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Tremella fuciformis is a dimorphic fungus which can undertake the reversible transition between yeast and pseudohypha forms. G protein α subunit (Gα) carries different signals to regulate a variety of biological processes in eukaryotes, including fungal dimorphism. In this study, a novel Gα subunit encoded gene, TrGpa1, was firstly cloned from T. fuciformis. The TrGpa1 open reading frame has 1059 nucleotides, and encodes a protein which belongs to the group I of Gαi superfamily. Furthermore, the role of TrGpa1 in the T. fuciformis dimorphism was analysed by gene overexpression and knockdown. Stable integration of the target gene into the genome was confirmed by PCR and Southern blot hybridization. Transformants with the highest and lowest TrGpa1 expression levels were selected via quantitative real-time PCR analysis and Western blot. Each transformant was compared with the wild-type strain about the morphological change under different environmental factors, including pH values, temperature, cultivation time, inoculum size, and quorum-sensing molecules (farnesol and tyrosol). Comparing with the wild-type strain, the overexpression transformant always had higher ratios of pseudohyphae, while the knockdown transformant had less proportions of pseudohyphae. Therefore, the TrGpa1 is involved in the dimorphism of T. fuciformis and plays a positive role in promoting pseudohyphal growth.

Annulohypoxylon stygium is an ascomycete that helps Tremella fuciformis produce the fruiting body in wild state or artificial cultivation. Although the interaction between these two fungi is well known, the underlying molecular mechanism is limited. In this study, the 981 bp and 886 bp promoter sequences of glyceraldehyde-3-phosphate dehydrogenase (gpd) gene and α-tubulin gene have been cloned, respectively. Sequence analysis showed that gpd promoter contained nine CAAT boxes, and single TGACG-motif, TATA box, ABRE motif, STRE motif, MYB motif, and W box. The α-tubulin promoter included eight CAAT boxes, three STRE, two TATA boxes and MYB, single Box 4, CAT-box, CCAAT-box, TGA-element, and ABRE. Subsequently, we evaluated the promoters' function by constructing four vectors pGEH, pGRH, pTEH, and pTRH to drive fused enhanced green fluorescent protein and hygromycin B phosphotransferase (egfp-hph) or red fluorescent protein and hygromycin B phosphotransferase (rfp-hph) expression under the control of gpd or α-tubulin promoters in A. stygium. The integration of the transformed DNA into A. stygium genome was verified by PCR, Southern blot, fluorescence microscopy, and quantitative real-time PCR (qRT-PCR). All the results indicated that the two promoters could drive egfp-hph and rfp-hph expression. This result could provide help in gene functional studies by using gpd and α-tubulin promoters to direct gene over-expression or build dual promoter silencing systems.

Tremella fuciformis is a dimorphic fungus which can undertake the reversible transition between yeast and pseudohypha forms. G protein α subunit (Gα) carries different signals to regulate a variety of biological processes in eukaryotes, including fungal dimorphism. In this study, a novel Gα subunit encoded gene, TrGpa1, was firstly cloned from T. fuciformis. The TrGpa1 open reading frame has 1059 nucleotides, and encodes a protein which belongs to the group I of Gαi superfamily. Furthermore, the role of TrGpa1 in the T. fuciformis dimorphism was analysed by gene overexpression and knockdown. Stable integration of the target gene into the genome was confirmed by PCR and Southern blot hybridization. Transformants with the highest and lowest TrGpa1 expression levels were selected via quantitative real-time PCR analysis and Western blot. Each transformant was compared with the wild-type strain about the morphological change under different environmental factors, including pH values, temperature, cultural time, inoculum size, and quorum-sensing molecules (farnesol and tyrosol). Comparing with the wild-type strain, the overexpression transformant always had higher ratios of pseudohyphae, while the knockdown transformant had less proportions of pseudohyphae. Therefore, the TrGpa1 involves in the dimorphism of T. fuciformis and plays a positive role in promoting pseudohyphal growth.

