Near-complete de novo assembly of Tricholoma bakamatsutake chromosomes revealed the structural divergence and differentiation of Tricholoma genomes.

Tricholoma bakamatsutake, which is an edible ectomycorrhizal fungus associated with Fagaceae trees, may have diverged before the other species in Tricholoma section Caligata. We generated a highly contiguous whole-genome sequence for T. bakamatsutake SF-Tf05 isolated in an Oak (Quercus salicina) forest in Japan. The assembly of high-fidelity long reads, with a median read length of 12.3 kb, resulted in 13 chromosome-sized contigs comprising 142,068,211 bases with an average guanine and cytosine (GC) content of 43.94%. The 13 chromosomes were predicted to encode 11,060 genes. A contig (122,566 bases) presumably containing the whole circular mitochondrial genome was also recovered. The chromosome-wide comparison of T. bakamatsutake and Tricholoma matsutake (TMA_r1.0) indicated that the basic number of chromosomes (13) was conserved, but the structures of the corresponding chromosomes diverged, with multiple inversions and translocations. Gene conservation and cluster analyses revealed at least 3 phylogenetic clades in Tricholoma section Caligata. Specifically, all T. bakamatsutake strains belonged to the "bakamatsutake" clade, which is most proximal to the "caligatum" clade consisting of Tricholoma caligatum and Tricholoma fulvocastaneum. The constructed highly contiguous nearly telomere-to-telomere genome sequence of a T. bakamatsutake isolate will serve as a fundamental resource for future research on the evolution and differentiation of Tricholoma species.

Identification of upregulated genes in Tricholoma matsutake mycorrhiza.

Many plant roots associate with fungi to form mycorrhizae; tree roots especially associate with ectomycorrhizal fungi, such as Tricholoma species. Tricholoma matsutake is an economically important fungus in Asian countries and usually inhabits forests primarily composed of Pinus densiflora (Japanese red pine). In this study, to understand the mycorrhizal association between T. matsutake and P. densiflora, genes specifically expressed in mycorrhiza compared with those expressed in mycelia and fruiting bodies were identified by RNA-seq. This revealed that genes for chromatin, proteasomes, signal transduction, pheromones, cell surface receptors, cytoskeleton, RNA processing and transporters from T. matsutake were highly expressed in mycorrhiza. It also identified 35 mycorrhiza-induced small secreted proteins (MiSSPs) that were highly expressed in mycorrhiza. Meanwhile, genes for proteases, defence-related proteins, cell-wall degradation, signal transduction, pinene synthesis, plant hormones and transporters from P. densiflora were highly expressed in mycorrhiza. These genes may be involved in mycorrhizal formation and maintenance. A MiSSP, 1460819, was highly expressed in mycorrhiza, and this expression was maintained for 24 months. These results provide insight into the mycorrhizal association between T. matsutake and P. densiflora.

The ectomycorrhizal association of Tricholoma matsutake and two allied species, T. bakamatsutake and T. fulvocastaneum, with native hosts in subtropical China.

Accurate identification of edible ectomycorrhizal (ECM) mushrooms and their host trees in nature is key to commercial production for consumption. For the first time we describe the ectomycorrhizal association of the three most common species of edible matsutake mushrooms with their native host trees in Yunnan Province in China. We collected ECM samples from three different localities in subtropical forests known to be highly productive areas of Tricholoma. Additionally, we collected basidiomata of Tricholoma from the field and markets in Yunnan. ECM samples were analyzed using morphological and molecular methods. We conducted phylogenetic analyses of nuc rDNA internal transcribed spacer region ITS1-5.8S-ITS2 (ITS) and analyzed the intergenic spacer of cpDNA psbA-trnH to identify basidiomata and plant hosts, respectively. Three species of Tricholoma were identified: T. bakamatsutake, T. fulvocastaneum, and T. matsutake. Four ECM associations in the study area were detected: Tricholoma bakamatsutake + Castanopsis tibetana, T. fulvocastaneum + C. tibetana, T. fulvocastaneum + Pinus yunnanensis, and T. matsutake + P. yunnanensis. Detailed descriptions and illustrations of the ECM associations are presented.

