The exp2 gene, which encodes a protein with two zinc finger domains, regulates cap expansion and autolysis in Coprinopsis cinerea.

Cap expansion in agaricoid mushroom species is an important event for sexual reproduction because meiosis occurs in basidia under the cap, and basidiospores can be released by opening the cap. However, molecular mechanisms underlying cap expansion in basidiomycetes remain poorly understood. We aimed to elucidate the molecular mechanisms of cap expansion in basidiomycetes by analyzing the unique cap-expansionless UV mutant #13 (exp2-1) in Coprinopsis cinerea. Linkage analysis and consequent genome sequence analysis revealed that the gene responsible for the mutant phenotypes encodes a putative transcription factor with two C2H2 zinc finger motifs. The mutant that was genome-edited to lack exp2 exhibited an expansionless phenotype. Some of the genes encoding cell wall degradation-related enzymes showed decreased expression during cap expansion and autolysis in the exp2 UV and genome-edited mutant. The exp2 gene is widely conserved in Agaricomycetes, suggesting that Exp2 homologs regulate fruiting body maturation in Agaricomycetes, especially cap expansion in Agaricoid-type mushroom-forming fungi. Therefore, exp2 homologs could be a target for mushroom breeding to maintain shape after harvest for some cultivating mushrooms, presenting a promising avenue for further research in breeding techniques.

Snowball: a novel gene family required for developmental patterning of fruiting bodies of mushroom-forming fungi (Agaricomycetes).

The morphogenesis of sexual fruiting bodies of fungi is a complex process determined by a genetically encoded program. Fruiting bodies reached the highest complexity levels in the Agaricomycetes; yet, the underlying genetics is currently poorly known. In this work, we functionally characterized a highly conserved gene termed snb1, whose expression level increases rapidly during fruiting body initiation. According to phylogenetic analyses, orthologs of snb1 are present in almost all agaricomycetes and may represent a novel conserved gene family that plays a substantial role in fruiting body development. We disrupted snb1 using CRISPR/Cas9 in the agaricomycete model organism Coprinopsis cinerea. snb1 deletion mutants formed unique, snowball-shaped, rudimentary fruiting bodies that could not differentiate caps, stipes, and lamellae. We took advantage of this phenotype to study fruiting body differentiation using RNA-Seq analyses. This revealed differentially regulated genes and gene families that, based on wild-type RNA-Seq data, were upregulated early during development and showed tissue-specific expression, suggesting a potential role in differentiation. Taken together, the novel gene family of snb1 and the differentially expressed genes in the snb1 mutants provide valuable insights into the complex mechanisms underlying developmental patterning in the Agaricomycetes. IMPORTANCE: Fruiting bodies of mushroom-forming fungi (Agaricomycetes) are complex multicellular structures, with a spatially and temporally integrated developmental program that is, however, currently poorly known. In this study, we present a novel, conserved gene family, Snowball (snb), termed after the unique, differentiation-less fruiting body morphology of snb1 knockout strains in the model mushroom Coprinopsis cinerea. snb is a gene of unknown function that is highly conserved among agaricomycetes and encodes a protein of unknown function. A comparative transcriptomic analysis of the early developmental stages of differentiated wild-type and non-differentiated mutant fruiting bodies revealed conserved differentially expressed genes which may be related to tissue differentiation and developmental patterning fruiting body development.

Comparative transcriptome analysis on candidate genes associated with fruiting body growth and development in Lyophyllum decastes.

Lyophyllum decastes is a mushroom that is highly regarded for its culinary and medicinal properties. Its delectable taste and texture make it a popular choice for consumption. To gain a deeper understanding of the molecular mechanisms involved in the development of the fruiting body of L. decastes, we used RNA sequencing to conduct a comparative transcriptome analysis. The analysis encompassed various developmental stages, including the vegetative mycelium, primordial initiation, young fruiting body, medium-size fruiting body, and mature fruiting body stages. A range of 40.1 to 60.6 million clean reads were obtained, and de novo assembly generated 15,451 unigenes with an average length of 1,462.68 bp. Functional annotation of transcriptomes matched 76.84% of the unigenes to known proteins available in at least one database. The gene expression analysis revealed a significant number of differentially expressed genes (DEGs) between each stage. These genes were annotated and subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses. Highly differentially expressed unigenes were also identified, including those that encode extracellular enzymes, transcription factors, and signaling pathways. The accuracy of the RNA-Seq and DEG analyses was validated using quantitative PCR. Enzyme activity analysis experiments demonstrated that the extracellular enzymes exhibited significant differences across different developmental stages. This study provides valuable insights into the molecular mechanisms that underlie the development of the fruiting body in L. decastes.

