Volatile sulfur compounds gradually develop in Lentinula edodes after hot-air drying, and many genes are involved in the generation of these sulfur compounds. The expression stability of reference genes may vary in a particular experimental treatment when analyzing their expressions by quantitative real-time polymerase chain reaction (qRT-PCR). In this study, the expression profile of 17 candidate genes was assessed in L. edodes under treatment at 50 °C for 0, 1, 2, and 3 h, and the expression stability of each reference gene was analyzed by three statistical algorithms, including geNorm, NormFinder, and BestKeeper. Results indicated that the two optimal reference genes for mycelium and fruiting body were CAC and DAHP as well as CAC and NUP, respectively. Additionally, CAC and DAHP were found to be the two most stable reference genes across the mycelium and fruiting body set. Our results will provide a genetic foundation for further research on the metabolism genes of sulfur compounds in L. edodes.

Lentinula edodes, one of the most popular, edible mushroom species with a high content of proteins and polysaccharides as well as unique aroma, is widely cultivated in many Asian countries, especially in China, Japan and Korea. As a white rot fungus with lignocellulose degradation ability, L. edodes has the potential for application in the utilization of agriculture straw resources. Here, we report its 41.8-Mb genome, encoding 14,889 predicted genes. Through a phylogenetic analysis with model species of fungi, the evolutionary divergence time of L. edodes and Gymnopus luxurians was estimated to be 39 MYA. The carbohydrate-active enzyme genes in L. edodes were compared with those of the other 25 fungal species, and 101 lignocellulolytic enzymes were identified in L. edodes, similar to other white rot fungi. Transcriptome analysis showed that the expression of genes encoding two cellulases and 16 transcription factor was up-regulated when mycelia were cultivated for 120 minutes in cellulose medium versus glucose medium. Our results will foster a better understanding of the molecular mechanism of lignocellulose degradation and provide the basis for partial replacement of wood sawdust with agricultural wastes in L. edodes cultivation.

Melatonin, a bioactive compound and an important signaling molecule produced in plants and animals, is involved in many biological processes. However, its function and synthetic pathways in fungi are poorly understood. Here, the samples from Tolypocladium guangdongense, a highly valued edible fungus with functional food properties, were collected under different experimental conditions to quantify the levels of melatonin and its intermediates. The results showed that the intracellular melatonin content was markedly improved by Congo red (CR), cold, and heat stresses; the levels of intracellular melatonin and its intermediates increased at the primordial (P) and fruiting body (FB) stages. However, the levels of most intermediates exhibited a notable decrease under CR stress. Several genes related to melatonin synthesis, excluding AADC (aromatic-L-amino-acid decarboxylase), were markedly upregulated at an early stage of CR stress but downregulated later. Compared to the mycelial stage, those genes were significantly upregulated at the P and FB stages. Additionally, exogenous melatonin promoted resistance to several abiotic stressors and P formation in T. guangdongense. This study is the first to report melatonin biosynthesis pathway in macro-fungi. Our results should help in studying the diversity of melatonin function and melatonin-synthesis pathways and provide a new viewpoint for melatonin applications in the edible-medicinal fungus.

Tolypocladium guangdongense has a similar metabolite profile to Ophiocordyceps sinensis, a highly regarded fungus used for traditional Chinese medicine with high nutritional and medicinal value. Although the genome sequence of T. guangdongense has been reported, relatively little is known about the regulatory networks for fruiting body development and about the metabolite biosynthesis pathways. In order to address this, an analysis of transcriptome and proteome at differential developmental stages of T. guangdongense was performed. In total, 9076 genes were found to be expressed and 2040 proteins were identified. There were a large number of genes that were significantly differentially expressed between the mycelial stage and the stages. Interestingly, the correlation between the transcriptomic and proteomic data was low, suggesting the importance of the post-transcriptional processes in the growth and development of T. guangdongense. Among the genes/proteins that were both differentially expressed during the developmental process, there were numerous heat shock proteins and transcription factors. In addition, there were numerous proteins involved in terpenoid, ergosterol, adenosine and polysaccharide biosynthesis that also showed significant downregulation in their expression levels during the developmental process. Furthermore, both tryptophan and tryptamine were present at higher levels in the primordium stage. However, indole-3-acetic acid (IAA) levels continuously decreased as development proceeded, and the enzymes involved in IAA biosynthesis were also clearly differentially downregulated. These data could be meaningful in studying the molecular mechanisms of fungal development, and for the industrial and medicinal application of macro-fungi.