Lectins, one of the most important bioactive compounds, are nonimmunoglobulin proteins that can bind carbohydrates specifically. However, few reports have been published on Pleurotus ostreatus lectin at the molecular level. Hence, in this study, seven lectins were identified based on transcriptomes in four developmental stages, i.e., mycelium, primordium, young fruiting body, and mature fruiting body. The expression profiles of the lectin genes were verified by quantitative real-time PCR. Lectin2-lectin6 had the highest expression in mycelium, while lectin1 was rich in mature fruiting body, and lectin7 was in primordium. We inferred that lectin2-lectin6 may take part in cell flocculation, lectin7 was the critical gene for primordium formation, and lectinl may be involved in fruiting body maturation, respectively. By in silico analysis, all lectins were divided into three distinct groups. Lectin1-Lectin5 were about 38.5-40.7 kDa as extracellular protein and belonged to the PCL-like lectins. Lectin6 (15.4 kDa) was predicted in nucleus and belonged to fungal fruit body lectins. Lectin7 (38.5 kDa) was a member of legume-like lectins and located in the plasma membrane. This study will help us understand how lectins mediate mushroom development.

Tremella fuciformis is an edible white jelly mushroom with medicinal qualities. The formation of T. fuciformis fruiting bodies is highly dependent on the presence of Annulohypoxylon stygium under natural conditions and during artificial cultivation. A lack of efficient transformation systems restricts the ability of researchers to functionally characterize the genes in these two interacting fungi. In this study, we tested the utility of the Agrobacterium tumefaciens-mediated transformation of T. fuciformis and A. stygium protoplasts. A. tumefaciens strain EHA105 cells harboring the pTrGEH plasmid, which contains genes for enhanced green fluorescent protein (egfp) and hygromycin B phosphotransferase (hph), was co-cultured with T. fuciformis protoplasts. Meanwhile, EHA105 cells harboring the pAnGRH plasmid, which contains the red fluorescent protein (rfp) and hph genes, was co-cultured with A. stygium protoplasts. The egfp, rfp, and hph genes were under the control of the promoter for gpd, which encodes glyceraldehyde-3-phosphate dehydrogenase. Optimal co-cultivation was achieved with a 1:1 mixture of bacteria (OD600 0.4-0.6) and fungal protoplasts (105/mL) incubated at 25°C in a medium containing 200 μM acetosyringone. The subsequent selection on hygromycin B-containing medium yielded 45 and 187 stable transformants per 105 protoplasts for T. fuciformis and A. stygium, respectively. The integration of the transformed DNA into the two fungal genomes was confirmed by polymerase chain reaction (PCR), Southern blot analysis, fluorescence imaging, and a quantitative real-time PCR. All results confirmed the feasibility of our transformation approach, which may facilitate future functional analyses of T. fuciformis and A. stygium genes.

Background: Hydrophobins are small proteins secreted by filamentous fungi, which show a highly surface activity. Because of the signally self-assembling abilities and surface activities, hydrophobins were considered as candidates in many aspects, for example, stabilizing foams and emulsions in food products. Lentinus tuber-regium, known as tiger milk mushroom, is both an edible and medicinal sclerotium-producing mushroom. Up to now, the hydrophobins of L. tuber-regium have not been identified. Results: In this paper, a Class I hydrophobin gene, Ltr.hyd, was cloned from L. tuber-regium and expressed in the yeast-like cells of Tremella fuciformis mediated by Agrobacterium tumefaciens. The expression vector pGEH-GH was under the control of T. fuciformis glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter. The integration of Ltr.hyd into the genome of T. fuciformis was confirmed by PCR, Southern blot, fluorescence observation and quantitative real-time PCR (qRT-PCR). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that recombinant hydrophobin rLtr.HYD with an expected molecular mass of 13 kDa was extracted. The yield of rLtr.HYD was 0.66 mg/g dry weight. The emulsifying activity of rLtr.HYD was better than the typical food emulsifiers sodium caseinate and Tween 20. Conclusions: We evaluated the emulsifying property of hydrophobin Ltr.HYD, which can be potentially used as a food emulsifier. Keywords: Agrobacterium tumefaciens, Emulsifier, Expression vector, Filamentous fungi, Gel electrophoresis, Glyceraldehyde-3-phosphate dehydrogenase, Heterogenous expression, Hydrophobin, Quantitative real-time PCR, Southern blot, Surface activity

