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 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, , 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 , 11 Rue in accordance with 18 U.S.C. §1734 solely to indicate this fact. Humann, 67085 Strasbourg Cedex, France.

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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). 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). bind ssDNA in the presence of 2% SDS, as shown by filter Either affinity-purified VirD2 antibody (from 2 ml of crude binding experiments with VirE2 and phage ssDNA in vitro serum) or 20 ,l of crude VirD2 or crude VirG antibody were (ref. 15 and data not shown). Phenol partitioning of T-strand reacted with 0.1 ml of protein A-Sepharose slurry. These from Pronase- and non-Pronase-treated extracts lysed and amounts of antibody reflect similar amounts of IgG. The phenol-extracted in the presence of 2% SDS was analyzed amounts of IgG were estimated by electrophoresis of serum (Fig. 1, lanes d and e). Under these conditions, T-strand from in SDS/polyacrylamide gels and staining with Coomassie non-Pronase-treated extracts continued to partition to the blue. DNA-protein complexes from extracts (lysed as in interface (Fig. 1, lane e). These data indicate that the T-strand sample a, Fig. 1) of AS-induced cells were purified on glass is tightly complexed in vivo to a protein other than VirE2. beads. Eight individual samples, each derived from 37 ml of T-Strand Is Tightly Associated with a 58-kDa Protein. To cells, were pretreated by incubation at 70°C for 10 min to identify the protein(s) tightly bound to T-strand, AS-induced inactivate . Each sample was added to 6.6 ,ul of Agrobacterium proteins were labeled in vivo and T-strand antibody-bound protein A-Sepharose in TSA buffer (21) con- with its associated protein was prepared. For these experi- taining 10% (vol/vol) glycerol and incubated at 4°C with ments, we enriched for proteins bound to DNA by isolating mixing for 2.5 hr. The immunoprecipitates were pooled and DNA on glass beads in the absence of Pronase; this proce- washed in TSA buffer. The bound antigen was eluted from the dure purifies DNA-associated proteins 100- to 1000-fold beads by boiling in SDS sample buffer. The beads were relative to bulk proteins. Under the high ionic conditions pelleted at 400 x g for 3 min, and the supernatant was analyzed used, VirE2 does not bind to ssDNA and is effectively for T-strand content by agarose gel electrophoresis and non- removed from the sample. The resulting samples were treated denatured transfer to nitrocellulose (4). with or without S1 and analyzed for their protein composition by SDS/polyacrylamide gel electrophoresis. A protein of =58 kDa was detected in the S1-treated AS- RESULTS induced sample (Fig. 2, lanes c and d) but not in the T-Strand Is Tightly Associated with Protein in Vivo. T- non-Si-treated induced sample or in either of the noninduced strand analysis to date has primarily examined DNA pre- samples (lanes d-f). This was the only protein that exhibited pared from extracts treated with Pronase. To analyze poten- this behavior. The 58-kDa protein was AS-induced and tial T-strand-protein complexes, vir-induced extracts were tightly associated with ssDNA; i.e., the protein was not prepared in the absence as well as the presence of Pronase. removed by SDS or boiling. A 58-kDa protein was also DNA molecules tightly associated with protein have previ- detected in total extracts from AS-induced cells (Fig. 2, lane ously been shown to partition to the interface during phenol a); presumably this protein was not DNA-associated. extraction, whereas free DNA molecules partition to the Identification of the Tightly Associated Protein as VirD2. aqueous phase (22). We therefore assayed the DNA recov- The border-specific endonuclease required for T-strand gen- ered