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International Journal of Medicinal Mushrooms
Facteur d'impact: 1.423 Facteur d'impact sur 5 ans: 1.525 SJR: 0.431 SNIP: 0.716 CiteScore™: 2.6

ISSN Imprimer: 1521-9437
ISSN En ligne: 1940-4344

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International Journal of Medicinal Mushrooms

DOI: 10.1615/IntJMedMushrooms.2019031600
pages 921-930

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

Dongmei Liu
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
Hanyu Zhu
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
Liesheng Zheng
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
Liguo Chen
College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China
Aimin Ma
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, P.R. China; Key Laboratory of Agro-Microbial Resources and Utilization, Ministry of Agriculture, Huazhong Agricultural University, Wuhan 430070, China


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.


  1. Hou LH, Chen Y, Ma CJ, Liu J, Chen LG, Ma AM. Effects of environmental factors on dimorphic transition of the jelly mushroom Tremella fuciformis. Cryptogam Mycol. 2011;32(4):421-28.

  2. Zhu H, Yuan Y, Liu J, Zheng L, Chen L, Ma A. Comparing the sugar profiles and primary structures of alkali-extracted water-soluble polysaccharides in cell wall between the yeast and mycelial phases from Tremella fuciformis. J Microbiol. 2016;54:381-86.

  3. Zou Y, Hou X. Extraction optimization, composition analysis, and antioxidation evaluation of polysaccharides from white jelly mushroom, Tremella fuciformis (Tremellomycetes). Int J Med Mushrooms. 2017;19:1113-21.

  4. Xu W, Shen X, Yang F, Han Y, Li R, Xue D, Jiang C. Protective effect of polysaccharides isolated from Tremella fuciformis against radiation-induced damage in mice. J Radiat Res. 2012;53:353-60.

  5. Kiho T, Tsujimura Y, Sakushima M, Usui S, Ukai S. Polysaccharides in fungi. XXXIII. Hypoglycemic activity of an acidic polysaccharide (AC) from Tremella fuciformis. Yakugaku Zasshi. 1994;114:308-15.

  6. Reshetnikov SR, Wasser SP, Duckman I, Tsukor K. Medicinal value of the genus Tremella Pers. (Heterobasidiomycetes) (review). Int J Med Mushrooms. 2000;2:169-93.

  7. Shi ZW, Liu Y, Xu Y, Hong YR, Liu Q, Li XL, Wang ZG. Tremella Polysaccharides attenuated sepsis through inhibiting abnormal CD4+CD25 (high) regulatory T cells in mice. Cell Immunol. 2014;288:60-5.

  8. Wen L, Gao Q, Ma C, Ge Y, You L, Liu RH, Fu X, Liu D. Effect of polysaccharides from Tremella fuciformis on UV-induced photoaging. J Funct Foods. 2016;20:400-10.

  9. Hsieh HM, Ju YM, Rogers JD. Molecular phylogeny of Hypoxylon and closely related genera. Mycologia. 2005;97:844-65.

  10. Deng Y, van Peer AF, Lan FS, Wang QF, Jiang Y, Lian LD, Lu DM, Xie B. Morphological and molecular analysis identifies the associated fungus ("Xianghui") of the medicinal white jelly mushroom, Tremella fuciformis, as Annulohypoxylon stygium. Int J Med Mushrooms. 2016;18:253-60.

  11. Rehman L, Su X, Guo H, Qi X, Cheng H. Protoplast transformation as a potential platform for exploring gene function in Verticillium dahlia. BMC Biotechnol. 2016;16:57.

  12. Chen L, Wang Q, Chen H, Sun G, Liu H, Wang H. Agrobacterium tumefaciens-mediated transformation of Botryosphaeria dothidea. World J Microbiol Biotechnol. 2016;32:106.

  13. Zhu H, Wang TW, Sun SJ, Shen YL, Wei DZ. Chromosomal integration of the Vitreoscilla hemoglobin gene and its physiological actions in Tremella fuciformis. Appl Microbiol Biotechnol. 2006;72:770-76.

  14. Sun SJ, Chen DX, Xie BG, Hu FP, Zheng JG. Isolation of GPD promoter from Tremella fuciformis and driving expression of EGFP gene. DNA Cell Biol. 2009; 28:65-70.

  15. Kang LZ, Li Q, Lin JF, Guo LQ. Biosynthesis of resveratrol in blastospore of the macrofungus Tremella fuciformis. Mol Biotechnol. 2015;57:675-84.

  16. Shin DI, Park HS. Mechanical wounding of yeast-like conidium cells of Tremella fuciformis makes them susceptible to Agrobacterium-mediated transformation. Biosci Biotechnol Biochem. 2013;77:2157-9.

  17. Zhu H, Liu D, Wang Y, Ren D, Zheng L, Chen L, Ma A. Use of the yeast-like cells of Tremella fuciformis as a cell factory to produce a Pleurotus ostreatus hydrophobin. Biotechnol Lett. 2017;39:1167-73.

  18. Liu D, Zhu H, Chen Y, Zheng L, Chen L, Ma A. Cloning and heterologous expression of a hydrophobin gene Ltr.hyd from the tiger milk mushroom Lentinus tuber-regium in yeast-like cells of Tremella fuciformis. Electron J Biotech. 2018;32:6-12.

