Electroporation Mediated DNA Transformation of Filamentous Fungi

  • B. N. Chakraborty
Part of the Fungal Biology book series (FUNGBIO)


The electroporation offers a simple, rapid, reproducible, and efficient transformation method for various species of filamentous fungi without making protoplast or without the need to use of toxic chemicals. This procedure is directly applicable to sporulating species and can also be used with either mycelia or non-conidiating species. Selection of the age for conidia and germination stage, nature of mycolytic enzyme(s), duration of treatment, choice of selectable markers and selection medium are to be considered as critical factors while yielding high transformants. Multiple copies of plasmids can integrate at unlinked sites and thus provide the potential for increased yield of the desired product. Repeat induced point mutation has proved to be useful in the mutagenesis of specific DNA fragments in vivo. The filamentous fungi provide potential for innovation to identify new promoters and regulatory sequences and the application of genetic transformation promises a healthy future for fungal biotechnology.


Electroporation Co-transformation β-glucuronidase Heat shock protein RIP Filamentous fungi 



Financial assistance received from the Department of Biotechnology, Ministry of Science and Technology, Government of India in the form of Long-term Biotechnology Associateship program under dynamic guidance of Professor Manju Kapoor at Cellular, Molecular and Microbial Biology Division, Department of Biological Sciences, The University of Calgary, Canada is gratefully acknowledged.


  1. Adachi K, Nelson GH, Peoples KA, Frank SA (2002) Efficient gene identification and targeted gene disruption in the wheat blotch fungus Mycosphaerella graminicola using TAGKO. Curr Genet 42:123–127PubMedCrossRefGoogle Scholar
  2. Ballance DJ, Buxton FP, Turner G (1983) Transformatio of Aspergillus nidulans by the orotine-5-phosphate decarboxylas gene of Neurospora crassa. Biochem Biophys Res Commun 112:284–289PubMedCrossRefGoogle Scholar
  3. Bechtold N, Ellis J, Pelletier G (1993) In-planta Agrobacterium-mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci III-VI E Paris 316:1194–1199Google Scholar
  4. Bellini C, Chupeau M, Guerche P, Vastra G, Chupeau Y (1989) Transformation of Lycopersicon peruvianum and Lycopersicon esculentum mesophyll protoplasts by electroporation. Plant Sci 65:63–75CrossRefGoogle Scholar
  5. Binninge DM, Skrznia C, Pukkila PJ, Casselton LA (1987) DNA-mediated transformation of the basidiomycete Coprinus cinereus. EMBO J 6:835–840Google Scholar
  6. Burns C, Geraghty R, Neville C, Murphy A, Kavanagh K, Doyle S (2005) Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigates. Fungal Genet Biol 42:319–327PubMedCrossRefGoogle Scholar
  7. Calvin MN, Hanawalt PC (1988) High-efficiency transformation of bacterial cells by electroporation. J Bacteriol 170:2796–2801PubMedPubMedCentralGoogle Scholar
  8. Cambareri EB, Jensen BC, Schabtach E, Selker EU (1989) Repeat-induced G-C to A-T mutations in Neurospora. Science 244:1571–1575PubMedCrossRefGoogle Scholar
  9. Case ME, Schweizer MSR, Giles NH (1979) Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci U S A 76:5259–5263PubMedCrossRefPubMedCentralGoogle Scholar
  10. Chakraborty BN, Patterson NA, Kapoor M (1991) An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi. Can J Microbiol 37:858–863PubMedCrossRefGoogle Scholar
  11. Chakraborty BN, Ouimet PM, Sreenivasan GM, Curle CA, Kapoor M (1995) Sequence repeat-induced disruption of the major heat-inducible HSP70 gene of Neurospora crassa. Curr Genet 29:18–26PubMedCrossRefGoogle Scholar
