Abstract
The telomerase reverse transcriptase (TERT) is the core catalytic subunit of telomerase. Its canonical function is synthesizing telomeric repeats to maintain telomere length and chromosomal stability. Accumulating evidence suggests that TERT has other important fundamental functions in addition to its catalytic telomere repeat synthesis activity. However, the non-canonical roles of TERT independent of its enzymatic activity are not clear in filamentous fungi. In the present study, we characterized the GlTert gene in Ganoderma lucidum. The non-canonical roles of GlTert were explored using GlTert-silenced strains (Terti8 and Terti25) obtained by RNA interference. Silencing GlTert delayed the fungal growth, decreased the length between hyphal branches, and induced fungal resistance to oxidative stress in G. ludicum. Further examination revealed that the intracellular ROS (reactive oxygen species) levels were increased while the enzyme activities of the antioxidant systems (superoxide dismutase, catalase, glutathione peroxidase, and ascorbate peroxidase) were decreased in GlTert-silenced strains. In addition, silencing GlTert decreased the ganoderic acid (GA) biosynthesis of G. lucidum. Taken together, our results indicate that GlTert plays a fundamental function on fungal growth, oxidative stress, and GA biosynthesis in G. lucidum, providing new insights for the canonical functions of TERT in filamentous fungi.
Key points
• GlTert affected fungal growth and hyphal branching of G. lucidum.
• Silencing GlTert increased the intracellular ROS levels of G. lucidum.
• GlTert regulated GA biosynthesis of G. lucidum.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files.
References
Boh B (2013) Ganoderma lucidum: a potential for biotechnological production of anti-cancer and immunomodulatory drugs. Recent Pat Anticancer Drug Discov 8(3):255–287. https://doi.org/10.2174/1574891x113089990036
Dai L, Zimmerly S (2002) The dispersal of five group II introns among natural populations of Escherichia coli. RNA 8(10):1294–1307. https://doi.org/10.1017/s1355838202023014
Daniel M, Peek GW, Tollefsbol TO (2012) Regulation of the human catalytic subunit of telomerase (hTERT). Gene 498(2):135–146. https://doi.org/10.1016/j.gene.2012.01.095
Ding D, ** P, Zhou J, Wang M, Cong YS (2013) Human telomerase reverse transcriptase regulates MMP expression independently of telomerase activity via NF-κB-dependent transcription. FASEB J 27(11):4375–4383. https://doi.org/10.1096/fj.13-230904
Ghosh A, Saginc G, Leow SC, Khattar E, Shin EM, Yan TD, Wong M, Zhang Z, Li G, Sung WK, Zhou J, Chng WJ, Li S, Liu E, Tergaonkar V (2012) Telomerase directly regulates NF-κB-dependent transcription. Nat Cell Biol 14(12):1270–1281. https://doi.org/10.1038/ncb2621
Greider CW, Blackburn EH (1989) A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337(6205):331–337. https://doi.org/10.1038/337331a0
Harris SD (2008) Branching of fungal hyphae: regulation, mechanisms and comparison with other branching systems. Mycologia 100(6):823–832. https://doi.org/10.3852/08-177
Hsu KD, Cheng KC (2018) From nutraceutical to clinical trial: frontiers in Ganoderma development. Appl Microbiol Biotechnol 102(21):9037–9051. https://doi.org/10.1007/s00253-018-9326-5
Hu J, Hwang SS, Liesa M, Gan B, Sahin E, Jaskelioff M, Ding Z, Ying H, Boutin AT, Zhang H, Johnson S, Ivanova E, Kost-Alimova M, Protopopov A, Wang YA, Shirihai OS, Chin L, DePinho RA (2012) Antitelomerase therapy provokes ALT and mitochondrial adaptive mechanisms in cancer. Cell 148(4):651–663. https://doi.org/10.1016/j.cell.2011.12.028
Hung JH, Weng Z (2016) Sequence alignment and homology search with BLAST and ClustalW. Cold Spring Harb Protoc 11:1–6. https://doi.org/10.1101/pdb.prot093088
Indran IR, Hande MP, Pervaiz S (2011) hTERT overexpression alleviates intracellular ROS production, improves mitochondrial function, and inhibits ROS-mediated apoptosis in cancer cells. Cancer Res 71(1):266–276. https://doi.org/10.1158/0008-5472.CAN-10-1588
