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

2001; Springer Nature; Volume: 20; Issue: 13 Linguagem: Inglês

10.1093/emboj/20.13.3596

1460-2075

Tzvi Tzfira,

Plant-Microbe Interactions and Immunity

Article2 July 2001free access VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity Tzvi Tzfira Tzvi Tzfira Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY, 11794-5215 USA Search for more papers by this author Manjusha Vaidya Manjusha Vaidya Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY, 11794-5215 USA Search for more papers by this author Vitaly Citovsky Corresponding Author Vitaly Citovsky Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY, 11794-5215 USA Search for more papers by this author Tzvi Tzfira Tzvi Tzfira Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY, 11794-5215 USA Search for more papers by this author Manjusha Vaidya Manjusha Vaidya Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY, 11794-5215 USA Search for more papers by this author Vitaly Citovsky Corresponding Author Vitaly Citovsky Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY, 11794-5215 USA Search for more papers by this author Author Information Tzvi Tzfira1, Manjusha Vaidya1 and Vitaly Citovsky 1 1Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY, 11794-5215 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:3596-3607https://doi.org/10.1093/emboj/20.13.3596 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction Agrobacterium infection, the only known case of interkingdom DNA transfer (Stachel and Zambryski, 1989), elicits neoplastic growths on many plant species. This genetic transformation is achieved by transporting a single-stranded copy (T-strand) of the bacterial transferred DNA (T-DNA) from the tumor-inducing (Ti) plasmid into the plant cell nucleus followed by its integration into the host genome (reviewed by Gelvin, 2000; Tzfira et al., 2000; Zupan et al., 2000). The wild-type T-DNA carries genes involved in the synthesis of plant growth regulators and tumor-specific compounds, opines. Production of growth regulators in the transformed cell induces the formation of tumors, which then synthesize opines, a major carbon and nitrogen source for Agrobacterium. Thus, Agrobacteria are usually classified based on the type of opines specified by their T-DNA, the most common strains being nopaline- or octopine-specific. In addition to the T-DNA contents, nopaline and octopine Agrobacteria differ from each other in the composition and nucleotide sequence of the virulence (vir) region of their Ti-plasmids, which encodes the protein machinery of the T-DNA transfer (reviewed in Hooykaas and Beijersbergen, 1994). While only the wild-type T-DNA contains tumor-inducing genes, any DNA placed between the T-DNA borders will be transported into the plant cell nucleus (reviewed by Zambryski, 1992). This lack of sequence specificity implies that a T-DNA molecule itself does not encode specific signals for nuclear import and integration. Instead, these functions are probably performed by two Agrobacterium virulence proteins, VirD2 and VirE2, which are thought to associate directly with the T-strand, forming a transport (T) complex (Zupan and Zambryski, 1997). In the T-complex, one molecule of VirD2 is covalently attached to the 5′ end of the T-strand, while VirE2, a single-stranded (ss) DNA-binding protein (SSB), is presumed to coat the rest of the ssDNA molecule cooperatively (Gietl et al., 1987; Christie et al., 1988; Citovsky et al., 1988; Das, 1988; Sen et al., 1989) and package it into a rigid coiled structure (Citovsky et al., 1997). The need for active nuclear uptake is evident from the calculated diameter of VirE2–ssDNA complexes (12.6 nm; Citovsky et al., 1997), which exceeds the diffusion limit of the nuclear pore (9 nm; reviewed by Rout and Wente, 1994). Presumably, the T-complex nuclear import is mediated by VirD2 and VirE2 proteins, which localize to the plant cell nucleus (Herrera-Estrella et al., 1990; Citovsky et al., 1992, 1994; Howard et al., 1992; Shurvinton et al., 1992; Koukolikova-Nicola et al., 1993; Rossi et al., 1993; Zupan et al., 1996). Whereas VirD2 and VirE2 accumulate in the cell nucleus even in plant species that are recalcitrant to Agrobacterium-induced tumor formation (Citovsky et al., 1994), they probably employ different pathways for nuclear import. VirD2 is imported by a mechanism conserved between animal, yeast and plant cells (Herrera-Estrella et al., 1990; Howard et al., 1992; Koukolikova-Nicola et al., 1993; Rossi et al., 1993; Citovsky et al., 1994; Guralnick et al., 1996; Ziemienowicz et al., 1999; Rhee et al., 2000), while the nuclear import of VirE2 is plant specific