Skip to main content
SpringerLink
Log in

Electroporation Mediated DNA Transformation of Filamentous Fungi

  • Chapter
  • First Online:
Genetic Transformation Systems in Fungi, Volume 1

Part of the book series: Fungal Biology ((FUNGBIO))

  • 3048 Accesses

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  • 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 42:123–127

    Article  PubMed  CAS  Google Scholar 

  • 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 112:284–289

    Article  PubMed  CAS  Google Scholar 

  • 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 316:1194–1199

    CAS  Google Scholar 

  • Bellini C, Chupeau M, Guerche P, Vastra G, Chupeau Y (1989) Transformation of Lycopersicon peruvianum and Lycopersicon esculentum mesophyll protoplasts by electroporation. Plant Sci 65:63–75

    Article  CAS  Google Scholar 

  • Binninge DM, Skrznia C, Pukkila PJ, Casselton LA (1987) DNA-mediated transformation of the basidiomycete Coprinus cinereus. EMBO J 6:835–840

    Google Scholar 

  • 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 42:319–327

    Article  PubMed  CAS  Google Scholar 

  • Calvin MN, Hanawalt PC (1988) High-efficiency transformation of bacterial cells by electroporation. J Bacteriol 170:2796–2801

    PubMed  CAS  PubMed Central  Google Scholar 

  • Cambareri EB, Jensen BC, Schabtach E, Selker EU (1989) Repeat-induced G-C to A-T mutations in Neurospora. Science 244:1571–1575

    Article  PubMed  CAS  Google Scholar 

  • Case ME, Schweizer MSR, Giles NH (1979) Efficient transformation of Neurospora crassa by utilizing hybrid plasmid DNA. Proc Natl Acad Sci U S A 76:5259–5263

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Chakraborty BN, Patterson NA, Kapoor M (1991) An electroporation-based system for high-efficiency transformation of germinated conidia of filamentous fungi. Can J Microbiol 37:858–863

    Article  PubMed  CAS  Google Scholar 

  • 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 29:18–26

    Article  PubMed  CAS  Google Scholar 

  • Charaborty BN, Kapoor M (1990) Transformation of filamentous fungi by electroporation. Nucleic Acids Res 18:673–677

    Article  Google Scholar 

  • Chu G, Hayakawa H, Berg P (1987) Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15:1311–1326

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • 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 219:270–276

    Article  PubMed  CAS  Google Scholar 

  • Costaglioli P, Meilhoc E, Mason JM (1994) High-efficiency electrotransformation of the yeast Schwanniomyces accidentalis. Curr Genet 27:26–30

    Article  PubMed  CAS  Google Scholar 

  • Delorme E (1989) Transformation of Saccharomyces cerevisiae by electroporation. Appl Environ Microbiol 55:2242–2246

    PubMed  CAS  PubMed Central  Google Scholar 

  • Dhawale SS, Paietta JV, Marzluf GA (1984) A new, rapid and efficient transformation procedure for Neurospora. Curr Genet 8:77–79

    Article  PubMed  CAS  Google Scholar 

  • Dombrowski JE, Baldwin JC, Alderman SC, Martin RC (2011) Transformation of Epichloe typhina by electroporation of conidia. BMC Res Notes 4(46):1–7

    Google Scholar 

  • Dower WJ, Miller JF, Ragsdale CW (1988) High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 16:6127–6145

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Faber KN, Haima P, Harder W, Veenhuis M, Geert AB (1994) Highly-efficient electrotransformation of the yeast Hansenula polymorpha. Curr Genet 25:305–310

    Article  PubMed  CAS  Google Scholar 

  • 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 124:585–591

    PubMed  CAS  PubMed Central  Google Scholar 

  • Fincham JRS (1989) Transformation of fungi. Microbiol Rev 53:148–170

    PubMed  CAS  PubMed Central  Google Scholar 

  • Friedler S, Wirth R (1988) Transformation of bacteria with plasmid DNA by electroporation. Anal Biochem 170:38–44

