Expression of marY1, a gypsy-type LTR-retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake, in the budding yeast Saccharomyces cerevisiae

marY1 is a gypsy-type LTR-retroelement present in the genome of the ectomycorrhizal homobasidiomycete Tricholoma matsutake. We document here that a marY1-lacZ gene fusion was expressed in the budding yeast Saccharomyces cerevisiae. The finding strongly suggests that marY1 is activated by trans-regulatory factors common to higher fungi, and may be useful for the development of new recombinant systems in ectomycorrhizal fungi and homobasidiomycetes.     

The long terminal repeat (LTR) sequence of marY1, a retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake, is highly conserved in various higher fungi.

marY1 is an LTR-retroelement from the homobasidiomycete Tricholoma matsutake. Nucleotide sequences that correspond to the putative U3-R region and the R-U5 region of marY1 are highly conserved in various higher fungi. Data suggest that the LTR sequence of marY1 originated early in the evolution of higher fungi and has become widely distributed. Therefore, it may be useful for the construction of an LTR-mediated transformation system in basidiomycetes.     

marY2N, a LINE-like non-long terminal repeat (non-LTR) retroelement from the ectomycorrhizal homobasidiomycete Tricholoma matsutake.

A LINE-like non-LTR retroelement designated marY2N was cloned from the ectomycorrhizal homobasidiomycete Tricholoma matsutake. marY2N has open reading frames that correspond to gag and pol, and a putative promoter and consensus sequences common to those of the mutators from fruit flies. While it is common to T. matsutake and Tricholoma magnivelare, marY2N does not reside in any other species of Tricholoma tested.     

DNA Polymorphisms in Lentinula edodes, the Shiitake Mushroom

DNA restriction fragment length polymorphisms (RFLPs) were examined in Lentinula edodes strains. Genomic DNA from strain 70 was cloned in plasmid vector pUC19, and 18 random clones containing low-copy DNA sequences were used to probe seven strains in Southern DNA-DNA hybridizations. Each cloned fragment revealed DNA polymorphism. An RFLP genotype was determined for each strain and the genetic relatedness was assessed. The coefficients of genetic similarity among the seven strains ranged from 0.43 to 0.90. The inheritance of RFLP markers was examined in single spore isolates. Homokaryons displayed a loss of polymorphic bands compared with the parent dikaryon. Hybrids constructed by crossing compatible homokaryons displayed the inheritance of RFLP markers from each parent homokaryon.     

Characterization of subrepeat regions within rDNA intergenic spacers of the edible basidiomycete Lentinula edodes

For 16 commercial cultivars of Lentinula edodes, DNA fragments for the nuclear rDNA intergenic spacers IGS1 and IGS2 were amplified and analyzed. IGS1 contained a subrepeat region, named SR1, and IGS2 contained a pair of direct repeats and a subrepeat region, named SR2. Three and five types of subrepeats were found in SR1 and SR2, respectively. Heterogeneity in the lengths of IGS1 and IGS2 arose mainly from the number of different kinds of subrepeats within SR1 and SR2. The DNA fingerprints from the PCR products targeting SR1 and SR2 were specific for each of the 16 cultivars, and had enough variation for discrimination among the cultivars. This result suggests that the DNA fingerprints targeting SR1 and SR2 are useful for investigations of L. edodes cultivars.     

Cloning and characterization of the gene encoding beta subunit of mitochondrial processing peptidase from the basidiomycete Lentinula edodes

We have isolated the gene encoding the beta subunit of mitochondrial processing peptidase (β-MPP) from the shiitake mushroom Lentinula edodes. It is a nuclear gene with two small introns. Comparison with known β-MPP genes revealed that the L. edodes gene is most closely related with that from Neurospora crassa, with 60.8% identities and 87% similarity in the amino-acid sequences. The deduced L. edodes β-MPP peptide sequence contains the inverse zinc-binding motif (H–X–E–H) that has been found in a large family of zinc-binding metalloproteinases including bacterial proteinases, insulin degrading enzymes and β-MPPs. The two histidines are thought to contribute two of the three residues for zinc binding. The expression of L. edodes β-MPP is higher during the development of the fruiting bodies, suggesting that higher mitochondrial activities may be required to meet the energy demand in the rapid growth of the fruiting bodies.     

