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Disulfide-Linked Protein Complex (Plant Tumor/Virulence Genes/Virb Proteins/Virb7-Virb9 Complex/Disulfide Bond) LORRAINE B

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Disulfide-Linked Protein Complex (Plant Tumor/Virulence Genes/Virb Proteins/Virb7-Virb9 Complex/Disulfide Bond) LORRAINE B Proc. Natl. Acad. Sci. USA Vol. 93, pp. 8889-8894, August 1996 Biochemistry Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex (plant tumor/virulence genes/VirB proteins/VirB7-VirB9 complex/disulfide bond) LORRAINE B. ANDERSON*, ANN VOGEL HERTZEL*, AND ANATH DAS*t# *Department of Biochemistry and tPlant Molecular Genetics Institute, University of Minnesota, 1479 Gortner Avenue, St. Paul, MN 55108 Communicated by Ronald L. Phillips, University ofMinnesota, St. Paul, MN, May 28, 1996 (received for review March 11, 1996) ABSTRACT Agrobacterium tumefaciens VirB proteins are macromolecule export. The requirement of the virB genes in essential for gene transfer from bacteria to plants. These tumor formation, in DNA transfer to plants in a transient DNA proteins are postulated to form a transport pore to allow transfer assay, in conjugal transfer of an IncQ plasmid transfer of the T-strand DNA intermediate. To study the RSF1010 between twoAgrobacterium strains, and most recently function of the VirB proteins in DNA transfer, we developed in DNA transfer from Agrobacterium to yeast provides experi- an expression system in A. tumefaciens. Analysis of one VirB mental support for the transport pore model (13-17). In addition protein, VirB9, by Western blot assays showed that under to the VirB proteins, the vir-mediated DNA transfer processes nonreducing conditions VirB9, when expressed alone, mi- require VirD4, an essential virulence protein (13, 16, 18). grates as a -31-kDa band but that it migrates as a -36-kDa While the requirement for VirB proteins and VirD4 in DNA band when expressed with all other VirB proteins. The 36-kDa transfer is well-established, the role of each individual protein band is converted to the 31-kDa band by the reducing agent in this process is not clear. Two VirB proteins, VirB4 and 2-mercaptoethanol. Using strains that contain a deletion in a VirB11, contain a nucleotide-binding motif that is essential for defined virB gene and strains that express specific VirB tumor formation (19, 20). These proteins are postulated to proteins, we demonstrate that the 36-kDa band is composed participate in an energy-requiring step in DNA transfer. VirB4 ofVirB9 and VirB7 that are linked to each other by a disulfide is also essential for the localization of VirB3 into the outer bond. Mutational studies demonstrate that cysteine residues membrane (21). VirB7 stabilizes VirB9 and probably a few at positions 24 of VirB7 and 262 of VirB9 participate in the other VirB proteins, and VirB9, VirB10, and VirB1l are formation of this complex. required in a stoichiometric ratio since coordinated expression of all three proteins are essential for the relief of RSF1010- The transformation of a susceptible plant cell by the soil mediated inhibition of T-DNA transfer (3, 22). To understand bacteriumAgrobacterium tumefaciens results from the transfer how these proteins carry out their function and to define the and integration of a segment of the tumor-inducing (T,) constituents and the assembly of the putative transport pore, plasmid DNA into the plant nuclear genome. The virulence it is necessary to define the interactions among the various (vir) genes of the Ti plasmid play an essential role in the DNA VirB proteins. To simplify these studies, we developed a system transfer and integration processes. The portion of the T1 that allows overproduction of a protein or a set of proteins in plasmid that is transferred to plant cells (T-DNA) is defined by Agrobacterium. Using this system, we demonstrate that migra- a 24-bp direct repeat sequence known as the border sequence. tion of VirB9 in SDS/polyacrylamide gels under a nonreducing Two proteins of the virD operon, VirDl and VirD2, initiate the environment is affected by the presence ofother VirB proteins. processing of the T-DNA by introducing a site- and strand- We demonstrate that this aberrant migration results from the specific nick at the T-DNA borders, leading to the formation formation of a complex between VirB9 and VirB7, and a of a single-stranded T-strand DNA composed of the bottom disulfide linkage mediates the formation of this complex. The strand of the T-DNA. The T-strand DNA contains a VirD2 cysteine residues involved in this process were identified by molecule covalently attached to its 5'-end and is believed to be site-specific mutagenesis. an intermediate in DNA transfer to plants (for reviews, see refs. 1 and 2). Little is known about the mechanism of DNA transfer from METHODS bacteria to plants. It is postulated that DNA transfer occurs Construction of a VirB Deletion Plasmid. To introduce a through a transport pore primarily composed of the