Jie MM, Chang X, Zeng S, Liu C, Liao GB, Wu YR, Liu CH, Hu CJ, Yang SM, Li XZ (2019) Diverse regulatory manners of human telomerase reverse transcriptase. Cell Commun Signal 17(1):63. https://doi.org/10.1186/s12964-019-0372-0
Khattar E, Tergaonkar V (2017) Transcriptional regulation of telomerase reverse transcriptase (TERT) by MYC. Front Cell Dev Biol 5:1. https://doi.org/10.3389/fcell.2017.00001
Letunic I, Khedkar S, Bork P (2021) SMART: recent updates, new developments and status in 2020. Nucleic Acids Res 49(D1):D458–D460. https://doi.org/10.1093/nar/gkaa937
Li C, Shi L, Chen D, Ren A, Gao T, Zhao M (2015a) Functional analysis of the role of glutathione peroxidase (GPx) in the ROS signaling pathway, hyphal branching and the regulation of ganoderic acid biosynthesis in Ganoderma lucidum. Fungal Genet Biol 82:168–180. https://doi.org/10.1016/j.fgb.2015.07.008
Li M, Chen T, Gao T, Miao Z, Jiang A, Shi L, Ren A, Zhao M (2015b) UDP-glucose pyrophosphorylase influences polysaccharide synthesis, cell wall components, and hyphal branching in Ganoderma lucidum via regulation of the balance between glucose-1-phosphate and UDP-glucose. Fungal Genet Biol 82:251–263. https://doi.org/10.1016/j.fgb.2015.07.012
Liang C, Tian D, Liu Y, Li H, Zhu J, Li M, **n M, **a J (2019) Review of the molecular mechanisms of Ganoderma lucidum triterpenoids: ganoderic acids A, C2, D, F, DM, X and Y. Eur J Med Chem 174:130–141. https://doi.org/10.1016/j.ejmech.2019.04.039
Lin Z, Sun L (2019) Antitumor effect of Ganoderma (Lingzhi) mediated by immunological mechanism and its clinical application. Adv Exp Med Biol 1182:39–77. https://doi.org/10.1007/978-981-32-9421-9_2
Liu L, DiGirolamo CM, Navarro PA, Blasco MA, Keefe DL (2004) Telomerase deficiency impairs differentiation of mesenchymal stem cells. Exp Cell Res 294(1):1–8. https://doi.org/10.1016/j.yexcr.2003.10.031
Liu N, Ding D, Hao W, Yang F, Wu X, Wang M, Xu X, Ju Z, Liu JP, Song Z, Shay JW, Guo Y, Cong YS (2016) hTERT promotes tumor angiogenesis by activating VEGF via interactions with the Sp1 transcription factor. Nucleic Acids Res 44(18):8693–8703. https://doi.org/10.1093/nar/gkw549
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408. https://doi.org/10.1006/meth.2001.1262
Lu X, **e C, Wang Y, Liu Y, Han J, Shi L, Zhu J, Yu H, Ren A, Zhao M (2020) Function of ceramide synthases on growth, ganoderic acid biosynthesis and sphingolipid homeostasis in Ganoderma lucidum. Phytochemistry 172:112283. https://doi.org/10.1016/j.phytochem.2020.112283
Maida Y, Masutomi K (2015) Telomerase reverse transcriptase moonlights: therapeutic targets beyond telomerase. Cancer Sci 106(11):1486–1492. https://doi.org/10.1111/cas.12806
Mu D, Shi L, Ren A, Li M, Wu F, Jiang A, Zhao M (2012) The development and application of a multiple gene co-silencing system using endogenous URA3 as a reporter gene in Ganoderma lucidum. PLoS ONE 7(8):e43737. https://doi.org/10.1371/journal.pone.0043737
Ouellette MM, Liao M, Herbert BS, Johnson M, Holt SE, Liss HS, Shay JW, Wright WE (2000) Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. J Biol Chem 275(14):10072–10076. https://doi.org/10.1074/jbc.275.14.10072
Park JI, Venteicher AS, Hong JY, Choi J, Jun S, Shkreli M, Chang W, Meng Z, Cheung P, Ji H, McLaughlin M, Veenstra TD, Nusse R, McCrea PD, Artandi SE (2009) Telomerase modulates Wnt signalling by association with target gene chromatin. Nature 460(7251):66–72. https://doi.org/10.1038/nature08137
Perrault SD, Hornsby PJ, Betts DH (2005) Global gene expression response to telomerase in bovine adrenocortical cells. Biochem Biophys Res Commun 335(3):925–936. https://doi.org/10.1016/j.bbrc.2005.07.156
Ren A, Qin L, Shi L, Dong X, da Mu S, Li YX, Zhao MW (2010) Methyl jasmonate induces ganoderic acid biosynthesis in the basidiomycetous fungus Ganoderma lucidum. Bioresour Technol 101(17):6785–6790. https://doi.org/10.1016/j.biortech.2010.03.118
