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Agrobacterium T-DNA in the eukaryotic cell: nuclear import and integration into the plant genome

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Agrobacterium tumefaciens, a gram-negative soil bacterium, is able to transfer DNA to most plant species causing crown gall disease in dicotyledonous plants. Due to this activity Agrobacterium is widely used for plant transformation. The transferred DNA (T-DNA) that resides on a large Ti plasmid is processed within the bacterium and is exported to the plant where it is integrated into the chromosome. DNA transfer requires plasmid encoded virulence (vir) genes as well as several chromosomal genes. In vivo studies suggested that Agrobacterium proteins are involved in T-DNA transfer and integration. We study the function of virulence proteins VirD2 and VirE2 in T-DNA nuclear import and integration using in vitro systems. We found that the T-DNA is imported into the plant cell nucleus as a complex with VirD2 and VirE2 proteins. The C-terminal NLS of VirD2 has a piloting function in this process. Import of the T-DNA follows the classical NLS- and importin-dependent nuclear import pathway for proteins. For studies of integration of T-DNA into the plant DNA an in vitro integration/ligation assay has been designed. We have found out that VirD2 is not able to ligate the T-DNA to the plant DNA in vitro. Consequently, plant enzymes must be involved in this process. Indeed, we found an activity responsible for the ligation of T-DNA in extracts from tobacco BY2 suspension cultured cells and from pea axes. This activity is likely to originate from plant DNA ligase, since the T-DNA ligation shows the same requirements for hydrolysis of ATP to AMP as ligation mediated by any ATP-dependent DNA ligase. This does not, however, exclude the involvement of other plant enzymes in T-DNA integration.
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A. Ziemienowicz, Zaklad Ochrony i Biotechnologii Roslin, Katedra Biotechnologii, Miedzyuczelniany Wydzia? Biotechnologii, ul. Kladki 24, 80-822 Gdansk, Poland, e-mail:
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