  12. Charaborty BN, Kapoor M (1990) Transformation of filamentous fungi by electroporation. Nucleic Acids Res 18:673–677CrossRefGoogle Scholar
  13. Chu G, Hayakawa H, Berg P (1987) Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15:1311–1326PubMedCrossRefPubMedCentralGoogle Scholar
  14. Coppin-Raynal E, Picard M, Arnaise S (1989) Transformation by integration in Podospora anserine iii. Replacement of a chromosome segment by a two-step process. Mol Gen Genet 219:270–276PubMedCrossRefGoogle Scholar
  15. Costaglioli P, Meilhoc E, Mason JM (1994) High-efficiency electrotransformation of the yeast Schwanniomyces accidentalis. Curr Genet 27:26–30PubMedCrossRefGoogle Scholar
  16. Delorme E (1989) Transformation of Saccharomyces cerevisiae by electroporation. Appl Environ Microbiol 55:2242–2246PubMedPubMedCentralGoogle Scholar
  17. Dhawale SS, Paietta JV, Marzluf GA (1984) A new, rapid and efficient transformation procedure for Neurospora. Curr Genet 8:77–79PubMedCrossRefGoogle Scholar
  18. Dombrowski JE, Baldwin JC, Alderman SC, Martin RC (2011) Transformation of Epichloe typhina by electroporation of conidia. BMC Res Notes 4(46):1–7Google Scholar
  19. Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145PubMedCrossRefPubMedCentralGoogle Scholar
  20. Faber KN, Haima P, Harder W, Veenhuis M, Geert AB (1994) Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr Genet 25:305–310PubMedCrossRefGoogle Scholar
  21. Faugeron G, Rhounim L, Rossignol JL (1990) How does the cell count the number of ectopic copies of a gene in the premeiotic inactivation process acting in Ascobolus immersus? Genetics 124:585–591PubMedPubMedCentralGoogle Scholar
  22. Fincham JRS (1989) Transformation of fungi. Microbiol Rev 53:148–170PubMedPubMedCentralGoogle Scholar
  23. Friedler S, Wirth R (1988) Transformation of bacteria with plasmid DNA by electroporation. Anal Biochem 170:38–44CrossRefGoogle Scholar
  24. Fromm ME, Taylor LP, Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319:791–793PubMedCrossRefGoogle Scholar
  25. Goldman GH, van Montagu M, Herra-Estrella A (1990) Transformation of Trichoderma harzianum by high-voltage electric pulse. Curr Genet 17:169–174CrossRefGoogle Scholar
  26. Goyon C, Faugeron G (1989) Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences. Mol Cell Biol 9:2818–2827PubMedPubMedCentralGoogle Scholar
  27. Gurpilharesa DB, Hasmann FA, Pessoa A, Roberto IC (2006) Optimization of glucose-6-phosphate dehydrogenase releasing from Candida guilliermondii by disruption with glass beads. Enzyme Microb Technol 39:591–595CrossRefGoogle Scholar
  28. Gutierrez A, Lopez-Garcia S, Garre V (2011) High reliability transformation of the basal fungus Mucor circinelloides by electroporation. J Microbiol Methods 84:442–446PubMedCrossRefGoogle Scholar
  29. Hama-Inaba H, Takahashi M, Kasai M, Shiomi T, Ito A, Hanaoka F, Sato K (1987) Optimum conditions for electric pulse mediated gene transfer to mammalian cells in suspension. Cell Struct Funct 12:173–180PubMedCrossRefGoogle Scholar
  30. Hatterman DR, Stacey G (1990) Efficient DNA transformation of Bradyrhizobium japonicum by electroporation. Appl Environ Microbiol 56:833–836Google Scholar
  31. Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci U S A 75:1929PubMedCrossRefPubMedCentralGoogle Scholar
  32. Hood MT, Stachow C (1990) Transformation of Schizosaccharomyces pombe by electroporation. Nucleic Acids Res 18:688PubMedCrossRefPubMedCentralGoogle Scholar
  33. Howard PK, Ahren KG, Firtel RA (1988) Establishment of a transient expression system for Dictyostelium discoideum. Nucleic Acids Res 16:2613–2623PubMedCrossRefPubMedCentralGoogle Scholar
