Lessons on fruiting body morphogenesis from genomes and transcriptomes of Agaricomycetes

Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are among the most complex structures produced by fungi. Unlike vegetative hyphae, fruiting bodies grow determinately and follow a genetically encoded developmental program that orchestrates tissue differentiation, growth and sexual sporulation. In spite of more than a century of research, our understanding of the molecular details of fruiting body morphogenesis is limited and a general synthesis on the genetics of this complex process is lacking. In this paper, we aim to comprehensively identify conserved genes related to fruiting body morphogenesis and distill novel functional hypotheses for functionally poorly characterized genes. As a result of this analysis, we report 921 conserved developmentally expressed gene families, only a few dozens of which have previously been reported in fruiting body development. Based on literature data, conserved expression patterns and functional annotations, we provide informed hypotheses on the potential role of these gene families in fruiting body development, yielding the most complete description of molecular processes in fruiting body morphogenesis to date. We discuss genes related to the initiation of fruiting, differentiation, growth, cell surface and cell wall, defense, transcriptional regulation as well as signal transduction. Based on these data we derive a general model of fruiting body development, which includes an early, proliferative phase that is mostly concerned with laying out the mushroom body plan (via cell division and differentiation), and a second phase of growth via cell expansion as well as meiotic events and sporulation. Altogether, our discussions cover 1480 genes of Coprinopsis cinerea, and their orthologs in Agaricus bisporus, Cyclocybe aegerita, Armillaria ostoyae, Auriculariopsis ampla, Laccaria bicolor, Lentinula edodes, Lentinus tigrinus, Mycena kentingensis, Phanerochaete chrysosporium, Pleurotus ostreatus, and Schizophyllum commune, providing functional hypotheses for ~10% of genes in the genomes of these species. Although experimental evidence for the role of these genes will need to be established in the future, our data provide a roadmap for guiding functional analyses of fruiting related genes in the Agaricomycetes. We anticipate that the gene compendium presented here, combined with developments in functional genomics approaches will contribute to uncovering the genetic bases of one of the most spectacular multicellular developmental processes in fungi. Key words: functional annotation; comparative genomics; cell wall remodeling; development; fruiting body morphogenesis; mushroom; transcriptome

Whole-Genome and Transcriptome Sequencing of Phlebopus portentosus Reveals Its Associated Ectomycorrhizal Niche and Conserved Pathways Involved in Fruiting Body Development

Phlebopus portentosus (Berk. and Broome) Boedijin, a widely consumed mushroom in China and Thailand, is the first species in the order Boletaceae to have been industrially cultivated on a large scale. However, to date, the lignocellulose degradation system and molecular basis of fruiting body development in P. portentosus have remained cryptic. In the present study, genome and transcriptome sequencing of P. portentosus was performed during the mycelium (S), primordium (P), and fruiting body (F) stages. A genome of 32.74 Mb with a 48.92% GC content across 62 scaffolds was obtained. A total of 9,464 putative genes were predicted from the genome, of which the number of genes related to plant cell wall-degrading enzymes was much lower than that of some saprophytic mushrooms with specific ectomycorrhizal niches. Principal component analysis of RNA-Seq data revealed that the gene expression profiles at all three stages were different. The low expression of plant cell wall-degrading genes also confirmed the limited ability to degrade lignocellulose. The expression profiles also revealed that some conserved and specific pathways were enriched in the different developmental stages of P. portentosus. Starch and sucrose metabolic pathways were enriched in the mycelium stage, while DNA replication, the proteasome and MAPK signaling pathways may be associated with maturation. These results provide a new perspective for understanding the key pathways and hub genes involved in P. portentosus development.

Endogenous bacteria inhabiting the Ophiocordyceps highlandensis during fruiting body development

Background The genus Ophiocordyceps, which includes Ophiocordyceps sinensis, has been demonstrated to be one of the most valuable medicinal taxa. The low rate of larval infection and slow development that characterize the cultivation of this genus should be urgently addressed. To identify potential bioinoculants that stimulate the growth of Ophiocordyceps, O. highlandensis was selected as a model system, and a total of 72 samples were collected to systematically compare the microbial communities present during fruiting body development. By applying high-throughput 16S and ITS2 amplicon sequencing technology, the bacterial and fungal communities were identified in O. highlandensis and its surrounding soil, and the functional dynamics of the bacteria were explored. Results The results indicate that the most abundant bacteria across all the samples from O. highlandensis were Proteobacteria, Firmicutes and Bacteroidetes, while members of Ascomycota were detected among the fungi. The pathways enriched in the developmental stages were associated with carbohydrate degradation, nucleotides and pyridoxal biosynthesis, and the TCA cycle. Compared with that in the fungal community, an unexpectedly high taxonomic and functional fluctuation was discovered in the bacterial community during the maturation of O. highlandensis. Furthermore, bipartite network analysis identified four potential supercore OTUs associated with O. highlandensis growth. Conclusions All the findings of this study suggest unexpectedly high taxonomic and functional fluctuations in the bacterial community of O. highlandensis during its maturation. O. highlandensis may recruit different endogenous bacteria across its life cycle to enhance growth and support rapid infection. These results may facilitate Ophiocordyceps cultivation and improve the development of strategies for the identification of potential bioinoculant resources.