from the aqueous phase of extracts prepared in the eration is composed of two polypeptides, VirD1 and VirD2; presence or absence of Pronase for T-strand content (Fig. 1, the VirD2 component migrates as a 58-kDa protein in SDS lanes a and b). In the absence of Pronase, the aqueous phase gels (9, 18, 24). To test whether the protein tightly associated contained negligible levels of T-strand, whereas expected with ssDNA in AS-induced Agrobacterium is VirD2, we used levels of T-strand were recovered from the aqueous phase of an approach similar to that described above but in a heter- Pronase-treated extracts. T-strand can be recovered from the ologous system with an introduced, inducible virD2 gene. For nonproteolyzed sample if the aqueous/phenol interface is these experiments E. coli that had been cotransformed with treated with Pronase (Fig. 1, lane c). a plasmid carrying a T-DNA substrate and another plasmid The AS-induced VirE2 protein binds tightly to ssDNA and carrying the virD operon was used as a T-strand-synthesizing is therefore likely to be bound to T-strand in vivo. To test system (18). One plasmid contained the entire nopaline- whether VirE2 protein is responsible for partitioning of the specific virD operon fused to the E. coli tac inducible T-strand to the aqueous/phenol interface in the absence of promoter 25 bp upstream of the virDi gene. A second, Downloaded by guest on September 26, 2021 Biochemistry: Howard et al. Proc. Nadl. Acad. Sci. USA 86 (1989) 4019

a b c d e f Aa b c d e f g h i j - 97

-68 A. . -43

-43

Ba b c d e f g h i j

-25

FIG. 3. VirD2 is tightly associated with ssDNA after induction of T-strand synthesis in E. coli. Total protein was labeled with L 18 [35S]methionine. DNA plus tightly associated protein was purified by using glass beads as described in Fig. 2. (A) Total protein composi- FIG. 2. SI nuclease reveals protein tightly associated with ss- tion of DNA-protein complexes. Lanes a and b, c and d, e and f, and DNA in vir-induced cells. The DNA-protein complexes were puri- g and h: Si-treated and untreated extracts after 1, 4, 14, or 23 hr of fied by using glass beads (Gene Clean, Bio 101) as follows. First, IPTG, respectively. Lanes i andj: Si-treated and untreated extracts Agrobacterium cells grown in the presence or absence of AS were from uninduced cultures. Samples were analyzed by SDS gel elec- labeled by the addition of [35S]methionine (3 ,Ci/ml, 1100 Ci/mmol; trophoresis and autoradiography. Arrowheads, the 43-kDa protein 1 Ci = 37 GBq) for 30 min. Pellets from 1 ml ofcells were resuspended present only in the Si-treated samples from IPTG-induced cells. (B) in 100 ,ul of 1x SDS sample buffer (23) and stored at -70°C; this Immunoblot analysis of proteins in DNA-protein complexes. Lanes sample represented the protein present in the total extract. To purify a and b: total extracts from uninduced and from AS-induced Agro- DNA-protein complexes, pellets from 10 ml of cells were resus- bacterium. Lanes c-j: aliquots from samples prepared as in A; lanes pended in 300 pl of TE and lysed by the addition of 100 ,ul of 5% c and d, untreated and Si-treated from uninduced cells; lanes e and sarkosyl in TE and 400 ,ul of 8 M urea in TE for 5 min at 37°C. The f, untreated and Si-treated from cells induced with IPTG for 23 hr; lysates were incubated with 2 volumes of 4 M Nal and 10 p1 of glass lanes g and h, untreated and Si-treated from cells induced with IPTG beads for 5 min at room temperature. The glass-bead pellets were for 4 hr; lanes i and j, untreated and SI-treated from cells induced washed three times with ethanol wash solution (Gene Clean) and with IPTG for 0.5 hr. After electrophoresis the samples were DNA-protein complexes were eluted with double-distilled H20 by transferred to nitocellulose and probed with a 1:3000 dilution of two 3-min incubations at 55°C. To analyze the protein composition