Skip to main content
SpringerLink
Log in

Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae

  • Methods
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The list of fungal species with known complete genome and/or expressed sequence tag collections is extending rapidly during the last couple of years. Postgenomic gene function assignment is an obvious follow-up and depends on methodologies to test gene function in vivo. One of such methods is the generation of null mutants via homologous recombination at the wild–type loci by using inactivation cassettes. In this paper, the ability of Agrobacterium tumefaciens to genetically transform filamentous fungi was exploited to drive homologous recombination at the trp1 locus of the enthomopathogenic fungus Metarhizium anisopliae. The trp1 disruptants exhibited a clearly distinguishable phenotype from wild-type cells and were recovered with high efficiency of homologous recombination (22%). The complementation of such mutants with the wild-type gene generates only transformants with homologous integration.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Arruda W, Lubeck I, Schrank A, Vainstein MH (2005) Morphological alterations of Metarhizium anisopliae during penetration of Boophilus microplus ticks. Exp Appl Acarol 37:231–244

    Article  Google Scholar 

  • Bird D, Bradshaw R (1997) Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans. Mol Gen Genet 255:219–225

    Article  CAS  Google Scholar 

  • Bogo MR, Vainstein MH, Aragao FJ, Rech E, Schrank A (1996) High frequency gene conversion among benomyl resistant transformants in the entomopathogenic fungus Metarhizium anisopliae. FEMS Microbiol Lett 142:123–127

    Article  CAS  Google Scholar 

  • Casselton LA, De La Fuente-Herce A (1989) Heterologous gene expression in the basidiomycete Coprinus cinereus. Curr Genet 16:35–40

    Article  CAS  Google Scholar 

  • Combier JP, Melayah D, Raffier C, Gay G, Marmeisse R (2003) Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMS Microbiol Lett 220:141–148

    Article  CAS  Google Scholar 

  • Covert SF, Kapoor P, Lee M, Briley A, Nairn CJ (2001) Agrobacterium-mediated transformation of Fusarium circinatum. Mycol Res 105:259–264

    Article  CAS  Google Scholar 

  • de Groot MJ, Bundock P, Hooykaas PJ, Beijersbergen AG (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839–842

    Article  Google Scholar 

  • Duarte RTD, Staats CC, Fungaro MHP, Schrank A, Vainstein MH, Furlaneto-Maia L, Nakamura CV, Souza W, Furlaneto MC (2007) Development of a simple and rapid Agrobacterium tumefaciens-mediated transformation system for the enthomopathogenic fungus Metarhizium anisopliae var. acridum. Lett Appl Microbiol 44:248–254

    Article  CAS  Google Scholar 

  • Dutra V, Nakazato L, Broetto L, Schrank IS, Vainstein MH, Schrank A (2004) Application of representational difference analysis to identify sequence tags expressed by Metarhizium anisopliae during the infection process of the tick Boophilus microplus cuticle. Res Microbiol 155:245–251

    Article  CAS  Google Scholar 

  • Fang W, Pei Y, Bidochka MJ (2006) Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens. Can J Microbiol 52:623–626

    Article  CAS  Google Scholar 

  • Fang W, Zhang Y, Yang X, Zheng X, Duan H, Li Y, Pei Y (2004) Agrobacterium tumefaciens-mediated transformation of Beauveria bassiana using an herbicide resistance gene as a selection marker. J Invertebr Pathol 85:18–24

    Article  CAS  Google Scholar 

  • Freimoser FM, Screen S, Bagga S, Hu G, St. Leger RJ (2003) Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts. Microbiology 149:239–247

    Article  CAS  Google Scholar 

  • Godio RP, Fouces R, Gudina EJ, Martin JF (2004) Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium. Curr Genet 46:287–294

    Article  CAS  Google Scholar 

  • Goosen T, van Engelenburg F, Debets F, Swart K, Bos K, van den Broek H (1989) Tryptophan auxotrophic mutants in Aspergillus niger: inactivation of the trpC gene by cotransformation mutagenesis. Mol Gen Genet 219:282–288

    Article  CAS  Google Scholar 

  • Hoffman B, Breuil C (2004) Disruption of the subtilase gene, albin1, in Ophiostoma piliferum. Appl Environ Microbiol 70:3898–3903

    Article  CAS  Google Scholar 

  • Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST (1994) Bergey’s manual of determinative microbiology. Williams and Wilkins, Maryland

    Google Scholar 

  • Kassa A, Stephan D, Vidal S, Zimmermann G (2004) Laboratory and field evaluation of different formulations of Metarhizium anisopliae var. acridum submerged spores and aerial conidia for the control locusts and grasshoppers. Biocontrol 49:63–81

