[Show abstract][Hide abstract] ABSTRACT: A suitable screening method for heterozygous DNA markers in shiitake,Lentinula edodes (Berk.) Pegler, is reported. Monokaryons were derived from a dikaryon by de-dikaryotization via protoplast formation. Compatibility
of the monokaryons was determined by pairwise culture on agar plates. We selected the primers to amplify polymorphic fragments
among the original strain (Hokken600∶H600) and two monokaryons (H600PP-39 and H600PP-67) showing compatibility. A total of
135 fragments were selected as specific random amplified polymorphic DNAs (RAPDs) resulting from 56 primers of the 147 primers
tested. Furthermore, we tested whether the polymorphic fragments segregated into 2∶2 among four strains isolated from a basidium.
Most of the polymorphic fragments (about 97.8%) showed 2∶2 segregation among the four strains. We concluded that the polymorphic
fragments were heterozygous if they were detected in either of the monokaryons (H600PP-39 and H600PP-67) and segregated to
2∶2 among four meiotic strains (H600B-1,-2, -3, and -4). A total of 132 heterozygous DNA markers were therefore selected from
a dikaryon of shiitake (Hokken600∶H600).
[Show abstract][Hide abstract] ABSTRACT: The purpose of this research was to analyze the karyotype of the interspecific fusants of twoPleurotus species. Auxotrophic mutants derived from the cultivated strain ofP. ostreatus andP. cornucopiae were used. Protoplasts were fused electrically, and the fusants were selected under auxotrophic complementation. Esterase
isozyme analysis showed that several fusants had isozyme bands originating from both parental strains, and others had unilateral
isozyme bands. The fusant that had expressed isozyme bands of both parental strains showed chromosomal DNA bands of both of
the parental strains in pulsedfield gel electrophoresis analysis. Despite the above results, the chromosomal composition of
the fusants obtained by the pulsed-field gel electrophoresis did not exhibit all of the bands of both fusion parents.
[Show abstract][Hide abstract] ABSTRACT: A mycovirus previously identified in brown discolored fruiting bodies of the cultivated mushroom Flammulina velutipes was characterized. We tentatively named the virus the F. velutipes browning virus (FvBV).
Purified FvBV particles contained two dsRNA genomes (dsRNA1 and 2). The complete sequence of dsRNA1 was 1,915 bp long, containing a single open reading frame (ORF) that encoded 580 amino acids of a putative 66-kDa RNA-dependent RNA polymerase (RdRp). dsRNA2 was 1,730 bp long containing a single ORF encoding 541 amino acids of a putative 60-kDa coat protein (CP1). Phylogenetic analysis of the RdRp sequences revealed FvBV to be a Partitivirus, most closely related to Chondrostereum purpureum cryptic virus. An RT-PCR assay was developed for the amplification of a 495-bp cDNA fragment from dsRNA encoding the CP1. When wild F. velutipes isolated from various parts of Japan were examined by RT-PCR assay, three isolates from the central region of Japan contained FvBV. One wild strain infected with FvBV was isolated in Nagano prefecture, where brown discoloration of white cultivated strains has occurred. Fruiting bodies produced by virus-harboring and virus-free F. velutipes were compared.
Cap color of the fruiting bodies of F. velutipes that contained Partitivirus FvBV was darker than FvBV-free fruiting bodies. The use of RT-PCR enabled association of FvBV and dark brown color of the fruiting body produced by F. velutipes strains.
[Show abstract][Hide abstract] ABSTRACT: In response to a change in the direction of gravity, morphogenetic changes of fruiting bodies of fungi are usually observed as gravitropism. Although gravitropism in higher fungi has been studied for over 100 years, there is no convincing evidence regarding the graviperception mechanism in mushrooms. To understand gravitropism in mushrooms, we isolated differentially expressed genes in Pleurotus ostreatus (oyster mushroom) fruiting bodies developed under three-dimensional clinostat-simulated microgravity. Subtractive hybridization, cDNA representational difference analysis was used for gene analysis and resulted in the isolation of 36 individual genes (17 upregulated and 19 downregulated) under clinorotation. The phenotype of fruiting bodies developed under simulated microgravity vividly depicted the gravitropism in mushrooms. Our results suggest that the differentially expressed genes responding to gravitational change are involved in several potential cellular mechanisms during fruiting body formation of P. ostreatus.
