produce fragments of approximately 400 bp. Next, to amplify the whole genomic clone of PnGlu1, oligonucleotide primer PnGP2F/ PnGP2R were designed based on the nucleotide sequence of DNA fragments amplified by cassette PCR method. The produced fragment has significant homology with glucoamylase of L. bicolor. To investigate the relationship between different composition of medium and glucoamylase expression, we checked the expression level of glucoamylase gene by realtime RT-PCR and measurement of glucoamylase enzyme activity.

Detections of reactive oxygen species (ROS) during ectomycorrhiza establishment between Rhizopogon roseolus (shoro) and Pinus thunbergii were made microscopically using a nitro blue tetrazolium (NBT) staining. Roots of P. thunbergii were aseptically infected with R. roseolus mycelium by using a Petri dish technique. From 2- to 4-week period after inoculation, initial mycorrhizal formation could be observed. Lateral root tips were treated with NBT and then observed under a light microscope. Depositions of blue formazan indicating O2- accumulation were detected mainly hyphal cells contacting with the roots surface. Observations of transverse section of the root revealed that depositions of blue formazan were also detected at the plasma membranes of the epidermal cells where the fungal hyphae were adhesively contacted. In the non-inoculated P. thunbergii roots, depositions of formazan were observed in root hair cells but not in epidermal cells. From 4- to 8-week period after inoculation, dichotomous mycorrhizas and extraradical mycelia were clearly observed. A section from the mycorrhiza treated with NBT showed that root tissue was surrounded by fungal mantle sheath, in which highly intensive reaction with NBT was demonstrated. The reactive formazan complexes were apparent in Hartig net hyphae between epidermal and cortical cells of the root. After 16 weeks following inoculation, morphology of mycorrhizas became variable, viz., initial, dichotomous and browned mycorrhizas. The browned mycorrhizas were characterized by wrinkled surfaces and sparse extraradical mycelia. The browned mycorrhizas were collected and treated with NBT. A section from the specimen showed that depositions were slightly observed only in the part of extraradical mycelia. These results suggest that O2- generations from both fungus and plant are involved with the early establishment of ectomycorrhizas between R. roseolus and P. thunbergii.

Rhizopogon roseolus (Corda) Th. M. Fr. (=R. rubescens Tul. & Tul.), known as “shoro” in Japan, is a hypogeous basidiomycete that is an important ectomycorrhizal symbiont of the Pinaceae. Rhizopogon roseolus produced a fruiting body with a basic globose to subglobose shape. Basidiospores were encompassed in a glebal chamber in the fruiting body. However, little is known about basidiosporogenesis and nuclear behavior after karyogamy. We treated R. roseolus glebar chambers with Gimsa acid and observed their hymenium microscopically to characterize nuclear behavior and basidiosporogenesis. Our observations revealed the following five characteristics: ⅰ) meiosis and postmeiotic mitosis took place in the basidium; ⅱ) meiosis occurred in the center of the basidium; ⅲ) the sterigma appeared when the first meiotic division occurred; ⅳ) the center of the basidium constricted slightly when the second meiotic division occurred; ⅴ) after postmeiotic mitosis, asynchronous nuclear migration from the basidium to the basidiospores took place, producing eight uninucleate basidiospores. However, unusual nuclear behavior was frequently observed, indicating that regulations of the timing and the way of nucleus entering to the spores were not exact in R. roseolus.

Rhizopogon roseolus (Corda) Th. M. Fr. (=R. rubescens Tul. & Tul.), known as “shoro” in Japan, is a hypogeous basidiomycete that is an important ectomycorrhizal symbiont of Pinaceae. In order to cultivate this edible ectomycorrhizal mushroom, several researches have tried to promote mycorrhization of this mushroom on host Pinus thunbergii roots: Pine seedlings were inoculated with mycelium in vitro, or with crushed fruiting bodies in nature. However, successful cultivation of this mushroom has not been fully refined. We have developed the useful mycelial inoculum that enable to produced abundant ectomycorrhizas and then to form fruiting body under greenhouse nursery conditions. We selected the superior strain that rapidly colonized and produced a lot of ectomycorrhizas in root of P. thunbergii． The mycelial inoculum was composed of mineral solution and homogenate of mycelium that had been cultured in liquid medium. Addition of surfactant in the mycelial inoculum resulted in stimulation of mycorrhzal formations in host roots. When the mycelial inoculum containng surfactant were introduced to the mother plant system in which the colonized seedling had been planted into in the nursery, stimulatally effects were observed on not only mycorrhzation of the seedlings but also fruiting body formation. Genotype analysis using microsatellite markers for R. roseolus showed that fruiting bodies produced in the nursery were originated from the inoculated strain. These results suggest that the mycelial inoculum containg surfactant could be the model of mycelial spawn for “shoro”.

The genes encoding cellulases, which belong to glycosyl hydrolase families have been cloned from the basidiomycetous mushrooms. The transcripts of cellulase genes are strongly induced when the mycelia are grown in medium containing crystalline cellulose, and they are not expressed in medium containing glucose, but how insoluble substrates such as microcrystalline cellulose are recognized by these fungal cells is not clear. The polypore mushroom Polyporus arcularius is a wood-decomposing basidiomycete that produces at least three types (I, II, and IIIa) of carboxymethyl cellulase (CMCase) when the medium contains crystalline cellulose as the sole carbon source and produced mainly cellobiose in the medium. The genomic and cDNA clones encoding the family 12 endoglucanase (CMCase IIIa) gene (cel3A) of P. arcularius have been sequenced, and Cel3A has been expressed as an active enzyme in Escherichia coli. To determine the role and function of each type of cellulase in the degradation of crystalline cellulose by basidiomycetous mushrooms, the structure of all of the cellulase genes should be investigated, but the nucleotide sequences of the other cellulase genes in P. arcularius have not yet been reported. In the current study, the genomic and cDNA clones encoding the endoglucanases (cel4), and the two cellobiohydrolases (cel1 and cel2) of P. arcularius sequenced and characterized. The predicted amino acid sequence of Cel1 Cel2, Cel3a and Cel4 are similar to glycosyl hydrolase family 7, 6 12 and 5 protein, respectively. The expressions of the all cellulase genes (cel1 cel2, cel3a and cel4) were induced by Avicel (microcrystalline cellulose) and cellopentaose but repressed by glucose, cellobiose, cellotriose, and cellotetraose. There was a low level of transcription of both genes regardless of the carbon source. These results suggest that P. arcularius cells constitutively express a very low level of cellulase that can degrade insoluble crystalline cellulose and that the transcription of celluases in the cells is induced by products produced by these endoglucanases such as cellooligosaccharides. From our findings, we propose a possible mechanism for the recognition and degradation of insoluble crystalline cellulose by fungal cells.