    Article  Google Scholar 

  • Fromm ME, Taylor LP, Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319:791–793

    Article  PubMed  CAS  Google Scholar 

  • Goldman GH, van Montagu M, Herra-Estrella A (1990) Transformation of Trichoderma harzianum by high-voltage electric pulse. Curr Genet 17:169–174

    Article  CAS  Google Scholar 

  • Goyon C, Faugeron G (1989) Targeted transformation of Ascobolus immersus and de novo methylation of the resulting duplicated DNA sequences. Mol Cell Biol 9:2818–2827

    PubMed  CAS  PubMed Central  Google Scholar 

  • 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 39:591–595

    Article  Google Scholar 

  • Gutierrez A, Lopez-Garcia S, Garre V (2011) High reliability transformation of the basal fungus Mucor circinelloides by electroporation. J Microbiol Methods 84:442–446

    Article  PubMed  CAS  Google Scholar 

  • 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 12:173–180

    Article  PubMed  CAS  Google Scholar 

  • Hatterman DR, Stacey G (1990) Efficient DNA transformation of Bradyrhizobium japonicum by electroporation. Appl Environ Microbiol 56:833–836

    Google Scholar 

  • Hinnen A, Hicks JB, Fink GR (1978) Transformation of yeast. Proc Natl Acad Sci U S A 75:1929

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Hood MT, Stachow C (1990) Transformation of Schizosaccharomyces pombe by electroporation. Nucleic Acids Res 18:688

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Howard PK, Ahren KG, Firtel RA (1988) Establishment of a transient expression system for Dictyostelium discoideum. Nucleic Acids Res 16:2613–2623

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • 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, DC

    Google Scholar 

  • Kasuske A, Wedler H, Schulze S, Becher D (1992) Efficient electropulse transformation of intact Candida maltosa cells by different homologous vector plasmids. Yeast 8:691–697

    Article  PubMed  CAS  Google Scholar 

  • Kerridge D (1986) Mode of action of clinically important anti-fungal drugs. Adv Microb Physiol 27:1–72

    Article  PubMed  CAS  Google Scholar 

  • 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 4:17–122

    Google Scholar 

  • Knight DE (1981) Rendering cells permeable by exposure to electric fields. Tech Cell Phsiol 113:1–20

    Google Scholar 

  • Knight DE, Scrutton MC (1986) Gaining access to cytosol: the technique and some applications of electropermeabilization. Biochem J 234:497–506

    PubMed  CAS  PubMed Central  Google Scholar 

  • Knutson JC, Yee D (1987) Electroporation: parameters affecting transfer of DNA into mammalian cells. Anal Biochem 164:44–52

    Article  PubMed  CAS  Google Scholar 

  • Kothe GO, Free SJ (1996) Protocol for the elctroporation of Neurospora spheroplasts. FGN 43:31–33

    Google Scholar 

  • Kumar M, Sharma R, Dua M, Tuteja N, Johri AK (2013) “Electrotransformation” transformation system for root endophytic fungus Piriformospora indica. Soil Biol 33:309–321

    Article  Google Scholar 

  • Kwon-Chung KJ, Goldman WE, Klein B, Szaniszlo PJ (1998) Fate of transforming DNA in pathogenic fungi. Med Mycol 36:38–44

    PubMed  CAS  Google Scholar 

  • Lauer U, Burgelt E, Squire Z, Messmer K, Hofschneider PH, Gregor M (1997) Shock wave permeabilization as a new gene transfer method. Gene Ther 4:710715

    Article  Google Scholar 

  • Le Chevanton L, Leblon G, Lebilcot S (1989) Duplication in Sordaria macrospora are not inactivated during meiosis. Mol Gen Genet 218:390–396