Quantitative transcriptomic and metabolomic analyses reveal the changes in Tricholoma matsutake fruiting bodies during cold storage.

A combination of transcriptomic and metabolomic analyses was performed to systematically understand the metabolic changes in Tricholoma matsutake fruiting bodies during cold storage. In total, 800 metabolites were identified and 19,964 annotated unigenes were quantified. The unigenes related to the catabolism of proteins, carbohydrates, and lipids were mainly upregulated during cold storage, but the related primary metabolites were not accumulated, which indicated complete degradation and loss of nutrients. Concurrently, the synthesis and metabolism of the main components of the cell wall, chitin and ß-1,3-glucan, were regulated, indicating the dynamic remodeling of the T. matsutake cell wall structure. Additionally, indole-3-acetic acid and components of its synthesis pathway were found in T. matsutake, indicating their potential role as a communicator between T. matsutake and its symbiotic plants. The results provide new information to improve the understanding of the metabolic mechanism of T. matsutake fruit bodies during postharvest cold storage.

Tricholoma alpinum, sp. nov., under five-needle pines in alpine and subalpine zones in Japan.

Alpine regions in Japan are characterized by the Siberian dwarf pine, Pinus pumila. Although these regions are conserved as national parks due to their unique biome, few reports of the fungal biota are available. We examined mushroom fungi under such vegetation and found a unique yellowish-capped Tricholoma species. Multilocus molecular phylogenetic analyses and morphological observation of specimens showed that the Tricholoma species is very similar to T. fumosoluteum known in North America. The Japanese yellowish-capped T. aff. fumosoluteum had larger basidiospores and basidia and shorter pileipellis hyphae compared with T. fumosoluteum. Therefore, we describe the Japanese entity as a new species, T. alpinum.

Dynamic evolution of eukaryotic mitochondrial and nuclear genomes: a case study in the gourmet pine mushroom Tricholoma matsutake.

Fungi, as eukaryotic organisms, contain two genomes, the mitochondrial genome and the nuclear genome, in their cells. How the two genomes evolve and correlate to each other is debated. Herein, taking the gourmet pine mushroom Tricholoma matsutake as an example, we performed comparative mitogenomic analysis using samples collected from diverse locations and compared the evolution of the two genomes. The T. matsutake mitogenome encodes 49 genes and is rich of repetitive and non-coding DNAs. Six genes were invaded by up to 11 group I introns, with one cox1 intron cox1P372 showing presence/absence dynamics among different samples. Bioinformatic analyses suggested limited or no evidence of mitochondrial heteroplasmy. Interestingly, hundreds of mitochondrial DNA fragments were found in the nuclear genome, with several larger than 500 nt confirmed by PCR assays and read count comparisons, indicating clear evidence of transfer of mitochondrial DNA into the nuclear genome. Nuclear DNA of T. matsutake showed a higher mutation rate than mitochondrial DNA. Furthermore, we found evidence of incongruence between phylogenetic trees derived from mitogenome and nuclear DNA sequences. Together, our results reveal the dynamic genome evolution of the gourmet pine mushroom.

Biosynthesis of (R)-(-)-1-Octen-3-ol in Recombinant Saccharomyces cerevisiae with Lipoxygenase-1 and Hydroperoxide Lyase Genes from Tricholoma matsutake.

Tricholoma matsutake is an ectomycorrhizal fungus, related with the host of Pinus densiflora. Most of studies on T. matsutake have focused on mycelial growth, genes and genomics, phylogenetics, symbiosis, and immune activity of this strain. T. matsutake is known for its unique fragrance in Eastern Asia. The most major component of its scent is (R)-(-)-1-octen-3-ol and is biosynthesized from the substrate linoleic acid by the sequential reaction of lipoxygenase and peroxide lyase. Here, we report for the first time the biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake using the yeast Saccharomyces cerevisiae as a host. In this study, cDNA genes correlated with these reactions were cloned from T. matsutake, and expression studies of theses genes were carried out in the yeast Saccharomyces cerevisiae. The product of these genes expression study was carried out with Western blotting. The biosynthesis of (R)-(-)- 1-octen-3-ol of T. matsutake in recombinant Saccharomyces cerevisiae was subsequently identified with GC-MS chromatography analysis. The biosynthesis of (R)-(-)-1-octen-3-ol with S. cerevisiae represents a significant step forward.

