A 14-bp stretch plays a critical role in regulating gene expression from β1-tubulin promoters of basidiomycetes

  • Dong Xuan Nguyen
  • Taku Sakaguchi
  • Takehito Nakazawa
  • Masahiro Sakamoto
  • Yoichi HondaEmail author
Original Article


Cis-acting elements play a vital role in regulation of transcription initiation. Several cis-acting elements have been identified in filamentous fungi; however, the fundamental requirements for basic promoter function in basidiomycetes are obscure. In this study, core elements in β1-tubulin promoters of basidiomycetes were functionally characterized. Using transient transformation in Ceriporiopsis subvermispora as a promoter assay, we found that a 14-bp region (β1-tubulin core promoter element, BCE), as well as CT-rich stretch, in the β1-tubulin promoter of the species played a critical role in the expression of a recombinant hph as a reporter gene. In addition, in silico analysis revealed other members of basidiomycetes also harboured the BCE motif as well as CT-rich stretch in the β1-tubulin promoter region, suggesting their functional conservation among the species of basidiomycetes. To confirm the function of BCE, we investigated the effects of BCE motif deletion in the Pleurotus ostreatus β1-tubulin promoter on expression levels of a recombinant luminous shrimp luciferase reporter gene, which was targeted into the Pofcy1 locus. Intriguingly, luciferase activity was abolished when the BCE motif was deleted in the β1-tubulin promoter, strongly demonstrating its essential function in transcription from this promoter on the chromosome. This study clearly demonstrates the crucial role of the BCE as well as the CT-rich stretch regions in the β1-tubulin promoter among basidiomycetes and provides new insights into the fundamental mechanism of transcription initiation in this group.


Basidiomycetes Ceriporiopsis subvermispora Pleurotus ostreatus β1-tubulin gene Transient transformation Core promoter element 



This work was supported in part by the Noda Institute for Scientific Research (to Y. H.), and JSPS KAKENHI [15K14771 and 18H02254 to Y. H.]. We would like to thank Prof. Yitzhak Hadar (Hebrew University of Jerusalem, Israel) for providing P. ostreatus strains 20b, Profs. Kazumitsu Ueda and Noriyuki Kioka (Kyoto University, Japan) for help in measurement of luciferase activity, Prof. Keishi Osakabe (Tokushima University, Japan) for providing the plasmid containing the luciferase reporter gene.

