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. 2002 Feb;14(2):451-63.
doi: 10.1105/tpc.010360.

The protein encoded by oncogene 6b from Agrobacterium tumefaciens interacts with a nuclear protein of tobacco

Saeko Kitakura  1 , Tomomichi Fujita, Yoshihisa Ueno, Shinji Terakura, Hiroetsu Wabiko, Yasunori Machida
Affiliations

The protein encoded by oncogene 6b from Agrobacterium tumefaciens interacts with a nuclear protein of tobacco

Saeko Kitakura et al. Plant Cell. 2002 Feb.

Abstract

The 6b gene in the T-DNA from Agrobacterium has oncogenic activity in plant cells, inducing tumor formation, the phytohormone-independent division of cells, and alterations in leaf morphology. The product of the 6b gene appears to promote some aspects of the proliferation of plant cells, but the molecular mechanism of its action remains unknown. We report here that the 6b protein associates with a nuclear protein in tobacco that we have designated NtSIP1 (for Nicotiana tabacum 6b-interacting protein 1). NtSIP1 appears to be a transcription factor because its predicted amino acid sequence includes two regions that resemble a nuclear localization signal and a putative DNA binding motif, which is similar in terms of amino acid sequence to the triple helix motif of rice transcription factor GT-2. Expression in tobacco cells of a fusion protein composed of the DNA binding domain of the yeast GAL4 protein and the 6b protein activated the transcription of a reporter gene that was under the control of a chimeric promoter that included the GAL4 upstream activating sequence and the 35S minimal promoter of Cauliflower mosaic virus. Furthermore, nuclear localization of green fluorescent protein-fused 6b protein was enhanced by NtSIP1. A cluster of acidic residues in the 6b protein appeared to be essential for nuclear localization and for transactivation as well as for the hormone-independent growth of tobacco cells. Thus, it seems possible that the 6b protein might function in the proliferation of plant cells, at least in part, through an association with NtSIP1.

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Figures

Figure 1.
Figure 1.
The Acidic Region of the 6b Protein Is Essential for the Hormone-Independent Formation of Callus and the Interaction with NtSIP1. (A) Scheme of the structure of the 6b protein. The amino acid (a.a.) sequences around the acidic regions (positions 151 to 200) of 6b and 6b(B6), which were derived from pTiAKE10 and pTiB6S3trac, respectively, are shown below the domain organization. The region deleted in the 6bΔA mutant is indicated by a bracket, and the deletion point at residue 174 in the 6bΔC(B6) mutant is indicated by an arrow. Acidic amino acid residues are underlined. (B) The hormone-independent formation of shoot-bearing calli from tobacco leaf discs requires the acidic region of 6b. Tobacco leaf discs were infected with Agrobacterium cells that harbored the pBI121 vector plasmid with the His-T7-6b gene (a), the His-T7-6bΔA gene (b), or no additional gene (c) and cultured on phytohormone-free medium for 21 days. (d) shows a protein gel blot of the His-T7-6b and His-T7-6bΔA proteins synthesized in BY-2 cells. Protein extracts were prepared from BY-2 cells that had been transfected with pBI221 that included the His-T7-6b gene (lane 1) or the His-T7-6bΔA gene (lane 2) or with the empty vector (lane 3). Ten micrograms of total protein was subjected to SDS-PAGE, electroblotted, and analyzed by protein gel blotting with anti-T7 antibodies.
Figure 2.
Figure 2.
Analysis of the Role of the Acidic Region of 6b in the Interaction of 6b with NtSIP1 Using the Yeast Two-Hybrid System.
Figure 3.
Figure 3.
Alignment of the Deduced Amino Acid Sequences of NtSIP1 and Homologs from Arabidopsis and the Nuclear Localization of NtSIP1. (A) The deduced amino acid sequence of NtSIP1 is aligned with the sequences of F14P22.220, MOP10_9, F9F8.9, and F11B9.6 from Arabidopsis. The triple helix motif that is predicted to form amphipathic helices (helix) and intervening nonhelical regions (dotted lines above sequences) are indicated. Sequences that resemble NLS are indicated by asterisks. Residues are highlighted in white letters on black if NtSIP1 and other sequences have identical residues, and gray shading indicates similar residues at the same position. (B) Subcellular localization of sGFP-NtSIP1 in BY-2 cells. Plasmids that carried P35S-linked sGFP:cNtSIP1 (a), P35S-linked sGFP (b), and P35S-linked NLS:sGFP (c) were introduced into BY-2 cells, and transfected cells were cultured for 16 hr at 26°C. Fluorescence was monitored with a fluorescence microscope (top panels). Nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) are shown in the bottom panels. Bar = 10 μm.
Figure 4.
Figure 4.
Interaction between 6b and NtSIP1. Appropriate combinations of recombinant proteins that had been produced in E. coli were immunoprecipitated with anti-T7 antibodies and subjected to protein gel blot analysis with His tag–specific antibodies. Details are given in the text. Lane 1, His-6b protein as a marker; lane 2, His-6b alone; lane 3, His-T7-NtSIP1 alone; lane 4, His-6b and His-T7-NtSIP1; lane 5, His-6b and His-T7-NRK1 (NRK1 is a mitogen-activated protein kinase that was used as a negative control; our unpublished data). Positions of these recombinant proteins are indicated by arrows. Two bands are apparent in the lane that corresponds to His-T7-NtSIP1: the band with greater mobility might represent a degradation product that was generated during immunoprecipitation.
Figure 5.
Figure 5.
Cotransfection of BY-2 Cells with P35S-Linked sGFP:6b and P35S-Linked cNtSIP1. (A) Nuclear localization of sGFP-6b in mesophyll protoplasts of tobacco. P35S-linked sGFP:6b (a), P35S-linked sGFP (b), and P35S-linked NLS:sGFP (c) were introduced into tobacco mesophyll protoplasts with polyethylene glycol. Fluorescence from sGFP (top panels) and from DAPI (bottom panels) was monitored with a fluorescence microscope. (d) shows an RNA gel blot showing the levels of transcripts of the NtSIP1 gene in leaves and BY-2 cells. (B) Stimulation of the nuclear localization of the sGFP-6b protein by coexpression of NtSIP1. Gold particles that carried the indicated combinations of constructs were introduced into BY-2 cells by particle bombardment, and cells were cultured for 16 hr at 26°C. Fluorescence from sGFP (left panels) and DAPI (right panels) was monitored with a fluorescence microscope. Bar = 10 μm. (C) Quantitative analysis of the nuclear localization of sGFP-6b. Results indicated by bars a through h correspond to the same panels in (B). We chose nine sGFP-positive cells at random in each experiment. Fluorescence per unit area of nuclei and per unit area of cytoplasmic regions was measured with the software program IPLab (Scanalytics, Fairfax, VA), and average values with standard deviations were calculated for each experiment. Relative values were calculated by dividing the nuclear values by the cytoplasmic values.
Figure 6.
Figure 6.
Transactivation Activity of the GALDBD-Fused 6b Protein. Control GALDBD, effector GALDBD:6bΔA, and GALDBD:6b genes were introduced individually with the GALUAS35S:LUC reporter gene and a reference reporter gene (luciferase gene from Renilla) into protoplasts of BY-2 cells by polyethylene glycol–mediated transfection. After culture for 24 hr, luciferase activities were assayed as described in the text. The activity of firefly luciferase was normalized in each case by reference to the activity of the reference luciferase. The average results of five independent determinations with standard deviations were calculated, and values given are relative to the activity obtained without an effector gene.
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