The Role of Chromatin and Transcriptional Control in the Formation of Sexual Fruiting Bodies in Fungi.

Fungal fruiting bodies are complex, three-dimensional structures that arise from a less complex vegetative mycelium. Their formation requires the coordinated action of many genes and their gene products, and fruiting body formation is accompanied by major changes in the transcriptome. In recent years, numerous transcription factor genes as well as chromatin modifier genes that play a role in fruiting body morphogenesis were identified, and through research on several model organisms, the underlying regulatory networks that integrate chromatin structure, gene expression, and cell differentiation are becoming clearer. This review gives a summary of the current state of research on the role of transcriptional control and chromatin structure in fruiting body development. In the first part, insights from transcriptomics analyses are described, with a focus on comparative transcriptomics. In the second part, examples of more detailed functional characterizations of the role of chromatin modifiers and/or transcription factors in several model organisms (Neurospora crassa, Aspergillus nidulans, Sordaria macrospora, Coprinopsis cinerea, and Schizophyllum commune) that have led to a better understanding of regulatory networks at the level of chromatin structure and transcription are discussed.

Effects of ß-1,6-Glucan Synthase Gene (FfGS6) Overexpression on Stress Response and Fruit Body Development in Flammulina filiformis.

ß-1, 6-glucan synthase is a key enzyme of ß-1, 6-glucan synthesis, which plays a vital role in the cell wall cross-linking of fungi. However, the role of the ß-1, 6-glucan synthase gene in the development of the fruiting body and the stress response of macrofungi is largely unknown. In this study, four overexpression transformants of the ß-1, 6-glucan synthase gene (FfGS6) were successfully obtained, and gene function was studied in Flammulina filiformis. The overexpression of FfGS6 can increase the width of mycelium cells and improve the tolerance ability under mechanical injury and oxidative stress. Moreover, FfGS6 gene expression fluctuated in up-regulation during the recovery process of mycelium injury but showed a negative correlation with H2O2 concentration. Fruiting body phenotype tests showed that mycelia's recovery ability after scratching improved when the FfGS6 gene was overexpressed. However, primordia formation and the stipe elongation ability were significantly inhibited. Our findings indicate that FfGS6 is involved in regulating mycelial cell morphology, the mycelial stress response, and fruit body development in F. filiformis.

Transcriptome and Differentially Expressed Gene Profiles in Mycelium, Primordium and Fruiting Body Development in Stropharia rugosoannulata.

Stropharia rugosoannulata uses straw as a growth substrate during artificial cultivation and has been widely promoted in China. However, its fruiting body formation and development processes have not been elucidated. In this study, the developmental transcriptomes were analyzed at three stages: the mycelium (G-S), primordium (P-S) and fruiting body (M-F) stages. A total of 9690 differentially expressed genes (DEGs) were identified in the different developmental stages. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses showed that these DEGs were involved mainly in hydrolase activity, structural molecule activity and oxidoreductase activity as well as xenobiotic biodegradation and metabolism and energy metabolism pathways. We further found that the higher expression of most carbohydrate enzyme (i.e., GH, CE, CBM, AA and PL) genes in the hyphal (i.e., G-S) stage was related mainly to substrate degradation, while the upregulation of glycosyltransferase (GT) gene expression in the P-S and M-F stages may be related to cell wall synthesis. In addition, we found that CO2-sensing-related genes (i.e., CA-2, CA-3, PKA-1 and PKA-2) were upregulated in the P-S and M-F stages, heat shock protein genes (HSP60 and HSP90) were significantly downregulated in the P-S stage and upregulated in the M-F stage and the transcription factors (i.e., steA, MYB, nosA, HAP1, and GATA-4/5/6) involved in growth and development were significantly upregulated in the P-S stage. These results suggest that environmental factors (i.e., CO2 and temperature) and transcription factors may play a key role in primordium formation. In short, this study provides new insights into the study of stimulating primordia formation affecting the development of fruiting bodies of S. rugosoannulata.

