Search

The effect of oxygen on the germination and culturability of aerobic Bacillus atrophaeus spores was investigated in this study. Under oxic or anoxic conditions, various nutritional and non-nutritional germinants were utilized to induce germination. Tb3+-dipicolinic acid fluorescence assay and phase-contrast microscopy were used to track the germination process. The final germination level, germination half time, and germination speed were used to define germination kinetics. Colony-forming unit enumeration was used to assess the culturability of germinated spores germinated with or without oxygen. The results show that in the absence of oxygen, the final germination level was unaffected, germination half time decreased by up to 35.0%, germination speed increased by up to 27.4%, and culturability decreased by up to 95.1%. It is suggested that oxygen affects some germinant receptor-dependent germination pathways, implying that biomolecules engaged in these pathways may be oxygen-sensitive. Furthermore, spores that have completed the germination process in either anoxic or oxic conditions may have different culturability. This research contributed to a better understanding of the fundamental mechanism of germination.

Coprinopsis cinereais a model mushroom for studying the developmental processes of homobasidiomycetous fungi. The development ofC. cinereadepends on the sensing of environmental conditions, such as light, temperature, humidity and nutrients. The signal transduction pathway from the environmental condition sensors to the regulators and reproduction is an important research topic as the knowledge on it is still fragmentary. Protein kinases not only are crucial to the signal transduction, but also can be master switches of development. However, information on their role in basidiomycetous fungi is still limited. Using the effective gene-targeting of the current gene modification, a system of CRISPR/ Cas9 RNP with double strain template donor that contained a selective mark hygromycin cassette was developed in this study. In this system,CcPka1gene knockout was successfully generated through homologues repair together with Cas9 RNP complexes. This was the first report to use CRISPR/ Cas9 RNP approach with C. cinerea. ΔCcPka1mutants showed slow mycelial growth and rapid fruiting body induction compared to the wild type even when under high glucose condition. In addition, ΔCcPka1mutants had significantly increased intracellular glucose level and decreased glycogen level. Moreover, ΔCcPka1mutants regulated the gene expression of components in cAMP-PKA pathway. All these results demonstrated that PKA played an important role in the nutrient signal transduction and negatively regulated the fruiting body formation. This study also confirmed that GSK3 could be the downstream target of PKA, but their interaction requires further investigation.

The “developmental hourglass” describes the morphological convergence to a conserved form at mid-stages of animal embryogenesis. The molecular hourglass pattern during embryogenesis was also identified across kingdoms. Previously, we reported young fruiting body as the conserved “waist” in mushroom-forming “developmental hourglass”. However, its robustness is doubted because of the fungal diversity. Additionally, fungi lack embryogenesis, and develop directly from spore to hyphae with morphological similarities during the transition. Here, we updated the “developmental hourglass” model in the life cycle of fungi, namely, spore germination, vegetative growth, and sexual reproduction. Germinating spores, both sexual and asexual, showed the strongest transcriptomic conservation signals across the phyla Mucoromycota, Ascomycota and Basidiomycota. Cross kingdom comparisons revealed high expression levels of “information storage and processing” genes at the waist stages of embryonic and non-embryonic developments in animals, plants, and fungi. The “developmental hourglass” might reflect the mutual transcriptome switches on developmental transitions in eukaryotes that are additional to embryonic organogenesis.HighlightsUpdated fungal molecular “developmental hourglass” model in the life cycle of fungiGerminating spores are the evolutionarily conserved “waist” across fungal phylaHigh expression levels of “information storage and processing” genes at the waist stages in the embryonic and non-embryonic hourglasses across kingdoms“Developmental hourglass” may reflect the mutual transcriptome switches on developmental transitions in eukaryotes

The homokaryoticCoprinopsis cinereastrainA43mut B43mut pab1-1#326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factorsAandBresult in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with theC. cinereastrain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein(RasGAP)was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.HighlightA chromosome-level genome assembly ofC. cinerea #326A fast and efficient high-molecular-weight fungal genomic DNA isolation protocolStructural variant and single nucleotide variant calling using Nanopore readsA series of solutions and reference parameters for fungal genomic analysis on MinION