    Article  PubMed  Google Scholar 

  • 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 131:289–294

    Article  Google Scholar 

  • 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 134:81–86

    Article  PubMed  CAS  Google Scholar 

  • 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 56:9–16

    Article  PubMed  CAS  Google Scholar 

  • May GS (1992) Fungal technology. In: Kinghorn JR (ed) Appiled molecular genetics of filamentous fungi. Blackie Academic and Professional, Glasgow, pp 1–27

    Chapter  Google Scholar 

  • Mclntyre DA, Harlander SK (1989) Genetic transformation of intact Lactococcus lactis subsp. lactis by high-voltage electroporation. Appl Environ Microbiol 55:604–610

    Google Scholar 

  • Meyer V (2008) Genetic engineering of filamentous fungi – progress, obstacles and future trends. Biotechnol Adv 26:177–185

    Article  PubMed  CAS  Google Scholar 

  • Meyer V, Mueller D, Strowig T, Stahl U (2003) Comparison of different transformation methods for Aspergillus giganteus. Curr Genet 43:371–377

    Article  PubMed  CAS  Google Scholar 

  • Michielse CB, Hooykaas PJJ, van den Hondel CAM, Ram AFJ (2005) Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet 48:1–17

    Article  PubMed  CAS  Google Scholar 

  • 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 85:856–860

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Munoz-Rivas A, Specht CA, Drummond BJ, Froelinger E, Novotny CP (1986) Transformation of the basidiomycete, Schizophyllum commune. Mol Gen Genet 205:103–106

    Article  PubMed  CAS  Google Scholar 

  • 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 141:1018–1024

    Article  PubMed  CAS  Google Scholar 

  • 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 71:868–877

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • 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 9:507–517

    Article  CAS  Google Scholar 

  • 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 6:2452–2461

    PubMed  CAS  PubMed Central  Google Scholar 

  • Ozeki K, Kyoya F, Hizume K, Kanda A, Hamachi M, Nunokawa Y (1994) Transformation of intact Aspergillus niger by electroporation. Biosci Biotechnol Biochem 58:2224–2227

    Article  PubMed  CAS  Google Scholar 

  • Piredda S, Gaillardin C (1994) Development of a transformation system for the yeast Yamadazyma(Pichia) ohmeri. Yeast 10:1601–1612

    Article  PubMed  CAS  Google Scholar 

  • 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 54:655–660

    PubMed  CAS  PubMed Central  Google Scholar 

  • Prasanna GL, Panda T (1997) Electroporation: basic principles, practical considerations and applications in molecular biology. Bioprocess Eng 16:261–264

    Article  CAS  Google Scholar 

  • 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 468–472

    Google Scholar 

  • 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 209–233

    Google Scholar 

  • Richley MG, Marek ET, Schardl CL, Smith DA (1989) Transformation of filamentous fungi with plasmid DNA by electroporation. Phytopathology 79:844–847

    Article  Google Scholar 

  • Riggs CD, Bates GW (1986) Stable transformation of tobacco by electroporation: evidence for plasmid concatenation. Proc Natl Acad Sci U S A 83:5602–5606

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Rivera AL, Gomez-Lim M, Fernandez F, Loske AM (2012) Physical methods for genetic plant transformation. Phys Life Rev 9:308–345

    Article  PubMed  Google Scholar 

  • 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

    Article  PubMed  Google Scholar 

  • Robinson M, Sharon A (1999) Transformation of the bioherbicide Colletotrichum gloeosporioidesf. sp. aeschynomene by electroporation of germinated conidia. Curr Genet 36:98–104

    Article  PubMed  CAS  Google Scholar 

  • Rohrer TL, Picataggio SK (1992) Targeted integrative transformation of Candida tropicalis by electroporation. Appl Microbiol Biotechnol 36:650–654

    PubMed  CAS  Google Scholar 

  • Rossier C, Pugin A, Turian G (1985) Genetic analysis of transformation in a microconidiating strain of Neurospora crassa. Curr Genet 10:313–320