Lentinula edodes (shiitake mushrooms) is heavily affected by the infection of Trichoderma atroviride, causing yield loss and decreases quality in shiitake mushrooms. The selection and breeding of fungal-resistant L. edodes species are an important approach to protecting L. edodes from T. atroviride infection. Herein, a highly resistant L. edodes strain (Y3334) and a susceptible strain (Y55) were obtained by using a resistance evaluation test. Transcriptome analyses and qRT-PCR detection showed that the expression level of LeTLP1 (LE01Gene05009) was strongly induced in response to T. atroviride infection in the resistant Y3334. Then, LeTLP1-silenced and LeTLP1-overexpression transformants were obtained. Overexpression of LeTLP1 resulted in resistance to T. atroviride. Compared with the parent strain Y3334, LeTLP1-silenced transformants had reduced resistance relative to T. atroviride. Additionally, the LeTLP1 protein (Y3334) exhibited significant antifungal activity against T. atroviride. These findings suggest that overexpression of LeTLP1 is a major mechanism for the resistance of L. edodes to T. atroviride. The molecular basis provides a theoretical basis for the breeding of resistant L. edodes strains and can eventually contribute to the mushroom cultivation industry and human health.

Volatile organosulfur compounds are the main components that contribute to the unique aroma of dried Lentinula edodes. They are mainly generated during the hot-air drying process, and cysteine desulfurase is the key enzyme in this process. Temperature may be an essential factor of volatile organosulfur compound production by influencing the expression of the cysteine desulfurase gene. In this study, the promoter sequence of the cysteine desulfurase gene (pCS) was cloned and analyzed using bioinformatics tools. A series of 5'deletion fragments and site-directed mutations of pCS were constructed to identify the element that responds to heat stress. Six heat shock transcription factor (HSTF) binding sites were predicted by SCPD (The Promoter Database of Saccharomyces cerevisiae) and three of the binding sites were predicted by Yeastract (Yeast Search for Transcriptional Regulators and Consensus Tracking) in pCS. The results indicated that pCS was able to drive the expression of the EGFP (Enhanced Green Fluorescent Protein) gene in L. edodes. Moreover, the fluorescence intensity increased after heat stress. The changes in fluorescence intensity of different 5'deletion fragments showed that the heat response region was located between -500 bp and -400 bp in pCS. The site-directed mutation analysis further showed that the heat-inducible element was between -490 bp and -500 bp (TTTCTAGAAT) in pCS. Our results provide molecular insight for studying the formation of volatile organosulfur compounds in dried L. edodes.

Our previous study found that LeDnaJ07 RNAi decreased Lentinula edodes resistance to heat stress and Trichoderma atroviride infection. In this study, the structure and function of the LeDnaJ07 gene was analyzed by gene cloning and overexpression in L. edodes stress-sensitive strain YS55 via the Agrobacterium-mediated transformation method. Transformants were confirmed by qRT-PCR, fluorescence observation and Southern blotting. Overexpression of LeDnaJ07 in YS55 not only enhanced L. edodes mycelial resistance to heat stress but also facilitated mycelial growth. In the presence of heat stress, the intracellular IAA content showed a significant increase in the two LeDnaJ07 overexpression strains but only a slight change in the YS55 wild type strain. Moreover, the interaction between LeDnaJ07 and LetrpE was demonstrated via Y2H and BiFC assays. These results suggested that LeDnaJ07 may be involved in regulating IAA biosynthesis and the resistance of L. edodes to heat stresses via interacting with LetrpE.

DnaJ is an important molecular chaperone, with significant roles in growth, development, and stress resistance. Studies on the DnaJ gene family in macro-fungi such as Cordyceps spp. s.l. is scare. In this study, 22, 20, and 24 putative DnaJ genes were identified in Tolypocladium guangdongense, Ophiocordyceps sinensis, and C. militaris, respectively. They were classified into four groups based on the presence of the J, zinc finger, and C-terminal domains. We mainly studied the T. guangdongense DnaJ genes being located in the endoplasmic reticulum, cytoplasm, mitochondrion, and nucleus. Phylogenetic analysis revealed gene duplications during the evolutionary process. Multiple cis-elements and transcription factor binding sites were observed in the promoter, suggesting their involvement in the response to multiple stresses. qRT-PCR analysis showed that 63.63% and 45.45% of T. guangdongense DnaJ genes were differentially expressed under cold and heat stress, respectively, indicating their involvement in the response to temperature stress. Many T. guangdongense DnaJ genes in the primordium and fruiting body exhibited differential expression, in comparison to those in the mycelium, suggesting a regulatory role in its growth and development process. These findings will facilitate further functional analysis, and provide information on the classification and conservative functions of DnaJ proteins in macro-fungi.

