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Journal of Bacteriology, April 2004, p. 2038-2045, Vol. 186, No. 7
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.7.2038-2045.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Agrobacterium-Mediated Transformation of Aspergillus awamori in the Absence of Full-Length VirD2, VirC2, or VirE2 Leads to Insertion of Aberrant T-DNA Structures

Caroline B. Michielse,* Arthur F. J. Ram, Paul J. J. Hooykaas, and Cees A. M. J. J. van den Hondel

Clusius Laboratory, Institute of Biology, Leiden University, 2333 AL, Leiden, The Netherlands

Received 27 August 2003/ Accepted 16 December 2003

Reductions to 2, 5, and 42% of the wild-type transformation efficiency were found when Agrobacterium mutants carrying transposon insertions in virD2, virC2, and virE2, respectively, were used to transform Aspergillus awamori. The structures of the T-DNAs integrated into the host genome by these mutants were analyzed by Southern and sequence analyses. The T-DNAs of transformants obtained with the virE2 mutant had left-border truncations, whereas those obtained with the virD2 mutant had truncated right ends. From this analysis, it was concluded that the virulence proteins VirD2 and VirE2 are required for full-length T-DNA integration and that these proteins play a role in protecting the right and left T-DNA borders, respectively. Multicopy and truncated T-DNA structures were detected in the majority of the transformants obtained with the virC2 mutant, indicating that VirC2 plays a role in correct T-DNA processing and is required for single-copy T-DNA integration.

* Corresponding author. Mailing address: Fungal Genetics Research Group, Clusius Laboratory, Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands. Phone: 31 71 5274861. Fax: 31 71 5274999. E-mail: Michielse{at}rulbim.leidenuniv.nl.

Journal of Bacteriology, April 2004, p. 2038-2045, Vol. 186, No. 7
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.7.2038-2045.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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