Vir-induced recombination in Agrobacterium: Physical characterization of precise and imprecise T-circle formation

Abstract

Induction of Ti plasmid virulence ( vir) gene expression during the early stages of plant cell transformation by Agrobacterium tumefaciens initiates the generation of several T-DNA-associated molecular events: (1) site-specific nicks at T-DNA border sequences (border nicks); (2) free, unipolar, linear, single-stranded T-DNA copies (T-strands); and (3) double-stranded, circular T-DNA molecules (T-circles). The first two T-DNA products have been detected in A. tumefaciens, while T-circles have only been detected following Escherichia coli transformation or transduction. The relationship between the three events has not been evaluated since the genesis of T-circles in A. tumefaciens has not been clarified. Evidence is presented here that T-circles are not an artefact of E. coli transformation, but are present as free, double-stranded molecules in A. tumefaciens resulting from site-specific reciprocal recombination between the left and right 25-base-pair border sequences that flank the T-DNA. Furthermore, the frequency of T-circle formation correlates with the frequency of formation of its reciprocal product, the Ti plasmid deleted in the T-DNA region. Several types of recombinant T-DNA circles arise after activation of vir gene expression, a major class representing precise site-specific recombination between both T-DNA borders, and a minor class representing recombination events either utilizing only one T-DNA border sequence and other Ti plasmid sequences, or utilizing only Ti plasmid sequences (i.e. no T-DNA borders). Nucleotide sequence analyses show that when one (nicked) border recombines with other Ti plasmid sequences, a small stretch (16 to 17 base-pairs) of local homology suffices to allow crossing over.