  34. Kapoor M, Chakraborty BN, Machwe A, Patterson NA, Vijayaraghavan Y, Curle CA (1993) Electroporation mediated gene transfer and expression in Neurospora crassa. In: Baltz RH, Hegeman GD, Skaturd PL (eds) Industrial microorganisms: basic and applied molecular genetics. American Society of Microbiology, Washington, DCGoogle Scholar
  35. Kasuske A, Wedler H, Schulze S, Becher D (1992) Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids. Yeast 8:691–697PubMedCrossRefGoogle Scholar
  36. Kerridge D (1986) Mode of action of clinically important anti-fungal drugs. Adv Microb Physiol 27:1–72PubMedCrossRefGoogle Scholar
  37. Kinnard JH, Keighren MA, Kinsey JA, Eaton M, Fincham JR (1982) Cloning of the glutamate dehydrogenase gene of Neurospora crassa through the use of a synthetic DNA probe. Mol Cell Biol 4:17–122Google Scholar
  38. Knight DE (1981) Rendering cells permeable by exposure to electric fields. Tech Cell Phsiol 113:1–20Google Scholar
  39. Knight DE, Scrutton MC (1986) Gaining access to cytosol: the technique and some applications of electropermeabilization. Biochem J 234:497–506PubMedPubMedCentralGoogle Scholar
  40. Knutson JC, Yee D (1987) Electroporation: parameters affecting transfer of DNA into mammalian cells. Anal Biochem 164:44–52PubMedCrossRefGoogle Scholar
  41. Kothe GO, Free SJ (1996) Protocol for the elctroporation of Neurospora spheroplasts. FGN 43:31–33Google Scholar
  42. Kumar M, Sharma R, Dua M, Tuteja N, Johri AK (2013) “Electrotransformation” transformation system for root endophytic fungus Piriformospora indica. Soil Biol 33:309–321CrossRefGoogle Scholar
  43. Kwon-Chung KJ, Goldman WE, Klein B, Szaniszlo PJ (1998) Fate of transforming DNA in pathogenic fungi. Med Mycol 36:38–44PubMedGoogle Scholar
  44. Lauer U, Burgelt E, Squire Z, Messmer K, Hofschneider PH, Gregor M (1997) Shock wave permeabilization as a new gene transfer method. Gene Ther 4:710715CrossRefGoogle Scholar
  45. Le Chevanton L, Leblon G, Lebilcot S (1989) Duplication in Sordaria macrospora are not inactivated during meiosis. Mol Gen Genet 218:390–396PubMedCrossRefGoogle Scholar
  46. St. Leger RJ, Shimizu S, Joshi L, Bidochka MJ, Roberts DW (1995) Co-transformation of Metarhizium anisopliae by elctroporation or using the gene gun to produce stable GUS transformants. FEMS Microbiol Lett 131:289–294CrossRefGoogle Scholar
  47. Lu L, Wang TN, Xu TF, Wang JY, Wang CL, Zhao M (2013) Cloning and expression of thermo-alkali-stable laccase of Bacillus locheniformis in Pichia pastoris and its characterization. Bioresour Technol 134:81–86PubMedCrossRefGoogle Scholar
  48. Magana-Ortiz D, Coconi-Linares N, Ortiz-Vezquez E, Fernandez F, Loske AM, Gomez-Lim MA (2013) A novel and highly efficient method for genetic transformation of fungi employing shock waves. Fungal Genet Biol 56:9–16PubMedCrossRefGoogle Scholar
  49. May GS (1992) Fungal technology. In: Kinghorn JR (ed) Appiled molecular genetics of filamentous fungi. Blackie Academic and Professional, Glasgow, pp 1–27CrossRefGoogle Scholar
  50. Mclntyre DA, Harlander SK (1989) Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation. Appl Environ Microbiol 55:604–610Google Scholar
  51. Meyer V (2008) Genetic engineering of filamentous fungi – progress, obstacles and future trends. Biotechnol Adv 26:177–185PubMedCrossRefGoogle Scholar
  52. Meyer V, Mueller D, Strowig T, Stahl U (2003) Comparison of different transformation methods for Aspergillus giganteus. Curr Genet 43:371–377PubMedCrossRefGoogle Scholar
  53. Michielse CB, Hooykaas PJJ, van den Hondel CAM, Ram AFJ (2005) Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet 48:1–17PubMedCrossRefGoogle Scholar