iTRAQ-based comparative proteome analyses of different growth stages revealing the regulatory role of reactive oxygen species in the fruiting body development of Ophiocordyceps sinensis

In this study, using an isobaric tags for relative and absolute quantitation (iTRAQ ) approach coupled with LC-MS / MS and bioinformatics, the proteomes were analyzed for the crucial three stages covering the fruiting body development of Ophiocordyceps sinensis, including sclerotium (ST), primordium (PR) and mature fruiting body (MF), with a focus on fruiting body development-related proteins and the potential mechanisms of the development. A total of 1,875 proteins were identified. Principal Component Analysis (PCA) demonstrated that the protein patterns between PR and MF were more similar than ST. Differentially accumulated proteins (DAPs) analysis showed that there were 510, 173 and 514 DAPs in the comparisons of ST vs. PR, PR vs. MF and ST vs. MF, respectively. A total of 62 shared DAPs were identified and primarily enriched in proteins related to ‘carbon transport and mechanism’, ‘the response to oxidative stress’, ‘antioxidative activity’ and ‘translation’. KEGG and GO databases showed that the DAPs were enriched in terms of ‘primary metabolisms (amino acid/fatty acid/energy metabolism)’, ‘the response to oxidative stress’ and ‘peroxidase’. Furthermore, 34 DAPs involved in reactive oxygen species (ROS) metabolism were identified and clustered across the three stages using hierarchical clustering implemented in hCluster R package . It was suggested that their roles and the underlying mechanisms may be stage-specific. ROS may play a role in fungal pathogenicity in ST, the fruit-body initiation in PR, sexual reproduction and highland adaptation in MF. Crucial ROS-related proteins were identified, such as superoxide dismutase (SOD, T5A6F1), Nor-1 (T5AFX3), electron transport protein (T5AHD1), histidine phosphotransferase (HPt, T5A9Z5) and Glutathione peroxidase (T5A9V1). Besides, the accumulation of ROS at the three stages were assayed using 2,7-dichlorofuorescin diacetate (DCFH-DA) stanning. A much stronger ROS accumulation was detected at the stage MF, compared to the stages of PR and ST. Sections of ST and fruit-body part of MF were stained by DCFH-DA and observed under the fluorescencemicroscope, showing ROS was distributed within the conidiospore and ascus. Besides, SOD activity increased across the three stages, while CAT activity has a strong increasement in MF compared to the stages of ST and PR. It was suggested that ROS may act in gradient-dependent manner to regulate the fruiting body development. The coding region sequences of six DAPs were analyzed at mRNA level by quantitative real-time PCR (qRT-PCR). The results support the result of DAPs analysis and the proteome sequencing data. Our findings offer the perspective of proteome to understand the biology of fruiting body development and highland adaptation in O. sinensis, which would inform the big industry of this valuable fungus.

The Glyoxysomal Protease LON2 Is Involved in Fruiting-Body Development, Ascosporogenesis and Stress Resistance in Sordaria macrospora

Microbodies, including peroxisomes, glyoxysomes and Woronin bodies, are ubiquitous dynamic organelles that play important roles in fungal development. The ATP-dependent chaperone and protease family Lon that maintain protein quality control within the organelle significantly regulate the functionality of microbodies. The filamentous ascomycete Sordaria macrospora is a model organism for studying fruiting-body development. The genome of S. macrospora encodes one Lon protease with the C-terminal peroxisomal targeting signal (PTS1) serine-arginine-leucine (SRL) for import into microbodies. Here, we investigated the function of the protease SmLON2 in sexual development and during growth under stress conditions. Localization studies revealed a predominant localization of SmLON2 in glyoxysomes. This localization depends on PTS1, since a variant without the C-terminal SRL motif was localized in the cytoplasm. A ΔSmlon2 mutant displayed a massive production of aerial hyphae, and produced a reduced number of fruiting bodies and ascospores. In addition, the growth of the ΔSmlon2 mutant was completely blocked under mild oxidative stress conditions. Most of the defects could be complemented with both variants of SmLON2, with and without PTS1, suggesting a dual function of SmLON2, not only in microbody, but also in cytosolic protein quality control.