Abstract
BackgroundSpecies 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.ResultsWe 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.ConclusionThe 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.
Highlights
Species of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, and widely used as biocontrol agents (BCAs) in agriculture
For the genetic manipulation of T. atroviride various transformation techniques, inlcuding biolistic, Agrobacterium-mediated, and protoplast-based methods have been established [11]. These approaches basically rely on only two dominant selection markers, the Hygromycin B resistance-conferring hph gene and the acetamidaseencoding amdS gene enabling the fungus to grow on acetamide as sole nitrogen source [12]
Fungal growth from mycelial agar plug inocula placed on potato dextrose agar (PDA) plates supplemented with increasing amounts of geneticin were effectively impaired by concentrations of 10–60 μg/ml
Summary
Species of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, and widely used as biocontrol agents (BCAs) in agriculture. In T. atroviride, the Tga Gα protein was shown to regulate mycoparasitismrelevant processes, such as the attachment to the host fungus, and the production of cell wall-degrading enzymes and antifungal secondary metabolites According to these essential functions, tga gene deletion mutants were avirulent, i.e. unable to attack and lyse host fungi [10]. For the genetic manipulation of T. atroviride various transformation techniques, inlcuding biolistic, Agrobacterium-mediated, and protoplast-based methods have been established [11] These approaches basically rely on only two dominant selection markers, the Hygromycin B resistance-conferring hph gene and the acetamidaseencoding amdS gene enabling the fungus to grow on acetamide as sole nitrogen source [12]. In order to facilitate serial gene deletions and re-transformations in this important biocontrol fungus, the establishment of additional selection markers is crucial
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