title: Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants.
abstract: The Agrobacterium tumefaciens VirG response regulator of the VirA/VirG two-component system was adapted to function in tobacco protoplasts. The subcellular localization of VirG and VirA proteins transiently expressed in onion cells was determined using GFP fusions. Preliminary studies using Gal4DBD-VP16 fusions with VirG and Escherichia coli UhpA, and NarL response regulators indicated compatibility of these bacterial proteins with the eukaryotic transcriptional apparatus. A strong transcriptional activator based on tandem activation domains from the Drosophila fushi tarazu and Herpes simplex VP16 was created. Selected configurations of the two-site Gal4-vir box GUS reporters were activated by chimeric effectors dependent on either the yeast Gal4 DNA-binding domain or that of VirG. Transcriptional induction of the GUS reporter was highest for the VirE19-element promoter with both constitutive and wild-type VirG-tandem activation domain effectors. Multiple VirE19 elements increased the reporter activity proportionately, indicating that the VirG DNA binding domain was functional in plants. The VirG constitutive-Q-VP16 effector was more active than the VirG wild-type. In both the constitutive and wild-type forms of VirG, Q-VP16 activated transcription of the GUS reporter best when located at the C-terminus, i.e. juxtaposed to the VirG DNA binding domain. These results demonstrate the possibility of using DNA binding domains from bacterial response regulators and their cognate binding elements in the engineering of plant gene expression.
authors.affiliation: Department of Microbiology and Cell Science, Program of Plant Molecular and Cellular Biology, University of Florida, Bldg. 981, 1355 Museum Drive, P.O. Box 110700, Gainesville, FL, 32611-0700, USA. evaczar@ufl.edu.
authors.forename: Eva
authors.initials: E
authors.lastname: Czarnecka-Verner
authors.affiliation: Department of Microbiology and Cell Science, Program of Plant Molecular and Cellular Biology, University of Florida, Bldg. 981, 1355 Museum Drive, P.O. Box 110700, Gainesville, FL, 32611-0700, USA.|Molecular Biology Department, Genetic Engineering and Biotechnology Institute, University of Sadat City, Sadat City, Egypt.
authors.forename: Tarek A
authors.initials: TA
authors.lastname: Salem
authors.affiliation: Department of Microbiology and Cell Science, Program of Plant Molecular and Cellular Biology, University of Florida, Bldg. 981, 1355 Museum Drive, P.O. Box 110700, Gainesville, FL, 32611-0700, USA. wgurley@ufl.edu.
authors.forename: William B
authors.initials: WB
authors.lastname: Gurley
mesh_terms: D016960:Agrobacterium tumefaciens / Q000378:metabolism* / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011523:Protoplasts / Q000378:metabolism / Q000382:microbiology; D014026:Nicotiana / Q000378:metabolism / Q000382:microbiology; D015533:Transcriptional Activation; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D037521:Virulence Factors
keywords: Bacterial response regulator; Fushi tarazu Q; Particle bombardment; Protoplasts; Synthetic transactivator; Two-component system; VP16