Streptomyces-derived induction system for gene expression in cultured plant cells

Abstract

We have constructed an induction system for plant gene expression using an operator/repressor gene pair of Streptomyces virginiae. In this system, the repressor protein BarA dissociates from the operator sequence BARE in the presence of an inducer virginiae butanolide (VB), resulting in the induction of the transcription of the operator's downstream genes required for virginiamycin biosynthesis [Kinoshita et al., J. Bacteriol., 179, 6986-6993 ((1997))]. Two vectors were constructed: one was an effector plasmid, in which BarA was driven by plant promoters, and the other was a reporter plasmid, in which the BARE sequence was incorporated into the cauliflower mosaic virus 35S promoter to express the Escherichia coli beta-glucuronidase gene (GUS). An electroporation-mediated gene expression assay with cultured tobacco cells showed that GUS expression from the reporter plasmid was repressed upon coexpression with the effector plasmid and that the repression was relieved by VB. The result of electroporation to insert the reporter plasmid with various numbers and positions of BAREs into tobacco cells that had been transformed with the effector plasmid showed that the GUS induction by derepression increases with the number of BAREs and with BAREs downstream rather than upstream of the TATA box. Double transformants with the effector and reporter plasmids showed 30-fold induction with VB. The induction appeared within 8 h after VB addition, maximum induction being observed with 1 microM VB.