affinity-purified VirD2 antibody. Arrowhead, the 43-kDa protein. of the glass-bead-purified samples, eluants were made 1x in Si buffer (280 mM NaCl/50 mM NaOAc, pH 4.6/4.5 mM ZnSO4) and appearance of this protein correlated with the induction of half of each sample was incubated with 50 units of Si for 30 min at room temperature in a total volume of 200 ,ul. Si digestion was VirD2 in whole cell extracts (data not shown). No 43-kDa terminated by precipitation with trichloroacetic acid. Samples were protein was detected either in the non-Sl-treated samples resuspended in 1x SDS sample buffer, adjusted to pH 7, and (lanes b, d, f, and h) or in the uninduced samples, with or analyzed by SDS/polyacrylamide gel electrophoresis (23). Lanes a without S1 treatment (lanes i andj). There was no detectable and b: total extracts from AS-induced cells and from uninduced cells, protein of 58 kDa that exhibited similar behavior. However, respectively. Lanes c-f, protein present in DNA samples recovered other studies have suggested that the 58-kDa VirD2 protein on glass beads from cells lysed in the absence of Pronase; lanes c and has a breakdown or processed form that migrates as a 43-kDa d, Si-treated and untreated glass-purified extract from AS-induced protein (18, 24). Here we confirm that the 43-kDa protein is cells; lanes e and f, Si-treated and untreated glass-purified extract from uninduced cells. Large arrowheads, the 58-kDa protein present a form of VirD2, since affinity-purified nopaline VirD2 anti- in total extract from AS-induced cells and in the Si-treated sample body reacted with a 43-kDa protein in total extracts of from glass-purified extracts of AS-induced cells. Small arrowhead, a AS-induced Agrobacterium (Fig. 3B, lane a). In this exper- protein of unknown identity that is enriched in the non-Si-treated iment, no 58-kDa protein was detected. In other total extracts glass-purified extract of induced cells. Open arrowhead, VirE2 ofAS-induced Agrobacterium, either only the 58-kDa protein protein. Scaling on the right is molecular mass in kilodaltons. was detected or both the 43-kDa and 58-kDa proteins were detected (data not shown). Affinity-purified nopaline VirD2 compatible plasmid, containing T-DNA flanked by the left also reacted with a 43-kDa protein in Si-treated extracts from and right borders of the octopine Ti plasmid, was used as the heterologous T-strand-synthesizing E. coli cells that had T-strand substrate. Following induction of virD expression, been induced with IPTG for 4 hr or longer (Fig. 3B, lanes h T-strands are actively produced (18); thus, this system can be and f). No 43-kDa protein was detected by immunoblotting used to directly test for VirD2 binding to T-strand in the in the non-Si-treated samples from induced cells (Fig. 3B, absence of other vir- or Agrobacterium-specific proteins. lanes e, g, and i) or either Si-treated or untreated samples E. coli cells containing both plasmids were induced with from uninduced cells (lanes c and d). These data confirm that isopropyl /3-D-thiogalactoside (IPTG), labeled with [35S]me- the VirD2 protein is tightly associated with a ssDNA mole- thionine, and lysed. DNA, as well as any DNA-protein cule. Further, the 43-kDa form of the VirD2 protein must complexes, was purified by adsorption to glass beads. Sam- retain the DNA-binding domain of the intact VirD2 protein. ples were treated with or without S1 nuclease and analyzed Stoichiometry ofVirD2 Bound to ssDNA. Ifthe bound VirD2 by SDS/polyacrylamide gel electrophoresis; proteins were is part of the T-strand complex, the number of bound VirD2 detected by both autoradiography and immunoblotting. A molecules per cell is expected to be close to the number of labeled inducible protein of 43 kDa was present only in T-strands per cell. Bound VirD2 molecules are detectable Si-treated samples (Fig. 3A, lanes c, e, and g). This protein