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.