  54. Miller JF, Dower WJ, Tompkins LS (1988) High voltage electroporation of bacteria: genetic transformation of Campylobacter jejuni with plasmid DNA. Proc Natl Acad Sci U S A 85:856–860PubMedCrossRefPubMedCentralGoogle Scholar
  55. Munoz-Rivas A, Specht CA, Drummond BJ, Froelinger E, Novotny CP (1986) Transformation of the basidiomycete, Schizophyllum commune. Mol Gen Genet 205:103–106PubMedCrossRefGoogle Scholar
  56. Narayanan R, Jastreboff MM, Chiu CF, Bertino JR (1986) In vivo expression of a nonselected gene transferred into murine hematopoietic stem cells by electroporation. Biochem Biophys Res Commun 141:1018–1024PubMedCrossRefGoogle Scholar
  57. Neumann E, Kakorin S, Tsoneva I, Nikolva B, Tomov T (1996) Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation. Biophys J 71:868–877PubMedCrossRefPubMedCentralGoogle Scholar
  58. Nuttley WM, Brade AM, Eitzen GA, Glover JR, Aiitchison JD, Rachubinski RA (1993) Rapid identification and characterization of peroxisomal assembly mutants in Yarrowia lipolytica. Yeast 9:507–517CrossRefGoogle Scholar
  59. Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for ß-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461PubMedPubMedCentralGoogle Scholar
  60. Ozeki K, Kyoya F, Hizume K, Kanda A, Hamachi M, Nunokawa Y (1994) Transformation of intact Aspergillus niger by electroporation. Biosci Biotechnol Biochem 58:2224–2227PubMedCrossRefGoogle Scholar
  61. Piredda S, Gaillardin C (1994) Development of a transformation system for the yeast Yamadazyma(Pichia) ohmeri. Yeast 10:1601–1612PubMedCrossRefGoogle Scholar
  62. Powell IB, Achen MG, Hillier AJ, Davidson BE (1988) A simple and rapid method for genetic transformation of lactic streptococci by electroporation. Appl Environ Microbiol 54:655–660PubMedPubMedCentralGoogle Scholar
  63. Prasanna GL, Panda T (1997) Electroporation: basic principles, practical considerations and applications in molecular biology. Bioprocess Eng 16:261–264CrossRefGoogle Scholar
  64. Queener SW, Ingolia TD, Skatud PL, Chapman JL, Kaster KR (1985) A system for genetic transformation of Cephalosporium acremonium. In: Schlessinger D (ed). American Society of Microbiology, Washington, DC, p 468–472Google Scholar
  65. Riach MBR, Kinghorn JR (1996) Genetic transformation and vector developments in filamentous fungi. In: Bos CJ (ed) Fungal genetics: principles and practice. Marcel Dekker Inc., p 209–233Google Scholar
  66. Richley MG, Marek ET, Schardl CL, Smith DA (1989) Transformation of filamentous fungi with plasmid DNA by electroporation. Phytopathology 79:844–847CrossRefGoogle Scholar
  67. Riggs CD, Bates GW (1986) Stable transformation of tobacco by electroporation: evidence for plasmid concatenation. Proc Natl Acad Sci U S A 83:5602–5606PubMedCrossRefPubMedCentralGoogle Scholar
  68. Rivera AL, Gomez-Lim M, Fernandez F, Loske AM (2012) Physical methods for genetic plant transformation. Phys Life Rev 9:308–345PubMedCrossRefGoogle Scholar
  69. Rivera AL, Magana-Ortiz D, Gomez-Lim M, Fernandez F, Loske AM (2014) Physical methods for genetic transformation of fungi and yeast. Phys Life Rev 11(2):184–203. doi: 10.1016/j.plrev.2014.01.007 PubMedCrossRefGoogle Scholar
  70. Robinson M, Sharon A (1999) Transformation of the bioherbicide Colletotrichum gloeosporioidesf. sp. aeschynomene by electroporation of germinated conidia. Curr Genet 36:98–104PubMedCrossRefGoogle Scholar
  71. Rohrer TL, Picataggio SK (1992) Targeted integrative transformation of Candida tropicalis by electroporation. Appl Microbiol Biotechnol 36:650–654PubMedGoogle Scholar
  72. Rossier C, Pugin A, Turian G (1985) Genetic analysis of transformation in a microconidiating strain of Neurospora crassa. Curr Genet 10:313–320PubMedCrossRefGoogle Scholar
