IMPa-4, an Arabidopsis importin alpha isoform, is preferentially involved in agrobacterium-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 alpha 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 alpha family in Agrobacterium transformation. Yeast two-hybrid, plant bimolecular fluorescence complementation, and in vitro protein-protein interaction assays demonstrated that all tested Arabidopsis importin alpha 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 alpha 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.

Figure 1.

Cladogram of the Nine Importin α Proteins Investigated in This Study. The genes encoding the corresponding importin α proteins are indicated in parentheses. Alignments were performed using ClustalW. The alignment information was used to generate a cladogram using MegAlign 7.1 (DNASTAR) and a bootstrap value of 1000, with seed of 111. Asterisks denote the importin α isoforms investigated in this study. Sequences used to generate the cladogram are presented in Supplemental Data Set 1 online.

Figure 2.

KAPα, IMPa-2, IMPa-3, and IMPa-4 Interact with VirE2 and VirD2. (A) and (C) In yeast two-hybrid assays, yeast strains containing various bait/prey combinations were tested for LacZ activity using liquid ONPG as a β-galactosidase substrate. The black bars indicate LacZ activity in yeast strains containing importin α bait/VirD2 prey (A) or importin α bait/VirE2 prey (C) combinations. The gray bars show the LacZ activity in the yeast strains containing importin α bait/nonspecific GTPase prey. Error bars represent se obtained with three biological replicates. (B) and (D) KAPα, IMPa-2, IMPa-3, and IMPa-4 bind to VirD2 and VirE2 in vitro. GST-tagged importin α proteins and GST alone were linked to separate glutathione–Sepharose columns and treated with lysates from E. coli expressing VirD2 (B) or VirE2 (D). Bound proteins were eluted, and equimolar fraction volumes were analyzed by immunoblot using either rabbit anti-octopine VirD2 (B) or anti-nopaline VirE2 (D) polyclonal antiserum as the primary antibody (1:1000 dilution) and goat anti-rabbit IgG conjugated to horseradish peroxidase as the secondary antibody (1:1000 dilution). Lane 1, VirD2 (B) or VirE2 (D) crude lysate. Lanes 2 to 6, eluted fractions from different columns: lane 2, GST-KAPα; lane 3, GST-IMPa-2; lane 4, GST-IMPa-3; lane 5, GST-IMPa-4; lane 6, GST. The asterisk represents a lower molecular weight, partially degraded VirE2 protein previously reported in the lysates of VirE2-expressing E. coli cells (Dombek and Ream, 1997).

Figure 3.

IMPa-4–Interacting Domains of VirD2 and VirE2. (A) Schematic representation of the various VirD2 deletion mutants used. VirD2 protein or its truncated derivatives are shown as gray boxes. VirD2 contains two NLS, a N-terminal monopartite NLS (stippled box) and a C-terminal bipartite NLS (black boxes). Thin lines indicate deleted regions. The N- and C-terminal NLS residues are underlined. (B) Results of interaction assays in yeast with IMPa-4 bait and the various VirD2 deletion mutant preys. VirD2 interacts with IMPa-4 via residues Arg-372 to Arg-417. The LacZ activity was measured by liquid ONPG assays. Error bars represent se of three biological replicates. (C) Schematic representation of the various VirE2 deletion mutants used. VirE2 protein or its truncated derivatives are shown as gray boxes. VirE2 contains two central bipartite NLSs (black boxes). The NLS motifs are underlined. (D) Results of interaction assays in yeast with IMPa-4 bait and the various VirE2 deletion mutant preys. VirE2 interacts with IMPa-4 via its two central NLS domains and also via the last 158 amino acids in its C terminus. Full-length and various deletion constructions of VirD2 preys, VirE2 preys, or the nonspecific GTPase prey were tested for interaction in yeast with IMPa-4 as a bait. The LacZ activity was measured by liquid ONPG assays. The x axis indicates the various bait plasmids used. Error bars represent se of three biological replicates.

Figure 4.

SSB and Importin α Binding Activities of VirE2 Are Not Mutually Exclusive. (A) ssDNA-bound VirE2 protein binds importin α in vitro. ³²P end–labeled ssDNA complexed with 30 ng of His-VirE2 was incubated with increasing amounts (20 to 400 ng; amounts used are indicated below each lane) of T7–IMPa-4 protein. The samples were subjected to nondenaturing PAGE and visualized by autoradiography. Lane 1, labeled ssDNA; lanes 2 to 8, labeled ssDNA (5 ng) + VirE2 (30 ng) with increasing amounts of T7–IMPa-4 (20 to 400 ng); lane 9, labeled ssDNA (5 ng) + 400 ng of T7–IMPa-4. (B) An importin α-VirE2 complex binds ssDNA in vitro. A total of 400 ng of IMPa-4 was preincubated with 30 ng of VirE2, and 5 ng of ³²P end–labeled labeled ssDNA was added before electrophoresis. Lane 1, labeled ssDNA; lane 2, labeled ssDNA (5 ng) + VirE2 (30 ng); lane 3, preincubated T7–IMPa-4 (400 ng) + VirE2 (30 ng) followed by 5 ng of labeled ssDNA; lane 4, labeled ssDNA (5 ng) + 400 ng T7–IMPa-4.