Supplementary material

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  1. Akhtar M, Blanchette RA, Kirk TK (1997) Fungal delignification and biomechanical pulping of wood. Adv Biochem Eng Biotechnol 57:159–195Google Scholar
  2. Bertossa RC, Kües U, Aebi M, Künzler M (2004) Promoter analysis of cgl2, a galectin encoding gene transcribed during fruiting body formation in Coprinopsis cinerea (Coprinus cinereus). Fungal Genet Biol 41(12):1120–1131Google Scholar
  3. Burns C, Leach KM, Elliott TJ, Challen MP, Foster GD, Bailey A (2006) Evaluation of Agrobacterium-mediated transformation of Agaricus bisporus using a range of promoters linked to hygromycin resistance. Mol Biotechnol 32(2):129–138Google Scholar
  4. Carey M, Smale ST (2000) Transcriptional regulation in eukaryotes: concepts, strategies, and techniques. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 1–50Google Scholar
  5. Carnince P, Sandelin A, Albin LB, Katayama S, Shimokawa K, Ponjavic J, Semple CAM, Taylor MS, Pär EG, Frith MC, Forrest ARR, Alkema WB, Tan SL, Plessy C, Kodzius R, Ravasi T, Kasukawa T, Fukuda S, Kanamori-Katayama M, Kitazume Y, Kawaji H, Kai C, Nakamura M, Konno H, Nakano K, Mottagui-Tabar S, Arner P, Chesi AL, Gustincich S, Persichetti F, Suzuki H, Grimmond SM, Wells CA, Orlando V, Wahlestedt C, Liu ET, Harbers M, Kawai J, Bajic VB, Hume DA, Hayashizaki Y (2006) Genome-wide analysis of mammalian promoter architecture and evolution. Nat Genet 38(6):626–635Google Scholar
  6. Cohen R, Persky L, Hadar Y (2002) Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus. Appl Microbiol Biotechnol 58(5):582–594Google Scholar
  7. Cumming WL, Celerin M, Crodian J, Bronick JK, Jolan ME (1999) Insertional mutagenesis in Coprinus cinereus: use of a dominant selectable marker to generate tagged, sporulation-defective mutants. Curr Genet 36(6):371–382Google Scholar
  8. de Mattos-Shipley KMJ, Ford KL, Alberti F, Banks AM, Bailey AM, Foster GD (2016) The good, the bad and the tasty: the many role of mushrooms. Stud Mycol 85:125–157Google Scholar
  9. Doniger SW, Fay JC (2007) Frequent gain and loss of functional transcription factor binding sites. PLoS Comput Biol 3(5):e99Google Scholar
  10. Fei X, Zhao MW, Li YX (2006) Cloning and sequence analysis of a glyceraldehyde-3-phosphate dehydrogenase gene from Ganoderma lucidum. J Microbiol 44(5):515–522Google Scholar
  11. Feng J, Bhadauria V, Liu G, Selvaraj G, Hughes GR, Wei Y (2011) Analysis of the promoter region of the gene LIP1 encoding triglyceride lipase from Fusarium graminearum. Microbiol Res 166(8):618–628Google Scholar
  12. Gasch AP, Moses AM, Chiang DY, Fraser HB, Bernardini M, Eisen MB (2004) Conservation and evolution of cis-regulatory systems in ascomycetes fungi. PLoS Biol 2(12):e398Google Scholar
  13. Gross P, Oelgeschläger T (2006) Core promoter-selective RNA polymerase II transcription. Biochem Soc Symp 73:225–236Google Scholar
  14. Gurr SJ, Unkles SEU, Kinghorn JR (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn JR (ed) Gene structure in eukaryotic microbes. IRL Press, London, pp 93–139Google Scholar
  15. Hahn S (2004) Structure and mechanism of the RNA polymerase II transcription machinery. Nat Struct Mol Biol 11(5):394–403Google Scholar
  16. Hernandez-Garcia CM, Finer JJ (2014) Identification and validation of promoter and cis-regulatory elements. Plant Sci 217:109–119Google Scholar
  17. Hibbett DS, Donoghue MJ (2001) Analysis of character correlation among wood decay mechanisms, mating systems, and substrate ranges in homobasidiomycetes. Syst Biol 50(2):215–242Google Scholar
  18. Honda Y, Matsuyama T, Irie T, Watanabe T (2000) Carboxin resistance transformation of the homobasidiomycete fungus Pleurotus ostreatus. Curr Genet 37(3):209–212Google Scholar
  19. Honda Y, Tanigawa E, Tsukihara T, Nguyen XD, Kawabe H, Sakatoku N, Watari J, Sato H, Yano S, Tachiki T, Irie T, Watanabe T, Watanabe T (2019) Stable and transient transformation, and a promoter assay in the selective lignin-degrading fungus, Ceriporiopsis subvermispora. AMB Express 9:92Google Scholar
