T-DNA Agrobacterium w komórce eukariotycznej: import do jądra komórkowego i integracja do genomu roślinnego

Ziemienowicz, Alicja

doi:0860-7796

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Title: T-DNA Agrobacterium w komórce eukariotycznej: import do jądra komórkowego i integracja do genomu roślinnego

Abstract:

Agrobacterium tumefaciens, a gram-negative soil bacterium, is able to transfer DNA to most plant species causing crown gall disease in dicotyledonousplants. 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 intothe chromosome. DNA transfer requires plasmid encoded virulence (vir) genesas 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 plantcell nucleus as a complex with VirD2 and VirE2 proteins. The C-terminal NLS ofVirD2 has a piloting function in this process. Import of the T-DNA follows theclassical NLS- and importin-dependent nuclear import pathway for proteins. Forstudies 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 ligatethe T-DNA to the plant DNA in vitro. Consequently, plant enzymes must beinvolved in this process. Indeed, we found an activity responsible for the ligation of T-DNA in extracts from tobacco BY2 suspension cultured cells and frompea axes. This activity is likely to originate from plant DNA ligase, since the T-DNAligation shows the same requirements for hydrolysis of ATP to AMP as ligationmediated by any ATP-dependent DNA ligase. This does not, however, excludethe involvement of other plant enzymes in T-DNA integration.