Journal of Bacteriology, November 1999, p. 6850-6855, Vol. 181, No. 21
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Agrobacterium tumefaciens Chaperone-Like Protein, VirE1, Interacts with VirE2 at Domains Required for Single-Stranded DNA Binding and Cooperative Interaction

Christopher D. Sundberg^1, and Walt Ream^2,*

Program in Genetics¹ and Department of Microbiology,² Oregon State University, Corvallis, Oregon 97331

Received 17 May 1999/Accepted 13 August 1999

	ABSTRACT

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Agrobacterium tumefaciens transfers single-stranded DNA (ssDNA) into plants. Efficient tumorigenesis requires VirE1-dependent export of ssDNA-binding (SSB) protein VirE2. VirE1 binds VirE2 domains involved in SSB and self-association, and VirE1 may facilitate VirE2 export by preventing VirE2 aggregation and the premature binding of VirE2 to ssDNA.

	TEXT

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Agrobacterium tumefaciens transfers part of its tumor-inducing (Ti) plasmid, the T-DNA, into the plant nuclear genome (10). Border sequences delimit the T-DNA (41, 42, 49, 60). T-DNA transfer requires vir genes located elsewhere on the Ti plasmid (23, 52). The VirD1-VirD2 endonuclease nicks border sequences, and VirD2 attaches to the 5' end of the nicked strand (50). Subsequent events produce single-stranded DNA (ssDNA) called T strands (2, 31, 53).

A transmembrane channel, comprising 11 VirB proteins and VirD4, exports VirD2-T strand complexes and VirE2 into plants (11, 39, 50, 57, 62, 63). VirB proteins resemble Legionella pneumophila Icm (also called Dot), Helicobacter pylori Cag, and Bordetella pertussis Ptl toxin export proteins (1, 46, 47, 59, 62). Rickettsia prowazekii contains virD4 and virB homologs (3). Proteins that mediate plasmid conjugation resemble VirB and VirD4 (5, 32, 34, 35, 62). The VirB-VirD4 channel mediates plasmid conjugation from A. tumefaciens into other bacteria (6, 21, 22, 25, 56), plants (8), or fungi (9, 17). L. pneumophila Icm and Dot proteins mobilize plasmids into other bacteria (46, 59). This family of transmembrane proteins exports proteins and protein-DNA complexes into a broad range of recipients.

VirE2 binds ssDNA cooperatively (14, 48), contains two bipartite nuclear localization signals (NLSs) (15), and plays an important role in tumorigenesis (24, 45). The C-terminal half of VirE2 is essential for ssDNA binding (SSB) (15, 19, 48). Some alterations in the N-terminal half of VirE2 retain their function, while others affect cooperativity (15, 19). VirE2 protects T strands from nuclease attack (14, 45, 48, 63), but the absence of VirE2 does not diminish T-strand accumulation in A. tumefaciens (45, 54, 58). The NLSs, which overlap the SSB domain, function when VirE2 binds ssDNA (65). VirD2 NLS mutants transform plants, implicating VirE2 in the nuclear uptake of T strands (38, 51). Transgenic VirE2-producing tobacco plants are transformed by a virE mutant of A. tumefaciens (15), showing that VirE2 is necessary only inside plant cells.

View this table: [in this window] [in a new window]   TABLE 1.   Yeast strainsa

VirE2 export requires VirE1 (57). Although VirE2 is stable in A. tumefaciens without VirE1 (57), VirE1 stabilizes VirE2 in Escherichia coli (37), suggesting that these proteins interact. We used protein interaction cloning in yeast (Saccharomyces cerevisiae) to study this interaction. Recently, Deng et al. reported similar data (18).

Protein interaction assays. Table 1 describes the yeast strains used. Bait fusions contained virE2 sequences fused to lexA in pEG202 (26). Prey fusions contained virE1 or virE2 fused to a transcriptional activator (TA) domain in pJG4-5 (26); pSH18-34 contains lacZ downstream of the GAL1 promoter and four lexA operators (26). Recruitment of prey protein to the operators via interaction with the bait protein induces lacZ (26). -Galactosidase activity indicated protein interaction (26, 43).

