Sequence characterization of the vir region of a nopaline type Ti plasmid, pTi-SAKURA.

We isolated a crown gall tumor-inducing nopaline type Ti plasmid from Agrobacterium tumefaciens on a Sakura Japanese cherry tree, and designated it as pTi-SAKURA. By primer walking sequencing with long PCR and a newly developed PCR subcloning technique for long insert DNA, we completed DNA sequencing of the most important functional unit, the virulence (vir) region of pTi-SAKURA, which is indispensable for T-DNA transfer into the plant's chromosomes. By homology searches with the vir genes of other bacterial plasmids, we identified 11 open reading frames (orfs) and 31 genes and 11 vir box, which are 6 bp regulatory sequences. In total, 26 vir genes, including the putative virF and virK and the main vir region, were present as the vir gene cluster. The presence of vir box, GC content, codon usage and expression analysis in these genes led us to propose a new vir region.