Transcriptional Divergence Underpinning Sexual Development in the Fungal Class Sordariomycetes.

Gene expression divergence through evolutionary processes is thought to be important for achieving programmed development in multicellular organisms. To test this premise in filamentous fungi, we investigated transcriptional profiles of 3,942 single-copy orthologous genes (SCOGs) in five related sordariomycete species that have morphologically diverged in the formation of their flask-shaped perithecia. We compared expression of the SCOGs to inferred gene expression levels of the most recent common ancestor of the five species, ranking genes from their largest increases to smallest increases in expression during perithecial development in each of the five species. We found that a large proportion of the genes that exhibited evolved increases in gene expression were important for normal perithecial development in Fusarium graminearum. Many of these genes were previously uncharacterized, encoding hypothetical proteins without any known functional protein domains. Interestingly, the developmental stages during which aberrant knockout phenotypes appeared largely coincided with the elevated expression of the deleted genes. In addition, we identified novel genes that affected normal perithecial development in Magnaporthe oryzae and Neurospora crassa, which were functionally and transcriptionally diverged from the orthologous counterparts in F. graminearum. Furthermore, comparative analysis of developmental transcriptomes and phylostratigraphic analysis suggested that genes encoding hypothetical proteins are generally young and transcriptionally divergent between related species. This study provides tangible evidence of shifts in gene expression that led to acquisition of novel function of orthologous genes in each lineage and demonstrates that several genes with hypothetical function are crucial for shaping multicellular fruiting bodies. IMPORTANCE The fungal class Sordariomycetes includes numerous important plant and animal pathogens. It also provides model systems for studying fungal fruiting body development, as its members develop fruiting bodies with a few well-characterized tissue types on common growth media and have rich genomic resources that enable comparative and functional analyses. To understand transcriptional divergence of key developmental genes between five related sordariomycete fungi, we performed targeted knockouts of genes inferred to have evolved significant upward shifts in expression. We found that many previously uncharacterized genes play indispensable roles at different stages of fruiting body development, which have undergone transcriptional activation in specific lineages. These novel genes are predicted to be phylogenetically young and tend to be involved in lineage- or species-specific function. Transcriptional activation of genes with unknown function seems to be more frequent than ever thought, which may be crucial for rapid adaption to changing environments for successful sexual reproduction.

Sexual Crossing, Chromosome-Level Genome Sequences, and Comparative Genomic Analyses for the Medicinal Mushroom Taiwanofungus Camphoratus (Syn. Antrodia Cinnamomea, Antrodia Camphorata).