Plasmid construction. Portions of virE2 were amplified by PCR; primers contained NcoI (downstream) and BamHI (upstream) sites. virE2 contains BglII and BclI sites phased to allow their use instead of the BamHI site in the upstream primer. Some primers contained a BsaI site that generated NcoI- or BamHI-compatible ends. We inserted virE2 sequences into pEG202 at the BamHI and NcoI sites. Duplicate bait constructs were prepared in pCD2M1, a pEG202 derivative containing a simian virus 40 NLS (PKKKRKV [44]) fused to lexA at the EcoRI and BamHI sites. Bait fusions in both plasmids gave equivalent results. The TA sequence was fused to full-length virE1 or virE2 or truncated virE1 by inserting PCR amplicons with EcoRI- and XhoI-compatible ends into pJG4-5 at these sites.

PCR was performed with Stratagene Pfu DNA polymerase with a template (pGR1) containing the virE operon (57). Thermocycle ligations (36) were performed with T4 DNA ligase; DNA was transformed into E. coli as previously described (4). All gene fusions were sequenced by the dye terminator method (4). virE2 differed from the published sequence (61), with G-C and C-G transversions at positions 1073 and 1074. These changes were present in pGR1. This double mutation changed codon 358 from cysteine to serine, the residue present in nopaline-type VirE2 (27).

VirE1 interacts with VirE2. A bait fusion containing full-length VirE2 interacted with VirE1 prey (CSY1) (Fig. 1 to 3). This LexA-VirE2 protein did not activate lacZ in yeasts that harbor pJG4-5 without virE1 (Fig. 2), nor did VirE2 interact with the TA domain encoded by pJG4-5. Several bait fusions with only a portion of VirE2 did not interact with the TA-VirE1 prey (Fig. 1 and 2), proving that VirE1 did not bind the LexA moiety of the bait. The LexA-VirE1 fusion alone, in cells devoid of a prey plasmid, activated lacZ (data not shown). This often occurs when LexA is joined to an acidic protein (26) such as VirE1 (pI 4.7 [61]). VirE1-VirE2 interactions were characterized with virE2 bait fusions and virE1 prey.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Interaction of VirE2 bait proteins and VirE1 prey fusion proteins. The large box represents the genetic and physical map of VirE2. Domains involved in cooperativity (Coop.) and DNA binding (ssDNA binding) are overlined. The C-terminal VirE1-binding domain is underlined. Numbers above the map indicate codons. Triangles mark XhoI linker insertions after codons 378 and 472. Hatched boxes on the map represent the NLSs. Bars show the region of VirE2 present in each bait fusion protein. Solid bars indicate VirE2 bait-VirE1 prey interaction (shown under the column at the left as a +), open bars indicate no interaction (shown by a ), and the hatched bar indicates VirE2 bait self-activation (shown by s.a.), in which the bait plasmid activated transcription of lacZ in the presence of pJG4-5 (without virE1 sequences).

View larger version (46K): [in this window] [in a new window]   FIG. 2.   Interaction of VirE2 bait proteins with VirE1 or VirE2 prey proteins; -galactosidase assays. The top bar on the left represents the map of VirE2; hatched boxes are NLSs, triangles indicate XhoI linker insertions after codons 378 and 472, and the C-terminal VirE1-binding domain is underlined. Bars show the regions of VirE2 present in each bait fusion protein. Solid bars indicate VirE2 bait-VirE1 prey interaction, open bars indicate no interaction, and the hatched bar indicates VirE2 bait self-activation. On the right, the Bait/Prey column shows -galactosidase assays of yeast strains that harbored both bait and prey fusion plasmids, whereas the Bait column shows results for strains that harbored a bait fusion plasmid together with pJG4-5. Dark patches indicate -galactosidase activity corresponding to VirE2 bait-VirE1 prey interaction. Light patches indicate no activity. VirE2 bait self-activation is shown by dark patches in both the Bait/Prey and Bait columns. Vertical bars indicate whether VirE2 bait fusions were tested with VirE2 (top panel) or VirE1 (lower panels) prey fusions. Numbers on the left (Ile 190, etc.) indicate the VirE2 codons included in the bait fusion.