VirB deletion in the virB locus of the octopine Ti plasmid pTiA6, we proteins. Of the 11 VirB proteins, 10 (VirB2-VirB11) are constructed a plasmid pLA9 that contains an - 1.5 kb segment essential for DNA transfer. The other protein (VirBl) is of the 3' end of virA (a SacI-KpnI fragment) followed by the required for a high efficiency of DNA transfer (3). The kanamycin-resistance gene of the plasmid pUC4K (23) and a observations that the VirB proteins have no role in T-strand 1.3-kb BglII-PstI fragment encompassing the pTiA6 virG in the DNA synthesis and that most of the VirB proteins are mem- plasmid vector pTJS75 (24). Plasmid pLA9 was introduced into brane associated led to their proposed role in DNA transfer A. tumefaciens A348 by triparental mating (25) and colonies (4-10). Subsequently, DNA sequence analysis of the trb genes resistant to tetracycline, kanamycin, and carbenicillin were of the conjugative plasmid pRP4 and that of theptl operon of selected. Plasmid pPHlJI (which is resistant to gentamycin) the pathogenic bacteria Bordetella pertussis revealed that these was then introduced into A. tumefaciens A348/pLA9 and operons encode homologs of the VirB proteins (11, 12). The colonies resistant to gentamycin and kanamycin were selected. requirement of the trb genes in conjugal transfer of plasmid The resistant colonies were screened for sensitivity to carben- DNA, that of the ptl genes in the secretion of the Bordetella icillin. The carbenicillin-sensitive colonies are expected to toxin protein, and that of the virB genes in T-DNA transfer contain a substitution of the virB locus with the kanamycin- suggests the existence of a common transport pathway for Abbreviations: Ti, tumor-inducing; T-DNA, the portion of the T1 The publication costs of this article were defrayed in part by page charge plasmid that is transferred to plant cells; ,B-ME, 2-mercaptoethanol. payment. This article must therefore be hereby marked "advertisement" in TTo whom reprint requests should be addressed. e-mail: anath@ accordance with 18 U.S.C. §1734 solely to indicate this fact. biosci.cbs.umn.edu. 8889 Downloaded by guest on September 28, 2021 8890 Biochemistry: Anderson et al. Proc. Natl. Acad. Sci. USA 93 (1996) resistance gene resulting from homologous recombination at centrifugation at 6000 x g for 10 min, washed three times with the virA and the virG sequences. Several such colonies were 10 ml phosphate buffer (20 mM sodium phosphate, pH 7.6; 20 analyzed by Southern blot hybridization to confirm that these mM sodium chloride), and resuspended in the same buffer at cells had lost virB and one was designated A. tumefaciens a concentration of 4 A600 unit/ml. DTBP was added to the cell A348AB. suspension at a final concentration of 5 mM and the mixture Construction of A. tumefaciens A348AB2, A348AB3, and was incubated at room temperature for 30 min. The reaction A348AB7. The pTjA6 virB operon was cloned into the plasmid was terminated by the addition of 1 M Tris HCl (pH 7.6) to a vector pBlueScriptIl KS+ (Stratagene) as a 10.5-kb NdeI- final concentration of 20 mM. After an additional 15-min XhoI fragment to construct pAD1287. The 5' end of the virB incubation, the cells were collected by centrifugation, washed operon was subcloned by digesting pAD1287 with SstI followed with 1 ml of 50 mM Tris HCl (pH 7.6), and resuspended in 150 by ligation generating pAV128. The 3' end of the virB operon ,l of buffer (100 mM Tris HCl, pH 8.0/1 mM EDTA). The cell was subcloned as a 5.3-kb SstI fragment into pBlueScriptII gent suspension was aliquoted into two tubes, 0.5 vol 3 x Laemmli (a gentamycin resistant derivative of pBlueScriptIl) to con- protein denaturation buffer [with or without 2-mercaptoethanol struct pAV134R. A deletion of the ORF for virB2, virB3, or (13-ME); ref. 32] was added, and samples were denatured at 37°C virB7 in plasmids pAV128 or pAV134R was introduced by for 30 min. Proteins were analyzed by SDS/PAGE, followed by oligonucleotide-directed site-specific mutagenesis (26). The Western blot analysis using anti-VirB9 antibody (33). mutants, pAV128AB2, pAV128AB3, and pAV134RA&B7, were Anti-VirB9 antibody was raised in rabbits using a TrpE'- confirmed by DNA sequence analysis (27). The entire virB 'VirB9 fusion protein as an antigen. A trpE'-'virB9 fusion was operon was reconstructed by ligating the 3' SstI fragment of constructed by cloning a 2.8-kb BglII fragment (encodes the pAV134R into pAV128AB2 and pAV128AB3 creating C-terminal 219 residues of VirB9) into the BamHl site of pAD1287AB2 and pAD1287AB3, respectively. The SstI frag- plasmid pATH2 (34). The fusion protein was purified by ment from pAV134RAB7 was subcloned back into the SstI site SDS/PAGE and used as an antigen for the production of of pAV128 constructing pAD1287/B7. Plasmids polyclonal antibody in rabbits. pAD1287/B2, -AB3, and -AB7 were fused to the wide-host- range plasmid pAV140, a derivative of pTJS75 (24), at a RESULTS unique KpnI site.
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