Figure 5.

Arabidopsis Importin α Mutants kapα, impa-2, and impa-3 Show Wild-Type Agrobacterium-Mediated Transformation Efficiencies. (A) Root segments from Arabidopsis mutants homozygous for the disruption of KAPα, IMPa-2, or IMPa-3 and wild-type control plants were infected with the tumorigenic strain Agrobacterium A208 (for stable transformation assays; multipatterned bars) or Agrobacterium strain At849 (containing a plant-active gusA gene for transient transformation assays; stippled black bars). Stable transformation efficiency (indicated by the presence of tumors) was scored and categorized after 1 month. The sizes and colors of the tumors are indicated by bar patterns. The larger and more green the tumors, the more highly susceptible the plant tissue is for transformation. For transient transformation, root segments were stained with 5-bromo-4-chloro-3-indolyl-β-glucuronic acid (X-gluc) at 6 d after infection, and the percentage of roots showing GUS activity was calculated. For each assay, 10 to 15 plants and >80 segments per plant were used. Error bars represent se for the 10 to 15 plants assayed for each treatment. (B) Representative plates showing tumorigenesis efficiencies (stable transformation) and tumor morphology on wild-type and importin α mutant root segments. Bar = 2 cm.

Figure 6.

An Arabidopsis impa-4 Mutant Is a rat Mutant. (A) Schematic representation of the IMPa-4 gene. The Arabidopsis impa-4 mutant contains a T-DNA insertion in the seventh intron. A part of the T-DNA/plant DNA junction sequence is shown. The sequence corresponding to the T-DNA right border primer TR1 is underlined. The sequence for intron 7 (shaded box) is followed by a portion of exon 8 sequence. The position of the T-DNA right border TR1 primer is indicated with an arrow. (B) Protein blot analysis of extracts isolated from leaf and root tissue of Arabidopsis ecotypes Ws-2 (lanes 1 and 4), Columbia (lanes 2 and 5), and impa-4 mutant plants (lanes 3 and 6) with an antibody raised against an IMPa-4 peptide. Recombinant GST-tagged IMPa-4 protein was used as a control (lane 7). Top, immunostain; bottom, portion of a Coomassie blue–stained gel showing equal protein loading within each tissue sample. (C) The Arabidopsis impa-4 mutant shows reduced levels of transient and stable Agrobacterium-mediated root transformation. Root segments from wild-type and impa-4 mutant plants were infected either with the tumorigenic strain Agrobacterium A208 (for stable transformation assays; multipatterned bars) or Agrobacterium At849 (for transient transformation assays; gray bars). For stable transformation, the tumors were scored after 1 month. For transient transformation, root segments were stained with X-gluc at 6 d after infection, and the percentage of roots showing GUS activity as a result of infection by Agrobacterium At849 was calculated. For each assay, 10 to 15 plants and >80 segments per plant were used. Error bars represent se. Representative plates of tumorigenesis assays are shown. White bars = 1 cm.

Figure 7.

Inhibition of Agrobacterium-Mediated Root Transformation by Expression of an RNAi Construct Targeted Specifically against IMPa-4. Root segments from wild-type (ecotype Ws-2), impa-4 mutant, and RNAi plants were inoculated with Agrobacterium A208 at 10⁷ colony-forming units/mL, and tumorigenesis assays were conducted. Tumors were scored after 6 weeks. Each bar indicates data from an individual plant, and average values for each set are indicated. Numbers above the error bars indicate the number of plants analyzed. Error bars indicate se.

Figure 8.

Differences in Transcript Levels of Various Importin α Isoforms in Wild-Type and impa-4 Mutant Roots as Determined by RT-PCR. Total RNA from wild-type and impa-4 mutant roots was reverse-transcribed using oligo(dT) primers and then subjected to real-time PCR analysis using various importin α gene-specific primers. RT-PCR with ubiquitin primers was performed as a control to correct for sample concentrations. Gray and black bars represent the transcript levels of the indicated importin α isoforms in wild-type and impa-4 mutant roots, respectively. Error bars represent se of four technical replicates.

Figure 9.

