A T-DNA transfer stimulator sequence in the vicinity of the right border of pRi8196

An 8 bp sequence repeated 6 times is present to the right of the mannopine type pRi8196 T-DNA right-border sequence. Experiments were designed to test whether these repeats have a role in T-DNA transfer. Several constructs in which different lengths of pRi8196 right-border region were linked to the cucumopine synthesis gene on an Agrobacterium-Escherichia coli shuttle vector were made. The recombinant plasmids were tested for their efficiency to act as a source of T-DNA in a binary system in which a wild-type Ri plasmid provided virulence and root-inducing functions. The T-DNA transfer efficiency of the constructs was assessed by computing the relative frequency of roots containing cucumopine. Depending on the Ri plasmid used as source of virulence functions, a high level of T-DNA transfer was observed only if 6 (pRi8196) or 5 (pRiA4) repeats were present. These results were confirmed by looking for single-stranded T-DNA molecules (T-strands) in bacteria induced for virulence. The repetition of the 8 bp unit was named 'T-DNA transfer stimulator sequence' (TSS).