Ren A, Liu R, Miao ZG, Zhang X, Cao PF, Chen TX, Li CY, Shi L, Jiang AL, Zhao MW (2017) Hydrogen-rich water regulates effects of ROS balance on morphology, growth and secondary metabolism via glutathione peroxidase in Ganoderma lucidum. Environ Microbiol 19(2):566–583. https://doi.org/10.1111/1462-2920.13498
Ren A, Shi L, Zhu J, Yu H, Jiang A, Zheng H, Zhao M (2019) Shedding light on the mechanisms underlying the environmental regulation of secondary metabolite ganoderic acid in Ganoderma lucidum using physiological and genetic methods. Fungal Genet Biol 128:43–48. https://doi.org/10.1016/j.fgb.2019.03.009
Romaniuk A, Paszel-Jaworska A, Toton E, Lisiak N, Holysz H, Krolak A, Grodecka-Gazdecka S, Rubis B (2018) The non-canonical functions of telomerase: to turn off or not to turn off. Mol Biol Rep 46(1):1401–1411. https://doi.org/10.1007/s11033-018-4496-x
Rouda S, Skordalakes E (2007) Structure of the RNA-binding domain of telomerase: implications for RNA recognition and binding. Structure 15(11):1403–1412. https://doi.org/10.1016/j.str.2007.09.007
Sahin E, Colla S, Liesa M, Moslehi J, Muller FL, Guo M, Cooper M, Kotton D, Fabian AJ, Walkey C, Maser RS, Tonon G, Foerster F, **ong R, Wang YA, Shukla SA, Jaskelioff M, Martin ES, Heffernan TP, Protopopov A, Ivanova E, Mahoney JE, Kost-Alimova M, Perry SR, Bronson R, Liao R, Mulligan R, Shirihai OS, Chin L, DePinho RA (2011) Telomere dysfunction induces metabolic and mitochondrial compromise. Nature 470(7334):359–365. https://doi.org/10.1038/nature09787
Sanodiya B, Thakur G, Baghel R, Prasad G, Bisen P (2009) Ganoderma lucidum: a potent pharmacological macrofungus. Curr Pharm Biotechnol 10(8):717–742. https://doi.org/10.2174/138920109789978757
Sfeir A, de Lange T (2012) Removal of shelterin reveals the telomere end-protection problem. Science 336(6081):593–597. https://doi.org/10.1126/science.1218498
Shi L, Ren A, Mu D, Zhao M (2010) Current progress in the study on biosynthesis and regulation of ganoderic acids. Appl Microbiol Biotechnol 88(6):1243–1251. https://doi.org/10.1007/s00253-010-2871-1
Shi L, Fang X, Li M, Mu D, Ren A, Tan Q, Zhao M (2012) Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum. World J Microbiol Biotechnol 28(1):283–291. https://doi.org/10.1007/s11274-011-0818-z
Shi L, Gong L, Zhang X, Ren A, Gao T, Zhao M (2015) The regulation of methyl jasmonate on hyphal branching and GA biosynthesis in Ganoderma lucidum partly via ROS generated by NADPH oxidase. Fungal Genet Biol 81:201–211. https://doi.org/10.1016/j.fgb.2014.12.002
Szostak JW, Blackburn EH (1982) Cloning yeast telomeres on linear plasmid vectors. Cell 29(1):245–255. https://doi.org/10.1016/0092-8674(82)90109-x
Takemoto D, Tanaka A, Scott B (2006) A p67Phox-like regulator is recruited to control hyphal branching in a fungal-grass mutualistic symbiosis. Plant Cell 18(10):2807–2821. https://doi.org/10.1105/tpc.106.046169
Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729. https://doi.org/10.1093/molbev/mst197
Thompson CAH, Wong JMY (2020) Non-canonical functions of telomerase reverse transcriptase: emerging roles and biological relevance. Curr Top Med Chem 20(6):498–507. https://doi.org/10.2174/1568026620666200131125110
Wang Y, Feigon J (2017) Structural biology of telomerase and its interaction at telomeres. Curr Opin Struct Biol 47:77–87. https://doi.org/10.1016/j.sbi.2017.06.010
Wang Y, Susac L, Feigon J (2019) Structural biology of telomerase. Cold Spring Harbor Perspect Biol 11(12):a032383. https://doi.org/10.1101/cshperspect.a032383
Wei ZH, Duan YY, Qian YQ, Guo XF, Li YJ, ** SH, Zhou ZX, Shan SY, Wang CR, Chen XJ, Zheng Y, Zhong JJ (2014) Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods. Bioprocess Biosyst Eng 37(9):1789–1797. https://doi.org/10.1007/s00449-014-1152-2
Wu FL, Zhang G, Ren A, Dang ZH, Shi L, Jiang AL, Zhao MW (2016a) The pH-responsive transcription factor PacC regulates mycelial growth, fruiting body development, and ganoderic acid biosynthesis in Ganoderma lucidum. Mycologia 108(6):1104–1113. https://doi.org/10.3852/16-079