  19. Bundock P, den Dulk-Ras A, Beijersbergen A, and Hooykaas PJ. Trans-kingdom T-DNA transfer from Agrobacterium tume-faciens to Saccharomyces cerevisiae. EMBO J. 1995;14:3206-14.

  20. de Groot MJA, Bundock P, Hooykaas PJJ, and Beijersbergen AG. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol. 1998;16:839-42.

  21. Kunik T, Tzfira T, Kapulnik Y, Gafni Y, Dingwall C, Citovsky V. Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci U S A. 2001;98:1871-6.

  22. Chen X, Stone M, Schlagnhaufer C, Romaine CP. A fruiting body tissue method for efficient Agrobacterium-mediated trans-formation of Agaricus bisporus. Appl Environ Microbiol. 2000;66:4510-13.

  23. Heneghan MN, Porta C, Zhang C, Burton KS, Challen MP, Bailey AM, Foster GD. Characterization of serine proteinase expression in Agaricus bisporus and Coprinopsis cinerea by using green fluorescent protein and the A. bisporus SPR1 promoter. Appl Environ Microbiol. 2009;75:792-801.

  24. Zheng Z, Huang C, Cao L, Xie C, Han R. Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in medicinal fungus Cordyceps militaris. Fungal Biol. 2011;115:265-74.

  25. Hatoh K, Izumitsu K, Morita A, Shimizu K, Ohta A, Kawai M, Yamanaka T, Neda H, Ota Y, Tanaka C. Transformation of the mushroom species Hypsizigus marmoreus, Flammulina velutipes, and Grifola frondosa by an Agrobacterium-mediated method using a universal transformation plasmid. Mycoscience. 2013;54:8-12.

  26. Lu Z, Kong X, Lu Z, Xiao M, Chen M, Zhu L, Shen Y, Hu X, Song S. Para-aminobenzoic acid (PABA) synthase enhances thermotolerance of mushroom Agaricus bisporus. PLoS One. 2014;9:e91298.

  27. Zhang J, Shi L, Chen H, Sun Y, Zhao M, Ren A, Chen M, Wang H, Feng Z. An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus. Microbiol Res. 2014;169:741-48.

  28. Zubieta MP, da Silva Coelho I, de Queiroz MV, de Araujo EF. Agrobacterium tumefaciens-mediated genetic transformation of the ectomycorrhizal fungus Laccaria laccata. Ann Microbiol. 2014;64:1875-78.

  29. Kim S, Ha BS, Ro HS. Current technologies and related issues for mushroom transformation. Mycobiology. 2015;43:1-8.

  30. Cao Y, Jiao R, Xia Y. A strong promoter PMagpd provides a tool for high gene expression in entomopathogenic fungus Metarhizium acridum. Biotechnol Lett. 2012;34:557-62.

  31. Huang X, Lu X, Li JJ. Cloning, characterization and application of a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus terreus. J Ind Microbiol Biotechnol. 2014;41:585-92.

  32. Kuo CY, Chou SY, Huang CT. Cloning of glyceraldehyde-3-phosphate dehydrogenase gene and use of the gpd promoter for transformation in Flammulina velutipes. Appl Microbiol Biotechnol. 2004;65:593-99.

  33. Godio RP, Fouces R, Gudina EJ, Martin JF. Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium. Curr Genet. 2004;46:287-94.

  34. Li Q, Ma A. Studies on isolation and regeneration of protoplast from Tremella fuciformis mycelium. J Changjiang Vegetables. 2001;9:28-9. (In Chinese with an English abstract).

  35. Li S, Cai H, Xu W, Hu Y, Gao Y, Lin Z. Efficient transformation of the medicinal mushroom Ganoderma lucidum. Plant Mol Biol Rep. 2001;19:383-84.

  36. Li G, Li R, Liu Q, Wang Q, Chen M, Li B. A highly efficient polyethylene glycol-mediated transformation method for mushrooms. FEMS Microbiol Lett. 2006;256: 203-8.

  37. Shi L, Fang X, Li MJ, Mu D, Ren A, Tan Q, Zhao M. Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum. World J Microbiol Biotechnol. 2012;28:283-91.

  38. Duarte RT, Staats CC, Fungaro MH, Schrank A, Vainsten MH, Furlaneto-Maia L, Nakamura CV, de Souza W, Furlaneto MC. Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the entomopathogenic fungus Metarhizium anisopliae var. acridum. Lett Appl Microbiol. 2007;44:248-54.

  39. Michielse CB, Hooykaas PJ, van den Hondel CA, Ram AF. Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Gene. 2005;48:1-17.

  40. Priyanka D, Gurvinder K. Optimization of different factors for efficient protoplast release from entomopathogenic fungus Metarhizium anisopliae. Ann Microbiol. 2009;59:183-86.

  41. Li L, Yin Q, Liu X, Yang H. An efficient protoplast isolation and regeneration system in Coprinus comatus. Afr J Microbiol Res. 2010;4:459-65.

  42. Ando A, Sumida Y, Negoro H, Suroto DA, Ogawa J, Sakuradani E, Shimizu S. Establishment of Agrobacterium tumefaciens-mediated transformation of an oleaginous fungus, Mortierella alpina 1S-4, and its application for eicosapentaenoic acid producer breeding. Appl Environ Microbiol. 2009;75:5529-35.

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