Unusual genome expansion and transcription suppression in ectomycorrhizal Tricholoma matsutake by insertions of transposable elements.

Genome sequencing of Tricholoma matsutake revealed its unusually large size as 189.0 Mbp, which is a consequence of extraordinarily high transposable element (TE) content. We identified that 702 genes were surrounded by TEs, and 83.2% of these genes were not transcribed at any developmental stage. This observation indicated that the insertion of TEs alters the transcription of the genes neighboring these TEs. Repeat-induced point mutation, such as C to T hypermutation with a bias over "CpG" dinucleotides, was also recognized in this genome, representing a typical defense mechanism against TEs during evolution. Many transcription factor genes were activated in both the primordia and fruiting body stages, which indicates that many regulatory processes are shared during the developmental stages. Small secreted protein genes (<300 aa) were dominantly transcribed in the hyphae, where symbiotic interactions occur with the hosts. Comparative analysis with 37 Agaricomycetes genomes revealed that IstB-like domains (PF01695) were conserved across taxonomically diverse mycorrhizal genomes, where the T. matsutake genome contained four copies of this domain. Three of the IstB-like genes were overexpressed in the hyphae. Similar to other ectomycorrhizal genomes, the CAZyme gene set was reduced in T. matsutake, including losses in the glycoside hydrolase genes. The T. matsutake genome sequence provides insight into the causes and consequences of genome size inflation.

Exploiting the potential of metal and solvent tolerant laccase from Tricholoma giganteum AGDR1 for the removal of pesticides.

Laccase from previously reported hardwood degrading fungus, Tricholoma giganteum AGDR1, was isolated, identified at molecular level, biochemically characterized and also utilized for pesticide degradation. Laccase gene is comprised of 3752 bp, which encompassed 742-bp of 5' flanking upstream sequence with 12 introns and 12 exons. Mature enzyme possesses 391 amino acids and signal peptide, which is determined to be monomeric protein with an apparent molecular weight of 41 kDa and 6.45 pI. Higher optimal activities were observed at 45 °C and pH 3.0 and surprisingly, it exhibited more than 20% of relative activity at pH 1.5. Purified laccase was tolerant to 100 mM of metals (i.e. Se, Pb, Cu, Cr and Cd), organic solvents (ethyl acetate, methanol, ethanol and acetone) and potent inhibitors (hydroxylamine, thiourea, NaF and Na-azide) as compared to reported laccases. It was able to degrade 29%, 7% and 72% of chlorpyrifos, profenofos and thiophanate methyl within 15 h, respectively. Molecular docking analysis revealed that higher binding efficacy of these pesticides is observed with H83, H320, A95, V384, and P366 which are presented near to the catalytic site. Based on the results, T. giganteum AGDR1 laccase can be applied for the potential remediation and industrial applications under harsh conditions.

Edible ectomycorrhizal fungi and Cistaceae. A study on compatibility and fungal ecological strategies.