following isolation ofDNA-protein complexes on glass beads was not detectable after 1 hr of induction (lane a) but was and subsequent treatment with S1 nuclease to release pro- detectable after 4 hr of induction (lane c) and continued to teins bound to ssDNA (Fig. 2, lane c). Total unbound VirD2 increase in amount until 23 hr (lane g). The kinetics of can be estimated from the protein pattern in vir-induced Downloaded by guest on September 26, 2021 4020 Biochemistry: Howard et al. Proc. Natl. Acad. Sci. USA 86 (1989) whole cell extracts (Fig. 2, lane a). Densitometer tracings of A the bands migrating at 58 kDa were used to deduce the H1O pBR H23 amount of bound versus unbound VirD2; the amount in the * I * whole cell extract was determined after subtraction of the 6.5 4.3 3.3 noninduced component migrating at 58 kDa (Fig. 2, lane b), a d and the amount in the Si-treated sample was corrected by the amount in the non-Si-treated induced glass-bead sample . 5, 3 . 2.2 1. (Fig. 2, lane d). The data reveal that there is 3 times more 3.5 3.0 2.2 1.1 VirD2 in the whole-cell-extract lane than in the Si-treated- B c sample lane. As the sample loaded in the S1 lane was from 50 a b c d e f g h a b c d e f times as many cells as that loaded in the total-extract lane, there are about 150 times as many unbound VirD2 proteins as -a bound VirD2 proteins in induced Agrobacterium. The number of VirD2 molecules per cell can be estimated by comparison -b .... -c with a protein of defined concentration, VirE2, migrating at 69 w Iod kDa. The VirE2 band has an intensity of about 2.4-fold relative to the 58-kDa VirD2 band. Since there are 350-700 molecules -e of VirE2 per vir-induced cell (12), there are 146-292 molecules of VirD2 per cell. As there are 150-fold more unbound than 'F.;;r:..<; bound VirD2 molecules in the cell, there are 1-2 molecules of bound VirD2 per cell. There are estimated to be 0.5-1 mole- cules of T-strand per cell (4); therefore, there is about 1 -f molecule of bound VirD2 per T-strand. Ti Plasmid Is Tightly Associated with Protein at Its Borders. It is important to show that the nucleic acid in the DNA- protein complexes analyzed is indeed T-strand and not an- FIG. 4. Protein association to the 5' side of T-DNA border nicks other ssDNA that is bound to VirD2. The VirD1/VirD2 in vir-induced cells. (A) HindIII fragments produced by double- endonuclease is required not only for T-strand synthesis but stranded cleavages of the pGV3850 borders. Fragment sizes (kbp) are indicated. Arrowheads indicate T-DNA border sequences; bars for nicking at the borders of the T-DNA on the Ti plasmid. If indicate HindIl! sites. (B) Analysis of left border-protein associa- the Ti plasmid is bound to a protein at the borders after tion. Extracts from AS-induced and uninduced cells were prepared nicking and if the binding is only to a single specific strand of in the presence or absence of Pronase (as in samples a and b, Fig. 1), the plasmid (i.e., the nicked strand), one might expect to find phenol-extracted, digested with HindIlI, incubated with or without a local region of single-strandedness surrounding the binding S1 nuclease as described (4), and analyzed either directly or after site. Border-specific nicking can be assayed after S1 nuclease phenol extraction and ethanol precipitation. Lanes a and b: samples digestion of restriction fragments overlapping the T-DNA from Pronase-treated extracts from uninduced cells, after incubation border regions of the Ti plasmid; S1 cleaves opposite the without or with S1 nuclease. Lanes c-h: extracts from AS-induced nicked site, resulting in double-stranded cleavage products cells; lanes c and d, samples from Pronase-treated extracts, incu- bated without or with S1 nuclease before phenol extraction; lane e, (4, 5). The border nick products expected for Ti plasmid as in lane d, but not phenol-extracted after digestion with