VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity

Abstract

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. This event is thought to be mediated by two bacterial proteins, VirD2 and VirE2, which are associated with the transported T-DNA molecule. While VirD2 is imported into the nuclei of plant, animal and yeast cells, nuclear uptake of VirE2 occurs most efficiently in plant cells. To understand better the mechanism of VirE2 action, a cellular interactor of VirE2 was identified and its encoding gene cloned from Arabidopsis. The identified plant protein, designated VIP1, specifically bound VirE2 and allowed its nuclear import in non-plant systems. In plants, VIP1 was required for VirE2 nuclear import and Agrobacterium tumorigenicity, participating in early stages of T-DNA expression.

Fig. 1. Specific interaction between VIP1 and VirE2 in the two-hybrid system and in vitro. (A) Growth in the absence of histidine, tryptophan and leucine. (B) Growth in the absence of tryptophan and leucine. VIP1 was expressed from pGAD424 whereas VirE2 and negative control interactors lamin C and topoisomerase I (TOP I) were expressed from pBTM116. Growth in histidine-deficient medium represents selective conditions for protein–protein interactions. (C) VirE2 binding to immobilized VIP1 in vitro. VIP1 (lanes 1, 3, 5 and 7) and VirD2 (lanes 2, 4, 6 and 8) were electrophoresed, blotted onto a membrane, incubated with VirE2 (lanes 3 and 4), VirD2 (lanes 5 and 6) or TMV MP (lanes 7 and 8) and probed with anti-VirE2, anti-VirD2 or anti-TMV MP antibodies, respectively. Lanes 1 and 2, Coomassie blue staining of VIP1 and VirD2, respectively, after electrophoresis; lanes 3–8, autoradiographs of the binding assays. Protein molecular mass standards are indicated on the left in kDa.

Fig. 2. Alignment of the VIP1 bZIP domain with the four most homologous plant proteins identified by the BLASTA search (Altschul et al., 1990). The bZIP domain of VIP1 (DDBJ/EMBL/GenBank accession No. AF225983) was aligned using the clustal algorithm (Saitou and Nei, 1987) with similar motifs of its closest homologs from Arabidopsis thaliana (AtbZIP, accession No. AAB87576), Lycopersicon esculentum (tomato) (LebZIP, accession No. CAA52015), Paulownia kawakamii (PkbZIP, accession No. AAC04862), Oryza sativum (rice) (OsbZIP, accession No. AAC49832) and Nicotiana tabacum (tobacco) (NtbZIP, accession No. BAA97100). Regions of identity are indicated by unshaded boxes; gaps introduced for alignment are indicated by dashes. In the bZIP motif, the seven leucine repeats (leucine zipper) are indicated by shaded boxes and the basic domain is denoted by a horizontal bar above its sequence. The consensus bipartite NLS (Dingwall and Laskey, 1991) within the basic domain of the VIP1 bZIP motif is indicated by a black box.

Fig. 3. A functional genetic assay for VIP1-mediated VirE2 nuclear import in yeast cells. Yeast cells expressing the indicated proteins were grown under selective (histidine and tryptophan double-dropout medium) or non-selective conditions (tryptophan single-dropout medium) for nuclear import. For co-expression with VIP1, yeast cells expressing the indicated combinations of tested proteins were grown in a histidine, tryptophan and uracil triple-dropout medium supplemented either with galactose or glucose to induce or repress the VIP1 expression, respectively.

Fig. 4. VIP1-mediated nuclear import of VirE2 in mammalian cells. (A) COS-1 cells expressing GFP–VirE2. Dispersed fluorescence surrounding the signal-free, black cell nucleus represents the cytoplasmic localization of GFP–VirE2. (B) COS-1 cells expressing GFP–VIP1. The fluorescent signal is concentrated exclusively in the cell nucleus. (C) COS-1 cells co-expressing GFP–VirE2 and unlabeled VIP1. In most cells, part of the fluorescent signal enters the nucleus and part remains dispersed in the surrounding areas of the cytoplasm. Bar = 15 µm.

Fig. 5. Quantitative RT–PCR analysis of wild-type and VIP1 antisense plants. (A) Detection of sense and antisense VIP1 RNA. Lanes 1 and 2, RT–PCR of sense and antisense VIP1 RNA in wild-type plants; lanes 3 and 4, RT–PCR of sense and antisense VIP1 RNA in one line of Agrobacterium-resistant VIP1 antisense plants; lanes 5 and 6, RT–PCR of sense and antisense VIP1 RNA in another line of Agrobacterium-resistant VIP1 antisense plants; lanes 7 and 8, RT–PCR of sense and antisense VIP1 RNA in a line of Agrobacterium-sensitive VIP1 antisense plants. (B) Detection of sense actin RNA-specific product in the same samples shown in (A). (C) Quantification of sense and antisense VIP1 RNA. The amount of VIP1-specific RT–PCR products is expressed as a percentage of that obtained using sense VIP1-specific primers in wild-type plants. These data represent average values of three independent experiments with the indicated standard deviations.

Fig. 6. Reduced tumor formation in Agrobacterium-infected VIP1 antisense plants. (A) Leaf disks from the wild-type tobacco plants. (B) Leaf disks from the VIP1 antisense transgenic plants. (C) Leaf disks from the control transgenic plants. (D) Summary of the number and sizes of Agrobacterium-induced tumors developed on leaf disks from two independent lines of VIP1 antisense plants and two lines of transgenic control plants relative to the wild-type (wt) plants.

Fig. 7. Reduced transient expression of GUS activity contained within Agrobacterium T-DNA in VIP1 antisense plants. (A) Infected leaf disks from the wild-type tobacco plants. (B) Infected leaf disks from the VIP1 antisense transgenic plants. (C) Microbombarded leaf disk from the wild-type tobacco plants. (D) Microbombarded leaf disk from the VIP1 antisense transgenic plants. Note that microbombardment experiments (C and D) required larger leaf disks than that used in Agrobacterium inoculations (A and B). (E) Quantification of GUS activity. Black and white bars indicate transient GUS expression in Agrobacterium-infected and microbombarded tissues, respectively, derived from two independent lines of VIP1 antisense plants as compared with the wild-type control plants. GUS activity in control, wild-type plants was defined as 100%. All data represent average values of three independent experiments with the indicated standard deviations.

Fig. 8. Nuclear import of GUS–VirE2 and GUS–VirD2 in wild-type and VIP1 antisense plants. (A and B) GUS–VirE2 expressed in wild-type plants. (C and D) GUS–VirE2 expressed in VIP1 antisense plants. (E and F) GUS–VirD2 expressed in wild-type plants. (G and H) GUS–VirD2 expressed in VIP1 antisense plants. (I and J) Free GUS expressed in VIP1 antisense plants. (K and L) GUS–VIP1 expressed in wild-type plants. (A, C, E, G, I and K) GUS staining. (B, D, F, H, J and L) DAPI staining. Bar = 25 µm.

Fig. 9. In vitro formation of ternary complexes between VIP1, VirE2 and ssDNA. Gel shift assays were performed as described in Materials and methods. Lane 1, ssDNA incubated with VirE2; lane 2, ssDNA incubated alone; lane 3, ssDNA incubated with VIP1; lane 4, ssDNA incubated with VirE2 and VIP1; lane 5, ssDNA incubated with VirE2 and VIP1 and treated for 30 min at 37°C with 1 mg/ml of proteinase K.