The homokaryotic Coprinopsis cinerea strain A43mut B43mut pab1-1 #326 is a widely used experimental model for developmental studies in mushroom-forming fungi. It can grow on defined artificial media and complete the whole lifecycle within two weeks. The mutations in mating type factors A and B result in the special feature of clamp formation and fruiting without mating. This feature allows investigations and manipulations with a homokaryotic genetic background. Current genome assembly of strain #326 was based on short-read sequencing data and was highly fragmented, leading to the bias in gene annotation and downstream analyses. Here, we report a chromosome-level genome assembly of strain #326. Oxford Nanopore Technology (ONT) MinION sequencing was used to get long reads. Illumina short reads was used to polish the sequences. A combined assembly yield 13 chromosomes and a mitochondrial genome as individual scaffolds. The assembly has 15,250 annotated genes with a high synteny with the C. cinerea strain Okayama-7 #130. This assembly has great improvement on contiguity and annotations. It is a suitable reference for further genomic studies, especially for the genetic, genomic and transcriptomic analyses in ONT long reads. Single nucleotide variants and structural variants in six mutagenized and cisplatin-screened mutants could be identified and validated. A 66 bp deletion in Ras GTPase-activating protein (RasGAP) was found in all mutants. To make a better use of ONT sequencing platform, we modified a high-molecular-weight genomic DNA isolation protocol based on magnetic beads for filamentous fungi. This study showed the use of MinION to construct a fungal reference genome and to perform downstream studies in an individual laboratory. An experimental workflow was proposed, from DNA isolation and whole genome sequencing, to genome assembly and variant calling. Our results provided solutions and parameters for fungal genomic analysis on MinION sequencing platform.

Satellite communication networks, as an effective complement to terrestrial communication networks, enable global coverage communications. The Low-orbit satellite network has the characteristics of low transmission delay, short operation period and global coverage communication, it has become a popular research direction in satellite communication networks. With the continuous development of digital and intelligent communication in the world, satellite network services continue to expand, and higher requirements are placed on network transmission performance. The traditional single-structure low-orbit satellite network using centralized routing has been unable to meet current needs.. Therefore, with the low-orbit satellite network as the core, people have conducted in-depth research on satellite communication network routing algorithms that meet the requirements of high-quality, high-efficiency and largecapacity communication. Especially in the three hot issues of low-orbit satellite network routing algorithms, a lot of research results have emerged, including: QoS-based artificial intelligence algorithm routing algorithm; multi-layer satellite network routing algorithm; satellite network multipath routing algorithm. Through the analysis and discussion of these problems and achievements, the idea of combining the three algorithms on demand is proposed, and the challenges are analyzed.

The balance and interplay between sexual and asexual reproduction is one of the most attractive mysteries in fungi. The choice of developmental strategy reflects the ability of fungi to adapt to the changing environment. However, the evolution of developmental paths and the metabolic regulation during differentiation and morphogenesis are poorly understood. Here, we monitor the carbohydrate metabolism and gene expression regulation during the early differentiation process from the “fungal stem cell”, vegetative mycelium, to the highly differentiated tissue/cells, fruiting body, oidia or sclerotia, of a homokaryotic fruitingCoprinopsis cinereastrain A43mut B43mut pab1-1 #326, uncovering the systematic changes during morphogenesis and the evolutionary process of developmental strategies. Conversion between glucose and glycogen and conversion between glucose and beta-glucan are the main carbon flows in the differentiation processes. Genes related to carbohydrate transport and metabolism are significantly differentially expressed among paths. RNA editing, a novel layer of gene expression regulation, occurs in all four developmental paths and enriched in cytoskeleton and carbohydrate metabolic genes. It is developmentally regulated and evolutionarily conserved in basidiomycetes. Evolutionary transcriptomic analysis on four developmental paths showed that all transcriptomes are under purifying selection, and the more stressful the environment, the younger the transcriptome age. Oidiation has the lowest value of transcriptome age index (TAI) and transcriptome divergence index (TDI), while fruiting process has the highest of both indexes. These findings provide new insight to the regulations of carbon metabolism and gene expressions during fungal developmental paths differentiation.ImportanceFungi is a group of species with high diversity and plays essential roles to the ecosystem. The life cycle of fungi is complex in structure and delicate in function. Choice of developmental strategies and internal changes within the organism are both important for the fungus to fulfill their ecological functions, reflecting the relationship between environment and the population. This study put the developmental process of vegetative growth, sexual and asexual reproduction, resistant structure formation of a classical model basidiomycetes fungus,C. cinerea, together for the first time to view the developmental paths differentiation process with physiology, transcriptomics and evolutionary prospects. Carbohydrate assays and RNA-seq showed the changes of the fungus. Our results fill the gaps on gene expression regulation during the early stage of developmental paths differentiation, and expand our understanding of the evolutionary process of life history and reproductive strategy in fungi.