Tdd-4, a DNA transposon of Dictyostelium that encodes proteins similar to LTR retroelement integrases.

Tdd-4 is the first DNA transposon to be isolated from Dictyostelium discoideum. This element was isolated by insertion into a target plasmid. Two classes of elements were identified which include a 3.8 kb version and a 3.4 kb deleted version. Sequence analysis reveals that the 145 bp inverted terminal repeats contain the 5'-TGellipsisCA-3' conserved terminal dinucleotides found in prokaryotic transposons and integrated LTR retroelement DNA sequences. Tdd-4 open reading frames are assembled by removal of six introns. Introns 1-5 conform to the GT-AG rule, whereas intron 6 appears to be an AT-AA intron. Also, intron 6 undergoes an alternative 5' splicing reaction. The alternatively spliced region encodes 15 tandem SPXX repeats that are proposed to function as a DNA binding motif. By analogy to other transposons that encode two proteins from the same gene, the full-length Tdd-4 protein is the putative transposase and the truncated Tdd-4 protein is the putative transposition inhibitor. Protein database searches demonstrate Tdd-4 encoded proteins are unique for a DNA element by containing similarities to retroviral/retrotransposon integrases. The putative Tdd-4 transposase contains the same structural relationship as integrases by possessing an N-terminal HHCC motif, a central DDE motif and a C-terminal DNA-binding domain composed of the SPXX motif.     

Molecular cloning of developmentally specific genes by representational difference analysis during the fruiting body formation in the basidiomycete Lentinula edodes

To understand molecular mechanisms of the fruiting body development in basidiomycetes, we attempted to isolate developmentally regulated genes expressed specifically during the fruiting body formation of Lentinula edodes (Shiitake-mushroom). cDNA representational difference analysis (cDNA-RDA) between vegetatively growing mycelium and two developmental substages, primordium and mature fruiting body, resulted in an isolation of 105 individual genes (51 in primordium and 54 in mature fruiting body, respectively). A search of homology with the protein databases and two basidiomycetous genomes in Phanerochaete chrysosporium and Coprinopsis cinerea revealed that the obtained genes encoded various proteins similar to those involved in general metabolism, cell structure, signal transduction, and responses to stress; in addition, there were apparently several metabolic pathways and signal transduction cascades that could be involved in the fruiting body development. The expression products of several genes revealed no significant homologies to those in the databases, implying that those genes are unique in L. edodes and the encoding products may possess possible functions in the course of fruiting body development. RT-PCR analyses revealed that 20 candidates of the obtained genes were specifically or abundantly transcribed in the course of the fruiting body formation, suggesting that the obtained genes in this work play roles in fruiting body development in L. edodes.     

Overexpression of the laccase gene,lcc1, in Lentinula edodes using the pChG vector

Lentinula edodes secretes laccase (Lcc: EC 1.10.3.2), an industrially useful enzyme. In this study, we introduced and expressed the L. edodes Lcc gene, lcc1, driven by L. edodes glyceraldehyde-3-phosphate dehydrogenase gene promoter into L. edodes. The resulting transformants showed 2-fold Lcc activity than that of the host strain, and expression of the recombinant lcc1 was confirmed by RT-PCR.     