View larger version (60K): [in this window] [in a new window]   FIG. 3.   Interaction of VirE2 bait proteins with truncated or full-length VirE1 prey proteins. On the left, open bars show regions included in VirE2 bait and VirE1 prey proteins; numbers (Ser288, etc.) indicate VirE1 or VirE2 codons included in each fusion. Triangles mark XhoI linker insertions after codons 378 and 472. -gal, -galactosidase activity, indicated as positive (+) or negative (). Panels A to H (right) indicate the corresponding -galactosidase assays.

N-terminal VirE1-binding domain. VirE2 residues 2 to 386 interacted with VirE1 but not with the TA domain of pJG4-5 (CSY168) (Fig. 1). Baits containing shorter segments of VirE2 (residues 2 to 349 or 2 to 242) activated lacZ in the presence of pJG4-5 (CSY46 and CSY45) (Table 1; Fig. 2), which prevented further mapping of this domain's distal end. The first 73 residues of VirE2, although poorly conserved (27, 61), were required for the interaction of this domain with VirE1; residues 46 to 349 did not interact with VirE1 (CSY155) (Fig. 1). Because VirE2 residues 2 to 386 contain cooperativity and VirE1 interaction domains, VirE1 binding at the VirE2 N terminus may prevent VirE2 self-association by occupying domains crucial for self-interaction. Indeed, VirE1 prevents VirE2 aggregation in vitro (18).

C-terminal SSB and VirE1-binding domains overlap. VirE2 residues 288 to 533 interacted with VirE1, but residues 326 to 533 did not (CSY21 and CSY20) (Fig. 1), indicating that residues between 288 and 326 were necessary for binding VirE1. The C-terminal boundary of this VirE1-binding domain was established with VirE2 fusions that extended from residue 190 toward the C terminus. One fusion extended through residue 495 and interacted with VirE1 (CSY169) (Fig. 1). VirE2 residues 190 to 436 failed to interact with VirE1 (CSY76) (Fig. 1 and 2), indicating that residues between 436 and 495 were required for binding VirE1. Thus, residues 288 to 495 encompass this VirE1-binding domain. CSY23 contained this VirE2 bait and interacted with VirE1 (Fig. 1 to 3), confirming that a VirE2 domain within residues 288 to 495 bound VirE1. Mutations that abolish SSB activity define a domain that includes residues 287 to 497 (13, 19, 48). The smallest VirE2 bait that bound VirE1, residues 288 to 495, corresponds to the SSB domain (Fig. 1). XhoI linker insertions in the SSB domain destroyed SSB activity (19) and abolished its interaction with VirE1 (CSY109 and CSY110) (Fig. 1 to 3). VirE1 and ssDNA interact with VirE2 features disrupted by the same mutations, so VirE1 may compete with ssDNA to bind VirE2. Full-length VirE2 baits containing these insertions interacted with VirE1, indicating that these mutations did not affect the N-terminal VirE1-binding domain (CSY165 and CSY166) (Fig. 1 and 3).

Bait proteins are stable. Yeast lysates contained VirE2 bait proteins detectable in immunoblots probed with antibodies to LexA (data not shown).

The VirE1 C terminus interacts with VirE2. VirE1 contains two conserved domains, residues 1 to 28 and 35 to 62 (27, 61). We fused virE1 codons 25 to 65 to the TA domain in pJG4-5. This VirE1 prey interacted with bait proteins containing either full-length VirE2 or the SSB-VirE1-binding domain (Fig. 3D and H). Bait fusions containing the SSB-VirE1-binding domain disrupted by an XhoI linker insertion did not interact with the truncated VirE1 prey (Fig. 3B and C). In full-length VirE2, these insertions did not affect the binding of truncated VirE1 to the VirE2 N terminus (Fig. 3F and G). Thus, truncated and full-length VirE1 preys behaved the same. Because the same region of VirE1 binds both protein interaction domains of VirE2, separate VirE1 molecules probably bind each domain.

VirE2-VirE2 interaction requires N- and C-terminal sequences. VirE2 contains two regions important for cooperativity (15, 19). Mutations in these regions affect cooperativity without abolishing SSB activity, implicating them in self-interaction. We fused codons 2 to 533 to the TA domain in pJG4-5, creating a full-length VirE2 prey which interacted with full-length VirE2 bait (CSY28) (Table 1; Fig. 2). Removing the first 189 residues of the VirE2 bait eliminated one cooperativity domain and abolished VirE2-VirE2 interaction (CSY29) (Table 1). VirE2 bait that included both cooperativity domains (residues 46 to 533) failed to bind full-length VirE2 prey (CSY157) (Table 1). VirE2 bait (residues 2 to 495) missing the last 38 residues did not interact with VirE2 prey (CSY37 and CSY39) (Table 1). VirE2-VirE2 interaction required both N- and C-terminal VirE2 sequences outside known cooperativity domains.