Living fungal mycelium with suppressed or abolished fruit-forming ability is a self-healing substance particularly valuable biomaterial for further engineering and development in applications such as monitoring/sensing environmental changes and secreting signals. The ability to suppress fungal fruiting is also a useful tool for maintaining stability (e.g., shape, form) of a mycelium-based biomaterial with ease and lower cost. The objective of this present study is to provide a biochemical solution to regulate the fruiting body formation to replace heat killing of mycelium during production. We discovered that GSK-3 activity directly correlates with the development of fruiting bodies in fungi, especially mushroom forming fungi such as Coprinopsis cinerea. By regulating GSK-3 expression and activity, one can control the fungal fruiting body development. We successfully demonstrated that treatment of an inhibitor of GSK-3 kinase activity resulted in acceleration in mycelium growth rate, absence of fruiting body and general decrease in GSK-3 gene expression. Therefore, GSK-3 inhibitor is suggested to be included in the mycelium cultivation recipes for regulating the growth of fungal mycelium and for inhibiting the development of fruiting bodies. This is the first report of using a GSK-3 inhibitor, such as lithium or any other GSK-3 inhibitor, to suppress or abolish fruiting body formation in living fungal mycelium-based biomaterial. It also provides an innovative strategy for easy, reliable, and low cost maintenance of biomaterial containing live fungal mycelium.

The functions of glycogen synthase kinase 3 (GSK3) have been well-studied in animal, plant and yeast. However, information on its roles in basidiomycetous fungi is still limited. In this study, we used the model mushroom Coprinopsis cinerea to study the characteristics of GSK3 in fruiting body development. Application of a GSK3 inhibitor Lithium chloride (LiCl) induced enhanced mycelial growth and inhibited fruiting body formation in C. cinerea. RNA-Seq of LiCl-treated C. cinerea resulted in a total of 14128 unigenes. There were 1210 differentially expressed genes (DEGs) between the LiCl-treated samples and control samples in the mycelium stage (first time point), whereas 1402 DEGs were detected at the stage when the control samples formed hyphal knots and the treatment samples were still in mycelium (second time point). Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analysis of the DEGs revealed significant associations between the enhanced mycelium growth in LiCl treated C. cinerea and metabolism pathways such as “biosynthesis of secondary metabolite” and “biosynthesis of antibiotics”. In addition, DEGs involved in cellular process pathways, including “cell cycle-yeast” and “meiosis-yeast”, were identified in C. cinerea fruiting body formation suppressed by LiCl under favorable environmental conditions. Our findings suggest that GSK3 activity is essential for fruiting body formation as it affects the expression of fruiting body induction genes and genes in cellular processes. Further functional studies of GSK3 in basidiomycetous fungi may help understand the relationships between environmental signals and fruiting body development.