Wu JG, Kan YJ, Wu YB, Yi J, Chen TQ, Wu JZ (2016b) Hepatoprotective effect of Ganoderma triterpenoids against oxidative damage induced by tert-butyl hydroperoxide in human hepatic HepG2 cells. Pharm Biol 54(5):919–929. https://doi.org/10.3109/13880209.2015.1091481
Wu CG, Tian JL, Liu R, Cao PF, Zhang TJ, Ren A, Shi L, Zhao MW (2017) Ornithine decarboxylase-mediated production of putrescine influences ganoderic acid biosynthesis by regulating ROS in Ganoderma lucidum. Appl Environ Microbiol 83(20):e01289-e1317. https://doi.org/10.1128/AEM.01289-17
Xu JW, Xu YN, Zhong JJ (2012) Enhancement of ganoderic acid accumulation by overexpression of an N-terminally truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in the basidiomycete Ganoderma lucidum. Appl Environ Microbiol 78(22):7968–7976. https://doi.org/10.1128/AEM.01263-12
Xu YN, **a XX, Zhong JJ (2014) Induction of ganoderic acid biosynthesis by Mn2+ in static liquid cultivation of Ganoderma lucidum. Biotechnol Bioeng 111(11):2358–2365. https://doi.org/10.1002/bit.25288
Zhang G, Ren A, Wu F, Yu H, Shi L, Zhao M (2017a) Ethylene promotes mycelial growth and ganoderic acid biosynthesis in Ganoderma lucidum. Biotechnol Lett 39(2):269–275. https://doi.org/10.1007/s10529-016-2238-5
Zhang G, Sun Z, Ren A, Shi L, Shi D, Li X, Zhao M (2017b) The mitogen-activated protein kinase GlSlt2 regulates fungal growth, fruiting body development, cell wall integrity, oxidative stress and ganoderic acid biosynthesis in Ganoderma lucidum. Fungal Genet Biol 104:6–15. https://doi.org/10.1016/j.fgb.2017.04.004
Zhang G, Ren A, Shi L, Zhu J, Jiang A, Shi D, Zhao M (2018a) Functional analysis of an APSES transcription factor ( GlSwi6) involved in fungal growth, fruiting body development and ganoderic-acid biosynthesis in Ganoderma lucidum. Microbiol Res 207:280–288. https://doi.org/10.1016/j.micres.2017.12.015
Zhang TJ, Shi L, Chen DD, Liu R, Shi DK, Wu CG, Sun ZH, Ren A, Zhao MW (2018b) 14–3-3 proteins are involved in growth, hyphal branching, ganoderic acid biosynthesis, and response to abiotic stress in Ganoderma lucidum. Appl Microbiol Biotechnol 102(4):1769–1782. https://doi.org/10.1007/s00253-017-8711-9
Zhou J, Ding D, Wang M, Cong YS (2014) Telomerase reverse transcriptase in the regulation of gene expression. BMB Rep 47(1):8–14. https://doi.org/10.5483/bmbrep.2014.47.1.284
Zhu J, Wu F, Yue S, Chen C, Song S, Wang H, Zhao M (2019) Functions of reactive oxygen species in apoptosis and ganoderic acid biosynthesis in Ganoderma lucidum. FEMS Microbiol Lett 366(23):fnaa015. https://doi.org/10.1093/femsle/fnaa015
Ziv C, Feldman D, Aharoni-Kats L, Chen S, Liu Y, Yarden O (2013) The N-terminal region of the Neurospora NDR kinase COT1 regulates morphology via its interactions with MOB2A/B. Mol Microbiol 90(2):383–399. https://doi.org/10.1111/mmi.12371
Acknowledgements
We thank professor Mingwen Zhao for providing RNAi vectors.
Funding
This work was financially supported by the National Natural Science Foundation of China (No. 31902090), Science and Technology Project of Henan Province (No. 202102110019), and High-level Talent Program of Henan Institute of Science and Technology (No. 2018004).
Author information
Authors and Affiliations
Contributions
GZ and CHZ contributed equally to this work. GZ and CHZ planed and performed the experiments, and wrote the manuscript. DDL, PY, ZHW, MXZ, and ZWW participated in performing the experiments. GZ designed and coordinated the project. All authors commented on the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare no competing interests.
Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhang, G., Zhang, C., Leng, D. et al. The non-canonical functions of telomerase reverse transcriptase gene GlTert on regulating fungal growth, oxidative stress, and ganoderic acid biosynthesis in Ganoderma lucidum. Appl Microbiol Biotechnol 105, 7353–7365 (2021). https://doi.org/10.1007/s00253-021-11564-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-021-11564-9