Abstract
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 α-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.
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Acknowledgements
P.J.J. Hooykaas is appreciated for providing A. tumefaciens strain LBA1100 and plasmid pTAS5. C.P. Kubicek and R.L. Mach are acknowledged for providing the infrastructural facilities and for critically reviewing the manuscript. This work was supported by grants from the Austrian Programme for Advanced Research and Technology of the Österreichische Akademie der Wissenschaften and from Fonds zur Förderung Wissenschaftlicher Forschung (P15483).
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Communicated by J. Heitman
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Zeilinger, S. Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation. Curr Genet 45, 54–60 (2004). https://doi.org/10.1007/s00294-003-0454-8
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DOI: https://doi.org/10.1007/s00294-003-0454-8