The balance and interplay between sexual and asexual reproduction is one of the most intriguing mysteries in the study of fungi. The choice of developmental strategy reflects the ability of fungi to adapt to the changing environment. However, the evolution of developmental paths and the metabolic regulation during differentiation and morphogenesis are poorly understood. Here, an analysis was performed of carbohydrate metabolism and gene expression regulation during the early differentiation process from the vegetative mycelium, to the differentiated structures, fruiting body, oidia and sclerotia, of a homokaryotic fruiting Coprinopsis cinerea strain A43mutB43mut pab1-1 #326. Changes during morphogenesis and the evolution of developmental strategies were followed. Conversion between glucose and glycogen and between glucose and beta-glucan were the main carbon flows in the differentiation processes. Genes related to carbohydrate transport and metabolism were significantly differentially expressed among paths. Sclerotia displayed a set of specifically up-regulated genes that were enriched in the carbon metabolism and energy production and conversion processes. Evolutionary transcriptomic analysis of four developmental paths showed that all transcriptomes were under the purifying selection, and the more stressful the environment, the younger the transcriptome age. Oidiation has the lowest value of transcriptome age index (TAI) and transcriptome divergence index (TDI), while the fruiting process has the highest of both indexes. These findings provide new insights into the regulations of carbon metabolism and gene expressions during the early stages of fungal developmental paths differentiation, and improve our understanding of the evolutionary process of life history and reproductive strategy in fungi.

Understanding the DNA variation in regulation of carbohydrate-active enzymes (CAZymes) is fundamental to the use of wood-decaying basidiomycetes in lignocellulose conversion into renewable energy. Our goal is to identify the regulators of lignocellulolytic enzymes in Coprinopsis cinerea, of which the genome harbors high number of Auxiliary Activities enzymes.The DNA sequence of C. cinerea family including 46 single spore isolates (SSIs) from crosses of two homozygous strains are used to develop a panel of SNP markers. Then the RNA sequence were used to characterize the gene expression profiles. The RNA were extracted from cultures grown on softwood-enriched sawdust to induce lignocellulolytic enzymes and CCR de-repression genes. To assess the genetic contribution to enzyme expression variations among the 46 SSIs, associations between SNPs and gene expressions were examined genome-widely. 5148 local eQTLs and 7738 distant eQTLs were obtained. By analyzing these eQTLs, the potential regulatory factors of the CAZymes expression and the de-repression of Carbon Catabolism Repression (CCR) were identified,.The eQTL network is characterized in terms of hotspots, evolutionary age and post-translational modifications (PTMs). In the eQTL network of C. cinerea, the non-regulatory genes are younger than the regulatory genes. The proteins regulated by combinational multiple types of PTMs are more likely to function as super regulatory hotspots in protein-protein interactions. The evolutionary age analysis and the PTMome analysis could serve as alternative methods to identify master regulators from genomic data.This work demonstrates a comprehensive bioinformatics approach to identify regulatory factors with next-generation sequencing data. The results provide candidate genes for bioengineering to increase the enzyme production, which will practically benefit the bioethanol production from lignocellulose.SignificanceThis eQTL analysis is designed to study the fungal CAZymes and carbon catabolism repression, especially during the mycelium stage.In Coprinopsis cinerea, only the regions near two ends of the chromosomes have high recombination rate, and suitable for family based eQTL analysis.A sugar transporter is a hotspot controlling many CCR genes.CAZymes are not regulated by a master regulator, but by individual regulators. This indicates that CAZymes are under specific regulatory pathways, so can response to specific conditions.In the eQTL network, the rGenes are evolutionarily older, with more types of PTM sites than eGenes.In the eQTL network, the proteins with more types of PTM sites are more likely associated with Information Storage and Processing, and act as super-hub in the network.

Living fungal mycelium with abolished ability to form fruiting bodies is a self-healing substance, which is particularly valuable for further engineering and development as materials sensing environmental changes and secreting signals. Suppression of fruiting body formation is also a useful tool for maintaining the stability of a mycelium-based material with ease and lower cost. The objective of this study was to provide a biochemical solution to regulate the fruiting body formation, which may replace heat killing of mycelium in practice. The concentrations of glycogen synthase kinase-3 (GSK-3) inhibitors, such as lithium chloride or CHIR99021 trihydrochloride, were found to directly correlate with the development of fruiting bodies in the mushroom forming fungi such as Coprinopsis cinerea and Pleurotus djamor. Sensitive windows to these inhibitors throughout the fungal life cycle were also identified. We suggest the inclusion of GSK-3 inhibitors in the cultivation recipes for inhibiting fruiting body formation and regulating mycelium growth. This is the first report of using a GSK-3 inhibitor to suppress fruiting body formation in living fungal mycelium-based materials. It provides an innovative strategy for easy, reliable, and low cost maintenance of materials containing living fungal mycelium.