  20. Juven-Gershon T, Kadonaga JT (2010) Regulation of gene expression via the core promoter and the basal transcriptional machinery. Dev Biol 339(2):225–229Google Scholar
  21. Kajiwara S, Shishido K (1992) Characterization of the promoter region of the basidiomycete Lentinus edodes Le.ras gene. FEMS Microbiol Lett 92(2):147–150Google Scholar
  22. Kato M (2005) An overview of the CCAAT-box binding factor in filamentous fungi: assembly, nuclear translocation, and transcriptional enhancement. Biosci Biotechnol Biochem 69(4):663–672Google Scholar
  23. Kilaru S, Kües U (2005) Comparison of gpd genes and their protein products in basidiomycetes. Fungal Genet Newsl 52:18–23Google Scholar
  24. Kohler A, Kuo A, Nagy LG, Morin E, Barry KW, Buscot F, Canbäck B, Choi C, Cichocki N, Clum A, Colpaert J, Copeland A, Costa MD, Doré J, Floudas D, Gay G, Girlanda M, Henrissat B, Herrmann S, Hess J, Högberg N, Johansson T, Khouja HR, LaButti K, Lahrmann U, Levasseur A, Erika A, Lindquist EA, Lipzen A, Marmeisse R, Martino E, Murat C, Ngan CY, Nehls U, Plett JM, Pringle A, Ohm RA, Perotto S, Peter M, Riley R, Rineau F, Ruytinx J, Salamov A, Shah F, Sun H, Tarkka M, Tritt A, Fourrey VC, Zuccaro A, Mycorrhizal Genomics Initiative Consortium, Tunlid A, Grigoriev IV, Hibbett DS, Martin F (2015) Convergent losses of decay mechanisms and rapid turnover of symbiosis genes in mycorrhizal mutualists. Nat Genet 47(4):410–415Google Scholar
  25. Kondoh O, Shishido K (1995) Characterization of promoter region of cell-adhesion protein gene derived from the basidiomycete Lentinus edodes. FEMS Microbiol Lett 130:189–192Google Scholar
  26. Larraya LM, Pérez G, Peñas MM, Baars JJP, Mikosch TSP, Pisabarro AG, Ramírez L (1999) Molecular karyotype of the white rot fungus Pleurotus ostreatus. Appl Environ Microbiol 65(8):3413–3417Google Scholar
  27. Lee TI, Young RA (2000) Transcription of eukaryotic protein-coding genes. Annu Rev Genet 34:77–137Google Scholar
  28. Li JJ, Kim RH, Sodek J (1995) An inverted TATA box directs downstream transcription of the bone sialoprotein gene. Biochem J 310:33–40Google Scholar
  29. Lis M, Walther D (2016) The orientation of transcription factor binding site motifs in gene promoter regions: does it matter? BMC Genom 17:185Google Scholar
  30. Liu S, Duan Y, Ge C, Chen C, Zho M (2012) Functional analysis of the β 2-tubulin gene of Fusarium graminearum and the β-tubulin gene of Botrytis cinerea by homologous replacement. Pest Manag Sci 69(5):582–588Google Scholar
  31. Machida M, Chang YC, Manabe M, Yasukawa M, Kunihiro S, Jigami Y (1996) Molecular cloning of a cDNA encoding enolase from the filamentous fungus, Aspergillus oryzae. Curr Genet 30:423–431Google Scholar
  32. Matsunaga Y, Ando M, Izumitsu K, Suzuki K, Honda Y, Irie T (2017) A development and an improvement of selectable markers in Pleurotus ostreatus transformation. J Microbiol Methods 134:27–29Google Scholar
  33. Mayfield MB, Kishi K, Alic M, Gold MH (1994) Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium. Appl Environ Microbiol 60(12):4303–4309Google Scholar
  34. McNeil JB (1988) Functional characterization of a pyrimidine-rich element in the 5′-noncoding region of the yeast iso-1-cytochrome c gene. Mol Cell Biol 8(3):1045–1054Google Scholar
  35. Messner K, Srebotnik E (1994) Biopulping: an overview of developments in an environmentally safe paper-making technology. FEMS Microbiol Rev 13:351–364Google Scholar
  36. Muraguchi H, Ito Y, Kamada T, Yanagi SO (2003) A linkage map of the basidiomycete Coprinus cinereus based on random amplified polymorphic DNAs and restriction fragment length polymorphism. FG&B 40:93–102Google Scholar
  37. Nakazawa T, Tatsuta Y, Fujita T, Nakahori K, Kamada T (2010) Mutations in the Cc.rmt1 gene encoding a putative protein arginine methyltransferase alter developmental programs in the basidiomycete Coprinopsis cinerea. Curr Genet 56(4):361–367Google Scholar
