Zhiyong Feng

An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus

Hypsizygus marmoreus is one of the major edible mushrooms in East Asia. As no efficient transformation method, the molecular and genetics studies were hindered. The glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of H. marmoreus was isolated and its promoter was used to drive the hygromycin B phosphotransferase (HPH) and enhanced green fluorescent protein (EGFP) in H. marmoreus. Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied in H. marmoreus. The transformation parameters were optimized, and it was found that co-cultivation of bacteria with protoplast at a ratio of 1000:1 at a temperature of 26 °C in medium containing 0.3 mM acetosyringone resulted in the highest transformation efficiency for Agrobacterium strain. Besides, three plasmids, each carrying a different promoter (from H. marmoreus, Ganoderma lucidum and Lentinula edodes) driving the expression of an antibiotic resistance marker, were also tested. The construct carrying the H. marmoreus gpd promoter produced more transformants than other constructs. Our analysis showed that over 85% of the transformants tested remained mitotically stable even after five successive rounds of subculturing. Putative transformants were analyzed for the presence of hph gene by PCR and Southern blot. Meanwhile, the expression of EGFP in H. marmoreus transformants was detected by fluorescence imaging. This ATMT system increases the transformation efficiency of H. marmoreus and may represent a useful tool for molecular genetic studies in this mushroom species.

Transcriptome analysis and its application in identifying genes associated with fruiting body development in basidiomycete Hypsizygus marmoreus.

To elucidate the mechanisms of fruit body development in H. marmoreus, a total of 43609521 high-quality RNA-seq reads were obtained from four developmental stages, including the mycelial knot (H-M), mycelial pigmentation (H-V), primordium (H-P) and fruiting body (H-F) stages. These reads were assembled to obtain 40568 unigenes with an average length of 1074 bp. A total of 26800 (66.06%) unigenes were annotated and analyzed with the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Eukaryotic Orthologous Group (KOG) databases. Differentially expressed genes (DEGs) from the four transcriptomes were analyzed. The KEGG enrichment analysis revealed that the mycelium pigmentation stage was associated with the MAPK, cAMP, and blue light signal transduction pathways. In addition, expression of the two-component system members changed with the transition from H-M to H-V, suggesting that light affected the expression of genes related to fruit body initiation in H. marmoreus. During the transition from H-V to H-P, stress signals associated with MAPK, cAMP and ROS signals might be the most important inducers. Our data suggested that nitrogen starvation might be one of the most important factors in promoting fruit body maturation, and nitrogen metabolism and mTOR signaling pathway were associated with this process. In addition, 30 genes of interest were analyzed by quantitative real-time PCR to verify their expression profiles at the four developmental stages. This study advances our understanding of the molecular mechanism of fruiting body development in H. marmoreus by identifying a wealth of new genes that may play important roles in mushroom morphogenesis.

Pseudomonas zeshuii sp. nov., isolated from herbicide-contaminated soil

A Gram-stain-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain BY-1(T), was isolated from a soil sample from the city of Qiqihar in Heilongjiang Province, PR China. Strain BY-1(T) grew optimally at pH 7.0 and 30-35 °C in the presence of 0.5% (w/v) NaCl. Analysis of 16S rRNA gene sequences revealed that strain BY-1(T) fell within the radiation of the genus Pseudomonas, and showed highest 16S rRNA gene sequence similarities to Pseudomonas luteola IAM 13000(T) (99.5%) and Pseudomonas duriflava HR2(T) (97.3%); the levels of sequence similarity with respect to other recognized species of the genus Pseudomonas were <96.7%. Strain BY-1(T) showed low DNA-DNA relatedness values with Pseudomonas luteola IAM 13000(T) (29 ± 3.1%) and Pseudomonas duriflava HR2(T) (21 ± 1.5 %). The G+C content of the genomic DNA of strain BY-1(T) was 55.3 mol%. The major fatty acids were C(18:1), C(16:0) and summed feature 3 (C(16:1)ω6c and/or C(16:1)ω7c). Major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine, and the major ubiquinone was Q-9. Data obtained in this study indicated that this isolate represents a novel species of the genus Pseudomonas, for which the name Pseudomonas zeshuii sp. nov. is proposed. The type strain is BY-1(T) (=KACC 15471(T)=ACCC 05688(T)).

Cloning and functional analysis of a laccase gene during fruiting body formation in Hypsizygus marmoreus.

The Hypsizygus marmoreus laccase gene (lcc1) sequence was cloned and analyzed. The genomic DNA of lcc1 is 2336 bp, comprising 13 introns and 14 exons. The 1626-bp full-length cDNA encodes a mature laccase protein containing 542 amino acids, with a 21-amino acid signal peptide. Phylogenetic analysis showed that the lcc1 amino acid sequence is homologous to basidiomycete laccases and shares the highest similarity with Flammulina velutipes laccase. A 2021-bp promoter sequence containing a TATA box, CAAT box, and several putative cis-acting elements was also identified. To study the function of lcc1, we first overexpressed lcc1 in H. marmoreus and found that the transgenic fungus producing recombinant laccase displayed faster mycelial growth than the wild-type (wt) strain. Additionally, primordium initiation was induced 3-5 days earlier in the transgenic fungus, and fruiting body maturation was also promoted approximately five days earlier than in the wt strain. Furthermore, we detected that lcc1 was sustainably overexpressed and that laccase activity was also higher in the transgenic strains compared with the wt strain during development in H. marmoreus. These results indicate that the H. marmoreus lcc1 gene is involved in mycelial growth and fruiting body initiation by increasing laccase activity.

