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The T-DNA portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid integrates into plant nuclear DNA. Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand. Subsequent events generate linear sngestranded VirD2-bound DNA molecules that include the entire T-DNA (T-strands). VirD2 protein contains a nuclear localization signal (NLS) near the C terminus and may direct bound T-strands to plant nuclei. We constructed mutations in virD2 and showed that the NLS was important for tumorigenesis, although T-strand production occurred normally in its absence. A tobacco etch virus NLS, substituted for the VirD2 NLS, restored tumor-inducing activity. Amino acids (the omega sequence) at the C terminus of VirD2, outside the NLS and the endonuclease domain, contributed s fcantly to tumorigenesis, suggesting that VirD2 may serve a third important function in T-DNA transfer.Agrobacterium tumefaciens causes crown gall tumors on many plants when the bacteria infect wounded tissue (1). The Ti plasmid carries genes essential for tumorigenesis. Transferred DNA (T-DNA) enters plant cells and integrates into nuclear DNA (2) where expression of certain T-DNA genes leads to tumorous growth (1). Virulence genes (vir) necessary for T-DNA transfer lie elsewhere on the Ti plasmid; wounded plants produce phenolic compounds that induce vir expression (3). T-DNA transmission requires, in cis, the right-hand 25-base-pair (bp) border sequence, and deletions removing it abolish tumorigenesis (4-6). Loss of a nearby sequence, called overdrive, reduces tumorigenesis several hundredfold (7). The endonuclease encoded by virDI and virD2 nicks the bottom strand of each border sequence at a specific site (8, 9), and VirD2 protein attaches covalently at the 5' end of the nicked DNA strand (10-14). Subsequent events displace linear single-stranded DNAs composed of the bottom strand of the T-DNA (T-strands) (15-17). VirE2 single-strand DNA binding protein binds cooperatively to T-strands (18-23). A. tumefaciens probably transfers T-DNA into plant cells via T-strand intermediates (24).VirD2 contains at least two functional domains. The N-terminal 262 amino acids of VirD2 [424 amino acids (aa) total] perform border nicking and attachment and suffice for T-strand production (10), but mutations at the C terminus abolish or severely attenuate tumorigenesis (25,26). The N-terminal endonuclease domains of VirD2 proteins from three different strains show 85% sequence conservation, but elsewhere they share only short stretches of similarity (27). Among the short conserved regions, two resemble the Xenopus nucleoplasmin nuclear localization signal (NLS) (Fig. 1C) (28), and one of them ( Fig. 1A) mediates nuclear transport when fused to 8-glucuronidase or /3-galactosidase and synthesized in tobacco cells (29,30). Proteins enter nuclei by ATP-dependent active transport through nuclear pores (31). Nuclear import de...

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an omega mutation. In addition, a mutant VirD2 protein lacking the omega domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the omega and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the omega domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG RP4 ) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG RP4 . A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG RP4 was expressed in the donors. Mutations in traG RP4 with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG RP4 nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG RP4 mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG RP4 -mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG RP4 and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.Plasmid conjugation conceptually can be divided into two functions. In the first, the DNA is processed by a complex of proteins, one of which introduces a single-strand nick at the nic site within the oriT recognition sequence. Called the relaxosome, the proteins of this complex are coded for by genes of the Dtr (DNA transfer and replication) component of the transfer system. In the second, the nucleoprotein transfer intermediate comprised of the nicked strand covalently linked at the 5Ј end to the relaxase is secreted from the donor directly into the recipient via a bridge that forms between the mating pair. This translocation apparatus is a complex membraneassociated structure coded for by the Mpf (mating pair formation) genes.The relaxosome of one conjugal plasmid may or may not be transferrable by the Mpf system of another. Specificity is conferred, in part, by a...

Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells. VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis. We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.Agrobacterium tumefaciens causes crown gall tumors on many dicotyledonous plant species when the bacteria infect wounded tissue (18). The tumor-inducing (Ti) plasmid carries genes essential for tumorigenesis (63,72). The transferred DNA (T-DNA) portion of the Ti plasmid enters plant cells and integrates into nuclear DNA (8,9,74), in which expression of certain T-DNA genes leads to tumorous growth (2,5,32,46,59). Virulence (vir) genes necessary for T-DNA transmission (transfer and integration) lie elsewhere on the Ti plasmid (24, 55), and wounded plants release phenolic compounds that induce vir expression (54).Border sequences define the T-DNA ends (42,43,48,65). T-DNA transfer begins when the VirD2 endonuclease nicks the right-hand border sequence (66, 78) and attaches to the 5Ј end of the nicked DNA strand (22,27,30,67,79). Strand displacement continues leftward (to the nicked left-hand border sequence), generating linear VirD2-bound T strands (3,33,56), which the bacteria appear to export into plant cells (58, 80). VirE2 single-stranded DNA-binding (SSB) protein (11-13, 16, 26, 47) may also accompany T strands into plant cells. Both VirE2 and VirD2 contain plant nuclear localization signals and probably play important roles inside infected plant cells (14,28,31,51,62).Export of DNA and proteins from A. tumefaciens into plant cells depends on membrane-associated proteins encoded by the virB operon (10,23,34,49,50,60,61,(68)(69)(70)(71)) (for reviews, see references 29 and 81) and the virD4 gene (33, 38). The VirB proteins are similar to pertussis toxin liberation (Ptl) proteins of Bordetella pertussis that mediate export of pertussis toxin (15,73) and to proteins that facilitate conjugal transfer of IncP␣ plasmid RP4 (Trb proteins) (35) and IncN plasmid pKM101 (Tra proteins) (44). VirD4 protein has similarity to TraG, another protein required for conjugal transfer of RP4 (35). Thus, Agrobacterium proteins essential for tumorigenesis appear to facilitate export of both proteins and DNA into plant cells by using pathways that operate in other bacteria.VirE2 plays an important role inside plant cells but not inside Agrobacterium cells. T strands (56), which are widely accepted as intermediates of T-DNA transfer (45,75,81), accumulate to wild-type levels in virE2 mutants (57, 64), showing that VirE2 does not stabilize T strands inside bacterial cells. In addition, virE2 mutants can transfer T strands into plant cells (80), albeit with unknown efficiency, proving that VirE2 is not essential for export of T strands. Transgenic tobacco plants that produce VirE2 protein are susceptible to transformation by a virE mutant (14), indicating that VirE2 is necessary inside plant cells.

Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants.

Agrobacterium tumefaciens transfers the T-DNA portion of its Ti plasmid to the nuclear genome of plant cells. Upon cocultivation of A. tumefaciens A348 with regenerating tobacco leaf protoplasts, six distinct single-stranded T-DNA molecules (T strands) were generated in addition to double-stranded T-DNA border cleavages which we have previously reported (K. Veluthambi, R.K. Jayaswal, and S.B. Gelvin, Proc. Natl. Acad. Sci. USA 84:1881-1885, 1987). The T region of an octopine-type Ti plasmid has four border repeats delimiting three T-DNA regions defined as T left (TL), T center (TC), and T right (TR). The six T strands generated upon induction corresponded to the TL, TC, TR, TL + TC, TC + TR, and TL + TC + TR regions, suggesting that the initiation and termination of T-strand synthesis can occur at each of the four borders. Most TL + TC + TR T-strand molecules corresponded to the top T-DNA strand, whereas the other five T strands corresponded to the bottom T-DNA strand. Generation of T strands required the virA, virG, and virD operons. Extra copies of vir genes, harbored on cosmids within derivatives of A. tumefaciens A348, enhanced production of T strands. The presence of right and left border repeats in their native orientation is important for the generation of full-length T strands. When a right border repeat was placed in the opposite orientation, single-stranded T-DNA molecules that corresponded to the top strand were generated. Deletion of overdrive, a sequence that flanks right border repeats and functions as a T-DNA transmission enhancer, reduced the level of T-strand generation. Induction of A. tumefaciens cells by regenerating tobacco protoplasts increased the copy number of the Ti plasmid relative to the bacterial chromosome.

Three global regulators are known to control antibiotic production by Pseudomonas fluorescens. A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production. A mutation inrpoS, which encodes the stationary-phase sigma factor ςS, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of P. fluorescens to survive exposure to oxidative stress. ThegacA gene of P. fluorescens Pf-5 was isolated, and the influence of gacS and gacA onrpoS transcription, ςS levels, and oxidative stress response of Pf-5 was determined. We selected a gacAmutant of Pf-5 that contained a single nucleotide substitution within a predicted α-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, ςS content in gacS and gacAmutants of Pf-5 was less than 20% of the wild-type level. Transcription of rpoS, assessed with anrpoS-lacZ transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase. Mutations ingacS and gacA compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress. The results of this study provide evidence for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in P. fluorescens.