IMPa-4, anArabidopsisImportin α Isoform, Is Preferentially Involved inAgrobacterium-Mediated Plant Transformation

Abstract

Successful transformation of plants by Agrobacterium tumefaciens requires that the bacterial T-complex actively escorts T-DNA into the host's nucleus. VirD2 and VirE2 are virulence proteins on the T-complex that have plant-functional nuclear localization signal sequences that may recruit importin a proteins of the plant for nuclear import. In this study, we evaluated the involvement of seven of the nine members of the Arabidopsis thaliana importin a family in Agrobacterium transformation. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that all tested Arabidopsis importin a members can interact with VirD2 and VirE2. However, only disruption of the importin IMPa-4 inhibited transformation and produced the rat (resistant to Agrobacterium transformation) phenotype. Overexpression of six importin a members, including IMPa-4, rescued the rat phenotype in the impa-4 mutant background. Roots of wild-type and impa-4 Arabidopsis plants expressing yellow fluorescent protein-VirD2 displayed nuclear localization of the fusion protein, indicating that nuclear import of VirD2 is not affected in the impa-4 mutant. Somewhat surprisingly, VirE2-yellow fluorescent protein mainly localized to the cytoplasm of both wild-type and impa-4 Arabidopsis cells and to the cytoplasm of wild-type tobacco (Nicotiana tabacum) cells. However, bimolecular fluorescence complementation assays indicated that VirE2 could localize to the nucleus when IMPa-4, but not when IMPa-1, was overexpressed.

The Intrinsically Disordered N-terminal Region of AtREM1.3 Remorin Protein Mediates Protein-Protein Interactions

“…5B). Interestingly, this does not correlate with the phylogenetic relation between importin ␣ proteins (46).…”

“…Importin ␣ proteins are soluble receptors involved in nuclear trafficking. In A. thaliana, this family comprises 9 members, some of which are involved in interaction with VirD2 and VirE2 bacterial T-complex proteins and mediate nuclear localization of these bacterial proteins (46). Moreover, AtIMPa3 (MOS6) plays a role in plant innate immunity because it is a suppressor of resistance responses and immunity to pathogens (42), further supporting possible roles of remorin proteins during plant-microbe interactions and innate immunity.…”

Agrobacterium May Delay Plant Nonhomologous End-Joining DNA Repair via XRCC4 to Favor T-DNA Integration

“…To gain better understanding on the functional significance of the VirE2 interaction with XRCC4 in relation to Agrobacteriummediated transformation, we took advantage of transgenic Arabidopsis plants expressing VirE2-YFP (Bhattacharjee et al, 2008). We hypothesized that VirE2 can interact with XRCC4 in plants expressing VirE2-YFP, thereby delaying the NHEJ-mediated DSB repair and providing greater opportunity for T-DNA integration.…”

“…Many molecules of Agrobacterium VirE2 protein likely enter the plant cell independent of T-DNA (Sundberg et al, 1996;Gelvin, 1998) and may localize into the nucleus (Citovsky et al, 1992). VirE2 localization in plant cells, either to the nucleus or perinuclear vicinity, is controversial (Citovsky et al, 1992;Bhattacharjee et al, 2008). We propose that free VirE2 molecules can interact with XRCC4 and titrate/exclude active XRCC4 protein available for DSB repair in the cell.…”

Overexpression of SeveralArabidopsisHistone Genes IncreasesAgrobacterium-Mediated Transformation and Transgene Expression in Plants

“…Following transfer of T-DNA to the plant cytoplasm, it likely forms complexes with various transferred Agrobacterium virulence effector proteins, including VirE2, a single-stranded DNA binding protein (Gietl et al, 1987;Christie et al, 1988;Citovsky et al, 1988Citovsky et al, , 1992Das, 1988) and VirF, which may mediate proteolysis of VirE2 in the nucleus (Schrammeijer et al, 2001;Tzfira et al, 2004). In addition, plantencoded proteins, such as VIP1 and importin a, may form supercomplexes with the T-strand and help target T-DNA to the nucleus (Ballas and Citovsky, 1997;Tzfira et al, 2001;Bhattacharjee et al, 2008;Lee et al, 2008). The timing and subcellular location of VirE2 interaction with T-strands remains unknown.…”