Construction and application of a gene silencing system using a dual promoter silencing vector in Hypsizygus marmoreus.

As efficient reverse genetic tools are lacking, molecular genetics research has been limited in Hypsizygus marmoreus. In this study, we firstly constructed a gene‐silencing method using a dual promoter vector (DPV) which was driven by gpd and 35 S promoters. The DPV was introduced into H. marmoreus via a simple electroporation procedure and the highest silenced rate of ura3 gene was 76.6%, indicating that the DPV might be suitable for gene silencing in basidiomycete. In this silencing system, the endogenous orotidine 5′‐monophosphate decarboxylase gene (ura3) was used as a selectable marker. Besides, we also constructed another silencing system which could silence the ura3 and other genes (lcc1 encoded laccase1) together in H. marmoreus, and named it as co‐silencing system. In the co‐silenced transformants, we found that the mycelia were thinner and the growth was slower than in the wild‐type and control2 strains, which was accordant with the previous study of lcc1 gene, indicating that the selective efficiency of the RNAi‐mediated silencing of several genes might be increased by co‐silencing ura3. The development of this molecular tool might improve functional studies of multiple genes in the basidiomycete H. marmoreus and also provide a reference for studies of other basidiomycetes.

Kojic acid-mediated damage responses induce mycelial regeneration in the basidiomycete Hypsizygus marmoreus

Mechanical damage can induce fruiting body production in fungi. In this study, the antioxidant kojic acid (KA) was found to enhance injured mycelial regeneration and increase fruiting body production in Hypsizygus marmoreus. KA reduced the level of reactive oxygen species (ROS), which are harmful to mycelia when excessively generated by mechanical damage. Moreover, KA increased catalase and superoxide dismutase activities and glutathione and ascorbic acid contents by up-regulating antioxidant gene expression. These results suggest that KA promotes mycelial regeneration in response to damage by activating a “stress signal” and enhances the ability of H. marmoreus to resist oxidative damage by invoking the antioxidant system. In addition, KA increased the content of extracellular ATP, which serves as a “stress signal” in response to injury, and modulated ROS signaling, decreasing NADPH oxidase gene expression and ROS levels in the mycelial-regeneration stage. KA treatment also up-regulated the MAPK, Ca2+ and oxylipin pathways, suggesting their involvement in the damage response. Furthermore, laccase and cellulase activities were stimulated by KA at different developmental stages. These results demonstrate that KA regulates gene expression and activates pathways for mycelial wound healing, regeneration of damaged mycelia and reproductive structure formation in the basidiomycete H. marmoreus.

Mechanism of Glucose Regulates the Fruiting Body Formation in the Beech Culinary-Medicinal Mushroom, Hypsizygus marmoreus (Agaricomycetes)

To understand the fruiting process of Hypsizygus marmoreus, a synthetic liquid medium (SLM) was optimized to induce fruiting body initiation. Dependent on the SLM, the effect of a monofactor (glucose) on the fruiting bodies of H. marmoreus was studied at different concentrations (10 and 40 g/L). Primordia appeared approximately 10 days earlier in low-glucose media (LGM) than in high-glucose media (HGM), whereas mature fruiting bodies formed on mushrooms approximately 7 days earlier and more primordia developed into mature fruiting bodies when cultured in HGM. In addition, the morphogenesis of the primordia was clustered in HGM, which was different than what was observed in LGM. Furthermore, differentially expressed genes (DEGs) that encoded various proteins involved in cell structure, general metabolism, signal transduction, and transcription and translation were analyzed by transcriptome sequencing. Six DEGs were detected by quantitative reverse-transcriptase polymerase chain reaction, and the results were consistent with the altered patterns of gene expression revealed by the transcriptome. This study not only identifies new candidate genes involved in the development of H. marmoreus but also provides a new research platform for studying the development of other edible mushrooms.

Hydrogen-rich water mediates redox regulation of the antioxidant system, mycelial regeneration and fruiting body development in Hypsizygus marmoreus

Hypsizygus marmoreus is an important industrialized mushroom, yet the lack of basic research on this fungus has hindered further development of its economic value. In this study, mycelia injured by scratching were treated with hydrogen-rich water (HRW) to investigate the involvement of the redox system in fruiting body development. Compared to the control group, damaged mycelia treated with HRW regenerated earlier and showed significantly enhanced fruiting body production. Antioxidant capacity increased significantly in damaged mycelia after HRW treatment, as indicated by higher antioxidant enzyme activities and antioxidant contents; the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were also reduced at the mycelial regeneration stage after treatment with HRW. Furthermore, genes involved in ROS, Ca2+, MAPK and oxylipin signaling pathways were up-regulated by HRW treatment. In addition, laccase and manganese peroxidase activities and mycelial biomass were higher after HRW treatment, suggesting that HRW might enhance the substrate-degradation rate to provide more carbon sources for fruiting body production.