Comparative analysis of genome-wide transcriptional responses to continuous heat stress in Pleurotus tuoliensis.

Temperature plays an impactful role in mushroom cultivation. To obtain insights of transcriptomic response in macrofungi against heat stress, we performed RNA-seq analysis using Pleurotus tuoliensis mycelium cells that were treated under 32 °C and 36 °C for consecutive 96 h. By comparing the growth rate data, we found mycelium cells could maintain normal growth rate almost the same as control under 32 °C, yet halted the growths under 36 °C. In total, 2724 differential expressed genes were identified from the three pair-wise comparisons, which were classified to four clusters based on their expression patterns. We also performed gene set enrichment analysis using both GO and KEGG databases, and revealed 48, 113 and 105 enriched GO terms, and 1, 5, and 6 enriched KEGG pathways for three pair-wise comparisons accordingly. In addition, we identified 9 overlapping GO terms and 1 overlapping KEGG pathway shared by the three comparisons. Differentially expressed genes (DEGs) involved in cell communication, amino acid metabolic process, intracellular signal transduction and small molecule biosynthesis were identified in two heat stress treatments despite of the stress intensity. However, the expression of two heat shock protein genes (HSP10 and HSP60) were induced by increasing temperature. Our findings also suggested the DEGs associated with cell cycle regulation had various expression patterns under two heat stress conditions possibly due to different functions. Furthermore, 11 DEGs related to ergosterol biosynthesis were identified with similar expression trends, indicating the ergosterol levels and cell membrane composition may have a tight connection to the acquisition of thermotolerance, which warrant further investigations for deeper understanding of molecular mechanisms in fungal stress responses.

De Novo Assembly Transcriptome Analysis Reveals the Preliminary Molecular Mechanism of Primordium Formation in Pleurotus tuoliensis.

Primordium formation is extremely important for yield of Pleurotus tuoliensis. However, the molecular mechanism underlying primordium formation is largely unknown. This study investigated the transcriptional properties during primordium formation of P. tuoliensis by comparing transcriptome. Clean reads were assembled into 57,075 transcripts and 6874 unigenes. A total of 1397 differentially expressed genes were identified (26 DEGs altered in all stages). GO and KEGG enrichment analysis showed that these DEGs were involved in "oxidoreductase activity", "glycolysis/gluconeogenesis", "MAPK signaling pathways", and "ribosomes". Our results support further understanding of the transcriptional changes and molecular processes underlying primordium formation and differentiation of P. tuoliensis.

TMT-MS/MS proteomic analysis of the carbohydrate-active enzymes in the fruiting body of Pleurotus tuoliensis during storage.

BACKGROUND: The fruiting body of Pleurotus tuoliensis deteriorates rapidly after harvest, causing a decline in its commercial value and a great reduction in its shelf life. According to the present research, carbohydrate-active enzymes (CAZymes) may cause the softening, liquefaction and autolysis of mature mushrooms after harvest. To further understand the in vivo molecular mechanism of CAZymes affecting the postharvest quality of P. tuoliensis fruiting bodies, a tandem mass tags labelling combined liquid chromatography-tandem mass spectrometry (TMT-MS/MS) proteomic analysis was performed on P. tuoliensis fruiting bodies during storage at 25 °C. RESULTS: A total of 4737 proteins were identified, which had at least one unique peptide and had a confidence level above 95%. Consequently, 1307 differentially expressed proteins (DEPs) were recruited using the criteria of abundance fold change (FC) >1.5 or < 0.67 and P < 0.05. The identified proteins were annotated by dbCAN2, a meta server for automated CAZymes annotation. Subsequently, 222 CAZymes were obtained. Several CAZymes participating in the cell wall degradation process, including ß-glucosidase, glucan 1,3-ß-glucosidase, endo-1,3(4)-ß-glucanase and chitinases, were significantly upregulated during storage. The protein expression level of CAZymes, such as xylanase, amylase and glucoamylase, were upregulated significantly, which may participate in the P. tuoliensis polysaccharide degradation. CONCLUSIONS: The identified CAZymes degraded the polysaccharides and lignin, destroying the cell wall structure, preventing cell wall remodeling, causing a loss of nutrients and the browning phenomenon, accelerating the deterioration of P. tuoliensis fruiting body. © 2020 Society of Chemical Industry.

