Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants
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.
Learn more about Dr. Gurley!
Department of Microbiology and Cell Science University of Florida
Ph. D. (1976) Department of Botany
University of Georgia, Athens Georgia
Postdoctoral: (1976-1979) Department of Plant Pathology
University of Wisconsin, Madison, Wisconsin
Plant and General Molecular Biology
PCB 4522, Molecular Genetics (Spring)
Description of Research
General areas: Transcriptional regulation in plants and other eukaryotes; the heatshock response, general transcription factors and upstream activator proteins; the basic mechanisms involved in activated transcription.
Research in my laboratory is focused on two main topics of study: one involving heat shock transcription factors, and the other, transcription factor IIB (TFIIB). All known organisms respond to a sudden elevation in temperature (ca. 10°C) by turning off most gene expression and activating a group of genes called “heat shock genes” that encode proteins that protect the cell against the deleterious effects of high temperature. The protein that regulates the induction of heat shock genes is the heat shock transcription factor (HSF). We have cloned 6 HSFs from soybean and 2 from Arabidopsis .Much to our surprise, only one of these transcription factors has been able to stimulate transcription when assayed in eitheryeast or plant cells.We now believe that we have isolated a novel class of repressor HSFs thatensure that heat shock genes are not expressed under non-heatshock conditions in plants. We are currently testing this hypothesisby expressing combinations of active HSFs and repressor HSFs in transient assays involving the introduction of the test genes (DNA) into plantcells by a variety of means including particle bombardment and PEG-mediated transformation of protoplasts prepared from leaf mesophyll cells.
Another objective of the heat shock research is to identify general transcription factors of the preinitiation complex that serve as targets of interaction for the HSFs. It is anticipated that identification of such targets will provide insights into the basic mechanisms of activated transcription in higher organisms and shed light on the process signal transduction, from the perception of environmental stress to activation of heat shock genes. We are conducting preliminary experiments using human HSFs since much more is known regarding the process of transcription in animal cells. We have identified regions of human HSF1 that make contact in vitro with several general transcription factors including TFIIA (smallsubunit), TFIIB, TAF55, TATA binding protein, and PC4 (a coactivator).This line of investigation will also be pursued in plants.
The second research area involves characterization of general transcription factors in plants including TBP, plant TAFs, and transcription factor IIB (TFIIB). TFIIB is a key member of the preinitiation complex and servestoattach the RNA polymerase II/TFIIF complex to the TATA binding protein which, in turn, is bound to the promoter DNA. There are several points of difference between plant TFIIB proteins and those from animals and yeast.The next step is to determine whether these differences interfere with function when TFIIBs from plants are mixed in vitro with the transcription machinery from fungal and metazoan cells. In our continuing efforts to study the fundamental mechanisms of activated transcription, we have begun to clone and characterize TBP associated factors (TAFs) from Arabidopsis . Antisense, T-DNA knockout, and RNAi approaches will be employedto deplete specific TAFs and determine the effects on global transcription.
Characterization of TBP associated proteins (TAFs) in plants: This project will assess the role of transcription factor TFIID and TATAA binding protein (TBP)-associated factors (TAFIIs) in the transcriptional expression of plant genes. The overall goal is to determine whether TFIID is required for the expression of most genes transcribed by RNA polymerase II or is needed for only a minor subset of genes. This issue is being addressed in animal and fungal systems, but almost no information is available regarding higher plants. Major objectives: 1) Clone and characterize the major TAFIIs from Arabidopsis. Function will be assessed by substitution of plant TAFIIs in yeast and by monitoring activity of tethered-TAFII activity in yeast and in plant cells using a double-site promoter. In addition, the pattern of TAFII mRNA and protein expression will be determined in various organs and at several developmental stages. 2) Evaluate the role of TFIID in global transcription. The major TAFIIs will be depleted either through RNAi (RNA interference) silencing of transformed Arabidopsis or using T-DNA knockout mutants. Transcriptional expression will be monitored by semi-quantitative RT-PCR and RNA profiling using oligonucleotide chip-based microarrays.3) Obtain antibodies to the AtTAFIIs and TBP to determine TFIID composition by coimmunoprecipitation with TBP. These results will not only confirm predictions regarding the identity of the cDNA clones, but immuno precipitation of TFIID complexes in TAFII-depleted lines will identify key TAFIIs required for TFIID stability. The identification and characterization of coactivator proteins is vital to understanding the pathways of activated transcription in all higher organisms. The cloning and characterization of Arabidopsis TAFIIsrepresents a logical first step in the study of co-adaptor complexesin plants. This information will not only provide added context tothe animal and yeast studies, but will generally facilitate the development of strategies to beneficially engineer plant gene expression. A detailed understanding of plant gene activation should facilitate the design of specifically tailored pathways for precise control of gene expressionand may contribute towards the development of high expression systems for the synthesis of novel gene products in plants such as peptidehormones or antibodies.
