title: Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Research Area Molecular Biotechnology and Microbiology, Institute of Chemical Engineering, Vienna University of Technology, Gumpendorferstrasse 1a, Wien, Austria.
authors.forename: Sabine
authors.initials: S
authors.lastname: Gruber
authors.forename: Markus
authors.initials: M
authors.lastname: Omann
authors.forename: Carolina Escobar
authors.initials: CE
authors.lastname: Rodrìguez
authors.forename: Theresa
authors.initials: T
authors.lastname: Radebner
authors.forename: Susanne
authors.initials: S
authors.lastname: Zeilinger
mesh_terms: D000890:Anti-Infective Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D061046:Biological Control Agents; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D005818:Genetic Engineering; D016681:Genome, Fungal; D005839:Gentamicins / Q000494:pharmacology; D006921:Hygromycin B / Q000494:pharmacology; D019868:Kanamycin Kinase / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D015398:Signal Transduction / Q000187:drug effects; D014170:Transformation, Genetic; D014242:Trichoderma / Q000187:drug effects / Q000235:genetics*
chemical_list: D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D061046:Biological Control Agents; D005656:Fungal Proteins; D005839:Gentamicins; D006921:Hygromycin B; C010680:antibiotic G 418; D019868:Kanamycin Kinase; D019204:GTP-Binding Proteins