Taiwanofungus camphoratus mushrooms are a complementary and alternative medicine for hangovers, cancer, hypertension, obesity, diabetes, and inflammation. Though Taiwanofungus camphoratus has attracted considerable biotechnological and pharmacological attention, neither classical genetic nor genomic approaches have been properly established for it. We isolated four sexually competent monokaryons from two T. camphoratus dikaryons used for the commercial cultivation of orange-red (HC1) and milky-white (SN1) mushrooms, respectively. We also sequenced, annotated, and comparatively analyzed high-quality and chromosome-level genome sequences of these four monokaryons. These genomic resources represent a valuable basis for understanding the biology, evolution, and secondary metabolite biosynthesis of this economically important mushrooms. We demonstrate that T. camphoratus has a tetrapolar mating system and that HC1 and SN1 represent two intraspecies isolates displaying karyotypic variation. Compared with several edible mushroom model organisms, T. camphoratus underwent a significant contraction in the gene family and individual gene numbers, most notably for plant, fungal, and bacterial cell-wall-degrading enzymes, explaining why T. camphoratus mushrooms are rare in natural environments, are difficult and time-consuming to artificially cultivate, and are susceptible to fungal and bacterial infections. Our results lay the foundation for an in-depth T. camphoratus study, including precise genetic manipulation, improvements to mushroom fruiting, and synthetic biology applications for producing natural medicinal products. IMPORTANCETaiwanofungus camphoratus (Tc) is a basidiomycete fungus that causes brown heart rot of the aromatic tree Cinnamomum kanehirae. The Tc fruiting bodies have been used to treat hangovers, abdominal pain, diarrhea, hypertension, and other diseases first by aboriginal Taiwanese and later by people in many countries. To establish classical genetic and genomic approaches for this economically important medicinal mushroom, we first isolated and characterized four sexually competent monokaryons from two dikaryons wildly used for commercial production of Tc mushrooms. We applied PacBio single molecule, real-time sequencing technology to determine the near-completed genome sequences of four monokaryons. These telomere-to-telomere and gapless haploid genome sequences reveal all genomic variants needed to be studied and discovered, including centromeres, telomeres, retrotransposons, mating type loci, biosynthetic, and metabolic gene clusters. Substantial interspecies diversities are also discovered between Tc and several other mushroom model organisms, including Agrocybe aegerita, Coprinopsis cinerea, and Schizophyllum commune, and Ganoderma lucidum.

Gene age shapes the transcriptional landscape of sexual morphogenesis in mushroom-forming fungi (Agaricomycetes).

Multicellularity has been one of the most important innovations in the history of life. The role of gene regulatory changes in driving transitions to multicellularity is being increasingly recognized; however, factors influencing gene expression patterns are poorly known in many clades. Here, we compared the developmental transcriptomes of complex multicellular fruiting bodies of eight Agaricomycetes and Cryptococcus neoformans, a closely related human pathogen with a simple morphology. In-depth analysis in Pleurotus ostreatus revealed that allele-specific expression, natural antisense transcripts, and developmental gene expression, but not RNA editing or a 'developmental hourglass,' act in concert to shape its transcriptome during fruiting body development. We found that transcriptional patterns of genes strongly depend on their evolutionary ages. Young genes showed more developmental and allele-specific expression variation, possibly because of weaker evolutionary constraint, suggestive of nonadaptive expression variance in fruiting bodies. These results prompted us to define a set of conserved genes specifically regulated only during complex morphogenesis by excluding young genes and accounting for deeply conserved ones shared with species showing simple sexual development. Analysis of the resulting gene set revealed evolutionary and functional associations with complex multicellularity, which allowed us to speculate they are involved in complex multicellular morphogenesis of mushroom fruiting bodies.

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.

Transcriptome analysis of genes associated with autolysis of Coprinus comatus.

Coprinus comatus, widely known as "Jituigu", is an important commodity and food in China. The yield of C. comatus, however, is substantially reduced by the autolysis of the fruiting bodies after harvest. To gain insight into the molecular mechanism underlying this autolysis, we divided the growth of C. comatus fruiting bodies into four stages: infant stage (I), mature stage (M), discolored stage (D), and autolysis stage (A). We then subjected these stages to de novo transcriptomic analysis using high-throughput Illumina sequencing. A total of 12,946 unigenes were annotated and analyzed with the Gene Ontology (GO), Clusters of Orthologous Groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG). We analyzed the differentially expressed genes (DEGs) between stages I and M, M and D, and D and A. Because the changes from M to D are thought to be related to autolysis, we focused on the DEGs between these two stages. We found that the pathways related to metabolic activity began to vary in the transition from M to D, including pathways named as autophagy-yeast, peroxisome, and starch and sucrose metabolism. This study also speculates the possible process of the autolysis of Coprinus comatus. In addition, 20 genes of interest were analyzed by quantitative real-time PCR to verify their expression profiles at the four developmental stages. This study, which is the first to describe the transcriptome of C. comatus, provides a foundation for future studies concerning the molecular basis of the autolysis of its fruiting bodies.

High Throughput Identification of the Potential Antioxidant Peptides in Ophiocordyceps sinensis.

