The basidiomycete fungus Coprinopsis cinerea is an important model system for multicellular development. Fruiting bodies of C. cinerea are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.

No abstract

Lentinan is an antitumor product that is purified from fresh Lentinula edodes fruiting bodies. It is a cell wall component, comprising b-1,3-glucan with b-1,6-linked branches, which becomes degraded during postharvest preservation as a result of increased glucanase activity. In this study, we used N-terminal amino acid sequence to isolate tlg1, a gene encoding a thaumatin-like (TL) protein in L. edodes. The cDNA clone was approximately 1.0 kb whereas the genomic sequence was 2.1 kb, and comparison of the two indicated that tlg1 contains 12 introns. The tlg1 gene product (TLG1) was predicted to comprise 240 amino acids, with a molecular mass of 25 kD and isoelectric point value of 3.5. The putative amino acid sequence exhibits approximately 40% identity with plant TL proteins, and a fungal genome database search revealed that these TL proteins are conserved in many fungi including the basidiomycota and ascomycota. Transcription of tlg1 was not detected in vegetative mycelium or young and fresh mushrooms. However, transcription increased following harvest. Western-blot analysis demonstrated a rise in TLG1 levels following harvest and spore diffusion. TLG1 expressed in Escherichia coli and Aspergillus oryzae exhibited b-1,3-glucanase activity and, when purified from the L. edodes fruiting body, demonstrated lentinan degrading activity. Thus, we suggest that TLG1 is involved in lentinan and cell wall degradation during senescence following harvest and spore diffusion.

Lentinula edodes is a popular, cultivated edible and medicinal mushroom. Lentinula edodes is susceptible to postharvest problems, such as gill browning, fruiting body softening, and lentinan degradation. We constructed a de novo assembly draft genome sequence and performed gene prediction for Lentinula edodes. De novo assembly was carried out using short reads from paired-end and mate-paired libraries and by using long reads by PacBio, resulting in a contig number of 1,951 and an N 50 of 1 Mb. Furthermore, we predicted genes by Augustus using transcriptome sequencing (RNA-seq) data from the whole life cycle of Lentinula edodes, resulting in 12,959 predicted genes. This analysis revealed that Lentinula edodes lacks lignin peroxidase. To reveal genes involved in the loss of quality of Lentinula edodes postharvest fruiting bodies, transcriptome analysis was carried out using serial analysis of gene expression (SuperSAGE). This analysis revealed that many cell wall-related enzymes are upregulated after harvest, such as ␤-1,3-1,6-glucan-degrading enzymes in glycoside hydrolase (GH) families GH5, GH16, GH30, GH55, and GH128, and thaumatin-like proteins. In addition, we found that several chitin-related genes are upregulated, such as putative chitinases in GH family 18, exochitinases in GH20, and a putative chitosanase in GH family 75. The results suggest that cell wall-degrading enzymes synergistically cooperate for rapid fruiting body autolysis. Many putative transcription factor genes were upregulated postharvest, such as genes containing high-mobility-group (HMG) domains and zinc finger domains. Several cell deathrelated proteins were also upregulated postharvest.IMPORTANCE Our data collectively suggest that there is a rapid fruiting body autolysis system in Lentinula edodes. The genes for the loss of postharvest quality newly found in this research will be targets for the future breeding of strains that keep fresh longer than present strains. De novo Lentinula edodes genome assembly data will be used for the construction of a complete Lentinula edodes chromosome map for future breeding.

An exo-beta-1,3-glucanase-encoding gene was isolated from Lentinula edodes to investigate the relationship between the cell wall lytic enzyme and mushroom morphogenesis. The deduced amino acid sequence of the protein corresponding to the exg1 gene displayed 67% identity to AbEXG1 of Agaricus bisporus and approximately 40% identity to yeast exo-beta-1,3-glucanases. Two conserved glutamic acids within the catalytic active site in yeast exo-beta-1,3-glucanases were conserved in exg1 of L. edodes. The exg1 gene was expressed in fruiting bodies, but not in vegetative mycelia. Expression was higher in the stipe than in the pileus of young fruiting bodies. The gene was additionally expressed in the gills of mature fruiting bodies. We purified a glucanase from the stipes of young fruiting bodies that had an N-terminus identical to that of the putative exg1 product. These results collectively indicate that exg1 is involved in L. edodes fruiting body development, including stipe elongation.