Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium

Abstract

Agrobacterium-mediated genetic transformation of plants, a unique example of transkingdom DNA transfer, requires the presence of several proteins encoded by the host cell. One such cellular factor is VIP1, an Arabidopsis protein proposed to interact with and facilitate import of the bacterial DNA-protein transport (T) complexes into the plant cell nucleus. Thus, VIP1 is required for transient expression of the bacterial DNA, an early step in the transformation process. However, the role of VIP1 in subsequent transformation events leading to the stable expression of bacterial DNA was unexplored. Here, we used reverse genetics to dissect VIP1 functionally and demonstrate its involvement in the stable genetic transformation of Arabidopsis plants by Agrobacterium. Our data indicate that the ability of VIP1 to interact with the VirE2 protein component of the T-complex and localize to the cell nucleus is sufficient for transient genetic transformation, whereas its ability to form homomultimers and interact with the host cell H2A histone in planta is required for tumorigenesis and, by implication, stable genetic transformation.T-complex ͉ VirE2 ͉ nuclear import ͉ histones ͉ chromatin targeting

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF VBF ubiquitin–ligase complex pathway

“…Potentially, before the plant defense response is fully mounted, the invading T-complexes are uncoated more rapidly, leading to T-DNA expression before the T-complex can be targeted by VIP1 to the chromatin for integration (22), whereas later the uncoating slows, allowing the T-complex to reach the target chromatin. Furthermore, it is tempting to speculate that, once at the chromatin, the SCF VBF pathway can expose not only the T-DNA by degrading VirE2 attached to VIP1, but also the target host DNA by degrading histone molecules to which VIP1 also attaches (21,22). Thus, disassembly of the T-complex via the host UPS may represent a more general mechanism for uncoating of DNA molecules within eukaryotic cells; for example, proteasomal degradation of H2A/ H2B histones from promoter regions has been shown to promote chromatin disassembly during transcriptional activation (39).…”

“…Because VirE2 is associated with the T-strand, VIP1 effectively mediates nuclear import of the entire T-complex. Once inside the nucleus, VIP1 mediates chromatin association of the T-complex by acting as a molecular link between VirE2 and nucleosomes via interactions with the core histones (21,22).…”

Divide and conquer: development and cell cycle genes in plant transformation

“…Although the regeneration capacity of explants overexpressing VIP1 is not increased [34], its overexpression increased the rate of transient and stable plant transformation [35] (Figure 1). VIP1 has a nuclear localization signal, and interacts not only with VirE2 but also with the plant H2A [34], H2B, H3 and H4 [33]. It has been proposed that the VIP1-histone association might trigger the proteolytic uncoating of the T-DNA, enabling its integration [33].…”

“…Further research determined that VIP1 interacts with VirE2 from Agrobacterium and with several histones, facilitating the integration of the foreign DNA into the plant genome [33,34]. Although the regeneration capacity of explants overexpressing VIP1 is not increased [34], its overexpression increased the rate of transient and stable plant transformation [35] (Figure 1). VIP1 has a nuclear localization signal, and interacts not only with VirE2 but also with the plant H2A [34], H2B, H3 and H4 [33].…”

The Role of the Ubiquitin-Proteasome System in Agrobacterium tumefaciens-Mediated Genetic Transformation of Plants

“…Among the exported proteins, VirE2 serves to package the mobile single-stranded T-DNA molecule, covalently associated with a single molecule of the bacterial endonuclease VirD2 (Dürrenberger et al, 1989), into a nucleoprotein complex (T-complex), in which numerous VirE2 molecules cover the entire length of the T-DNA molecule (Citovsky et al, 1997;Abu-Arish et al, 2004). Moreover, A. tumefaciens also utilizes the plant factor VIP1 (for VirE2-interacting protein1), which directly binds to VirE2 and facilitates the nuclear import and chromatin targeting of the entire T-complex (Tzfira et al, 2001;Li et al, 2005;Djamei et al, 2007;Lacroix et al, 2008). In the current model, the T-complex is most likely uncoated of its protein components by the VirF-mediated proteasomal degradation before the T-DNA becomes integrated into the host genome .…”