Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins - PubMed (original) (raw)

Import of Agrobacterium T-DNA into plant nuclei: two distinct functions of VirD2 and VirE2 proteins

A Ziemienowicz et al. Plant Cell. 2001 Feb.

Abstract

To study the mechanism of nuclear import of T-DNA, complexes consisting of the virulence proteins VirD2 and VirE2 as well as single-stranded DNA (ssDNA) were tested for import into plant nuclei in vitro. Import of these complexes was fast and efficient and could be inhibited by a competitor, a nuclear localization signal (NLS) coupled to BSA. For import of short ssDNA, VirD2 was sufficient, whereas import of long ssDNA additionally required VirE2. A VirD2 mutant lacking its C-terminal NLS was unable to mediate import of the T-DNA complexes into nuclei. Although free VirE2 molecules were imported into nuclei, once bound to ssDNA they were not imported, implying that when complexed to DNA, the NLSs of VirE2 are not exposed and thus do not function. RecA, another ssDNA binding protein, could substitute for VirE2 in the nuclear import of T-DNA but not in earlier events of T-DNA transfer to plant cells. We propose that VirD2 directs the T-DNA complex to the nuclear pore, whereas both proteins mediate its passage through the pore. Therefore, by binding to ssDNA, VirE2 may shape the T-DNA complex such that it is accepted for translocation into the nucleus.

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Figures

Figure 1.

Figure 1.

Localization of VirD2 and VirE2 in Plant Nuclei. (A) Localization of VirD2. (B) Localization of mutant VirD2ΔNLS. (C) Localization of VirE2. Cy3.5-labeled proteins (red fluorescence) were incubated with permeabilized tobacco protoplasts for 20 min at room temperature in the dark. The nuclei were stained with SYTO 13 dye (green fluorescence). Fluorescently labeled components are indicated by asterisks.

Figure 2.

Figure 2.

Import of a 25-Nucleotide Oligonucleotide into Plant Nuclei Requires the Functional NLS of the VirD2 Protein. (A) Oligonucleotide alone. (B) Oligonucleotide bound to VirD2wt. (C) Oligonucleotide bound to mutant VirD2ΔNLS. The positions of the nuclei are indicated by staining with SYTO 13 dye (green fluorescence). The plant nuclear import assay was performed using rhodamine-labeled oligonucleotide (red fluorescence), as described in Methods. Fluorescently labeled components are indicated by asterisks.

Figure 3.

Figure 3.

Import of a 250-Nucleotide ssDNA into Plant Nuclei Requires Both VirD2 and VirE2 Proteins. (A) ssDNA alone. (B) VirD2wt–ssDNA complex. (C) VirD2wt–ssDNA–VirE2 complex. (D) VirD2ΔNLS–ssDNA–VirE2 complex. (E) VirE2–ssDNA complex. (F) VirD2wt–ssDNA–VirE2 complex and wheat germ agglutinin (WGA; final concentration 0.5 mg/mL). The positions of the nuclei are indicated by staining with SYTO 13 dye (green fluorescence). In vitro import of different ssDNA–protein complexes into plant nuclei was performed as described in Methods. DNA was labeled with rhodamine (red fluorescence). Fluorescently labeled components are indicated by asterisks.

Figure 4.

Figure 4.

Localization of the VirE2 Protein Associated with ssDNA in Plant Nuclei. (A) A 38-nucleotide oligonucleotide reacted with labeled VirE2. (B) VirD2wt–ssDNA–VirE2-labeled complex quenched with excess of 38-nucleotide oligonucleotide. (C) VirD2ΔNLS–ssDNA–VirE2-labeled complex quenched with excess of 38-nucleotide oligonucleotide. In vitro nuclear import of different complexes of Cy3.5-labeled VirE2 (red fluorescence) with ssDNAs was performed as described in Methods. The positions of the nuclei are indicated by staining with SYTO 13 dye (green fluorescence). Fluorescently labeled components are indicated by asterisks.

Figure 5.

Figure 5.

Model for the Nuclear Import of the T-DNA Complex. (A) Docking of the VirD2–T-DNA–VirE2 complex at the nuclear pore. (B) and (C) Passage through the nuclear pore channel. The nuclear import machinery and NPC are simplified in this model. Sizes are not to scale. Nuclear import intermediates are indicated by black triangles. See text for more details.

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