Ophiocordyceps sinensis, an ascomycete caterpillar fungus, has been used as a Traditional Chinese Medicine owing to its bioactive properties. However, until now the bio-active peptides have not been identified in this fungus. Here, the raw RNA sequences of three crucial growth stages of the artificially cultivated O. sinensis and the wild-grown mature fruit-body were aligned to the genome of O. sinensis. Both homology-based prediction and de novo-based prediction methods were used to identify 8541 putative antioxidant peptides (pAOPs). The expression profiles of the cultivated mature fruiting body were similar to those found in the wild specimens. The differential expression of 1008 pAOPs matched genes had the highest difference between ST and MF, suggesting that the pAOPs were primarily induced and play important roles in the process of the fruit-body maturation. Gene ontology analysis showed that most of pAOPs matched genes were enriched in terms of 'cell redox homeostasis', 'response to oxidative stresses', 'catalase activity', and ' integral component of cell membrane'. A total of 1655 pAOPs was identified in our protein-seqs, and some crucial pAOPs were selected, including catalase, peroxiredoxin, and SOD [Cu-Zn]. Our findings offer the first identification of the active peptide ingredients in O. sinensis, facilitating the discovery of anti-infectious bio-activity and the understanding of the roles of AOPs in fungal pathogenicity and the high-altitude adaptation in this medicinal fungus.

Evolutionary Morphogenesis of Sexual Fruiting Bodies in Basidiomycota: Toward a New Evo-Devo Synthesis.

The development of sexual fruiting bodies is one of the most complex morphogenetic processes in fungi. Mycologists have long been fascinated by the morphological and developmental diversity of fruiting bodies; however, evolutionary developmental biology of fungi still lags significantly behind that of animals or plants. Here, we summarize the current state of knowledge on fruiting bodies of mushroom-forming Basidiomycota, focusing on phylogenetic and developmental biology. Phylogenetic approaches have revealed a complex history of morphological transformations and convergence in fruiting body morphologies. Frequent transformations and convergence is characteristic of fruiting bodies in contrast to animals or plants, where main body plans are highly conserved. At the same time, insights into the genetic bases of fruiting body development have been achieved using forward and reverse genetic approaches in selected model systems. Phylogenetic and developmental studies of fruiting bodies have each yielded major advances, but they have produced largely disjunct bodies of knowledge. An integrative approach, combining phylogenetic, developmental, and functional biology, is needed to achieve a true fungal evolutionary developmental biology (evo-devo) synthesis for fungal fruiting bodies.

Transcriptomics Analysis of Primordium Formation in Pleurotus eryngii.

Primordium formation is an important stage preceding the growth and development of the Pleurotus eryngii fruiting body. However, the molecular mechanisms underlying primordium formation remain unclear. In the present study, comparative transcriptomics was performed between mature mycelia and primordium to analyze the transcriptional properties during primordium formation in P. eryngii. A total of 19,655 differentially expressed genes (10,718 upregulated genes and 8937 downregulated genes) were identified. These differentially expressed genes were involved in cell wall degradation, carbohydrate hydrolysis, light perception, and cAMP signal transduction. These results aid further understanding of the transcriptional changes and the molecular processes underlying primordium formation and differentiation, which may lay the foundation for improving the cultivation and quality control of P. eryngii.

Functional characterization of the developmental genes asm2, asm3, and spt3 required for fruiting body formation in the filamentous ascomycete Sordaria macrospora.

