The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA

Yamamoto, Akiko; Iwahashi, Motoko; Yanofsky, Martin F.; Nester, Eugene W.; Takebe, Itaru; Machida, Yasunori

doi:10.1007/bf00326554

Cited by 38 publications

(21 citation statements)

References 31 publications

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“…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 transferred into plant cells is not yet clearly established, since doublestranded linear (23,49) and circular (2,29,54) T-DNA molecules also have been identified as products of the cleavage reaction.…”

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

et al. 1988

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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...

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

et al. 1988

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“…This gene presumably encodes a transcriptional activator that induces its own expression and the expression of the genes 1i'rB, virC, virD, virE, and pinF (27,42,51). The virD locus encodes a T-DNA border-specific endonuclease (1,17,41,49,52,53) that nicks the T-DNA border repeat sequences on the bottom strand, generating single-stranded T-DNA molecules called T-strands (1,40,48). T-strands can be coated with a virE-encoded singlestranded-DNA-binding protein (6,8,11,15) and have been hypothesized to be the form of the T-DNA transferred to the plant cell (43).…”

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Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid

et al. 1989

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Upon incubation of Agrobacterium tumefaciens A348 with acetosyringone, the vir genes encoded by the Ti (tumor-inducing) plasmid are induced. The addition of certain opines, including octopine, nopaline, leucinopine, and succinamopine, enhanced this induction 2-to 10-fold. The compounds mannopine, acetopine, arginine, pyruvate, and leucine did not stimulate the induction of the vir genes to such an extent. The enhancement of vir gene induction by opines depended on acetosyringone and the genes virA and virG. Opines stimulated the activity of the vir genes, the double-stranded cleavage of the T (transferred)-DNA at the border repeat sequences, and the production of T-strands by the bacterium. The transformation efficiency of cotton shoot tips was markedly increased by the addition of acetosyringone and nopaline at the time of infection.Phenolic compounds secreted by wounded plant cells can induce the activity of certain genes encoded by the Ti (ttmor-inducing) plasmid of Agrobacteriiin tuimeficiens (3,37,39 (42, 51) for such phenolic inducers as acetosyringone (37). The virG locus, which is expressed at a low basal level, is subsequently induced to higher levels of expression (38). This gene presumably encodes a transcriptional activator that induces its own expression and the expression of the genes 1i'rB, virC, virD, virE, and pinF (27,42,51). The virD locus encodes a T-DNA border-specific endonuclease (1,17,41,49,52,53) that nicks the T-DNA border repeat sequences on the bottom strand, generating single-stranded T-DNA molecules called T-strands (1,40,48). T-strands can be coated with a virE-encoded singlestranded-DNA-binding protein (6,8,11,15) and have been hypothesized to be the form of the T-DNA transferred to the plant cell (43). The virD endonuclease also generates doublestranded scissions at the T-DNA borders (17,47,48). The regulation of the genes *irC and virD by the chromosomal locus ros has also been described (9,10,44).Following insertion into plant nuclear DNA, T-DNA genes are transcribed and direct the synthesis of enzymes involved in the production of phytohormones and unusual low-molecular-weight compounds termed opines (for reviews, see references 2, 16, and 31). The phytohormones stimulate the uncontrolled proliferation of plant cells, resulting in tumors called crown gall tumors. Opines, which are crown gall tumor-specific compounds, are secreted into the rhizosphere where they can be used as an energy source by the inciting bacterium to the exclusion of most other soil microorganisms. It (45,46).We are interested in the relationship, if any, between opines and vir gene induction. We initially hypothesized that opines may inhibit the induction of these genes by acetosyringone. The rationale was that the bacterium would not induce the T-DNA transfer machinery if it sensed that a tumor, recognized by the presence of opines, had already been formed. The results presented below suggest that many opines do affect the acetosyringone induction of the vir genes. The effect, however, is to enhance r...

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“…Several potential intermediates in T-DNA transmission accumulate in A. tumefaciens after induction of the vir genes. Various groups have reported the generation of circular forms of the T-DNA (2,17,20,49). Induction ofA.…”

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“…Recently, we reported that double-stranded cleavages occur at each of the T-DNA borders upon cocultivation of A. tumefaciens with regenerating tobacco protoplasts (43). All of these different reactions require virA, virG, and the first two open reading frames of the virD locus (2,13,37,43,49,50).…”

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Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens

1988

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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.

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The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA

Cited by 38 publications

References 31 publications

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

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

Opines stimulate induction of the vir genes of the Agrobacterium tumefaciens Ti plasmid

Virulence genes, borders, and overdrive generate single-stranded T-DNA molecules from the A6 Ti plasmid of Agrobacterium tumefaciens

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