Efficient Transformation of the White Jelly Mushroom Tremella fuciformis (Tremellomycetes) and Its Companion Fungus Annulohypoxylon stygium (Ascomycetes) Mediated by Agrobacterium tumefaciens.

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.

Identification and In Silico Analysis of Lectins in Gray Oyster Culinary-Medicinal Mushroom Pleurotus ostreatus (Agaricomycetes) Based on the Transcriptomes.

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.

Agrobacterium tumefaciens-Mediated Transformation of the King Tuber Medicinal Mushroom Lentinus tuber-regium (Agaricomycetes).

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.