    Article  PubMed  CAS  Google Scholar 

  • Ruiz-Diez B (2002) Strategies for the transformation of filamentous fungi. J Appl Microbiol 92:189–195

    Article  PubMed  CAS  Google Scholar 

  • Ruiz-Diez B, Martinez-Suarez JV (1999) Electrotransformation of the human pathogenic fungus Scedosporium prolificans mediated by repetitive rDNA sequences. FEMS Immunol Med Microbiol 25:275–282

    Article  PubMed  CAS  Google Scholar 

  • Sanchez O, Aguirre J (1996) Efficient transformation of Aspergillus nidulans by electroporation of germinated conidia. FGN 43:48–51

    Google Scholar 

  • Selker EU (1990) Premeiotic instability of repeated sequences in Neurospora crassa. Annu Rev Genet 24:579–613

    Article  PubMed  CAS  Google Scholar 

  • Selker EU, Garrett PW (1988) DNA sequence duplications trigger gene inactivation in Neurospora crassa. Proc Natl Acad Sci U S A 85:6870–6874

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Selker EU, Cambareri EB, Jensen BC, Haack KR (1987) Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 51:741–752

    Article  PubMed  CAS  Google Scholar 

  • Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus I (1985) High efficiency direct gene transfer to plants. Nat Biotechnol 3:1099–1103

    Article  Google Scholar 

  • 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 

  • Spandidos DA (1987) Electric-field mediated gene transfer (electroporation) into mouse Friend and human K 562 erythroleukemic cells. Gene Anal Tech 4:50–56

    Article  PubMed  CAS  Google Scholar 

  • Theil T, Poo H (1989) Transformation of a filamentous Cyanobacterium by electroporation. J Bacteriol 171:5743–5746

    Google Scholar 

  • 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 14:565–571

    Article  PubMed  CAS  Google Scholar 

  • 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 83:3496–3499

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • 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 6:703–706

    PubMed  CAS  PubMed Central  Google Scholar 

  • Toriyama K, Arimoto Y, Uchimiya H, Hinata K (1988) Transgenic rice plants after direct gene transfer into protoplasts. Nat Biotechnol 6:1072–1074

    Article  CAS  Google Scholar 

  • 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 6:716–718

    PubMed  CAS  PubMed Central  Google Scholar 

  • Ward M, Kodama KH, Wilson LJ (1989) Transformation of Aspergillus awamori and A. niger by electroporation. Exp Mycol 13:289–293

    Article  Google Scholar 

  • Wen-Jun S, Forde BG (1989) Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res 17:83–85

    Article  Google Scholar 

  • Wu SX, Letchworth GJ (2004) High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques 36:152–154

    PubMed  CAS  Google Scholar 

  • Yelton MM, Hamer JE, Timberlake WE (1984) Transformation of Aspergillus nidulans by a trp plasmid. Proc Natl Acad Sci U S A 81:1470–1474

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  • Zerbib D, Amalric F, Teissie J (1985) Electric field mediated transformation: isolation and characterization of a TK + subclone. Biochem Biophys Res Commun 129:611–618

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

  1. Department of Botany, University of North Bengal, Siliguri, West Bengal, India

    B. N. Chakraborty M.Sc., Ph.D.

Authors
  1. B. N. Chakraborty M.Sc., Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Applied Biochemistry Department, DSM Biotechnology Center, Delft, The Netherlands

    Marco A. van den Berg

  2. Global Marker Technology Lab (DuPont-Pioneer), E.I.DuPont India Pvt Ltd, DuPont Knowledge Center, Hyderabad, Telangana, India

    Karunakaran Maruthachalam

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer International Publishing Switzerland

About this chapter

Cite this chapter

Chakraborty, B.N. (2015). Electroporation Mediated DNA Transformation of Filamentous Fungi. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 1. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10142-2_6

Download citation

Publish with us

Policies and ethics

Search

Navigation