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Open Access Highly Accessed Research article

Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker

Sabine Gruber1, Markus Omann12, Carolina Escobar Rodrìguez1, Theresa Radebner1 and Susanne Zeilinger1*

Author affiliations

1 Research Area Molecular Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstrasse 1a, Wien, Austria

2 Current address: Zuckerforschung Tulln GmbH, Josef-Reiter-Strasse 21-23, Tulln, Austria

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Citation and License

BMC Research Notes 2012, 5:641  doi:10.1186/1756-0500-5-641

Published: 17 November 2012

Abstract

Background

Species of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, but also widely used as biocontrol agents (BCAs) in agriculture. In the latter function Trichoderma species stimulate plant growth, induce plant defense and directly antagonize plant pathogenic fungi through their mycoparasitic capabilities. The recent release of the genome sequences of four mycoparasitic Trichoderma species now forms the basis for large-scale genetic manipulations of these important BCAs. Thus far, only a limited number of dominant selection markers, including Hygromycin B resistance (hph) and the acetamidase-encoding amdS gene, have been available for transformation of Trichoderma spp. For more extensive functional genomics studies the utilization of additional dominant markers will be essential.

Results

We established the Escherichia coli neomycin phosphotransferase II-encoding nptII gene as a novel selectable marker for the transformation of Trichoderma atroviride conferring geneticin resistance. The nptII marker cassette was stably integrated into the fungal genome and transformants exhibited unaltered phenotypes compared to the wild-type. Co-transformation of T. atroviride with nptII and a constitutively activated version of the Gα subunit-encoding tga3 gene (tga3Q207L) resulted in a high number of mitotically stable, geneticin-resistant transformants. Further analyses revealed a co-transformation frequency of 68% with 15 transformants having additionally integrated tga3Q207L into their genome. Constitutive activation of the Tga3-mediated signaling pathway resulted in increased vegetative growth and an enhanced ability to antagonize plant pathogenic host fungi.

Conclusion

The neomycin phosphotransferase II-encoding nptII gene from Escherichia coli proved to be a valuable tool for conferring geneticin resistance to the filamentous fungus T. atroviride thereby contributing to an enhanced genetic tractability of these important BCAs.

Keywords:
Fungi; Trichoderma; Genetic transformation; Geneticin; G protein signaling; Mycoparasitism