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Activation of the T-DNA Transfer Process in Agrobacterium Results

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Activation of the T-DNA Transfer Process in Agrobacterium Results Proc. Nati. Acad. Sci. USA Vol. 86, pp. 4017-4021, June 1989 Biochemistry Activation of the T-DNA transfer process in Agrobacterium results in the generation of a T-strand-protein complex: Tight association of VirD2 with the 5' ends of T-strands (DNA-protein interaction/VirD endonuclease) ELIZABETH A. HOWARD*t, BARBARA A. WINSOR*t, GUIDO DE VOS*, AND PATRICIA ZAMBRYSKI* *Division of Molecular Plant Biology, Hilgard Hall, University of California, Berkeley, CA 94720; and tPlant Gene Expression Center, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710 Communicated by Marc Van Montague, March 2, 1989 (received for review December 29, 1988) ABSTRACT The T-DNA transfer process of Agrobacte- transfer intermediate, it must be protected from exo- and rium is activated following induction of expression of the Ti endonucleolytic degradation, targeted to and through the plasmid virulence (vir) genes. The virDI and virD2 gene prod- bacterial cell membranes and cell wall and the plant cell and ucts are required for the production of nicks at the T-DNA nuclear membranes, and integrated into the plant genome. It borders and for the generation of free linear single-stranded is unlikely that the T-strand itself can accomplish all these copies of the T-DNA region, T-strands. T-strands are com- steps; instead the T-strand is probably incorporated into a plexed with proteins in vir-induced bacteria, since T-strands DNA-protein complex soon after its synthesis in the bacterial partition to the aqueous/phenol interface in non-Pronase- cell. While some of the later steps of transfer and integration treated total cell extracts. To determine whether the proteins must be mediated by plant-encoded proteins not intrinsic to are tightly associated with T-strands, DNA-protein complexes the T-strand-protein complex, the bacterial T-strand com- were purified away from bulk proteins by adsorption to glass plex must be capable of finding and interacting with the beads. A 58-kDa protein was specifically released from vir- proteins responsible for the various steps of transfer. One induced DNA-protein complexes after treatment with S1 nu- protein that is likely part ofthe T-strand complex is the VirE2 clease to digest single-stranded DNA. The 58-kDa protein was protein, a ssDNA-binding protein (11-14). Presumably, identified as VirD2 by using VirD2-specific antibodies. The VirE2 can protect the T-strand from degradation in vivo, tight association ofVirD2 with T-strands was shown directly by since recent results show that VirE2 binds tightly and coop- using VirD2-specific antibody to isolate T-strands. The 5' side eratively to ssDNA in vitro and that such VirE2-coated DNA of the borders nick sites on the Ti plasmid was also shown to be is highly resistant to exo- and endonucleases (15). tightly associated with protein. The data suggest that after Whereas VirE2 coats ssDNA along its entire length, T- VirDl/VirD2-dependent nicking at the T-DNA borders, the strand generation and integration into the plant genome are VirD2 protein remains bound to the 5' end of the nick, and the polar processes. (i) The T-strand is homologous to the bottom VirD2 protein continues to bind tightly to this 5' end during strand of the T-DNA region (4); this polarity implies that unwinding (or displacement) of the T-strand from the Ti T-strands are generated in a 5' to 3' direction, initiating with plasmid T-DNA region. The tight binding ofVirD2 to T-strands a nick at the right border and terminating with a nick at the suggests that this protein has additional functions in T-strand left border. (ii) Analyses of cloned T-DNA plant junctions generation and potentially in the later steps ofT-DNA transfer. show that integration is more precise at the right border than at the left border: on the T-DNA side, junctions on the right Agrobacterium tumefaciens is a soil phytopathogen that end of the T-DNA are within or a few bases from the 25-bp elicits tumors on plants by the transfer of a specific region of border repeat, while junctions at the left end of the T-DNA DNA (T-DNA) from its tumor-inducing (Ti) plasmid to the are spread over a 100-bp region internal to and including the plant cell genome (1). The T-DNA region is delimited by two left 25-bp repeat (1), and when a plant integration target site 25-base-pair (bp) direct repeats at each end. The DNA was analyzed, the transition between T-DNA and plant DNA internal to the direct repeats is not involved in the transfer was precise at the right border whereas plant sequences process; i.e., any DNA can be substituted for the DNA adjacent to the left border were extensively rearranged (16). between the borders without affecting the efficiency oftrans- Therefore, the T-strand might be associated with a protein(s) fer. The genes responsible for the transfer process reside that confers the property of polarity on the transfer process outside the T-DNA, in the 35-kbp virulence (vir) region ofthe by binding the T-strand asymmetrically. Here we show that Ti plasmid (2). The vir genes are induced in response to small VirD2 is a candidate for this role, since it is found tightly phenolic compounds, such as acetosyringone (AS), excreted bound to the 5' end of the T-strand in vivo. by wounded but otherwise actively metabolizing plant cells (3). Upon activation of vir gene expression, a linear single- MATERIALS AND METHODS stranded DNA (ssDNA) molecule (T-strand) is generated from the T-DNA region (4-6). This process involves nicking Bacterial Strains, Plasmids, and General Procedures. A. between the third and fourth base pairs of the 25-bp direct tumefaciens containing the nopaline Ti plasmid pGV3850 (17) repeat of the left and right borders (4, 7, 8). The products of was used for all in vivo analyses of T-strand. The heterolo- the virDI and virD2 genes are required both for nicking and gous T-strand-synthesizing system used E. coli RR1, T- for T-strand synthesis (5, 9, 10). strand substrate plasmid pGS112, and plasmid pGS360 car- The T-strand is presumed to be the intermediate that is rying the virD operon (18). E. coli strain BL21DE3 (19) was transferred from Agrobacterium to the plant cell. As the Abbreviations: ssDNA, single-stranded DNA; AS, acetosyringone; IPTG, isopropyl /3-D-thiogalactoside. The publication costs of this article were defrayed in part by page charge tPresent address: Centre National de la Recherche Scientifique, payment. This article must therefore be hereby marked "advertisement" Laboratoire de Genetique Moleculaire des Eukaryotes, 11 Rue in accordance with 18 U.S.C. §1734 solely to indicate this fact. Humann, 67085 Strasbourg Cedex, France. Downloaded by guest on September 26, 2021 4017 4018 Biochemistry: Howard et al. Proc. Natl. Acad. Sci. USA 86 (1989) used to overexpress VirD2. Agrobacteria were grown either a b c d e in YEB (25) or M9 minimal medium at pH 5.5 (12). Conditions for AS induction were as described (4, 12). E. coli cells were grown as described (18). Enzymes were used according to suppliers' specifications. VirD2 Antibody Preparation and Affinity Purification. VirD2 coding sequences were cloned in a T7 expression plasmid. The VirDl deletion plasmid pGS380 (18) was used FIG. 1. Phenol partitioning of T-strand. Lane a: sample was as a source of VirD2 sequences; a Bgl II-BamHI fragment prepared following lysis at 370C for 15 min with sarkosyl (N- was cloned into the BamHI site of the T7 vector pET3c (19). lauroylsarcosine, 0.5%) and Pronase (1 mg/ml) in TE (10 mM Tris, The resulting plasmid, pGS377, synthesizes VirD2 as a fusion pH 8.0/1 mM EDTA) (4). Lane b: as in a, except that lysozyme (10 to the first 12 amino acids of the T7 gene 10 protein. mg/ml) was used instead of Pronase. Lane c: the aqueous/phenol Overexpressed VirD2 protein was prepared from plasmid interface was recovered from the sample in b, dialyzed overnight pGS377 exactly as described for isolation of overexpressed against TE, and treated with Pronase. Lanes d and e: samples were VirE2 protein (15). Virtually all of the overexpressed VirD2 prepared following lysis for 5 min at 370C in Laemmli sample buffer was found in an insoluble fraction after lysis. A 35,000 x g containing 2.3% SDS (23); in addition, the d sample contained Pronase (1 mg/ml). All samples were phenol-extracted, and the pellet of the insoluble fraction, containing 500 gg of (>90% aqueous phase was recovered and ethanol-precipitated prior to pure) VirD2, was resuspended in lysis buffer (15) and mixed electrophoresis and transfer to nitrocellulose under nondenaturing 1:1 with Freund's complete adjuvant (Sigma) prior to injec- conditions (4) to assay for T-strands. Only the region of the gel tion into rabbits for antibody production. Affinity purification corresponding to the T-strand is shown; the mobility ofthe T-strand of VirD2 antibody was essentially as described (20). produced from the pGV3850 Ti plasmid corresponds to 4.5 kilobases Immunoblotting and Immunoprecipitation. Immunoblot- (4). The probe used was homologous to HindIll fragment 10 cloned ting was performed as described by Ausubel et al. (21), with in pBR322 (Fig. 4). a 1:3000 dilution of antibody and bacterial alkaline phospha- tase conjugated to secondary antibody (Bio-Rad) at a 1:7500 Pronase, we performed experiments under conditions in dilution as secondary reagent. Protein A-Sepharose beads which VirE2 does not bind to ssDNA. VirE2 protein does not were prepared and reacted with antibody as described (21).
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