Site-Specific Nick in the T-DNA Border Sequence as a Result of Agrobacterium vir Gene Expression

Abstract

The T-DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products act in trans to mobilize the T-DNA element from the bacterial Ti plasmid. The T-DNA is bounded by 25-base pair direct repeat sequences, which are the only sequences on the element essential for transfer. Thus, specific reactions must occur at the border sites to generate a transferable T-DNA copy. The T-DNA border sequences were shown in this study to be specifically nicked after vir gene activation. Border nicks were detected on the bottom strand just after the third or fourth base (+/- one or two nucleotides) of the 25-base pair transferpromoting sequence. Naturally occurring and base-substituted derivatives of the 25-base pair sequences are effective substrates for acetosyringone-induced border cleavage, whereas derivatives carrying only the first 15 or last 19 base pairs of the 25-base pair sequence are not. Site-specific border cleavages occur within 12 hours after acetosyringone induction and probably represent an early step in the T-DNA transfer process.

The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights

“…In type IV secretion systems that transfer DNA, synthesis of the transfer intermediate is initiated by strand-speci®c nicks in particular sequences, oriT for conjugation and T-DNA right border for T-strand Wang et al, 1987). Four groups of transfer origins can be distinguished by sequence analysis (reviewed in Pansegrau and Lanka, 1996).…”

Three-dimensional Reconstruction of Agrobacterium VirE2 Protein with Single-stranded DNA

“…4,6, and 7). The tDNA itself does not carry any signals for its processing, transport, nuclear import, and integration; the only cis elements that define the tDNA region are specific 25-bp direct repeats at its left and right borders (14,15). For this reason primarily, tDNA transfer is not sequence-dependent, leading to the emergence of Agrobacterium as a powerful tool for modifications of plants both in the laboratory and in molecular plant breeding (16 -18).…”

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

“…Products of the vir (virulence) genes direct the processing of the T-DNA from the Ti-(tumor inducing) plasmid and its subsequent export from the bacterial cell. Two of the products of the virD locus, VirD1 and VirD2, nick the T-DNA between nucleotides 3 and 4 of the 25 bp directly repeated sequences (the T-DNA borders) that flank the T-DNA (AIbright eta/., 1987;Wang et al, 1987). VirD2 protein tightly associates with the 5' end of the T-DNA and presumably acts as a 'pilot protein' to lead the single-stranded T-DNA molecule (the 'T-strand') from the bacterial cell into the plant cell (Durrenberger eta/., 1989;Herrera-Estrella et al, 1988;Howard et a/., 1989;Ward and Barnes, 1988;Young and Nester, 1988).…”