VirE2 and T-strand transfer may occur independently. VirE2 export requires VirE1, but T-strand transfer does not (57). VirB-VirD4-dependent mobilization of RSF1010 from A. tumefaciens into plants abolishes tumorigenesis by preventing VirE2 export; RSF1010 reduces but does not eliminate T-strand transfer (7, 55). The oncogenesis-suppressing Osa protein inhibits VirE2 export but not T-strand transfer (33). Thus, T-strand transfer occurs even though VirE2 export is blocked. A. tumefaciens lacking VirE1 and VirE2 transfers T strands into plants (24, 57, 63), and VirE2 export does not require T-strand production (12, 40, 57). Tumors form readily when a single plant wound is inoculated with two nonpathogenic strains of A. tumefaciens: one lacking T-DNA and another lacking virE2 (12, 40, 57). Because both strains must bind plant cells (12), VirE2 and T strands are probably exported directly, and independently, into plants.

VirE1 is a secretory chaperone for VirE2. Others have suggested that VirE1 may promote the proper folding of VirE2 (64). This seems unlikely because transgenic plants express functional VirE2 in the absence of VirE1 (15). Instead, VirE1 may act as a molecular escort of VirE2. Because VirE1 binds VirE2 at sites critical for SSB and self-interaction, VirE1 may hinder these interactions. VirE1 may promote the export of VirE2 by limiting its association with ssDNA and by preventing VirE2 aggregation. Similar molecular escorts participate in protein secretion in many pathogenic bacteria, where they prevent the premature association of secreted proteins with each other or with the translocation machinery (16, 20, 28). Yersinia outer membrane protein (Yop) export requires a specific Yop chaperone (Syc), which binds its target Yop (29, 30). The physical properties of Syc proteins resemble those of VirE1: all are small (123 to 168 amino acids), acidic (pI 4.5 to 4.88) proteins with a hydrophobic C terminus (16). Syc proteins release their Yop as it exits the cell (30). The export of monomer subunits that form a complex following secretion is a common strategy for assembling large extracellular complexes. If formed inside the cell, complexes might be too large to pass through the transmembrane channel. Secretory chaperones promote protein export by preventing the assembly of large complexes in the cytoplasm. VirE1 appears to act as a secretory chaperone for VirE2.

	ACKNOWLEDGMENTS

We thank Mark Ptashne for antibodies to LexA, Roger Brent for two-hybrid strains, Jay Evans and Dan Rockey for advice, and Laurie Bissonette, Hyewon Lee, and Sarah Andrews for critiques of this paper.

This work was supported by USDA grant 96-35301-3178. C. Sundberg was supported by a USDA National Needs fellowship and an OSU Minority Pipeline Fellowship.

	FOOTNOTES

^* Corresponding author. Mailing address: Department of Microbiology, Oregon State University, Corvallis, OR 97331. Phone: (541) 737-1791. Fax: (541) 737-0496. E-mail: reamw{at}bcc.orst.edu.

Oregon State University Agricultural Experiment Station paper 11,549.

Present address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305.