A Comparative Transcriptome Analysis Reveals Physiological Maturation Properties of Mycelia in Pleurotus tuoliensis.

Pleurotus tuoliensis is a precious edible fungus with extremely high nutritive and medicinal value. The cultivation period of P. tuoliensis is longer than those of other Pleurotus species, which is mainly due to a longer mycelium physiological maturation period (30-60 days). Currently, the molecular processes underlying physiological maturation of the mycelium remain unclear. We performed a comparative transcriptomic analysis of immature and mature mycelia using RNA-seq. De novo transcriptome assembly resulted in identification of 17,030 unigenes. 451 differentially expressed genes-including those encoding nucleoside diphosphate kinase (NDPK), glycoside hydrolase family proteins, exopolygalacturonase, and versatile peroxidases-were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that nucleotide synthesis and energy metabolism are highly active during the physiological maturation of mycelia, and genes related to these pathways were significantly upregulated in mature mycelia. NDPK is predicted to be essential for mycelia maturation. Our findings contribute to a comprehensive understanding of mycelia maturation in a commercially important fungal species. Future efforts will focus on the function of NDPK and the mechanism by which it regulates mycelia maturation.

Transcriptome Changes during Major Developmental Transitions Accompanied with Little Alteration of DNA Methylome in Two Pleurotus Species.

Pleurotus tuoliensis (Pt) and P. eryngii var. eryngii (Pe) are important edible mushrooms. The epigenetic and gene expression signatures characterizing major developmental transitions in these two mushrooms remain largely unknown. Here, we report global analyses of DNA methylation and gene expression in both mushrooms across three major developmental transitions, from mycelium to primordium and to fruit body, by whole-genome bisulfite sequencing (WGBS) and RNA-seq-based transcriptome profiling. Our results revealed that in both Pt and Pe the landscapes of methylome are largely stable irrespective of genomic features, e.g., in both protein-coding genes and transposable elements (TEs), across the developmental transitions. The repressive impact of DNA methylation on expression of a small subset of genes is likely due to TE-associated effects rather than their own developmental dynamics. Global expression of gene orthologs was also broadly conserved between Pt and Pe, but discernible interspecific differences exist especially at the fruit body formation stage, and which are primarily due to differences in trans-acting factors. The methylome and transcriptome repertories we established for the two mushroom species may facilitate further studies of the epigenetic and transcriptional regulatory mechanisms underpinning gene during development in Pleurotus and related genera.

Enzymatic gene expression by Pleurotus tuoliensis (Bailinggu): differential regulation under low temperature induction conditions.

Pleurotus tuoliensis is a valuable, rare and edible mushroom that is been commercially cultivated and is rapidly developing in China markets. Low temperatures are required to induces primordia initiation for the successful production of fruiting bodies (basidiomes) during commercial cultivation. In this work, we investigated the enzymatic activities and performed transcription profiling analysis of enzymatic genes under different low temperature conditions. The results suggest that the enzymatic activities and transcription levels decrease or increase significantly at 4 and 13 °C. Lacc10 and mnp6 seems to play a dominant role during nutrition growth. Furthermore, the expression of laccase and peroxidase genes was highly correlated to the detected extracellular enzymatic activity. Cold stress genes expression profiles were upregulated under 4 °C/13 °C (3 days), while only the Hsp70 gene was downregulated (at the stage of fruiting bodies production) at 13 °C (12 days). Our results showed that the transcriptional regulation of laccase and ligninolytic peroxidase genes plays an important role in the fruiting bodies of Bailinggu under low temperature induction (4 °C). Induction at low temperatures was a highly important cultivation condition in Bailinggu.

Cloning, purification and characterization of trehalose-6-phosphate synthase from Pleurotus tuoliensis.

