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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...

Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2.Agrobacterium rhizogenes root-inducing (Ri) plasmids show many similarities to Agrobacterium tumefaciens tumor-inducing (Ti) plasmids, including nearly identical organizations of the vir operons (6,32,35,36,44,80). One notable exception to this rule is the absence of virE1 and virE2 from the Ri plasmid (and the rest of the genome) in some strains of A. rhizogenes (2,6,36,44). This raises an important question which is the subject of our paper. How can A. rhizogenes transfer DNA into plant cells efficiently when two critical virulence proteins are missing: the single-stranded (ss) DNA-binding protein VirE2 (10-12, 16, 19, 28, 56, 76) and its secretory chaperone, VirE1 (17, 66, 67, 85)? Our work shows that the GALLS protein encoded by the Ri plasmid can replace VirE2 and VirE1 and that the GALLS gene is essential for the virulence of A. rhizogenes strains that lack virE1 and virE2.

During crown gall tumorigenesis, part of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid, the T-DNA, integrates into plant DNA. Direct repeats define the left and right ends of the T-DNA, but tumorigenesis requires only the right-hand repeat. Virulence (vir) genes act in trans to mobilize the T-DNA into plant cells. Transfer of T-DNA begins when the VirD endonuclease cleaves within the right-hand border repeat. Although the T-DNA right-border repeat promotes T-DNA transmission best in its normal orientation, an inverted right border exhibits reduced but significant activity. Two models may account for this diminished tumorigenesis. The right border may function bidirectionally, with strong activity only in its wild-type orientation, or it may promote T-DNA transfer in a unidirectional manner such that, with an inverted right border, transfer proceeds around the entire Ti plasmid before reaching the T-DNA. To determine whether a substantial portion of the Ti plasmid is transferred to plant cells, as predicted by the unidirectional-transfer hypothesis, we examined T-DNAs in tumors induced by strains containing a Ti plasmid with a right border inverted with respect to the T-DNA oncogenes. These tumors contained extremely long T-DNAs corresponding to most or all of the Ti plasmid. To test whether the right border can function bidirectionally, we inserted T-DNAs with either a properly oriented or an inverted right border into a specific site in the A. tumefaciens chromosome. A border situated to transfer the oncogenes first directed T-DNA transfer even from the bacterial chromosome, whereas a border in the opposite (inverted) orientation did not transfer the oncogenes to plant cells. Our results indicate that the right-border repeat functions in a unidirectional manner.

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of singlestranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.