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Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants

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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.

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Acknowledgments

We thank UF undergraduates Jennifer Cheeseman and Denys Ganyc for excellent technical assistance. We also thank Rob Ferl’s laboratory for help with the confocal microscope. This project was funded by a Grant to L. Ingram and WBG from the United States Defense Advanced Research Project Agency (DARPA) and Office of Naval Research.

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Authors and Affiliations

  1. 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

    Eva Czarnecka-Verner, Tarek A. Salem & William B. Gurley

  2. Molecular Biology Department, Genetic Engineering and Biotechnology Institute, University of Sadat City, Sadat City, Egypt

    Tarek A. Salem

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  1. Eva Czarnecka-Verner
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  2. Tarek A. Salem
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  3. William B. Gurley
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Correspondence to Eva Czarnecka-Verner or William B. Gurley.

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Supplemental Fig. 1

VirA-VirG two-component system of Agrobacterium tumefaciens. VirA is a membrane-imbedded sensor histidine kinase that detects phenolic compounds produced by wounded plant cells (Lee et al. 1995). Detection of acetosyringone ultimately induces a conformational change in VirA resulting in autophosphorylation that triggers a subsequent phosphor residue transfer from His474 of VirA to Asp52 of the VirG response regulator receiver domain (Jin et al. 1990a, 1990b; Lin et al. 2014). The details and identity of other proteins that may be involved in the sensing of phenolics and delivery of that signal to VirA are still unclear (hypothetical proteins X and Y) (Lee et al. 1992). The phosphorylation of VirG facilitates binding of VirG to the vir box promoter elements leading to increased transcription of the vir regulon (Tamamoto et al. 1990). ChvE interacts with the periplasmic domain of VirA and plays an auxiliary role in the induction by increasing the sensitivity of phenolic sensing by VirA (Cangelosi et al. 1990; Nair et al. 2011). The affinity of ChvE for VirA is modulated through ChvE binding to aldose monosaccharaides produced by the host plant (Hu et al. 2013)

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Czarnecka-Verner, E., Salem, T. & Gurley, W.B. Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants. Plant Mol Biol 90, 217–231 (2016). https://doi.org/10.1007/s11103-015-0407-x

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