Wild edible mycorrhizal mushrooms are among the most appreciated and prized mushrooms in the world. Despite the cultivation of ectomycorrhizal (ECM) mushrooms has been a growing subject of study worldwide, it has been hampered by the mutualistic lifestyle of the fungi. Although not being obligate symbionts, most of the species of ECM mushrooms only produce fruit bodies in association with trees or shrubs. In the present study, we aimed at understanding certain aspects of the ecology of four different edible ECM fungi: Lactarius deliciosus, Tricholoma equestre, T. portentosum and Boletus fragrans. Despite having a broad distribution worldwide, these fungi inhabit also Mediterranean habitats with understories typically dominated by rockroses (Cistaceae). Studying the ecology of these mutualistic fungi as well as the interaction with these species of shrubs is not only scientifically relevant but also pivotal for the discovery of profitable cultivation protocols. We evaluated the compatibility of these ECM species with five species within Cistaceae family - Cistus ladanifer, C. psilosepalus, C. salviifolius, Halimium halimifolium and Tuberaria lignosa. Each species of fungi proved to be able to establish mycorrhizas with at least 2 different plants species but varied in their host range of the tested Cistaceae. The dissimilarity in terms of host specificity between some fungal species seemed to be connected with the phylogenetic distances of the fungi. A correlation between the colonization percentage of the root systems and the mycelial growth rates in pure culture was found. The connection of these traits might be an important key to understanding the ecological competitor-colonizer tradeoffs of these ECM fungal species. Altogether, our study reports unknown plant-fungi combinations with economical relevance and also adds new insights about the ecology of these species of ECM fungi.

Purification and characterization of a glycoside hydrolase family 5 endoglucanase from Tricholoma matsutake grown on barley based solid-state medium.

An endoglucanase was isolated from solid-state culture of the ectomycorrhizal fungus Tricholoma matsutake (TmEgl5A) grown on rolled barley and vermiculite. The enzyme was purified by ammonium sulfate fractionation, ion-exchange, hydrophobic, and gel filtration. TmEgl5A showed a molecular mass of approximately 40 kDa as determined by SDS-PAGE. The single band of the protein was analyzed by peptide-mass-finger-printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and the trypsin-digested peptide sequences were matched to a putative endoglucanase sequence (protein ID1465229) in the JGI T. matsutake 945 v3.0 genome database. Based on the sequence information, the gene encoding TmEgl was cloned and expressed in Pichia pastoris KM71H. The deduced amino acid sequence was similar to GH5 family endoglucanases from Basidiomycetes. The enzyme acts on barley ß-glucan, lichenan, and CMC-Na. The hydrolyzation products from these substrates were detected by thin-layer chromatography as oligosaccharides with minimal disaccharides. These results suggested that T. matsutake produces a typical endoglucanase in solid-state culture, and the fungus has the potential to degrade ß-linkage polysaccharides.

Identification of the Pol Gene as a Species-Specific Diagnostic Marker for Qualitative and Quantitative PCR Detection of Tricholoma matsutake.

Tricholoma matsutake is a rare, precious, and wild edible fungus that could not be cultivated artificially until now. This situation has given way to the introduction of fake T. matsutake commodities to the mushroom market. Among the methods used to detect food adulteration, amplification of species-specific diagnostic marker is particularly important and accurate. In this study, the Pol gene is reported as a species-specific diagnostic marker to identify three T. matsutake varieties and 10 other types of edible mushrooms through qualitative and quantitative PCR. The PCR results did not reveal variations in the amplified region, and the detection limits of qualitative and quantitative PCR were found to be 8 ng and 32 pg, respectively. Southern blot showed that the Pol gene exists as a single copy in the T. matsutake genome. The method that produced the purest DNA of T. matsutake in this study was also determined, and the high-concentration salt precipitation method was confirmed to be the most suitable among the methods tested. The assay proposed in this work is applicable not only to the detection of raw materials but also to the examination of processed products containing T. matsutake.

Diversity and taxonomy of Tricholoma species from Yunnan, China, and notes on species from Europe and North America.

Although taxonomic knowledge on Tricholoma (Agaricales, Basidiomycota) is fairly comprehensive in northwest Europe, knowledge of the global diversity and distribution of Tricholoma spp. is still sparse. In this study, the diversity and distribution of some Tricholoma spp. are analyzed by morphological and molecular methods based on 70 collections from Yunnan, China, 45 from central Europe, 32 from Colorado, USA, 9 from Japan, and 3 from Ukraine. A Holarctic distribution is suggested for several species, based on collections and nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) sequences. Six species new to science are formally described from Yunnan: five in existing sections, Tricholoma forteflavescens, T. olivaceoluteolum, T. melleum, T. olivaceum, and T. sinoportentosum, and one, T. muscarioides, in the newly described section Muscaria alongside several previously described species. Additional putatively new species cannot be formally described because they lack sufficient material. Tricholoma foliicola is recognized as a species of the genus Gerhardtia.