Tremella fuciformis is an edible and medicinal white jelly mushroom. It has a life cycle that interacts with its companion fungus Annulohypoxylon stygium, both in natural conditions and artificial cultivation. RNA sequencing (RNA-Seq) was used to study the interaction between T. fuciformis and A. stygium by constructing 5 libraries, including the individual T. fuciformis mycelium (1), the T. fuciformis mycelium after interaction with A. stygium (2), the dual mycelia after interaction (3), the A. stygium mycelium after interaction with T. fuciformis (4), and the individual A. stygium mycelium (5). 33.4 G data and 46,871 Unigenes were generated from de novo splicing. For identification of differentially expressed genes (DEGs) related to interaction, we analyzed the expression data of DEGs1-vs-2 ∩ DEGs1-vs-3, and DEGs5-vs-4 ∩ DEGs5-vs-3. DEGs1-vs-2 ∩ DEGs1-vs-3, and DEGs5-vs-4 ∩ DEGs5-vs-3 data showed 614 DEGs and 1537 DEGs, respectively. The 614 DEGs for T. fuciformis and 1537 DEGs for A. stygium were analyzed by GO annotation and were assigned to biology process, cell composition, and molecular functions. The DEGs were used to match the KEGG database. In T. fuciformis, the pathways are primarily enriched various amino acids metabolism, pentose and glucuronate interconversions. In A. stygium, the pathways are primarily enriched in the biosynthesis of secondary metabolites, biosynthesis of antibiotics, starch and sucrose metabolism. The expression patterns of DEGs determined by qRT-PCR were consistent with those obtained by RNA-Seq, thus validating the reliability of our RNA-Seq data. Future studies of the functions of these interesting genes will be helpful to understand the mechanisms by which T. fuciformis interacts with A. stygium. This will also provide a reference for other research on interacting microorganisms.

To gain insights into dimorphism, cell wall polysaccharides from Tremella fuciformis strains were obtained from alkali-extracted water-soluble fractions PTF-M38 (from the mycelial form), PTF-Y3 and PTF-Y8 (from the yeast form) of T. fuciformis strains were used to gain some insights into dimorphism study. Their chemical properties and structural features were investigated using gel permeation chromatography, gas chromatography, UV and IR spectrophotometry and Congo red binding reactions. The results indicated that the backbones of PTF-M38, PTF-Y3 and PTF-Y8 were configured with α-linkages with average molecular weights of 1.24, 1.08, and 1.19 kDa, respectively. PTF-M38 was mainly composed of xylose, mannose, glucose, and galactose in a ratio of 1:1.47:0.48:0.34, while PTF-Y3 and PTF-Y8 were mainly composed of xylose, mannose and glucose in a ratio of 1:1.65:4.06 and 1:1.21:0.44, respectively. The sugar profiles of PTF-M38, PTF-Y3 and PTF-Y8 were also established for further comparison. These profiles showed that all three polysaccharides contained the same sugars but in different ratios, and the carbon sources (xylose, mannose, glucose, and galactose) affected the sugar ratios within the polysaccharides.

