Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation

A modified Agrobacterium-mediated transformation method for the efficient disruption of two genes encoding signaling compounds of the mycoparasite Trichoderma atroviride is described, using the hph gene of Escherichia coli as selection marker. The transformation vectors contained about 1 kb of 5...

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Bibliographic Details
Published in:Current genetics 2004-02, Vol.45 (1), p.54-60
Main Author: Zeilinger, Susanne
Format: Article
Language:English
Subjects:
Agrobacterium
E coli
Gene Targeting - methods
Genes
Genes, Fungal - genetics
Genetic Vectors - genetics
hph gene
hygromycin
Mutagenesis, Insertional - methods
Mutagenesis, Site-Directed - genetics
Proteins
Recombination, Genetic - genetics
Rhizobium - genetics
RNA, Fungal - genetics
RNA, Fungal - metabolism
RNA, Messenger - genetics
RNA, Messenger - metabolism
tga3 gene
tmk1 gene
Transformation, Genetic
Trichoderma - genetics
Trichoderma atroviride
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Summary:A modified Agrobacterium-mediated transformation method for the efficient disruption of two genes encoding signaling compounds of the mycoparasite Trichoderma atroviride is described, using the hph gene of Escherichia coli as selection marker. The transformation vectors contained about 1 kb of 5' and 3' non-coding regions from the tmk1 (encoding a MAP kinase) or tga3 (encoding an alpha-subunit of a heterotrimeric G protein) target loci flanking a selection marker. Transformation of fungal conidia and selection on hygromycin-containing media applying an overlay-based procedure, which overcomes the lack of formation of distinct single colonies by the fungus, led to stable clones for both disruption constructs. Southern and PCR analyses proved gene disruption by single-copy homologous integration with a frequency of approximately 60% for both genes; and the loss of tmk1 and tga3 transcript formation in the disruptants was demonstrated by RT-PCR.
ISSN:0172-8083
1432-0983
DOI:10.1007/s00294-003-0454-8