Heavy-ion beam mutagenesis of the ectomycorrhizal agaricomycete Tricholoma matsutake that produces the prized mushroom "matsutake" in conifer forests.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom "matsutake" in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

North American matsutake: names clarified and a new species described.

Tricholoma matsutake, known widely as "matsutake," has great commercial and cultural significance in Japan. Because Japanese production is insufficient to meet the high domestic demand, morphologically similar mushrooms, thought by many to belong to T. magnivelare, are imported from western North America. However, molecular data produced since the early 2000s have indicated that more than one species of matsutake occur in North America and this raises the question of correct naming for the different species. To address this question, we assessed the phylogenetic diversity within North American matsutake based on nuc rDNA ITS1-5.8S-ITS2 (internal transcribed spacer [ITS] barcode) sequences, including newly obtained sequences from the type collections for Agaricus ponderosus and Armillaria arenicola, and morphological characters. Our results agree with earlier indications that three matsutake species occur in North America and allow us to clarify the correct application of names-T. magnivelare from the eastern USA and Canada, T. murrillianum from the western USA and Canada, and T. mesoamericanum from Mexico, newly described here. The existence of the three North American species is further supported by the results of evolutionary divergence analysis, geographical distributions, and morphological characters.

Hydrophobins in the Life Cycle of the Ectomycorrhizal Basidiomycete Tricholoma vaccinum.

Hydrophobins-secreted small cysteine-rich, amphipathic proteins-foster interactions of fungal hyphae with hydrophobic surfaces, and are involved in the formation of aerial hyphae. Phylogenetic analyses of Tricholoma vaccinum hydrophobins showed a grouping with hydrophobins of other ectomycorrhizal fungi, which might be a result of co-evolution. Further analyses indicate angiosperms as likely host trees for the last common ancestor of the genus Tricholoma. The nine hydrophobin genes in the T. vaccinum genome were investigated to infer their individual roles in different stages of the life cycle, host interaction, asexual and sexual development, and with respect to different stresses. In aerial mycelium, hyd8 was up-regulated. In silico analysis predicted three packing arrangements, i.e., ring-like, plus-like and sheet-like structure for Hyd8; the first two may assemble to rodlets of hydrophobin covering aerial hyphae, whereas the third is expected to be involved in forming a two-dimensional network of hydrophobins. Metal stress induced hydrophobin gene hyd5. In early steps of mycorrhization, induction of hyd4 and hyd5 by plant root exudates and root volatiles could be shown, followed by hyd5 up-regulation during formation of mantle, Hartig' net, and rhizomorphs with concomitant repression of hyd8 and hyd9. During fruiting body formation, mainly hyd3, but also hyd8 were induced. Host preference between the compatible host Picea abies and the low compatibility host Pinus sylvestris could be linked to a stronger induction of hyd4 and hyd5 by the preferred host and a stronger repression of hyd8, whereas the repression of hyd9 was comparable between the two hosts.

Identification of Mushroom Species by Automated rRNA Intergenic Spacer Analysis (ARISA) and Its Application to a Suspected Case of Food Poisoning with Tricholoma ustale.