Lentinus tuber-regium is a sclerotium-forming basidiomycetous mushroom. It has increasingly aroused people's attention for its medicinal effects. In this study, we investigated the applicability of the Agrobacterium tume-faciens-mediated transformation (ATMT) method in L. tuber-regium. A. tumefaciens strain GV 3101 harboring the vector pPEH was used to transform the mycelium of L. tuber-regium strain ACCC50657. The genes for hygromycin B phosphotransferase (hph) and enhanced green fluorescent protein (egfp) under the control of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene promoter of Pleurotus ostreatus were employed as the selection marker and reporter gene, respectively. The optimal cocultivation temperature and time for transformation were 3 days and 4 days at 25°C and 20°C, respectively. Southern blot analysis showed the variation in the copy number and position of hph, which indicated random integration of hph. Polymerase chain reaction and fluorescence microscopy indicated that the P. ostreatus gpd promoter can drive the fused hph-egfp gene expression in L. tuber-regium. This is the first report that the ATMT method was successfully applied to L. tuber-regium. This reliable and efficient transformation method could be a powerful tool for strain genetic improvement and gene function study in L. tuber-regium.

Proteins are subjected to modification by reactive oxygen species (ROS), and oxidation of specific amino acid residues can impair their biological functions. Methionine as a sulfur-containing amino acid is easily oxidized to methionine sulfoxide (MetSO). The modified methionine can be repaired by methionine sulfoxide reductase (Msr), an enzyme that reverses oxidation of methionine in proteins. In this study, a methionine sulfoxide reductase A (PoMsrA) gene from Pleurotus ostreatus was cloned and characterized. Furthermore, the function of PoMsrA gene was analyzed by overexpression in P. ostreatus via Agrobacterium-mediated transformation. Stable integration of the target gene into the genome of P. ostreatus was confirmed by PCR, fluorescence observation, and Southern blot hybridization. qRT-PCR analysis showed that PoMsrA was highly expressed in the stage of mature and young fruiting bodies as well as the osmotic stress condition of 0.3 M NaCl. Additionally, the transgenic strains with PoMsrA overexpression exhibited an enhanced tolerance to high temperature, high osmotic stress, and oxidative stress. This suggests that PoMsrA is an active player in the protection of the cellular proteins from oxidative stress damage.

To obtain hydrophobin, a Class I hydrophobin gene, Po.hyd from Pleurotus ostreatus, was transformed into the yeast-like cells of Tremella fuciformis using Agrobacterium tumefaciens. The hydrophobin Po.HYD from P. ostreatus was heterogeneously expressed by the yeast-like cells of T. fuciformis. Plasmids harboring the Po.hyd gene driven by endogenous glyceraldehyde-3-phosphate dehydrogenase promoter were transformed by A. tumefaciens. The integration and expression of the rPo.HYD in the T. fuciformis cells were confirmed by PCR, Southern blot, fluorescence microscopy and quantitative real-time PCR. SDS-PAGE demonstrated that the rPo.HYD was extracted with the expected MW of 14 kDa. The yield of purified rPo.HYD was 0.58 mg/g dry wt. The protein, with its ability to stabilize oil droplets, exhibited a better emulsifying activity than the typical food emulsifiers Tween 20 and sodium caseinate. Tremella fuciformis can be used as a cell factory to produce hydrophobin on a large scale for the food industry.

Manganese-containing superoxide dismutase (Mn-SOD) is one of the most important superoxide dismutases found in many eukaryotes and bacteria. In this study, the full-length cDNA of mitochondrial manganese superoxide dismutase from Pleurotus ostreatus (PoMn-SOD) was obtained. It contained 776 nucleotides with an open reading frame (ORF) of 663 bp, encoding 220 amino acid residues. The deduced amino acid sequence showed high identity with the sequences of other basidiomycetous Mn-SODs from Trametes versicolor (71 %) to Laccaria bicolor (77 %). Quantitative real-time PCR (RT-PCR) analysis revealed that PoMn-SOD gene transcripts were abundant in the stage of young fruit bodies and mature fruit bodies. The up-regulation of the PoMn-SOD gene suggested that it was developmental regulated and could play an important role in antagonizing environmental stresses. In addition, another isoform of Mn-SOD was detected by a non-denaturing polyacrylamide gel electrophoresis (PAGE) approach and the highest total Mn-SOD activity (203.9 U/mg) was observed in the stage of mature fruit bodies. These data could provide important reference for functional study of the PoMn-SOD gene and may benefit biotechnological production of Mn-SOD in the future.

