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Molecular and Cellular Biology, September 2000, p. 6317-6322, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

Alicja Ziemienowicz,1,2,* Bruno Tinland,1,dagger John Bryant,3 Veronique Gloeckler,1 and Barbara Hohn1

Friedrich Miescher-Institut, CH-4002 Basel, Switzerland1; Plant Pathology Laboratory, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk, Gdansk, Poland2; and Department of Biological Sciences, University of Exeter, EX4 4QG Exeter, United Kingdom3

Received 17 December 1999/Returned for modification 17 February 2000/Accepted 2 June 2000

Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.

* Corresponding author. Mailing address: Plant Pathology Laboratory, Department of Biotechnology, Intercollegiate Faculty of Biotechnology, University of Gdansk, ul. Kladki 24, 80-822 Gdansk, Poland. Phone: 0048-58-3012241, ext. 360 or 315. Fax: 0048-58-3012807. E-mail: ziemien{at}biotech.univ.gda.pl.

dagger Present address: Monsanto, 1150 Brussels, Belgium.

Molecular and Cellular Biology, September 2000, p. 6317-6322, Vol. 20, No. 17
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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