Expression of Agrobacterium rhizogenes rolB gene fusions in Escherichia coli and production of antibodies against the RolB protein

Expression of the rolB gene of Agrobacterium rhizogenes TL-DNA is sufficient to trigger root differentiation in transformed plant cells. To investigate the role of RolB in differentiation, a large portion of rolB, comprising about 90% of its C-terminal coding sequence, was cloned into vectors pEX34 and pEA305 in frame with the truncated N termini of the pL-MS2 phage DNA polymerase and, respectively, the ptac-c Its phage lambda repressor gene. Hybrid proteins were expressed from both fusions and the one from pMTBEX1 was utilized to raise antibodies. These antibodies specifically recognize the RolB moiety in both pL-MS2-rolB and ptac-cI-rolB fusions. Unfused, complete RolB protein was obtained by in vitro translation in a rabbit reticulocyte system of a transcript obtained by in vitro transcription of rolB. RolB protein is specifically immunoprecipitated by the antibodies raised against the hybrid protein MS2-RolB.