Hydrophobins are a family of small, moderately hydrophobic proteins with eight cysteine residues arranged in a conserved pattern. A full-length cDNA, designated Po.hyd, corresponding to a hydrophobin gene of Pleurotus ostreatus was obtained in our previous work. The Po.hyd gene contains a 333 bp open reading frame (ORF), which is interrupted by two typical classI introns. There was no consensus signal for a polyA tail detected in the 3′untranslated region. However, an analogous T- or TG-rich motif was observed that probably influence the formation of the mRNA 3′ end. We assign the putative Po.HYD protein to the classI hydrophobins since its sequence arrangement and hydropathy pattern has a high consensus to other known class I hydrophobins. Northern analysis showed that the Po.hyd gene was abundantly expressed throughout the fruiting process (from primordium to mature fruiting body) but silenced during vegetative growth of the mycelium. Southern blot analysis showed Po.hyd to be a single copy gene in the genome of dikaryotic strain likely to locate at the same locus within the two parental genomes. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Developing efficient native promoters is important for improving recombinant protein expression by fungal genetic engineering. The promoter region of glyceraldehyde-3-phosphate dehydrogenase gene in Pleurotus ostreatus (Po gpd ) was isolated and optimized by upstream truncation. The activities of these promoters with different lengths were further confirmed by fluorescence, quantitative real-time PCR and Western blot analysis. A truncated Po gpd -P2 fragment (795 bp) drove enhanced green fluorescence protein ( egfp ) gene expression in P. ostreatus much more efficiently than full-length Po gpd -P1. Further truncating Po gpd -P2 to 603, 403 and 231 bp reduced the eGFP expression significantly. However, the 403-bp fragment between –356 bp and the start codon was the minimal but sufficient promoter element for eGFP expression. Compact native promoters for genetic engineering of P. ostreatus were successfully developed and validated in this study. This will broaden the preexisting repertoire of fungal promoters for biotechnology application.

The full-length cDNA of Pleurotus ostreatus superoxide dismutase (PoMn-SOD) was cloned and successfully expressed by using the pPIC9K vector under the control of alcohol oxidase 1 promoter with a secretion signal peptide (α-factor) in Pichia pastoris. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting demonstrated that recombinant PoMn-SOD, a 21.8 kDa protein, was secreted into the culture medium. Nondenaturing PAGE experiments confirmed that recombinant PoMn-SOD was secreted in a functionally active form and the expression system did not require any acid activation process. The factors affecting the expression level were optimized in shaking flask cultures. The maximum enzyme activity (156.9 U/mg) was observed under the following conditions: Initial medium pH was 6.0, induction time point was at the 6th day, and methanol concentration was 0.7 % (v/v). This was the first report on secretory expression of recombinant PoMn-SOD in P. pastoris, which might provide a reference for further practical applications.

An aspartic protease gene from Pleurotus ostreatus (Po-Asp) had been cloned based on the 3′ portion of cDNA in our previous work. The Po-Asp cDNA contained 1,324 nucleotides with an open reading frame (ORF) of 1,212 bp encoding 403 amino acid residues. The putative amino acid sequence included a signal peptide, an activation peptide, two most possible N-glycosylation sites and two conserved catalytic active site. The mature polypeptide with 327 amino acid residues had a calculated molecular mass of 35.3 kDa and a theoretical isoelectric point of 4.57. Basic Local Alignment Search Tool analysis showed 68–80 % amino acid sequence identical to other basidiomycetous aspartic proteases. Sequence comparison and evolutionary analysis revealed that Po-Asp is a member of fungal aspartic protease family. The DNA sequence of Po-Asp is 1,525 bp in length without untranslated region, consisting of seven exons and six introns. The Po-Asp cDNA without signal sequence was expressed in Pichia pastoris and sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the molecular mass of recombinant Po-Asp was about 43 kDa. The crude recombinant aspartic protease had milk-clotting activity.