  73. Ruiz-Diez B (2002) Strategies for the transformation of filamentous fungi. J Appl Microbiol 92:189–195PubMedCrossRefGoogle Scholar
  74. Ruiz-Diez B, Martinez-Suarez JV (1999) Electrotransformation of the human pathogenic fungus Scedosporium prolificans mediated by repetitive rDNA sequences. FEMS Immunol Med Microbiol 25:275–282PubMedCrossRefGoogle Scholar
  75. Sanchez O, Aguirre J (1996) Efficient transformation of Aspergillus nidulans by electroporation of germinated conidia. FGN 43:48–51Google Scholar
  76. Selker EU (1990) Premeiotic instability of repeated sequences in Neurospora crassa. Annu Rev Genet 24:579–613PubMedCrossRefGoogle Scholar
  77. Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation in Neurospora crassa. Proc Natl Acad Sci U S A 85:6870–6874PubMedCrossRefPubMedCentralGoogle Scholar
  78. Selker EU, Cambareri EB, Jensen BC, Haack KR (1987) Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 51:741–752PubMedCrossRefGoogle Scholar
  79. Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus I (1985) High efficiency direct gene transfer to plants. Nat Biotechnol 3:1099–1103CrossRefGoogle Scholar
  80. Singh N, Rajam MV (2013) A simple and rapid glass bead transformation method for a filamentous fungus Fusarium oxysporum. Cell Dev Biol 2:115. doi: 10.4172/2168-9296.1000115 Google Scholar
  81. Spandidos DA (1987) Electric-field mediated gene transfer (electroporation) into mouse Friend and human K 562 erythroleukemic cells. Gene Anal Tech 4:50–56PubMedCrossRefGoogle Scholar
  82. Theil T, Poo H (1989) Transformation of a filamentous Cyanobacterium by electroporation. J Bacteriol 171:5743–5746Google Scholar
  83. Thompson JR, Register E, Curotto J, Kurtz M, Kelly R (1998) An improved protocol for the preparation of yeast cells for transformation by electroporation. Yeast 14:565–571PubMedCrossRefGoogle Scholar
  84. Toneguzzo F, Keating A (1986) Stable expression of selectable genes introduced into human hematopoietic stem cells by electric field mediated DNA transfer. Proc Natl Acad Sci U S A 83:3496–3499PubMedCrossRefPubMedCentralGoogle Scholar
  85. Toneguzzo F, Hayday AC, Keating A (1986) Electric field mediated DNA transfer: transient and stable expression in human and mouse lymphoid cells. Mol Cell Biol 6:703–706PubMedPubMedCentralGoogle Scholar
  86. Toriyama K, Arimoto Y, Uchimiya H, Hinata K (1988) Transgenic rice plants after direct gene transfer into protoplasts. Nat Biotechnol 6:1072–1074CrossRefGoogle Scholar
  87. Tur-kaspa R, Teicher L, Levine BJ, Skoultchi AI, Shafritz DA (1986) Use of electropration to introduce biologically active foreign genes into primary rat hepatocytes. Mol Cell Biol 6:716–718PubMedPubMedCentralGoogle Scholar
  88. Ward M, Kodama KH, Wilson LJ (1989) Transformation of Aspergillus awamori and A. niger by electroporation. Exp Mycol 13:289–293CrossRefGoogle Scholar
  89. Wen-Jun S, Forde BG (1989) Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res 17:83–85CrossRefGoogle Scholar
  90. Wu SX, Letchworth GJ (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36:152–154PubMedGoogle Scholar
  91. Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by a trp plasmid. Proc Natl Acad Sci U S A 81:1470–1474PubMedCrossRefPubMedCentralGoogle Scholar
  92. Zerbib D, Amalric F, Teissie J (1985) Electric field mediated transformation: isolation and characterization of a TK + subclone. Biochem Biophys Res Commun 129:611–618PubMedCrossRefGoogle Scholar

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© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • B. N. Chakraborty
    • 1
  1. 1.Department of BotanyUniversity of North BengalSiliguriIndia

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