Overexpression of Importin α Isoforms Rescues the Rat Phenotype of impa-4 Mutant Roots. (A) Transgenic plants individually overexpressing At KAPα, IMPa-2, IMPa-3, IMPa-4, IMPa-6, IMPa-7, or IMPa-9 cDNAs in the impa-4 mutant background show increased susceptibility to Agrobacterium-mediated root transformation compared with parent impa-4 mutant plants. Root segments from several T2 generation transgenic plants (2 to 3 independent lines each for KAPα, IMPa-2, IMPa-3, or IMPa-4 overexpression) or T1 generation transgenic plants (14 to 15 independent plants each for IMPa-6, IMPa-7, or IMPa-9 overexpression) were infected with either the tumorigenic strain Agrobacterium A208 (for stable transformation assays; multipatterned bars) or Agrobacterium At849 (for transient transformation assays; gray bars). Infected root segments from wild-type and impa-4 mutant plants were used as positive and negative controls, respectively. For stable transformation, the tumors were scored after approximately 1 month. For transient transformation, root segments were stained with X-gluc at 6 d after infection, and the percentage of roots showing GUS activity resulting from infection by Agrobacterium At849 was calculated. N.D., not determined. Error bars indicate se. (B) Representative plates of tumorigenesis assays. Bar = 2 cm.

Figure 10.

Localization of YFP-VirD2, YFP-VirE2, and VirE2-YFP in Wild-Type and impa-4 Mutant Roots. Single-plane confocal optical sections show YFP localization (green fluorescence) throughout cells (both in the cytoplasm and in the nucleus) in the root tip of wild-type ([A] and [B]) and impa-4 mutant ([C] and [D]) plants. YFP-VirD2 preferentially localizes to the nuclei of root tip cells of both wild-type ([E] and [F]) and impa-4 mutant ([G] and [H]) plants. YFP-VirD2 colocalizes with nuclear Hoechst 33242 stain (blue fluorescence). In both wild-type ([I] and [J]) and impa-4 mutant ([K] and [L]) root tip cells, YFP-VirE2 localizes at cell poles (arrowheads). VirE2-YFP also aggregates in the cytoplasm (arrowheads) in both wild-type ([M] and [N]) and impa-4 mutant ([O] and [P]) root tip cells. In (A) to (L), blue indicates Hoechst 33242 staining of nuclei. Bars = 15 μm in (A), (C), (E), (G), (I), (K), (M), and (O) and 50 μm in (B), (D), (F), (H), (J), (L), (N), and (P).

Figure 11.

Interaction and Localization of VirE2 with Itself and of VirD2 and VirE2 with Importin α Proteins in Plant Cells. BiFC constructs were electroporated into tobacco BY-2 cells, and the cells were visualized by fluorescence microscopy 48 h later. Constructs are as follows: VirE2-nYFP plus VirE2-cYFP (A); nYFP-VirD2 plus KAPα-cYFP (B); nYFP-VirD2 plus IMPa-4–cYFP (C); nYFP-VirD2 plus IMPa-7–cYFP (D); nYFP-VirD2 plus IMPa-9–cYFP (E); nYFP-VirD2 plus IMPa-4–cYFP genomic clone (F); VirE2-nYFP plus KAPα-cYFP (G); VirE2-nYFP plus IMPa-4–cYFP ([H] and [I]); VirE2-nYFP plus IMPa-7–cYFP (J); VirE2-nYFP plus IMPa-9–cYFP (K); VirE2-nYFP plus IMPa-4–cYFP genomic clone (L). In (A) and (E), blue indicates Hoechst 33242 staining of nuclei. In (B), (C), (D), (G), (J), and (K), blue indicates false-color image of the entire cell. In (F), (H), (I), and (L), red fluoresecent protein images (marking the entire cell) are merged with the YFP images. Bars = 20 μm.

Figure 12.

Wild-Type and impa-4 Mutant Roots Overexpressing VirE2-YFP, but Not YFP-VirE2, Complement a virE2 Mutant Agrobacterium in Transient Transformation Assays. Several independent T1 transgenic plants overexpressing YFP-VirE2 or VirE2-YFP in the wild type or in the impa-4 mutant background were infected with Agrobacterium At1565. Root segments were stained with X-gluc at 6 d after infection, and the percentage of roots showing GUS activity resulting from infection by Agrobacterium At89 was calculated. Root segments from wild-type plants overexpressing untagged VirE2 were used as positive controls (left bar). Nontransgenic wild-type plants and impa-4 mutant plants were used as negative controls. For each assay, >80 segments per plant were used. Error bars represent se.

Figure 13.

Multicolor BiFC Indicates the Site of Interaction of VirD2 and VirE2 with IMPa-1 (KAPα) and IMPa-4. VirD2-cCFP ([A] to [H]) and VirE2-cCFP ([I] to [P]) were used as baits to interact simultaneously with IMPa-1 or IMPa-4. mCherry indicates transfected BY-2 protoplasts; red fluorescence localizes both to the cytoplasm and to the nucleus. The various combinations of labeled proteins coexpressed in the protoplasts are indicated above each series of four panels. Cells were visualized using an epifluorescence microscope: (A), (E), (I), and (M), red (mCherry) channel; (B), (G), (J), and (O), yellow/green (Venus) channel; (C), (F), (K), and (N), blue (Cerulean) channel; (D), (H), (L), and (P), overlay of yellow and blue channels. Bars = 20 μm.