Automated rRNA intergenic spacer analysis (ARISA), a method of microbiome analysis, was evaluated for species identification of mushrooms based on the specific fragment sizes. We used 51 wild mushroom-fruiting bodies collected in the centre of Hokkaido and two cultivated mushrooms. Samples were hot-air-dried and DNA were extracted by a beads beating procedure. Sequencing analysis of portions of the rRNA gene (rDNA) provided 33 identifications of mushrooms by genus or species. The results of ARISA identification based on the combination of the fragment sizes corresponding to two inter spacer regions (ITS2 and ITS1) of rDNA within±0.1% accuracy showed that 27 out of the 33 species had specific fragment sizes differentiated from other species. The remaining 6 species formed 3 pairs that showed overlapping fragment sizes. In addition, within-species polymorphisms were observed as 1 bp differences among 32 samples of 13 species. ARISA was applied to investigate a case of suspected food poisoning in which the mushroom was thought to be a toxic Kakishimeji. The morphological identification of the mushroom was ambiguous since the remaining sample lacked a part of the fruiting body. Further, yeast colonies had grown on the surface of the fruiting body during storage. The ARISA fragment size of the mushroom showed 7 bp difference from that of the candidate toxic mushroom. Although ARISA could be a useful tools for estimation of mushroom species, especially in case where the fruiting bodies have deteriorated or been processed, further studies are necessary for reliable identification. For example, it may be necessary to adopt more informative genes which could provide clearer species-specific polymorphisms than the ITS regions.

Complete mitochondrial genome of the ectomycorrhizal fungus Tricholoma matsutake.

Tricholoma matsutake is an ectomycorrhizal fungus belonging to the class Agaricomycetes and the phylum Basidiomycota. Here, we report the complete mitochondrial genome sequence of T. matsutake. The mitochondrial DNA (mtDNA) of T. matsutake seems to be concatenated, and harbors 76,037 nucleotides, with an overall GC content of 20.69%. The mtDNA comprises 15 protein-coding genes, 28 tRNA genes, and 2 rRNA genes. A comparative mitogenomic analysis between Agaricomycetes and other classes (Exobasidiomycetes, Pucciniomycetes, Tremellomycetes, and Ustilaginomycetes) revealed that species belonging to the class Agaricomycetes preferably harbor the codon UUA (coding for leucine) in their mtDNAs, whereas other classes prefer the codon CUA. This bias in the usage of the leucine codon could be the result of evolutionary divergence between the Agaricomycetes and other classes. The T. matsutake mtDNA sequence thus provides insight into the evolutionary characteristics of the Agaricomycetes mtDNAs.

Dehydrogenase genes in the ectomycorrhizal fungus Tricholoma vaccinum: A role for Ald1 in mycorrhizal symbiosis.

Ectomycorrhizal symbiosis is important for forest ecosystem functioning with tree-fungal cooperation increasing performance and countering stress conditions. Aldehyde dehydrogenases (ALDHs) are key enzymes for detoxification and thus may play a role in stress response of the symbiotic association. With this focus, eight dehydrogenases, Ald1 through Ald7 and TyrA, of the ectomycorrhizal basidiomycete Tricholoma vaccinum were characterized and phylogenetically investigated. Functional analysis was performed through differential expression analysis by feeding different, environmentally important substances. A strong effect of indole-3-acetic acid (IAA) was identified, linking mycorrhiza formation and auxin signaling between the symbiosis partners. We investigated ald1 overexpressing strains for performance in mycorrhiza with the host tree spruce (Picea abies) and observed an increased width of the apoplast, accommodating the Hartig' net hyphae of the T. vaccinum over-expressing transformants. The results support a role for Ald1 in ectomycorrhiza formation and underline functional differentiation within fungal aldehyde dehydrogenases in the family 1 of ALDHs.

Tricholoma vaccinum host communication during ectomycorrhiza formation.

The genome sequence of Tricholoma vaccinum was obtained to predict its secretome in order to elucidate communication of T. vaccinum with its host tree spruce (Picea abies) in interkingdom signaling. The most prominent protein domains within the 206 predicted secreted proteins belong to energy and nutrition (52%), cell wall degradation (19%) and mycorrhiza establishment (9%). Additionally, we found small secreted proteins that show typical features of effectors potentially involved in host communication. From the secretome, 22 proteins could be identified, two of which showed higher protein abundances after spruce root exudate exposure, while five were downregulated in this treatment. The changes in T. vaccinum protein excretion with first recognition of the partner were used to identify small secreted proteins with the potential to act as effectors in the mutually beneficial symbiosis. Our observations support the hypothesis of a complex communication network including a cocktail of communication molecules induced long before physical contact of the partners.