Pleurotus tuoliensis, a kind of valuable and favorable edible mushroom in China, is always subjected to high environmental temperature during cultivation. In our previous study with P. tuoliensis, trehalose proved to be effective for tolerating heat stress. Trehalose-6-phosphate synthase (TPS; EC2.4.1.15) plays a key role in the biosynthesis of trehalose in fungi. In this study, a full-length of cDNA with 1,665 nucleotides encoding TPS (PtTPS) in P. tuoliensis was cloned. The PtTPS amino acid was aligned with other homologues and several highly conserved regions were analyzed. Thus, the TPS protein was expressed in Escherichia coli and purified by affinity chromatography to test its biochemical properties. The molecular mass of the enzyme is about 60 kDa and the optimum reaction temperature and pH is 30 °C and 7, respectively. The UDP-glucose and glucose-6-phosphate were the optimum substrates among all the tested glucosyl donors and acceptors. Metal cations like Mg2+, Co2+, Mn2+, Ni2+, K+, Ag+ stimulated PtTPS activity significantly. Metal chelators such as sodium citrate, citric acid, EDTA, EGTA and CDTA inhibited enzyme activity. Polyanions like heparin and chondroitin sulfate were shown to stimulate TPS activity.

Metabolic Profiling of Pleurotus tuoliensis During Mycelium Physiological Maturation and Exploration on a Potential Indicator of Mycelial Maturation.

Pleurotus tuoliensis is a valuable and rare edible fungus with extremely high nutritional and medicinal value. However, the relative immaturity of P. tuoliensis cultivation technology leads to fluctuating yields and quality. The main difficulty in P. tuoliensis cultivation is estimate of mycelial maturity. There is currently no measurable indicator that clearly characterizes the physiological maturation of mycelia. The aim of this study was to identify potential indicators of physiological maturation for P. tuoliensis mycelia by using metabolomics approach. A metabolite profiling strategy involving gas chromatography-mass spectrometry (GC/MS) was used to analyze changes to extracellular metabolites in mycelia collected at mycelium physiological maturation period (MPMP) day 0, MPMP day 35 at 17°C and MPMP day 35 at 29°C. 72 differential metabolites (37.8% up-regulated and 62.2% down-regulated) were identified based on the selected criteria [variable important in projection (VIP) greater than 1.0 and p < 0.01]. Metabolic pathways enrichment analysis showed that these metabolites are involved in glycolysis, organic acid metabolism, amino acid metabolism, tricarboxylic acid cycle (TCA), sugar metabolism, nicotinate and nicotinamide metabolism, and oxidative phosphorylation. In addition, the pyrimidine synthesis pathway was significantly activated during mycelium physiological maturation of P. tuoliensis. The abundance of N-carbamoyl-L-aspartate (CA-asp), a component of this pathway, was significantly increased at MPMP day 35, which motivated us to explore its potential as an indicator for physiological maturation of mycelia. The content of CA-asp in mycelia changed in a consistent manner during physiological maturation. The feasibility of using CA-asp as an indicator for mycelial maturation requires further investigation.

Development of Novel Polymorphic EST-SSR Markers in Bailinggu (Pleurotus tuoliensis) for Crossbreeding.

Identification of monokaryons and their mating types and discrimination of hybrid offspring are key steps for the crossbreeding of Pleurotus tuoliensis (Bailinggu). However, conventional crossbreeding methods are troublesome and time consuming. Using RNA-seq technology, we developed new expressed sequence tag-simple sequence repeat (EST-SSR) markers for Bailinggu to easily and rapidly identify monokaryons and their mating types, genetic diversity and hybrid offspring. We identified 1110 potential EST-based SSR loci from a newly-sequenced Bailinggu transcriptome and then randomly selected 100 EST-SSRs for further validation. Results showed that 39, 43 and 34 novel EST-SSR markers successfully identified monokaryons from their parent dikaryons, differentiated two different mating types and discriminated F1 and F2 hybrid offspring, respectively. Furthermore, a total of 86 alleles were detected in 37 monokaryons using 18 highly informative EST-SSRs. The observed number of alleles per locus ranged from three to seven. Cluster analysis revealed that these monokaryons have a relatively high level of genetic diversity. Transfer rates of the EST-SSRs in the monokaryons of closely-related species Pleurotuseryngii var. ferulae and Pleurotus ostreatus were 72% and 64%, respectively. Therefore, our study provides new SSR markers and an efficient method to enhance the crossbreeding of Bailinggu and closely-related species.