    Article  CAS  Google Scholar 

  • Lazzarini GM, Rocha LF, Luz C (2006) Impact of moisture on in vitro germination of Metarhizium anisopliae and Beauveria bassiana and their activity on Triatoma infestans. Mycol Res 110:485–492

    Article  Google Scholar 

  • Lee MH, Bostock RM (2006) Agrobacterium T-DNA-mediated integration and gene replacement in the brown rot pathogen Monilinia fructicola. Curr Genet 49:309–322

    Article  CAS  Google Scholar 

  • Lima IG, Duarte RT, Furlaneto L, Baroni CH, Fungaro MH, Furlaneto MC (2006) Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus with Agrobacterium tumefaciens. Lett Appl Microbiol 42:631–636

    CAS  PubMed  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  CAS  Google Scholar 

  • Michielse CB, Arentshorst M, Ram AF, van den Hondel CA (2005a) Agrobacterium–mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori. Fungal Genet Biol 42:9–19

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Mullin PG, Turgeon BG, Yoder OC (1993) Complementation of Cochliobolus heterostrophus trp-mutants produced by gene replacement. Fungal Genet Newsl 40:51–53

    Google Scholar 

  • Nakazato L, Dutra V, Broetto L, Staats CC, Vainstein MH, Schrank A (2006) Development of an expression vector for Metarhizium anisopliae based on the tef-1alpha homologous promoter. Appl Microbiol Biotechnol 72:521–528

    Article  CAS  Google Scholar 

  • Rauyaree P, Ospina-Giraldo MD, Kang S, Bhat RG, Subbarao KV, Grant SJ, Dobinson KF (2005) Mutations in VMK1, a mitogen-activated protein kinase gene, affect microsclerotia formation and pathogenicity in Verticillium dahliae. Curr Genet 48:109–116

    Article  CAS  Google Scholar 

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

    Article  Google Scholar 

  • Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Scholte EJ, Knols BG, Takken W (2006) Autodissemination of the entomopathogenic fungus Metarhizium anisopliae amongst adults of the malaria vector Anopheles gambiae s.s. Malar J 283:45

    Google Scholar 

  • St. Leger R, Shimizu S, Joshi L, Bidochka MJ, Roberts DW (1995) Co-transformation of Metarhizium anisopliae by electroporation or using the gene gun to produce stable GUS transformants. FEMS Microbiol Lett 131:289–294

    Article  CAS  Google Scholar 

  • Staats CC, Silva MS, Pinto PM, Vainstein MH, Schrank A (2004) The Metarhizium anisopliae trp1 gene: cloning and regulatory analysis. Curr Microbiol 49:66–70

    Article  CAS  Google Scholar 

  • Sugui JA, Chang YC, Kwon-Chung KJ (2005) Agrobacterium tumefaciens–mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Appl Environ Microbiol 71:1798–1802

    Article  CAS  Google Scholar 

  • Wang C, Hu G, St. Leger RJ (2005) Differential gene expression by Metarhizium anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals mechanisms of physiological adaptation. Fungal Genet Biol 42:704–718

    Article  CAS  Google Scholar 

  • Weld RJ, Plummer KM, Carpenter MA, Ridgway HJ (2006) Approaches to functional genomics in filamentous fungi. Cell Res 16:31–44

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by grants from CNPq, CAPES, and FAPERGS. The authors wish to thank W. Arruda for the SEM and the PIGS sequencing network at the CBiot-UFRGS. The authors thank G.H.Goldman and A. Zaha for critically reading of the manuscript.

Author information

Authors and Affiliations

  1. Centro de Biotecnologia, Programa de Pós-graduação em Biologia celular e Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

    Charley Christian Staats, Angela Junges, Mariana Fitarelli, Marilene Henning Vainstein & Augusto Schrank

  2. Centro de Ciências Biológicas, Universidade Estadual de Londrina, Porto Alegre, RS, Brazil

    Marcia Cristina Furlaneto

  3. Departamento de Microbiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

    Marilene Henning Vainstein

  4. Departamento de Biologia Molecular e Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil

    Augusto Schrank

Authors
  1. Charley Christian Staats
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Angela Junges
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mariana Fitarelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Marcia Cristina Furlaneto
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marilene Henning Vainstein
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Augusto Schrank
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Augusto Schrank.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Staats, C.C., Junges, A., Fitarelli, M. et al. Gene inactivation mediated by Agrobacterium tumefaciens in the filamentous fungi Metarhizium anisopliae . Appl Microbiol Biotechnol 76, 945–950 (2007). https://doi.org/10.1007/s00253-007-1043-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-007-1043-4

Keywords

Search

Navigation