[Show abstract][Hide abstract] ABSTRACT: To understand the molecular mechanisms of fruiting body formation of basidiomycetous mushrooms, we have isolated over a 100 of developmentally regulated genes that were specifically transcribed during fruiting body development in Lentinula edodes (Shiitake-mushroom) by a subtractive hybridization, cDNA-RDA (cDNA representational difference analysis). One of these genes, named Le.flp1, was isolated from the primordial cDNA library of L. edodes, and the expression product of Le.flp1 and putative fungal homologues contained a characteristic region, homologous to the Fas domain of fasciclin family proteins, which are capable of promoting cell adhesion through Fas domain-mediated homophilic interactions in various organisms. RT-PCR analyses suggested that Le.flp1 was specifically expressed in primordia and mature fruiting bodies. In situ hybridization indicated that Le.flp1 transcripts were distributed distinctly in the following tissues: the inside of gills of fruiting bodies, especially at the boundary between the subhymenium and trama, where there is active proliferation of basidium cells for producing basidiospores; peripheral regions of the primordium, pileus and stipe; and both inner tissue and outer regions of the stipe. Our results suggest the hypothesis that Le.flp1 plays a role in cellular differentiation and development in ubiquitous tissues during fruiting body formation in L. edodes, possibly through cell adhesion.
Current Genetics 07/2007; 51(6):367-75. DOI:10.1007/s00294-007-0133-2 · 2.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The basidiomycete Lyophyllum decastes was transformed by means of particle bombardment. We isolated five transformants under twelve conditions differing in the
two parameters of target distance and helium pressure. The transformation frequency was one transformant/µg DNA. In the transformants,
plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of L. decastes by particle bombardment.
[Show abstract][Hide abstract] ABSTRACT: Mycorrhizal basidiomycetes, Suillus grevillei and S. bovinus, were transformed by particle bombardment. We isolated eight and four transformants from S. grevillei and S. bovinus respectively under three conditions differing in terms of target distance and helium pressure. The transformation frequencies were 1.2 and 0.6 transformants/microg DNA. Plasmid DNAs were integrated into the genomic DNA of the transformants and stably maintained. This is the first description of the transformation of S. grevillei and S. bovinus by particle bombardment.
[Show abstract][Hide abstract] ABSTRACT: Recombinants were generated from the ectomycorrhizal basidiomycete, Suillus grevillei, through agroinfection using a binary vector carrying the hygromycin B resistance and the autofluorescent protein, DsRed2, markers. DsRed2 was driven by a cis-regulatory region of the glyceraldeyde-3-phosphate dehydrogenase gene (gpd) from the wood-rotting basidiomycete, Coriolus hirsutus, which contains promoters and 5' gpd sequences with first through fourth exons and expressed for the first time in Suillus spp. The transformation system and recombinants expressing an autofluorescent protein may be useful in genetic analysis of the symbiosis.
[Show abstract][Hide abstract] ABSTRACT: Using agroinfection with a T-DNA vector carrying a hygromycin resistance marker, the recombinants were generated for the first
time from the ectomycorrhizal basidiomycete Tricholoma matsutake, which produces commercially valuable fruit bodies, matsutake, during association with Pinus sp. plants. The transformation system may be useful in the genetic analysis of T. matsutake.
[Show abstract][Hide abstract] ABSTRACT: To analyze genes involved in fruit body development of Pleurotus ostreatus, mRNAs from three different developmental stages: i.e., vegetative mycelium, primordium, and mature fruit body, were isolated and reverse-transcribed to cDNAs. One hundred and twenty random PCR amplifications were performed with the cDNAs, which generated 382, 394, 393 cDNA fragments from each developmental stage. From these fragments, four cDNA clones specifically expressed in primordium or mature fruit body were detected. Sequence analysis and database searches revealed significant similarity with triacylglycerol lipase, cytochrome P450 sterol 14 alpha-demethylase and developmentally regulated genes of other fungi. Northern blot analyses confirmed that all of the four cDNAs were unexpressed in mycelium, thus stage-specific genes for fruit body formation of P. ostreatus were successfully isolated.
[Show abstract][Hide abstract] ABSTRACT: The physical properties of sawdust including porosity, water retention, and water drainage were analyzed to prove its suitability
for use as an artificial soil in the automatic decomposer-extinguisher (GADE) machine. The physical and chemical properties
of residual sawdust from the GADE machine were also analyzed, the mechanical abrasion of sawdust in the GADE machine was tested,
and the morphology of this residue was observed through a scanning electron microscope to investigate changes of these properties
in the medium of decomposing garbage. Sawdust, which showed a lower specific gravity and larger porosity than soil, is considered
capable of supplying air to bacteria. It was found that sawdust became worn from the operation of the machine. The spaces
of residual sawdust were still observed, but water drainage decreased. The portion of hollocellulose in residual sawdust decreased,
although the extractives in it increased. Results indicated that the capacity of sawdust to function as an artificial soil
in the GADE machine was decreased owing not only to the destruction of sawdust grain but also to the adherence of products
from decomposition, such that sawdust needed to be replaced every few months.