in living cells (Citovsky et al., 1992, 1994; Guralnick et al., 1996; Rhee et al., 2000). Consistent with this idea, VirE2 is not recognized by the Arabidopsis karyopherin α protein, AtKAPα, which has been shown to mediate nuclear import of VirD2 (Ballas and Citovsky, 1997). Interestingly, VirE2 nuclear localizing ability is sufficient for transport of ssDNA into the plant cell nucleus even in the absence of VirD2 (Zupan et al., 1996) or for genetic transformation of plant cells by an Agrobacterium mutant strain lacking the VirD2 nuclear localization signal (NLS) (Gelvin, 1998). To understand better the molecular mechanism by which VirE2 functions during the Agrobacterium–plant cell T-DNA transfer, it would be useful to identify and characterize plant cellular components that specifically associate with VirE2. Here, we used the yeast two-hybrid protein–protein interaction system (Fields and Song, 1989; Hollenberg et al., 1995) to identify and isolate a VirE2-interacting protein, designated VIP1, from Arabidopsis thaliana. VIP1 allowed VirE2 to be imported into the nuclei of living yeast and mammalian cells and was required for VirE2 nuclear import and Agrobacterium-induced tumor formation in tobacco plants, participating in early stages of T-DNA expression. Results Identification of VIP1 We used the yeast two-hybrid screen (Fields and Song, 1989; Hollenberg et al., 1995) with an Arabidopsis cDNA library and the Agrobacterium VirE2 protein as bait. Screening of ∼3 × 106 transformants resulted in identification and isolation of several independent cDNA clones producing VirE2 interactors. Two of these clones encoded the same cDNA, designated VIP1 (VirE2-interacting protein 1). The largest clone, representing the full-length cDNA of VIP1, was characterized in detail. The interaction of VIP1 with VirE2 was specific because it did not occur with DNA topoisomerase I and lamin C, known as non-specific activators in the two-hybrid system best suited to eliminate false-positive interactions (Bartel et al., 1993; Park and Sternglanz, 1998). Figure 1A shows that co-expression of VIP1 and VirE2, but not of topoisomerase I or lamin C, activated the HIS3 reporter gene. Furthermore, VIP1 did not interact with VirD2 (data not shown), which is thought to function differently from VirE2 during the T-DNA nuclear import (Guralnick et al., 1996). In control experiments, under the non-selective conditions, all combinations of the tested proteins resulted in efficient cell growth (Figure 1B). Figure 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. Download figure Download PowerPoint In an independent approach, VIP1–VirE2 binding was examined directly using a renatured blot overlay assay for protein–protein interactions (Dorokhov et al., 1999; Chen et al., 2000). In this approach, VIP1 and a negative control protein, VirD2, are electrophoresed (Figure 1C, lanes 1 and 2), immobilized on a PVDF membrane by electroblotting, reacted with purified VirE2, and VirE2 binding is detected using anti-VirE2 antibodies. Figure 1C shows that VirE2 specifically interacted with immobilized VIP1 (lane 3) but not with VirD2 (lane 4). Furthermore, when the blot was probed with unspecific ligands, i.e. purified VirD2 (lanes 5 and 6) or cell–cell movement protein (MP) of tobacco mosaic virus (TMV) (lanes 7 and 8), no binding to Vip1 was observed. These results strengthened the notion that VirE2 specifically recognizes and binds VIP1. Sequence analysis of the VIP1 cDNA showed that it contained a single open reading frame (ORF) encoding a protein of 261 amino acid residues (see DDBJ/EMBL/GenBank accession Nos AF225983 for VIP1 cDNA and AC009526 for the genomic sequence containing the VIP1 gene). The deduced amino acid sequence of VIP1 contained a conserved stretch of basic amino acids (basic domain) abutting a heptad leucine repeat (leucine zipper), two structural features characteristic of the basic-zipper (bZIP) proteins (Figure 2). Because plant bZIP proteins are known to localize to the cell nucleus (van der Krol and Chua, 1991), we hypothesized that binding of VIP1 to VirE2 may function to facilitate nuclear import of VirE2. Indeed, the basic domain of the VIP1 bZIP motif contained a consensus sequence for the bipartite NLS (Dingwall and Laskey, 1991) (Figure 2). Interestingly,VIP1 exhibited a modest homology to bZIP proteins from various plantspecies, such as Arabidopsis, tomato, Paulownia kawakamii, rice and tobacco (Figure 2), whereas no animal or yeast bZIP homologs of VIP1 were found. This finding supports the notion that VIP1 may, at least partly, be responsible for the plant-specific nuclear import of VirE2. To test this hypothesis, we examined whether expression