Fungal GATA-type transcription factors (GATA-TFs) are a class of transcriptional regulators involved in various biological processes. However, their functions are rarely analyzed systematically, especially in edible or medicinal fungi, such as Tolypocladium guangdongense, which has various medicinal and food safety properties with a broad range of potential applications in healthcare products and the pharmaceutical industry.

Obesity has caused serious health and economic problems in the world. Cordyceps guangdongensis is a high-value macrofungus with broad application potential in the food and bio-medicine industry. This current study aimed to estimate the role of C. guangdongensis lipid-lowering compound formula (CGLC) in regulating fat and lipid accumulation, gut microbiota balance, short-chain fatty acid (SCFA) contents, and expression levels of genes involved in fat and lipid metabolism in high-fat diet (HFD) mice. The results showed that CGLC intervention markedly reduced body weights and fat accumulation in HFD mice, improved glucose tolerance and blood lipid levels, and decreased lipid droplet accumulation and fat vacuole levels in the liver. CGLC decreased the ratio of Firmicutes and Bacteroidetes and increased the relative abundances of Bacteroides (B. acidifaciens) and Bifidobacterium (B. pseudolongum). In addition, CGLC treatment significantly promoted the production of SCFAs and regulated the relative expression levels of genes involved in fat and lipid metabolism in liver. Association analysis showed that several species of Bacteroides and most of SCFAs were significantly associated with serum lipid indicators. These results suggested that CGLC is a novel candidate formulation for treating obesity and non-alcohol fatty liver by regulating gut microbiota, SCFAs, and genes involved in fat and lipid metabolism.

Lentinula edodes is the most consumed mushroom in Asia due to its nutritional and medicinal values, and the optimal reference gene is crucial for normalization of its gene expression analysis. Here, the expression stability of 18 candidate reference genes (CRGs) in L. edodes was analyzed by three statistical algorithms (geNorm, NormFinder and BestKeeper) under different stresses (heat, cadmium excess and Trichoderma atroviride infection), different substrates (straw, sawdust and corn stalk) and different development stages (mycelia, primordia and fruit bodies). Among the 18 CRGs, 28S, Actin and α-tub exhibited the highest expression stability in L. edodes under all conditions, while GPD, SPRYP and MSF showed the least stable expression. The best reference gene in different conditions was different. The pairwise variation values showed that two genes would be sufficient for accurate normalization under different conditions of L. edodes. This study will contribute to more accurate estimation of the gene relative expression levels under different conditions using the optimal reference gene in qRT-PCR (quantitative reverse transcription polymerase chain reaction) analysis.

Tolypocladium guangdongense, formerly known as Cordyceps guangdongensis, is a widely cultivated fungus of the Cordyceps s.l. species that has been investigated over the last 12 years. It has the potential to be used in a number of applications in the health and pharmaceutical industries for it has shown its high nutritional and medicinal values according to previous animal studies. qRT-PCR (quantitative reverse transcription polymerase chain reaction) is extensively used to analyze the expression pattern and molecular mechanisms of functional genes under differentially experimental conditions. The expression stability of reference genes used for normalization determines the reliability of qRT-PCR results, indicating the importance of selection and validation of reference genes before gene expression analysis. In the present study, three statistical algorithms, geNorm, NormFinder and BestKeeper, were used for analyzing the expression stability of nineteen candidate reference genes (CRGs) in T. guangdongense. Investigation were carried out under differentially experimental conditions, which included differentially developmential stages (mycelia, primordia, young and mature fruiting bodies), different carbon sources, cold and heat stresses. The results showed that histone H4 and tubulin beta chain 2 (β-tub2) were the most and least stable genes, respectively, across all the experimental samples. Moreover, analysis of individual data sets exhibited different stability and expression profiles of reference genes. The vacuolar protein sorting gene VPS was the most stable gene expressed under the differentially developmental stages and temperature stresses, whereas H4 was the most stably expressed gene under different carbon sources. Therefore, it can be proposed that VPS and H4 are the preferred reference genes for normalization of gene expression under different experimental conditions. The results of our present study will enable more accurate evaluation of gene expression in T. guangdongense using the optimal reference gene for qRT-PCR analysis.