	REFERENCES

Top Abstract Text References

1.	Akopyants, N. S., S. W. Clifton, D. Kersulyte, J. E. Crabtree, B. E. Youree, C. A. Reece, N. O. Bukanov, E. S. Drazek, B. A. Roe, and D. E. Berg. 1998. Analyses of the cag pathogenicity island of Helicobacter pylori. Mol. Microbiol. 28:37-53[Medline].
2.	Albright, L. M., M. F. Yanofsky, B. Leroux, D. Ma, and E. W. Nester. 1987. Processing of the T-DNA of Agrobacterium tumefaciens generates border nicks and linear, single-stranded T-DNA. J. Bacteriol. 169:1046-1055[Abstract/Free Full Text].
3.	Andersson, S. G. E., A. Zomorodipour, J. O. Andersson, T. Sicheritz-Ponten, U. C. M. Alsmark, R. M. Podowski, A. K. Naslund, A. S. Eriksson, H. H. Winkler, and C. G. Kurland. 1998. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396:133-140[Medline].
4.	Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1987. Current protocols in molecular biology. Wiley, New York, N.Y
5.	Baron, C., and P. C. Zambryski. 1996. Plant transformation: a pilus in Agrobacterium T-DNA transfer. Curr. Biol. 6:1567-1569[Medline].
6.	Beijersbergen, A., A. D. Dulk-Ras, R. A. Schilperoort, and P. J. J. Hooykaas. 1992. Conjugative transfer by the virulence system of Agrobacterium tumefaciens. Science 256:1324-1327[Abstract/Free Full Text].
7.	Binns, A. N., C. E. Beaupre, and E. M. Dale. 1995. Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010. J. Bacteriol. 177:4890-4899[Abstract/Free Full Text].
8.	Buchanan-Wollaston, V., J. E. Passiatore, and F. Cannon. 1987. The mob and oriT mobilization functions of a bacterial plasmid promote its transfer to plants. Nature 328:172-175.
9.	Bundock, P., A. den Dulk-Ras, A. Beijersbergen, and P. J. J. Hooykaas. 1995. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J. 14:3206-3214[Medline].
10.	Chilton, M. D., M. H. Drummond, D. J. Merlo, D. Sciaky, A. L. Montoya, M. P. Gordon, and E. W. Nester. 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11:263-271[Medline].
11.	Christie, P. J. 1997. Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. J. Bacteriol. 179:3085-3094[Free Full Text].
12.	Christie, P. J., J. E. Ward, S. C. Winans, and E. W. Nester. 1988. The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA. J. Bacteriol. 170:2659-2667[Abstract/Free Full Text].
13.	Citovsky, V., D. Warnick, and P. Zambryski. 1994. Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco. Proc. Natl. Acad. Sci. USA 91:3210-3214[Abstract/Free Full Text].
14.	Citovsky, V., M. L. Wong, and P. Zambryski. 1989. Cooperative interaction of Agrobacterium VirE2 protein with single-stranded DNA: implications for the T-DNA transfer process. Proc. Natl. Acad. Sci. USA 86:1193-1197[Abstract/Free Full Text].
15.	Citovsky, V., J. Zupan, D. Warnick, and P. Zambryski. 1992. Nuclear localization of Agrobacterium VirE2 protein in plant cells. Science 256:1802-1805[Abstract/Free Full Text].
16.	Cornelis, G. R., A. Boland, A. P. Boyd, C. Geuijen, M. Iriarte, C. Neyt, M. P. Sory, and I. Stainier. 1998. The virulence plasmid of Yersinia, an antihost genome. Microbiol. Mol. Biol. Rev. 62:1315-1352[Abstract/Free Full Text].
17.	de Groot, M. J., P. Bundock, P. J. J. Hooykaas, and A. G. Beijersbergen. 1998. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat. Biotechnol. 16:839-842[Medline].
18.	Deng, W., L. Chen, W. T. Peng, X. Liang, S. Sekiguchi, M. P. Gordon, L. Comai, and E. W. Nester. 1999. VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium. Mol. Microbiol. 31:1795-1807[Medline].
19.	Dombek, P., and W. Ream. 1997. Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2. J. Bacteriol. 179:1165-1173[Abstract/Free Full Text].
20.	Finlay, B. B., and S. Falkow. 1997. Common themes in microbial pathogenicity revisited. Microbiol. Mol. Biol. Rev. 61:136-169[Abstract].
21.	Fullner, K. J., J. C. Lara, and E. W. Nester. 1996. Pilus assembly by Agrobacterium T-DNA transfer genes. Science 273:1107-1109[Abstract].