of VIP1 reconstructs nuclear import of VirE2 in non-plant systems. Figure 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. Download figure Download PowerPoint VIP1 facilitates transport of VirE2 into the nuclei of yeast and mammalian cells The ability of VIP1 to transport VirE2 into the yeast cell nucleus was examined using a recently developed genetic assay for functional nuclear import (Rhee et al., 2000). In this approach, a gene encoding the bacterial LexA protein was modified (mLexA), abolishing its intrinsic nuclear targeting activity, and fused to a sequence coding for the activation domain of the yeast Gal4p (Gal4AD). If a protein of interest fused to mLexA-Gal4AD (nuclear import assay hybrid, NIA) enters the yeast cell nucleus, it activates the expression of the reporter HIS3 gene, resulting in cell growth on a histidine-deficient medium (Rhee et al., 2000). First, VirE2 and VIP1 were tested for their own nuclear import capacity. Figure 3 shows that NIA-VirE2 expressed alone did not promote cell growth, indicating the lack of nuclear uptake. As expected, NIA-VirD2, which is known to function in non-plant systems (Guralnick et al., 1996; Ziemienowicz et al., 1999), induced cell growth following its nuclear import. NIA-VirD2 nuclear import was due to the presence of the VirD2 ORF because when the latter was fused to NIA in reverse orientation, producing the NIA-Vir2D hybrid, no cell growth was observed (Figure 3). Similarly to NIA-VirD2, expression of the NIA-VIP1 fusion promoted cell growth, indicating that VIP1 is imported into the cell nucleus in yeast. In control experiments, cells harboring all NIA fusions grew in the presence of histidine, indicating that the protein hybrids did not adversely and non-specifically affect cell physiology. Figure 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. Download figure Download PowerPoint Next, VIP1 was examined for its ability to assist nuclear import of VirE2. To this end, NIA-VirE2 was co-expressed with VIP1 driven by a galactose-inducible promoter. In the presence of galactose, cell growth was observed, indicating that NIA-VirE2 became capable of entering the cell nucleus and inducing expression of the HIS3 reporter (Figure 3). VirE2 nuclear import absolutely depended on the presence of VIP1 because, in the absence of galactose and, thus, VIP1 expression, no cell growth was observed (Figure 3). Facilitation of NIA-VirE2 nuclear import by VIP1 was due to VIP1–VirE2 interaction because co-expression of VIP1 and NIA-Vir2D did not result in nuclear import (Figure 3). These observations indicate that VIP1 facilitates nuclear import of VirE2 in yeast cells. Note that, in these experiments,VirE2 was fused to the mLexA-Gal4AD reporter while the VirE2–VIP1 binding experiments in the two-hybrid system utilized a relatively similar LexA-VirE2 fusion, providing compatibility between the protein–protein interaction and nuclear import data with respect to the VirE2 fusions used. To investigate the effect of VIP1 on VirE2 nuclear import in a mammalian system, these proteins were introduced into COS-1 cells and their intracellular localization determined using confocal microscopy. First, VirE2 and VIP1 were fused to the green fluorescent protein (GFP) and expressed separately. Figure 4 shows that GFP–VirE2 expressed alone remained completely cytoplasmic (dispersed fluorescent signal surrounding fluorescence-free, black nuclei in Figure 4A), whereas GFP–VIP1 efficiently localized to the cell nucleus (fluorescent signal exclusively concentrated within cell nuclei in Figure 4B). Then, GFP–VirE2 was co-expressed with unlabeled VIP1, resulting in entry of the fluorescent signal into the cell nucleus (Figure 4C). VIP1-facilitated nuclear import of GFP–VirE2 was incomplete because the fluorescent signal was found both accumulated within the cell nucleus and dispersed in the cytoplasm areas surrounding the nucleus (Figure 4C) (see also below). It is important to note that the confocal optical sections with the plane of focus through the cell nucleus detect intranuclear accumulation of the GFP label rather than its perinuclear binding. Thus, taken together, our functional genetic and microscopic data suggest that VIP1 plays an important role in the plant-specific nuclear import of VirE2. Figure 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. Download figure Download PowerPoint Quantification of GFP–VirE2 amounts on a per cell basis revealed that VIP1 redirected 40–60% of total expressed GFP–VirE2 to the cell nucleus. Because the VIP1 effect on VirE2 nuclear import is likely to be stoichiometric, it depends on the relative amounts of these proteins within the cell