The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA

Abstract

Agrobacterium tumefaciens transfers T-DNA into the plant genome by a process mediated by Ti plasmidencoded vir genes. Cleavage at T-DNA border sequences by the VirD endonuclease generates linear, single-stranded T-DNA molecules. In the work described in this report, we used electrophoretic mobility shift assays to show that the purified virE2 gene product binds to single-stranded DNA. VirE2 protein associates with T-DNA as shown by immunoprecipitation studies with VirE2-specific antiserum. The VirE2 protein was detected primarily in the cytoplasm, but also in the inner and outer membrane and periplasmic fractions. Virulence of a virE2 mutant was restored by mixed infection with strains carrying an intact vir region, but not with virA, virB, virD, virE, or virG mutants or chvA, chvB, or exoC mutants. We propose that the VirE2 protein is involved in the processing of T-DNA and in T-strand protection during transfer to the plant cell.Agrobacterium tumefaciens causes crown gall disease in dicotyledenous plants by transferring a segment of DNA (T-DNA) from a resident (Ti) tumor-inducing plasmid into plant cells, where the DNA becomes covalently integrated into the plant nuclear genome (for recent reviews, see references 28a, 35, and 47; see also A. Powell and M. P. Gordon, in S. Marcuo, ed., Biochemistry of Plants: a Comprehensive Treatise, vol. II, in press). The T-DNA then directs the synthesis of the plant growth hormones auxin and cytokinin, resulting in unregulated proliferation of plant tissue (35). Transfer of T-DNA is mediated by several trans-acting virulence loci, including at least three chromosomal loci involved in the initial attachment of the bacterium to the plant cell (8,11,12) and loci within the vir region, a ca. 35-kilobase (kb) region of the Ti plasmid outside of the T-DNA (15,27,43). Mutations in six operons within the vir region either abolish virulence (virA, virB, virD, and virG) or result in a sharply attenuated virulence phenotype (virC and virE) (21,27,43,55).Recently, workers in several laboratories have begun to elucidate the roles of vir proteins in the transfer process. Specific plant signals (4, 42) and the products of virA and virG are required for induction of transcription of the other vir operons (46,52). Products of the first two genes of the virD operon make up an endonuclease which recognizes and cleaves at specific sites within 24-base-pair (bp) border sequences that functionally define the boundaries of T-DNA (23,45,49,56). A strand-specific RNase protection assay (1, 56) and Southern hybridization studies (44, 45) have shown that a product of the cleavage event is a linear, singlestranded T-DNA molecule (T-strand) that corresponds to the bottom strand of T-DNA. One model for T-DNA processing is that initial border sequence cleavage by the virD endonuclease is followed by a replication event, which results in replacement synthesis of the bottom strand of T-DNA and the concomitant displacement of a T-strand molecule (1, 44). However, the structure of T-DNA molecules transferre...

Agrobacterium tumefaciens VirB11 protein requires a consensus nucleotide-binding site for function in virulence

“…The cells were passaged twice through a French pressure cell, and cell debris was removed by centrifugation (11). The cell lysate was applied to discontinuous sucrose gradients and processed as previously described (11,13). The fractions were analyzed to detect NADH oxidase, an inner membrane marker, and malate dehydrogenase, a cytoplasmic marker, by published procedures (13,27).…”

“…Strains of A. tumefaciens were assayed for virulence on Kalanchöe diagremontiana by inoculating 10 l of bacteria on three consecutive leaves of young plants which had been uniformly wounded (11). Bacteria were diluted into sterile H 2 O such that 10 l contained ϳ10 8 CFU (no dilution).…”

“…MgCl 2 was added to 5 mM, RNase A was added to 100 g/ml, and DNase I was added to 12.5 g/ml. The cells were passaged twice through a French pressure cell, and cell debris was removed by centrifugation (11). The cell lysate was applied to discontinuous sucrose gradients and processed as previously described (11,13).…”

Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins

“…Table 1 lists the bacterial strains and plasmids used in this study and their relevant characteristics. Growth of E. coli and A. tumefaciens cells was performed as described previously (14). For analysis of Vir protein content, cells were grown to an optical density at 600 nm (OD 600 ) of 0.5 in MG/L medium (22) with antibiotic selection.…”

“…This list includes DNA apparently of any length as long as it contains a recognition site for processing by the VirD2 endonuclease (34), the non-self-transmissible IncQ plasmid RSF1010, most likely also as a single-stranded DNA-protein particle (4, 9, 52), the VirE2 single-stranded DNA-binding protein (7,14,35), and VirF, which functions as an important virulence factor for infection of certain plant species (40). Second, recent studies have shown that the T-complex transporter delivers DNA to many dicotyledonous and monocotyledonous plant species as well as to agrobacteria (3,25) and yeast (10,11,36) recipient cells.…”