The formation of fruiting bodies is one of the most complex developmental processes in filamentous ascomycetes. It requires the development of sexual structures that give rise to meiosporangia (asci) and meiotic spores (ascospores) as well as surrounding structures for protection and dispersal of the spores. Previous studies have shown that these developmental processes are accompanied by significant changes of the transcriptome, and comparative transcriptomics of different fungi as well as the analysis of transcriptome changes in developmental mutants have aided in the identification of differentially regulated genes that are themselves involved in regulating fruiting body development. In previous analyses, we used transcriptomics to identify the genes asm2 and spt3, which result in developmental phenotypes when deleted in Sordaria macrospora. In this study, we identified another gene, asm3, required for fruiting body formation, and performed transcriptomics analyses of Δasm2, Δasm3, and Δspt3. Deletion of spt3, which encodes a subunit of the SAGA complex, results in a block at an early stage of development and drastic changes in the transcriptome. Deletion mutants of asm2 and asm3 are able to form fruiting bodies, but have defects in ascospore maturation. Transcriptomics analysis of fruiting bodies revealed a large overlap in differentially regulated genes in Δasm2 and Δasm3 compared to the wild type. Analysis of nuclear distribution during ascus development showed that both mutants undergo meiosis and postmeiotic divisions, suggesting that the transcriptomic and morphological changes might be related to defects in the morphogenesis of structural features of the developing asci and ascospores.

Changes of structures and biosynthesis/hydrolysis-associated genes expression of glucans at different Volvariella volvacea maturity stages.

In the present study, effects of maturity stage on structural characteristics and biosynthesis/hydrolysis-associated genes expression of glucans from Volvariella volvacea fruit body were well investigated. Elongation and pileus expansion stages decreased total soluble carbohydrate and protein contents to 17.09 mg/g and 8.33 mg/g, and significantly accumulated the total amino acids contents to 32.37 mg/g. Yields of crude polysaccharides significantly increased to 8.12% at egg stage and decreased to 3.72% at pileus expansion stage. Purified VVP I-a and VVP I-b were proved to be α-glucans. The maturity process affected the monosaccharide compositions, decreased the molecular weights of VVP I-a and VVP I-b with decreased transcription levels of glucan biosynthesis-associated enzyme genes vvugp and vvgls and increased glucan hydrolysis-associated glucanase gene vvexg2 expression with no significant effects on backbone structures including glycosidic linkages and configurations. The findings would benefit for understanding change patterns of V. volvacea glucan structures and their biosynthesis/hydrolysis-associated genes expression at maturity stages.

The Fvclp1 gene regulates mycelial growth and fruiting body development in edible mushroom Flammulina velutipes.

Fruiting body development in Agaricomycetes represents the most complex and unclear process in the fungi. Mating type pathways (A and B) and transcription factors are important regulators in the sexual development of mushrooms. It is known that clampless1 (clp1) is an additional gene that participate under the homeodomain (HD) genes in the matA pathway and clp1 inactivation blocks clamps formation in Coprinopsis cinerea. In this study we identified and analyzed a homologous Fvclp1 gene in the edible mushroom Flammulina velutipes. The coding sequence of the Fvclp1 was 1011 bp without intron interruption, encoding a protein of 336 amino acids. To exhibit the role of Fvclp1 in clamp development and fruiting body formation, knockdown and overexpression mutants were prepared. No significant difference was observed in the monokaryotic hyphal morphology of overexpression and knockdown transformants. In the dikaryotic hyphae from the compatible crossings between the wild-type L22 strain and Fvclp1 knockdown or overexpression mutants, clamp connections developed. However, knockdown mutants could generate fewer fruiting bodies than the wild-type strain. On the contrary, reduced mycelial growth rate but improved fruiting ability was observed in the dikaryotic Fvclp1 overexpression mutants as compared to the wild-type strain. These results indicate that Fvclp1 is necessary and actively involved in fruiting body development in F. velutipes. Overall, these findings suggest that further studies on the function of Fvclp1 would advance our understanding of sexual reproduction and fruiting body development in edible mushrooms.

Comparative transcriptome analysis of abnormal cap and healthy fruiting bodies of the edible mushroom Lentinula edodes.