22.	Fullner, K. J., and E. W. Nester. 1996. Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens. J. Bacteriol. 178:1498-1504[Abstract/Free Full Text].
23.	Garfinkel, D. J., and E. W. Nester. 1980. Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J. Bacteriol. 144:732-743[Abstract/Free Full Text].
24.	Gelvin, S. B. 1998. Agrobacterium VirE2 proteins can form a complex with T strands in the plant cytoplasm. J. Bacteriol. 180:4300-4302[Abstract/Free Full Text].
25.	Gelvin, S. B., and L. L. Habeck. 1990. vir genes influence conjugal transfer of the Ti plasmid of Agrobacterium tumefaciens. J. Bacteriol. 172:1600-1608[Abstract/Free Full Text].
26.	Golemis, E. A., J. Gyuris, and R. Brent. 1996. Interaction trap/two hybrid system to identify interacting proteins, p. 20.1.12-20.1.28. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, and J. A. Smith (ed.), Current protocols in molecular biology. John Wiley and Sons, New York, N.Y
27.	Hirooka, T., P. M. Rogowsky, and C. I. Kado. 1987. Characterization of the virE locus of Agrobacterium tumefaciens plasmid pTiC58. J. Bacteriol. 169:1529-1536[Abstract/Free Full Text].
28.	Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Biol. Rev. 62:379-433[Abstract/Free Full Text].
29.	Iriarte, M., and G. R. Cornelis. 1999. Identification of SycN, YscX, and YscY, three new elements of the Yersinia Yop virulon. J. Bacteriol. 181:675-680[Abstract/Free Full Text].
30.	Jackson, M. W., J. B. Day, and G. V. Plano. 1998. YscB of Yersinia pestis functions as a specific chaperone for YopN. J. Bacteriol. 180:4912-4921[Abstract/Free Full Text].
31.	Jayaswal, R. K., K. Veluthambi, S. B. Gelvin, and J. L. Slightom. 1987. Double-stranded cleavage of T-DNA and generation of single-stranded T-DNA molecules in Escherichia coli by a virD-encoded border-specific endonuclease from Agrobacterium tumefaciens. J. Bacteriol. 169:5035-5045[Abstract/Free Full Text].
32.	Kado, C. I. 1994. Promiscuous DNA transfer system of Agrobacterium tumefaciens: role of the virB operon in sex pilus assembly and synthesis. Mol. Microbiol. 12:17-22[Medline].
33.	Lee, L. Y., S. B. Gelvin, and C. I. Kado. 1999. pSa causes oncogenic suppression of Agrobacterium by inhibiting VirE2 protein export. J. Bacteriol. 181:186-196[Abstract/Free Full Text].
34.	Lessl, M., D. Balzer, W. Pansegrau, and E. Lanka. 1992. Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer. J. Biol. Chem. 267:20471-20480[Abstract/Free Full Text].
35.	Lessl, M., and E. Lanka. 1994. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell 77:321-324[Medline].
36.	Lund, A. H., M. Duch, and F. S. Pedersen. 1996. Increased cloning efficiency by temperature-cycle ligation. Nucleic Acids Res. 24:800-801[Free Full Text].
37.	McBride, K. E., and V. C. Knauf. 1988. Genetic analysis of the virE operon of the Agrobacterium Ti plasmid pTiA6. J. Bacteriol. 170:1430-1437[Abstract/Free Full Text].
38.	Narasimhulu, S. B., X. Deng, R. Sarria, and S. B. Gelvin. 1996. Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8:873-886[Abstract].
39.	Okamoto, S., A. Toyoda-Yamamoto, K. Ito, I. Takebe, and Y. Machida. 1991. Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane. Mol. Gen. Genet. 228:24-32[Medline].
40.	Otten, L., H. DeGreve, J. Leemans, R. Hain, P. J. J. Hooykaas, and J. Schell. 1984. Restoration of virulence of vir region mutants of Agrobacterium tumefaciens strain B653 by coinfection with normal and mutant Agrobacterium strains. Mol. Gen. Genet. 175:159-163.
41.	Peralta, E. G., R. Hellmiss, and W. Ream. 1986. Overdrive, a T-DNA transmission enhancer on the A. tumefaciens tumour-inducing plasmid. EMBO J. 5:1137-1142[Medline].
42.	Peralta, E. G., and L. W. Ream. 1985. T-DNA border sequences required for crown gall tumorigenesis. Proc. Natl. Acad. Sci. USA 82:5112-5116[Abstract/Free Full Text].
43.	Ream, W., and K. G. Field. 1999. Molecular biology techniques: an intensive laboratory course. Academic Press, San Diego, Calif