cytoplasm. Potentially, transient expression of GFP–VirE2 and VIP1 from separate plasmids does not generate or sustain protein concentrations necessary for the complete nuclear import of VirE2. Alternatively, in plants, VIP1 may be augmented by other cellular factors absent from the heterologous mammalian system. VIP1 antisense plants are resistant to Agrobacterium-induced tumor formation To study the biological role of VIP1 in Agrobacterium infection in planta, we generated transgenic tobacco plants expressing the VIP1 cDNA in the antisense orientation. A total of 10 independently transformed lines were produced and analyzed as described below. Five lines were not altered in their susceptibility to Agrobacterium infection, whereas the other five lines became largely resistant (data not shown). Here, we describe a detailed analysis of two of these resistant antisense lines, which were first examined for the presence of sense and antisense VIP1 RNA using quantitative RT–PCR and strand-specific oligonucleotide primers (Ni et al., 1998). Figure 5A shows that control, wild-type tobacco plants produced sense (lane 1), but not antisense (lane 2), VIP1 RNA, demonstrating the presence of VIP1 in tobacco (see also Figure 2). Two independent lines of VIP1 antisense plants, which exhibited resistance to Agrobacterium (see below), produced the antisense VIP1 RNA (Figure 5A, lanes 4 and 6) and significantly reduced amounts of the sense VIP1 RNA (Figure 5A, lanes 3 and 5). Conversely, VIP1 antisense lines that did not develop Agrobacterium, resistance retained high levels of the sense VIP1 transcript (Figure 5A, lane 7) even though the antisense transcript was also expressed (Figure 5A, lane 8). Figure 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. Download figure Download PowerPoint Quantification of these PCR products (Figure 5C) revealed that the sense VIP1 RNA, synthesized in the Agrobacterium-resistant antisense plants (bars 3 and 5), amounted to only 20% of that produced in the wild-type plants (bar 1), whereas the antisense RNA, undetectable in wild-type plants (bar 2), reached 25% of sense VIP1 RNA of the wild-type plants (bars 4 and 6). In Agrobacterium-sensitive antisense plants, the levels of the sense VIP1 RNA remained high (98% of the wild-type levels, bar 7) even in the presence of the antisense transcript (20% of sense VIP1 RNA of the wild-type plants, bar 8). In control experiments, analysis of actin-specific transcripts generated similar amounts of PCR products in all samples, indicating equal efficiencies of the RT–PCRs (Figure 5B). Thus, antisense expression of VIP1 cDNA in Agro bacterium-resistant transgenic tobacco substantially reduced transcription of the endogenous VIP1 gene and, by implication, synthesis of the VIP1 protein (data not shown). Furthermore, that Agrobacterium-sensitive antisense lines retained high levels of the sense VIP1 transcript supported the correlation between the RT–PCR data and the antisense phenotype. Next, we tested the ability of the VIP1 antisense plants to develop tumors following inoculation with wild-type, oncogenic Agrobacterium. Figure 6A shows that Agro bacterium elicited numerous and large tumors on leaf disks derived from the wild-type tobacco plants. Control transgenic plants transformed with an empty vector were equally susceptible to Agrobacterium-induced neoplastic growth (Figure 6C), indicating that the procedure used for generation of the transgenic plants did not render them resistant to the subsequent Agrobacterium infection. In contrast, two independent transgenic lines of VIP1 antisense plants exhibited a dramatic decrease in their susceptibility to Agrobacterium infection. Figure 6B shows that only very few and tiny tumors developed on leaf disks from one of these plants following inoculation with Agrobacterium. Figure 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. Download figure Download PowerPoint Agrobacterium infectivity was then quantified by the number and weight of tumors induced on the inoculated leaf disks. Figure 6D shows the results of these measurements averaged for both antisense lines as compared with the wild-type and transgenic control plants. Wild-type tobacco plants and transgenic control lines supported formation of multiple tumors (4–8 per leaf disk), which expanded and fused into large neoplastic growths (200–300 mg). The VIP1 antisense plants, on the other hand, developed only a very low number (1.0–1.5 per leaf disk) of small tumors (20–40 mg). Thus, the tumor-inducing activity of Agrobacterium in VIP1 antisense plants was reduced to ∼10% of that observed with the wild-type and transgenic control plants (Figure 6D). Progeny analysis demonstrated