  38. Nakazawa T, Tsuzuki M, Irie T, Sakamoto M, Honda Y (2016) Marker recycling via 5-fluoroorotic acid and 5-fluorocytosine counter-selection in the white-rot agaricomycete Pleurotus ostreatus. Fungal Biol 120(9):1146–1155Google Scholar
  39. Nakazawa T, Izuno A, Horii M, Kodera R, Nishimura H, Hirayama Y, Tsunematsu Y, Miyazaki Y, Awano T, Muraguchi H, Watanabe K, Sakamoto M, Takabe K, Watanabe T, Isagi Y, Honda Y (2017) Effects of pex1 disruption on wood lignin biodegradation, fruiting development and the utilization of carbon sources in the white-rot Agaricomycete Pleurotus ostreatus and non-wood decaying Coprinopsis cinerea. Fungal Genet Biol 109:7–15Google Scholar
  40. Oakley BR (2004) Tubulins in Aspergillus nidulans. Fungal Genet Biol 41(4):420–427Google Scholar
  41. Punt PJ, Dingemanse MA, Kuyvenhoven A, Soede DM, Pouwels PH, van den Hondel CA (1990) Functional elements in the promoter region of the Aspergillus nidulans gpdA gene encoding glyceraldehyde-3-phosphate dehydrogenase. Gene 93(1):101–109Google Scholar
  42. Ries LNA, Beattie SR, Espeso EA, Cramer RA, Goldman GH (2016) Diverse regulation of the CreA carbon catabolite repressor in Aspergillus nidulans. Genetics 203:335–352Google Scholar
  43. Roeder RG (1996) The role of general initiation factors in transcription by RNA polymerase II. Trends Biochem Sci 21(9):327–335Google Scholar
  44. Roy AL, Singer DS (2015) Core promoters in transcription: old problem, new insights. Trends Biochem Sci 40(3):165–171Google Scholar
  45. Sakamoto Y, Watanabe H, Nagai M, Nakade K, Takahashi M, Sato T (2006) Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence. Plant Physiol 141(2):793–801Google Scholar
  46. Salame T, Knop D, Tal DT, Levinson D, Yarden O, Hadar Y (2012) Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus. AEM 78(15):5341–5352Google Scholar
  47. Seizl M, Hartmann H, Hoeg F, Kurth F, Martin DE, Söding J, Cramer P (2011) A conserved GA element in TATA-less RNA polymerase II promoters. PLoS One 6(11):e27595Google Scholar
  48. Sharma KK, Kuhad RC (2010) Genetic transformation of lignin degrading fungi facilitated by Agrobacterium tumefaciens. BMC Biotechnol 10:67Google Scholar
  49. Sibthorp C, Wu H, Cowley G, Wong PWH, Palaima P, Morozov I, Weedall GD, Caddick MX (2013) Transcriptome analysis of the filamentous fungus Aspergillus nidulans directed to the global identification of promoters. BMC Genom 14:847Google Scholar
  50. Singh AP, Singh T (2014) Biotechnological applications of wood-rotting fungi: a review. Biomass Bioenergy 62:198–206Google Scholar
  51. Sugano SS, Suzuki H, Shnimokita E, Chiba H, Noji S, Osakabe Y, Osakabe K (2017) Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system. Sci Rep 7(1):1260Google Scholar
  52. Unkles SE (1992) Gene organization in industrial filamentous fungi. In: Kinghorn JR, Turner G (eds) Applied molecular genetics of filamentous fungi. Chapman and Hall, London, pp 28–53Google Scholar
  53. Wang J, Guo L, Zhang K, Wu Q, Lin J (2008) Highly efficient Agrobacterium-mediated transformation of Volvariella volvacea. Bioresour Technol 99:8524–8527Google Scholar
  54. Yin C, Robert R (1997) Identification of a functional CT-element in the Phytophthora infestans piypt1 gene promoter. Gene 198(1–2):159–164Google Scholar
  55. Yin C, Zheng L, Zhu J, Chen L, Ma A (2015) Characterization of the highly active fragment of glyceraldehyde-3-phosphate dehydrogenase gene promoter for recombinant protein expression in Pleurotus ostreatus. FEMS 362(5):fnv010Google Scholar
  56. Zhao Z, Liu H, Luo Y, Zhou S, An L, Wang C, Jin Q, Zhou M, Xu JR (2014) Molecular evolution and functional divergence of tubulin superfamily in the fungal tree of life. Sci Rep 4:6746Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Graduate School of AgricultureKyoto UniversityKyotoJapan
  2. 2.Biotechnology Center of Ho Chi Minh CityHo Chi Minh CityVietnam

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