HindIII and pGV3850 after HindIII digestion and S1 treatment are dia- incubation with S1 nuclease; lanes f and g, samples from cells lysed gramed in Fig. 4A. vir-induced DNA was prepared in the in the absence of Pronase (using lysozyme/sarkosyl as in Fig. 1), presence or absence of Pronase, digested with HindIII and incubated without or with S1 nuclease before phenol extraction; lane S1, and treated with phenol to assay for partitioning of h, as in lane g, but not phenol-extracted. Samples were analyzed by border-nicked fragments. The aqueous phase from phenol agarose gel electrophoresis followed by Southern blotting under extraction of the Pronase-treated extracts contained the denaturing conditions. Hybridization probe was nick-translated Hin- unnicked HindIII fragments from the left and right border (a dIII fragment 10 (H10 in A) cloned into pBR322. (C) Analysis of right and d, respectively), as well as each of the expected nicked border-protein association. Samples were prepared as in B, using AS-induced cells. Lanes a-f are as b-h in B. Samples were analyzed products (b and c from the left border and e and f from the for right-border fragment content as in B, except that the HindIll right border; Fig. 4 B and C, lane c). In contrast, the aqueous fragment 23 (H23 in A) cloned into pBR322 was used as probe. phase from non-Pronase-treated samples contained unnicked Autoradiograph in C was exposed 3 times as long as that in B. fragments and only the nicked product to the 3' side ofthe left (fragment b; Fig. 4B, lane g) or right (fragment d, Fig. 4C, lane rected against VirD2. For these experiments, cell extracts e) border nick sites. Interestingly, the 5' border nick products were immunoprecipitated with affinity-purified VirD2 anti- were detected in the non-Pronase-treated samples when they body and analyzed for full-length T-strand by Southern were electrophoresed directly, without prior phenol extrac- hybridization. Affinity-purified VirD2 antibody specifically tion (Fig. 4 B, lane h, and C, lane f). The phenol partitioning immunoprecipitated T-strand (Fig. 5, lane a). T-strand was of the 5' border-nicked products suggests that the Ti plasmid not specifically immunoprecipitated either by crude VirD2 in vir-induced cells contains protein associated with the 5' antibody (lane b) or by non-VirD antibody (VirG antibody; end of the nicks at the left and right T-DNA borders. Since lane c) under conditions in which the total levels of IgG in the border nicks arise as a function of the VirD1/VirD2 endo- three immunoprecipitations were equivalent. Since 1% or nuclease, it is likely that at least one or both of these less of the IgG in the crude antibody was VirD2-specific, polypeptides may remain bound to the 5' side of the nick site affinity-purified VirD2 antibody should be more efficient in following cleavage. These Ti plasmid virD-specific polypep- immunoprecipitating T-strand under these conditions, as tides might have contributed to the 58-kDa protein detected observed. These data confirm that free T-strand is tightly in the S1 analysis of DNA-protein complexes described associated with VirD2 in vivo. above. Thus, we chose an alternative method to test for VirD2 binding to T-strand. DISCUSSION T-Strand Is Complexed with VirD2 in Vivo. To prove directly that T-strand is tightly associated with VirD2, we Here we have shown that VirD2 is tightly associated with free asked whether full-length T-strand could be immunoprecip- T-strand in vivo. VirD2 is tightly associated with ssDNA, as itated from vir-induced cell extracts by using antibody di- shown by the S1 experiments in Agrobacterium and in the E. Downloaded by guest on September 26, 2021 Biochemistry: Howard et al. Proc. Natl. Acad. Sci. USA 86 (1989) 4021