Traceability of Asian Matsutake, specialty mushrooms produced by the ectomycorrhizal basidiomycete Tricholoma matsutake, on the basis of retroelement-based DNA markers

The ectomycorrhizal basidiomycete Tricholoma matsutake produces commercially valuable fruit bodies, matsutake, in forests. Here we report a PCR system targeting retroelement integration sites to differentiate among individual Asian isolates of T. matsutake based on their geographical origins, such as Japan, the area of South Korea through North Korea, the northeastern provinces of China, and the area of the southwestern provinces of China through Bhutan. The overall misjudgment rate of the analytical system was approximately 5% based on 95 samples of T. matsutake examined including those from cultures and from commodities. We also provide evidence that T. matsutake isolates grown throughout the Far East, including the northeastern provinces of China, are closely related to each other while distinct from those in the area of the southwestern provinces of China through Bhutan. The method allows us to trace back geographical origins of Asian matsutake, thus contributing to food safety, appropriate tariffs, and proper price setting.     

Reduction of organic and inorganic selenium compounds by the edible medicinal basidiomycete Lentinula edodes and the accumulation of elemental selenium nanoparticles in its mycelium

We report for the first time that the medicinal basidiomycete Lentinula edodes can reduce selenium from inorganic sodium selenite (Se^IV) and the organoselenium compound 1,5-diphenyl-3-selenopentanedione-1,5 (DAPS-25) to the elemental state, forming spherical nanoparticles. Submerged cultivation of the fungus with sodium selenite or with DAPS-25 produced an intense red coloration of L. edodes mycelial hyphae, indicating accumulation of elemental selenium (Se⁰) in a red modification. Several methods, including transmission electron microscopy (TEM), electron energy loss spectroscopy (EELS), and X-ray fluorescence, were used to show that red Se⁰ accumulated intracellularly in the fungal hyphae as electron-dense nanoparticles with a diameter of 180.51±16.82 nm. Under designated cultivation conditions, shiitake did not reduce selenium from sodium selenate (Se^VI).     

Purification, characterization, and molecular cloning of a pyranose oxidase from the fruit body of the basidiomycete, Tricholoma matsutake

A new H(2)O(2)-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H(2)O(2) and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V(max), K(m), and k(cat) for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50 degrees C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.     

Morphological mutation of Lentinula edodes mycelium, particularly detectable in the dikaryotic state

A morphological mutation particularly detectable in the dikaryotic state was found in Lentinula edodes. The mutant dikaryon was readily distinguishable from the normal dikaryon by the irregularly branched short hyphae, very slow hyphal growth, and sparse aerial hyphae. Genetic analysis revealed that expression of this mutation was controlled by a single recessive gene, mor-13. Linkage analysis showed that the mor-13 was not linked to either the incompatibility factors (A and B) or the five kinds of mor genes that were segregated independently of each other in a previous study. Contribution no. 380 from the Tottori Mycological Institute     

中国香菇种质资源中两种主要病毒的携带情况检测

香菇病毒病通常具有潜隐性特征,携带病毒的香菇菌株往往并不表现出明显症状,但一旦爆发常造成较大经济损失。本文采用RT-PCR检测技术,系统分析了中国香菇野生种质及栽培种质中两种主要病毒Lentinula edodes mycovirus HKB（LeV-HKB）和L. edodes partitivirus 1（LePV1）的携带情况。对来源于中国主要栽培省（市）的90个香菇栽培菌株的检测结果表明,有67.8%（61/90）的供试菌株单独或复合感染有LeV-HKB和LePV1中的1种或2种,LeV-HKB病毒携带率56.7%（51/90）,明显高于LePV1 35.6%（32/90）;栽培菌株两种病毒的复合感染率为24.4%（22/90）。对地理来源几乎覆盖整个中国的125个野生菌株的检测结果表明,有75.2%（94/125）的供试菌株至少感染了LeV-HKB和LePV1中的1种,8.8%（11/125）野生菌株同时感染了两种病毒,且野生菌株中LePV1病毒携带率70.4%（88/125）明显高于LeV-HKB 13.6%（17/125）。栽培菌株和野生菌株中LeV-HKB和LePV1病毒的存在与否与菌株地理来源没有明显相关性,显示香菇病毒极可能通过担孢子在自然界广泛传播。本研究为开展香菇菌种病毒病快速检测,从源头控制香菇病毒病传播,追踪病毒病的发生流行规律奠定了基础。