Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro

Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is inte grated into the genome. An in vitro ligation-integration assay has been des igned to investigate the mechanism of T-DNA Ligation and the factors involv ed in this process. The VirD2 protein, which is produced in Agrobacterium a nd is covalently attached to T-DNA, did not, under our assay conditions, li gate T-DNA to a model target sequence in vitro. We tested whether plant ext racts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presen ce of plant extracts. This reaction mas inhibited by dTTP, indicating invol vement of a plant DNA ligase. We found that prokaryotic DNA Ligases could s ubstitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less e fficient than ligation of a free oligonucleotide to the target. T-DNA ligat ion mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.