that this tumor-resistant phenotype of the VIP1 antisense plants co-segregated with the T-DNA inserts (data not shown). All VIP1 antisense plants were indistinguishable from the wild-type plants in their morphology and seed viability (data not shown). Furthermore, shoot regeneration from uninfected VIP1 antisense leaf disks cultured on tobacco regeneration medium (Horsch et al., 1985) was identical in its rate and efficiency to that of the wild-type plants (data not shown). Thus, VIP1 antisense expression most probably did not interfere with essential plant cellular functions. Early stages of T-DNA gene expression are blocked in VIP1 antisense plants Expression of genes contained on the Agrobacterium T-DNA takes place in two stages, early and late. Early gene expression, which reaches its maximum 2–4 days after infection (Janssen and Gardner, 1990; Nam et al., 1999), is transient, occurring from the T-DNA molecules that have not yet integrated into the plant genome. In contrast, late gene expression, which occurs 10–14 days after infection (Janssen and Gardner, 1990), is stable, resulting from the integrated T-DNA. If VIP1 indeed participates in nuclear import of the T-DNA, VIP1 antisense plants are expected to display reduced levels of T-DNA gene expression already early in the infection process, i.e. before the T-DNA integration can take place. To test this idea, we determined the efficiency of transient T-DNA gene expression by inoculating leaf disks derived from the wild-type and VIP1 antisense plants with Agrobacterium carrying on its T-DNA a uidA gene encoding a reporter enzyme β-glucuronidase (GUS). Figure 7A shows multiple areas of GUS histochemical staining on leaf disks derived from the wild-type plants, indicating transient T-DNA expression within the Agrobacterium-infected cells; identical results were obtained using control transgenic plants (data not shown). VIP1 antisense plants, on the other hand, failed transiently to express GUS contained on the T-DNA (Figure 7B). Both wild-type and VIP1 antisense plants supported GUS expression resulting from biolistic delivery of the uidA gene (Figure 7C and D). Figure 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. Download figure Download PowerPoint Next, early expression of GUS in two independent VIP1 antisense transgenic lines was quantified using a sensitive fluorimetric assay. Because the uidA gene contained on the T-DNA lacked regulatory sequences required for its expression in bacterial cells (Janssen and Gardner, 1990), our measurements represented the GUS activity directed by the T-DNA after its transfer to the plant rather than its potentially leaky expression in Agrobacterium. Figure 7E shows that VIP1 antisense plants inoculated with Agrobacterium displayed ∼20% transient GUS activity compared with the wild-type plants. No quantitative differences in GUS activity were detected when the uidA gene was introduced biolistically into the wild-type and VIP1 antisense plants (Figure 7E). Thus, VIP1 antisense plants specifically blocked early stages of the Agrobacterium-mediated uidA gene transfer but remained competent for efficiently expressing this gene when it was delivered by an Agrobacterium-independent technique. Nuclear import of VirE2 is impaired in VIP1 antisense transgenic plants To examine directly whether the reduced susceptibility of VIP1 antisense plants to Agrobacterium-mediated gene transfer was due to a decrease in VirE2 nuclear import, we compared the accumulation of VirE2 within the cell nuclei of the wild-type and VIP1 antisense tobacco tissues. Figure 8 shows that GUS–VirE2 expressed in the mesophyll of wild-type tobacco leaves, following biolistic delivery of its encoding gene, accumulated in the cell nucleus (Figure 8A), co-localizing with the nucleus-specific stain, 4′,6-diamidino-2-phenylindole (DAPI) (Figure 8B). However, GUS–VirE2 expressed in the leaf mesophyll cells of VIP1 antisense plants remained largely cytoplasmic (Figure 8C and D), supporting the notion of VIP1 involvement in VirE2 nuclear import. Importantly, nuclear import of GUS–VirD2 expressed in wild-type plants (Figure 8E and F) was identical to that in VIP1 antisense tissues (Figure 8G and H), indicating that VirE2 and VirD2 are imported into the host cell nucleus by different mechanisms and that antisense expression of VIP1 does not interfere non-specifically with the nuclear import reactions of the cell. As expected, free GUS expressed in wild-type (data not shown but see Table I, and Citovsky et al., 1992, 1994) and VIP1 antisense plants (Figure 8I and J) remained cytoplasmic. Similarly to our observations in yeast and COS cells (see Figures 3 and 4B, respectively),