Lentinula edodes, a commercially important mushroom, is cultivated worldwide. Artificially cultivated L. edodes often present with abnormal symptoms in the fruiting body, which affect their commercial value and reduce production efficiency. In this study, we carried out a comparative transcriptome analysis of normal fruiting body pileus (LeNP), normal margin in abnormal fruiting body pileus (LeAPNM), and abnormal margin in abnormal fruiting body pileus (LeAPAM). Metabolic pathways such as those involved in transmembrane transport, ribosome production, tryptophan metabolism, arginine and proline metabolism, and the metabolism of other amino acids were significantly enriched in LeAPAM. F-box, short-chain dehydrogenases/reductases, the major facilitator superfamily, and the FMN_red superfamily are related to malformation in L. edodes. Genes encoding heat shock proteins, G protein, and ß-1,3-glucanase in the GH5 family showed different expression patterns, suggesting that these genes are involved in the development of L. edodes fruiting bodies. In particular, CAZymes, which are involved in the development of cell walls in L. edodes, were highly expressed in LeAPAM. According to TEM observation, the cell wall of LeAPAM samples showed significant thickening compared to the other samples. These results suggested that cell wall anabolism in LeAPAM samples was more active than that in normal fruiting bodies, enhancing the environmental adaptability of the fungus. This study provides preliminary data for future research aimed at solving the phenomenon of abnormal fruiting bodies of L. edodes.

mRNA-seq and miRNA-seq profiling analyses reveal molecular mechanisms regulating induction of fruiting body in Ophiocordyceps sinensis.

Ophiocordyceps sinensis has been a source of valuable materials in traditional Asian medicine for over two thousand years. With recent global warming and overharvest, however, the availability of these wild fungi has decreased dramatically. While fruiting body of O. sinensis has been artificially cultivated, the molecular mechanisms that govern the induction of fruiting body at the transcriptional and post-transcriptional levels are unclear. In this study, we carried out both mRNA and small RNA sequencing to identify crucial genes and miRNA-like RNAs (milRNAs) involved in the development of fruiting body. A total of 2875 differentially expressed genes (DEGs), and 71 differentially expressed milRNAs (DEMs) were identified among the mycoparasite complex, the sclerotium (ST) and the fruiting body stage. Functional enrichment and Gene Set Enrichment Analysis indicated that the ST had increased oxidative stress and energy metabolism and that mitogen-activated protein kinase signaling might induce the formation of fruiting body. Integrated analysis of DEGs and DEMs revealed that n_os_milR16, n_os_milR21, n_os_milR34, and n_os_milR90 could be candidate milRNAs that regulate the induction of fruiting body. This study provides transcriptome-wide insight into the molecular basis of fruiting body formation in O. Sinensis and identifies potential candidate genes for improving induction rate.

Transcriptome of different fruiting stages in the cultivated mushroom Cyclocybe aegerita suggests a complex regulation of fruiting and reveals enzymes putatively involved in fungal oxylipin biosynthesis.

BACKGROUND: Cyclocybe aegerita (syn. Agrocybe aegerita) is a commercially cultivated mushroom. Its archetypal agaric morphology and its ability to undergo its whole life cycle under laboratory conditions makes this fungus a well-suited model for studying fruiting body (basidiome, basidiocarp) development. To elucidate the so far barely understood biosynthesis of fungal volatiles, alterations in the transcriptome during different developmental stages of C. aegerita were analyzed and combined with changes in the volatile profile during its different fruiting stages. RESULTS: A transcriptomic study at seven points in time during fruiting body development of C. aegerita with seven mycelial and five fruiting body stages was conducted. Differential gene expression was observed for genes involved in fungal fruiting body formation showing interesting transcriptional patterns and correlations of these fruiting-related genes with the developmental stages. Combining transcriptome and volatilome data, enzymes putatively involved in the biosynthesis of C8 oxylipins in C. aegerita including lipoxygenases (LOXs), dioxygenases (DOXs), hydroperoxide lyases (HPLs), alcohol dehydrogenases (ADHs) and ene-reductases could be identified. Furthermore, we were able to localize the mycelium as the main source for sesquiterpenes predominant during sporulation in the headspace of C. aegerita cultures. In contrast, changes in the C8 profile detected in late stages of development are probably due to the activity of enzymes located in the fruiting bodies. CONCLUSIONS: In this study, the combination of volatilome and transcriptome data of C. aegerita revealed interesting candidates both for functional genetics-based analysis of fruiting-related genes and for prospective enzyme characterization studies to further elucidate the so far barely understood biosynthesis of fungal C8 oxylipins.