a b c tions have been observed in replication complexes (e.g., 4X174-gene A protein complex), transfer complexes (e.g., the 5' end of F plasmid, which leads in transfer, is protein- bound), phage replication and maturation complexes (e.g., 0529-gene 3 protein complex), DNA-intermediate complexes affecting DNA conformation (e.g., DNA-bound topo- isomerases), and animal virus complexes that may participate in replication (e.g., adenovirus DNA is bound to a 75-kDa FIG. 5. VirD2 antibody immunoprecipitates T-strands from vir- protein at its 5' ends). The functions of the proteins in many induced Agrobacterium. Glass-bead-purified extracts of AS-induced of these complexes have only been inferred; for example, no Agrobacterium were immunoprecipitated with affinity-purified VirD2 antibody (lane a), crude VirD2 antibody (lane b), or crude protein has ever been directly shown to be a "pilot protein" non-VirD2 (VirG) antibody (lane c) under conditions in which the responsible for targeting a DNA molecule out ofor into a cell. total IgG in the three immunoprecipitations was equivalent. Immuno- Both nicking and T-strand formation in vir-induced Agrobac- precipitates were analyzed for T-strand content by gel electropho- terium require the products of the virDi and virD2 genes. resis followed by transfer to nitrocellulose under nondenaturing Identification of the enzymatic activities associated with conditions and hybridization to nick-translated HindlI fragment 10 these two proteins may contribute to our understanding not of pGV3850. only of T-strand generation but of more generalized mecha- nisms found in other systems as well. coli heterologous T-strand-synthesizing system. The partic- Recently, two papers have appeared that support our data. ular ssDNA associated with VirD2 is T-strand, since VirD2- Ward and Barnes (30) indirectly showed that T-strands are specific antibody purifies T-strand from bulk Ti plasmid and bound to VirD2, by using ,8-galactosidase antibody to immu- chromosomal DNA. Earlier studies that purported to show noprecipitate T-strand complexed to VirD2 fusion proteins. association of T-strand with VirD2 did not distinguish be- Herrera-Estrella (31) showed that T-strands are associated tween association of VirD2 on T-strand and association with with single-stranded regions ofthe Ti plasmid (25). This distinction proteins. is particularly important, given the evidence presented here We thank Vitaly Citovsky for invaluable discussions. This work that the T-DNA border regions of the Ti plasmid in vir- was supported by National Science Foundation Grant DMB-8617772 induced cells also are tightly associated with protein. This (P.Z.). E.A.H. was supported by a research fellowship from the Plant protein binding may distort duplex DNA at the borders, Gene Expression Center, U.S. Department of Agriculture. resulting in localized regions of ssDNA. 1. Zambryski, P. (1988) Annu. Rev. Genet. 22, 1-30. The protein associated with the T-DNA borders has been 2. Stachel, S. E. & Nester, E. W. (1986) EMBO J. 5, 1445-1454. localized to the 5' side 3. Stachel, S. E., Messens, E., Van Montagu, M. & Zambryski, P. (1985) of the border nick site. Since the Nature (London) 318, 624-629. VirDl/VirD2 endonuclease is responsible for border nicking, 4. Stachel, S. E., Timmerman, B. & Zambryski, P. (1986) Nature (London) most likely the protein bound to the 5' ends of the nicked site 322, 706-721. is VirD1 and/or VirD2. Since the T-strand must be displaced 5. Stachel, S. E., Timmerman, B. & Zambryski, P. (1987) EMBO J. 6, 857- from the 5' side of the border 863. nick, the protein complex at the 6. Veluthambi, K., Ream, W. & Gelvin, S. (1988) J. Bacteriol. 170, 1523- nick site may remain bound to the 5' end ofthe free T-strand. 1532. The data are consistent with the location of VirD2 on the 5' 7. Wang, K., Stachel, S. E., Timmerman, B., Van Montagu, M. & Zam- end of the T-strand. Potentially, VirD1 is also part of the bryski, P. (1987) Science 235, 587-591. 