Role of the overdrive sequence in T-DNA border cleavage in Agrobacterium

The T-DNA of the Ti plasmid of Agrobacterium is flanked by 25-base-pair imperfect direct repeats that are required in cis for transfer to the genome of the plant host. Another sequence, designated overdrive, is located adjacent to the right-border repeats and functions in cis to enhance tumor formation. We have examined the effect of the overdrive sequence on the early steps in T-DNA processing. We report here that overdrive greatly enhances cleavage by the site-specific endonuclease in Agrobacterium, perhaps by directing the endonuclease to the adjacent border sequences. We also show by a gel mobility-shift assay that overdrive affinity-purified proteins from acetosyringone-induced Agrobacterium cells interact with T-DNA border and overdrive sequences. Further, we show that in vivo the virC operon enhances cleavage at the T-DNA borders, most likely by interaction between the VirC1 protein and the overdrive sequence.