44.	Rihs, H. P., and R. Peters. 1989. Nuclear transport kinetics depend on phosphorylation-site-containing sequences flanking the karyophilic signal of the Simian virus 40 T-antigen. EMBO J. 8:1479-1484[Medline].
45.	Rossi, L., B. Hohn, and B. Tinland. 1996. Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 93:126-130[Abstract/Free Full Text].
46.	Segal, G., M. Purcell, and H. A. Shuman. 1998. Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome. Proc. Natl. Acad. Sci. USA 95:1669-1674[Abstract/Free Full Text].
47.	Segal, G., and H. A. Shuman. 1998. Intracellular multiplication and human macrophage killing by Legionella pneumophila are inhibited by conjugal components of IncQ plasmid RSF1010. Mol. Microbiol. 29:197-208.
48.	Sen, P., G. J. Pazour, D. Anderson, and A. Das. 1989. Cooperative binding of the VirE2 protein to single-stranded DNA. J. Bacteriol. 171:2573-2580[Abstract/Free Full Text].
49.	Shaw, C. H., M. D. Watson, G. H. Carter, and C. H. Shaw. 1984. The right hand copy of the nopaline Ti plasmid 25 bp repeat is required for tumour formation. Nucleic Acids Res. 12:6031-6041[Abstract/Free Full Text].
50.	Sheng, J., and V. Citovsky. 1996. Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. Plant Cell 8:1699-1710[Medline].
51.	Shurvinton, C. E., L. Hodges, and W. Ream. 1992. A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation. Proc. Natl. Acad. Sci. USA 89:11837-11841[Abstract/Free Full Text].
52.	Stachel, S. E., and E. W. Nester. 1986. The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J. 5:1445-1454[Medline].
53.	Stachel, S. E., B. Timmerman, and P. Zambryski. 1986. Generation of single-stranded T-DNA molecules during the initial stages of T-DNA transfer from Agrobacterium tumefaciens to plant cells. Nature 322:706-712.
54.	Stachel, S. E., B. Timmerman, and P. Zambryski. 1987. Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement of 5' virD gene products. EMBO J. 6:857-863[Medline].
55.	Stahl, L. E., A. Jacobs, and A. N. Binns. 1998. The conjugal intermediate of plasmid RSF1010 inhibits Agrobacterium tumefaciens virulence and VirB-dependent export of VirE2. J. Bacteriol. 180:3933-3939[Abstract/Free Full Text].
56.	Steck, T. R., and C. I. Kado. 1990. Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains. J. Bacteriol. 172:2191-2193[Abstract/Free Full Text].
57.	Sundberg, C., L. Meek, K. Carroll, A. Das, and W. Ream. 1996. VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells. J. Bacteriol. 178:1207-1212[Abstract/Free Full Text].
58.	Veluthambi, K., W. Ream, and S. B. Gelvin. 1988. Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens. J. Bacteriol. 170:1523-1532[Abstract/Free Full Text].
59.	Vogel, J. P., H. L. Andrews, S. K. Wong, and R. R. Isberg. 1998. Conjugative transfer by the virulence system of Legionella pneumophila. Science 279:873-876[Abstract/Free Full Text].
60.	Wang, K., L. Herrera-Estrella, M. Van Montagu, and P. Zambryski. 1984. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell 38:455-462[Medline].
61.	Winans, S. C., P. Allenza, S. E. Stachel, K. E. McBride, and E. W. Nester. 1987. Characterization of the virE operon of the Agrobacterium Ti plasmid pTiA6. Nucleic Acids Res. 15:825-838[Abstract/Free Full Text].
62.	Winans, S. C., D. L. Burns, and P. J. Christie. 1996. Adaptation of a conjugal transfer system for the export of pathogenic macromolecules. Trends Microbiol. 4:64-68[Medline].
63.	Yusibov, V. M., T. R. Steck, V. Gupta, and S. B. Gelvin. 1994. Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells. Proc. Natl. Acad. Sci. USA 91:2994-2998[Abstract/Free Full Text].
64.	Zupan, J., and P. Zambryski. 1997. The Agrobacterium DNA transfer complex. Crit. Rev. Plant Sci. 16:279-295.
65.	Zupan, J. R., V. Citovsky, and P. Zambryski. 1996. Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells. Proc. Natl. Acad. Sci. USA 93:2392-2397[Abstract/Free Full Text].