8. Albright, L. M., Yanofsky, M. F., Keroux, B., Ma, D. & Nester, E. W. T-strand-protein complex in vivo but is removed in vitro by (1987) J. Bacteriol. 169, 1046-1055. the harsh treatments (4 M urea, 4 M Nal, boiling with SDS) 9. Yanofsky, M. F., Porter, S. G., Young, C., Albright, L. M., Gordon, used. Alternatively, VirDl may not be part of the T-strand M. P. & Nester, E. W. (1986) Cell 47, 471-477. complex but instead be required only for nicking. 10. Jayaswal, R. K., Gelvin, S. B. & Slightom, J. L. (1987)J. Bacteriol. 169, VirD2 associated with serve 5035-5045. tightly T-strand may at least 11. Gietl, C., Koukolikova-Nicola, Z. & Hohn, B. (1987) Proc. Natl. Acad. four functions in vivo: (i) to act as an unwinding protein Sci. USA 84, 9006-9010. during T-strand synthesis; (ii) to protect the 5' end of the 12. Citovsky, V., De Vos, G. & Zambryski, P. (1988) Science 240, 501-504. complex from exonucleases; (iii) to "pilot" the complex to 13. Das, A. (1988) Proc. Natl. Acad. Sci. USA 85, 2909-2913. the in the 14. Christie, P. J., Ward, J. E., Winans, S. & Nester, E. W. (1988) J. specific proteins bacterial membrane that facilitate Bacteriol. 170, 2659-2667. its transfer to the plant cell; and (iv) to facilitate subsequent 15. Citovsky, V., Wong, M. L. & Zambryski, P. (1989) Proc. Natl. Acad. integration of T-DNA into the plant chromosome. Sci. USA 86, 1193-1197. Any model for the events involved with T-strand synthesis 16. Gheysen, G., Van Montagu, M. & Zambryski, P. (1987) Proc. Natl. must the existence of linked Acad. Sci. USA 84, 6169-6173. incorporate proteins tightly both 17. Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M. to the Ti plasmid and to the T-strand, as well as consider that & Schell, J. (1983) EMBO J. 2, 2143-2150. the left and right borders are not equivalent. Only the right 18. DeVos, G. & Zambryski, P. (1989) Mol. Plant-Microbe Interact. 2, border is absolutely required for tumor formation (26). Fur- 43-52. the context of the 19. Rosenberg, A. H., Lade, B. N., Chiu, D., Dunn, J. J. & Studier, W. ther, sequence right border promotes (27, (1987) Gene 56, 125-135. 28), whereas the sequence context of the left border de- 20. Smith, D. E. & Fischer, P. A. (1984) J. Cell Biol. 99, 20-28. creases (28), T-DNA transfer. Also, in the current study and 21. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Smith, J. A., earlier (4), the right border appeared to be less nicked than the Seidman, J. G. & Struhl, K. (1987) Current Protocols in Molecular left. One is that the border is more Biology (Greene, New York). explanation right used 22. Harding, N. E., Ito, J. & David, G. S. (1978) Virology 84, 279-292. efficiently for nicking and polymerization, possibly as the 23. Laemmli, U. K. (1970) Nature (London) 227, 680-685. result of a "loading site" [such as the "overdrive" sequence 24. Alt-Moerbe, J., Rak, B. & Schroeder, J. (1986) EMBO J. 5, 1129-1135. (27)] for the endonuclease downstream of the right border. 25. Young, C. & Nester, E. W. (1988) J. Bacteriol. 170, 3367-3374. The border nick would be extended as soon as 26. Wang, K., Herrera-Estrella, L., Van Montagu, M. & Zambryski, P. right it forms (1984) Cell 38, 35-41. and therefore appear less nicked. These observations indi- 27. Peralta, E. G., Hellmiss, R. & Ream, W. (1986) EMBO J. 5, 1137-1142. cate the complexity of T-strand synthesis. 28. Wang, K., Genetello, C., Van Montagu, M. & Zambryski, P. (1987) Mol. Proteins that are tightly bound to DNA have been de- Gen. Genet. 210, 338-346. scribed in a number of in ref. 29. Kornberg, A. (1980) DNA Replication (Freeman, New York). systems (reviewed 29). Many 30. Ward, E. R. & Barnes, W. M. (1988) Science 242, 927-930. of the tightly associated proteins found in DNA-protein 31. Herrera-Estrella, A., Chen, Z.,Van Montagu, M. & Wang, K. (1988) complexes are multifunctional. Tight DNA-protein associa- EMBO J. 7, 4055-4062. Downloaded by guest on September 26, 2021