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

Abstract

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.

VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals

“…2). This finding is consistent with an older report that the copy number of the Ti plasmid was increased upon cocultivation with tobacco protoplasts (30) and is considered further below.…”

Integration of T‐DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration

“…In this latter instance, however, the Ti-plasmid contained two sets of left and right borders, and the T-DNA presumably resulted from the integration of sequences extending from the leftmost border A to the right-most border D. Because of the likely polar generation and transport of the T-DNA, the tumor described by Ursic et al (1983) could result from T-DNA transfer initiating at the right border D, extending through and skipping borders C and B, and terminating at the left border A. Several groups have described T-strands in Agrobacterium cells induced with acetosyringone that extend between two T-DNA borders but encompass an intermediate border Veluthambi et al, 1988). We were, however, repeatedly unable to detect Tstrands containing gusA sequences in acetosyringoneinduced bacterial cells.…”

T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics

“…T-DNA regions beyond the border repeat were also found to be stably integrated into plant genomes at high frequencies. Read through of border sequences during T strand formation in the bacterium has been reported (Stachel et al 1987;Veluthambi et al 1988). Even DNA sequences of the vector, from far beyond the defined T-DNA region delimited by the border sequences have been detected.…”

“…A variety of problems are encountered while analyzing the T-DNA tagged lines, in particular while attempts are made to clone flanking sequences from a T-DNA tagged mutant. Difficulty can arise because of multiple insertions, complex arrangement of T-DNA, insertion of vector backbone sequences, chromosomal duplication and rearrangements or a combination of these (Jorgensen et al 1987, Veluthambi et al 1988, Tax and Vernon, 2001.…”