Abstract
Trametes sp. are among the most extensively studied basidiomycetes due to their importance in biotechnology. However, they are difficult to genetically modify. For instance, the low incidence of homologous integration hampers gene inactivation. To overcome this, we here constructed two post-transcriptional gene-silencing vectors that contain an antisense and a RNA interference mediating hairpin sequence, respectively. These vectors were used to knock down transcription of the heat shock protein 70 family gene HspA1. The two vectors were transformed into Trametes hirsuta AH28-2 by using a PEG/CaCl2 mediated transformation method. Based on Southern blot analysis, target sequences were integrated into the genome as multi-copies. Transcription analysis revealed that the antisense and hairpin sequences reduced the transcript number of HspA1 when compared to the wild type strain. Moreover, the antisense sequence resulted in a higher gene silencing efficiency when compared to the RNA interference vector. Together, antisense methodology provides a simple method for gene silencing in Trametes sp.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Aleksandar K, Ivan M, Mirjana S, Nikola L, Ilija B, Jelena V, Jasmina C (2013) Lignin degradation by selected fungal species. Bioresour Technol 138:117–123
Amoros M, Estruch F (2001) Hsf1p and Msn2/4p cooperate in the expression of Saccharomyces cerevisiae genes HSP26 and HSP104 in a gene- and stress type-dependent maner. Mol Microbiol 39:1523–1532
Binninger DM, Skrzynia C (1987) DNA-mediated transformation of the basidiomycete Coprinus cinerea. EMBO J 6:835–840
Case ME, Schweizer M, Kushner SR, Giles NH (1979) Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci USA 7610:5259–5263
Chang SS, Zhang Z, Liu Y (2012) RNA interference pathways in fungi: mechanisms and functions. Annu Rev Microbiol 66:305–323
De Jong JF, Deelstra HJ, Wösten HA, Lugones LG (2006) RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune. Appl Environ Microbiol 72:1267–1269
Dimitrios F, Manfred B, Robert R, Kerrie B et al (2012) The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes. Science 336:1715–1719
Eastwood DC, Challen MP, Zhang C, Jenkins H, Henderson J, Burton KS (2008) Hairpin mediated down-regulation of the urea cycle enzyme argininosuccinate lyase in Agaricus bisporus. Mycol Res 105:1223–1230
Flores C, Vidal C, Trejo-Hernández MR, Galindo E, Serrano-Carreón L (2009) Selection of Trichoderma strains capable of increasing laccase production by Pleurotus ostreatus and Agaricus bisporus in dual cultures. J Appl Microbiol 106:249–257
Glover JR, Lindquist S (1998) Hsp104, Hsp70, and Hsp40: a novel chaperone system that rescues previously aggregated proteins. Cell 94:73–84
Gritz L, Davies J (1983) Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene 25:179–188
Hauke H, Dietmar S, Lukas YW (2011) Untapped potential: exploiting fungi in bioremediation of hazardous chemicals. Nat Rev Microbiol 9:177–192
He LY, Feng J, Lu S, Chen ZW, Chen CM, He Y, Yi XW, Xi LY (2016) Genetic transformation of fungi. Int J Dev Biol. doi:10.1387/ijdb.160026lh
Keiko N, Hisayuki W, Yuichi S, Toshitsugu S (2011) Gene silencing of the Lentinula edodes lcc1 gene by expression of a homologous inverted repeat sequence. Microbiol Res 166:484–493
Kemppainen M, Duplessis S, Martin F, Pardo AG (2009) RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus. Environ Microbiol 11:1878–1896
Kim K, Leem Y, Kim K, Kim K, Choi HT (2002) Transformation of the medicinal basidiomycete Trametes versicolor to hygromycin B resistance by restriction enzyme mediated integration. FEMS Microbiol Lett 209:273–276
Koji Y, Kumiko Y, Hideki U, Masao H, Susumu K, Takashi Y, Kazuo S, Takashi A (1996) The integrative transformation of Pleurotus ostreatus using bialaphos resistance as a dominant selectable marker. Biosci Biotechnol Biochem 60:472–475
Kregel KC (2002) Invited review: heat shock proteins: modifying factors in physiological stress responses and acquired thermotolerance. J Appl Physiol 92:2177–2186
Mary NH, Ana MSBC, Michael PC, Peter RM, Andy B, Gary DF (2007) A comparison of methods for successful triggering of gene silencing in Coprinus cinereus. Mol Biotechnol 35:283–296
Namekawa SH, Iwabata K, Sugawara H, Hamada FN, Koshiyama A, Chiku H, Kamada T, Sakaguchi K (2005) Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing. Microbiology 151:3669–3678
Pollegioni L, Tonin F, Rosini E (2015) Lignin-degrading enzymes. FEBS J 282:1190–1213
Schuurs TA, Schaeffer EAM, Wessels JGH (1997) Homology dependent silencing of the SC3 gene in Schizophyllum commune. Genetics 147:589–596
Shraddha T, Raman T, Jata S (2015) Role of heat-shock proteins in cellular function and in the biology of fungi. Biotechnol Res Int. 2015:11
Sun Q, Wei W, Zhao J, Song J, Peng F, Zhang SP, Zheng YL, Chen P, Zhu WJ (2015) An efficient PEG/CaCl2-mediated transformation approach for the medicinal fungus Wolfiporia cocos. J Microbiol Biotechnol 25:1528–1531
Toshitsugu S, Kaori Y, Shizuko I, Tatsuya H, Susumu K, Kazuo S, Hitoshi E (1998) Transformation of the edible basidiomycete Lentinus edodes by restriction enzyme-mediated integration of plasmid DNA. Biosci Biotechnol Biochem 62:2346–2350
Wang JJ, Zhang YL, Xu Y, Fang W, Wang XT, Fang ZM, Xiao YZ (2015) Genome sequence of a laccase producing fungus Trametes sp. AH28-2. J Biotechnol 216:167–168
Werner-Washburne M, Stone DE, Craig EA (1987) Complex interactions among members of an essential subfamily of hsp70 genes in Saccharomyces cerevisiae. Mol Cell Biol 7:2568–2577
Xiao YZ, Hong YZ, Li JF, Hang J, Tong PG, Fang W, Zhou CZ (2006) Cloning of novel laccase iszoyme genes from Trametes sp. AH28-2 and analyses of their differential expression. Appl Microbiol Biotechnol 71:493–501
Zhang S, Hacham M, Panepinto J, Hu GW, Shin S, Zhu XD, Williamson PR (2006) The Hsp70 member, Ssa1, acts as a DNA-binding transcriptional co-activators of laccase in Cryptococcus neoformans. Mol Microbiol. doi:10.1111/j.1365-2958.2006.05422.x
Zhang JJ, Shi L, Chen H, Sun YQ, Zhao MW, Ren A, Chen MJ, Wang H, Feng ZY (2014) An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus. Microbiol Res 169:741–748
Acknowledgements
This work was supported by Grants from the Natural Science Foundation of China (31170056, 31300044), the Key Research Program of the Department of Education of Anhui Province (KJ2017ZD04), and the Introduction Project of Academic and Technology Leaders in Anhui University (32030066).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Wang, J., Chen, Y., Dong, Y. et al. A simple and efficient method for successful gene silencing of HspA1 in Trametes hirsuta AH28-2. Antonie van Leeuwenhoek 110, 1527–1535 (2017). https://doi.org/10.1007/s10482-017-0904-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10482-017-0904-9