Import of DNA into mammalian nuclei by proteins originating from a plant pathogenic bacterium

Abstract

Import of DNA into mammalian nuclei is generally inefficient. Therefore, one of the current challenges in human gene therapy is the development of efficient DNA delivery systems. Here we tested whether bacterial proteins could be used to target DNA to mammalian cells. Agrobacterium tumefaciens, a plant pathogen, efficiently transfers DNA as a nucleoprotein complex to plant cells. Agrobacterium-mediated T-DNA transfer to plant cells is the only known example for interkingdom DNA transfer and is widely used for plant transformation. Agrobacterium virulence proteins VirD2 and VirE2 perform important functions in this process. We reconstituted complexes consisting of the bacterial virulence proteins VirD2, VirE2, and single-stranded DNA (ssDNA) in vitro. These complexes were tested for import into HeLa cell nuclei. Import of ssDNA required both VirD2 and VirE2 proteins. A VirD2 mutant lacking its C-terminal nuclear localization signal was deficient in import of the ssDNA-protein complexes into nuclei. Import of VirD2-ssDNA-VirE2 complexes was fast and efficient, and was shown to depended on importin alpha, Ran, and an energy source. We report here that the bacterium-derived and plant-adapted protein-DNA complex, made in vitro, can be efficiently imported into mammalian nuclei following the classical importin-dependent nuclear import pathway. This demonstrates the potential of our approach to enhance gene transfer to animal cells.

Figure 1

Localization of VirD2 and VirE2 proteins in HeLa nuclei. Cy3.5-labeled proteins (red fluorescence) were incubated with digitonin-permeabilized HeLa cells for 20 min at room temperature in the dark. The nuclei were stained with SYTO 13 dye (green fluorescence). (A) Localization of VirD2 protein. (B) Localization of VirE2 protein. (C) Localization of BSA.

Figure 2

Formation of T-DNA complex in vitro. (A) Scheme of the cleavage assay. (B) Cleavage of 1-kb ssDNA by VirD2wt (lane 2) or mutant VirD2ΔNLS protein (lane 3), control without protein (lane 1). The efficiency of the cutting reaction by VirD2 proteins was tested on rhodamine-labeled 1-kb ssDNA. (C) ssDNA binding activity of VirE2. Increasing amounts of VirE2 protein were added to ssDNA in amounts indicated on the picture.

Figure 3

Import of 1-kb ssDNA into HeLa nuclei requires both VirD2 and VirE2 proteins. Different ssDNA–protein complexes containing rhodamine-labeled ssDNA (red fluorescence) were incubated with permeabilized HeLa cells for 20 min at room temperature in the dark. (A) ssDNA alone. (B) VirD2–ssDNA–VirE2 complex. The position of the nuclei is indicated by staining with SYTO 13 dye (green fluorescence). The fluorescently labeled component is indicated by ∗.