Arabidopsis TAFII225 as a ubiquitin domain protein: The proposed experiments bring together two fields of study that heretofore have had little overlap: transcriptional regulation and protein degradation. Analysis of genomic sequences for Arabidopsis TAFII225/205 indicate that these proteins contain an embedded ubiquitin domain (UbD) and a ubiquitin activating/conjugating domain (ubac). The experiments outlined here seek to clone the largest subunits of Arabidopsis TFIID, TAFII225/205, and characterize the role of the embedded ubiquitin domain. The main focus will be directed toward exploring the possible associationbetween TAFII225 and the proteasome under various conditions including progression through the cell cycle, heat stress, simulated pathogen-attack, and leaf senescence. Thesestudies will be conducted using Arabidopsis suspension cultures,transformed plants, tobacco BY-2 cells, and in vitro ubiquitinationreactions. The possibility that TFIID may be targeted for disassembly or degradation as a normal component of programmed cell death is especially intriguing since senescence and cell death play such an active rolein shaping the architecture of the plant throughout its vegetative and reproductive stages. These studies are the first to explore the possibility that ubiquitin-mediated processes may regulate the stability or activity of a general transcription factor.The identification and characterization co-adaptor complexes, such as the TAF subunits of TFIID, is vital to understanding the pathways of activated transcription in all higher organisms. The cloning and characterization of Arabidopsis TAFII225 will not only provide added context to the animal and yeast studies, but will generally facilitate the development of strategies to beneficially engineer plant gene expression. A detailed understanding of plant gene activation should facilitate the design of specifically tailored pathways for precise control of gene expression and may contribute towards the improvement of plant nutritional quality, or the development of high expression systems for the synthesis of novel gene products in plants such as peptide hormones or antibodies.
Title: A strategy for building an amplified transcriptional switch to detect bacterial contamination of plants
Author(s): Czarnecka, E (Czarnecka, Eva); Verner, FL (Verner, F. Lance); Gurley, WB (Gurley, William B.) Source: PLANT MOLECULAR BIOLOGY Volume: 78 Issue: 1-2 Pages: 59-75
Published: JAN 2012
Title: Parabolic Flight Induces Changes in Gene Expression Patterns in Arabidopsis thaliana
Author(s): Paul, AL (Paul, Anna-Lisa); Manak, MS (Manak, Michael S.); Mayfield, JD (Mayfield, John D.); Reyes, MF (Reyes, Matthew F.); Gurley, WB (Gurley, William B.); Ferl, RJ (Ferl, Robert J.)
Source: ASTROBIOLOGY Volume: 11 Issue: 8 Pages: 743-758 Published: OCT 2011
Lawit, SJ; O’Grady, K; Gurley, WB; Czarnecka-Verner, E. 2007. Yeast two-hybrid map of Arabidopsis TFIID. PLANT MOLECULAR BIOLOGY 64 (1-2): 73-87
Friedberg, JN; Bowley, SR; McKersie, BD; Gurley, WB; Czarnecka-Verner, E. 2006. Isolation and characterization of class A4 heat shock transcription factor from alfalfa. PLANT SCIENCE 171 (3): 332-344
Paul, AL; Popp, MP; Gurley, WB; Guy, C; Norwood, KL; Ferl, RJ. 2005. Arabidopsis gene expression patterns are altered during spaceflight. SPACE LIFE SCIENCES: GRAVITY-RELATED EFFECTS ON PLANTS AND SPACEFLIGHT AND MAN-MADE ENVIRONMENTS ON BIOLOGICAL SYSTEMS 36 (7): 1175-1181, Sp. Iss. 2005. edited by Hasenstein, KH; Levine, H; Porterfield, DM
Sehnke, PC; Laughner, BJ; Linebarger, CRL; Gurley, WB; Ferl, RJ. 2005. Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors. CELL RESEARCH 15 (8): 567-575
Czarnecka-Verner, E; Pan, SQ; Salem, T; Gurley, WB. 2004. Plant class BHSFs inhibit transcription and exhibit affinity for TFIIB and TBP. PLANT MOLECULAR BIOLOGY 56 (1): 57-75
Czarnecka-Verner, E; Yuan, CX; Scharf, KD; Englich, G; Gurley, WB. 2000. Plants contain a novel multi-member class of heat shock factors without transcriptional activator potential. PLANT MOLECULAR BIOLOGY 43 (4): 459-471.
Yuan, CX; Gurley, WB. 2000. Potential targets for HSF1 within the preinitiation complex. CELL STRESS & CHAPERONES 5 (3): 229-242.
Pan, SQ; Czarnecka-Verner, E; Gurley, WB. 2000. Role of the TATA binding protein-transcription factor IIB interaction in supporting basal and activated transcription in plant cells. PLANT CELL 12 (1): 125-135.
Pan, SQ; Sehnke, PC; Ferl, RJ; Gurley, WB. 1999. Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex. PLANT CELL 11 (8): 1591-1602.
Yuan, CX; Czarnecka-Verner, E; Gurley, WB. 1997. Expression of human heat shock transcription factors 1 and 2 in HeLa cells and yeast. CELL STRESS & CHAPERONES 2 (4): 263-275.
Czarnecka-Verner, E., Yuan, C.-X., Scharf, K.-D., Englich, K.-D. andGurley, W. B. (2000) Plants contain a novel multi-member class of heat shock transcription factors without activator potential, Plant Mol.Biol. 43: 459-471.
Czarnecka-Verner, E., Pan, S., Yuan, C. -X., and Gurley, W.B. (2000). Functional specialization of Plant Class A and B HSFs, In: Plant Tolerance to Abiotic Stresses in Agriculture: Role of Genetic Engineering, J. H. Cherry, ed., Kluewer Academic Publishers, Netherlands, pp. 3-28.
Yuan, C. -X. and Gurley, W.B. (2000). Potential targets for HSF1 within the preinitiation complex, Cell Stress & Chaperones 5: 229-242.
Pan, S., Czarnecka, E. and Gurley, W.B. (1999). Role o fthe TBP-TFIIB interaction in supporting basal and activated transcription in plant cells, Plant Cell 12: 125-135.
Czarnecka, E. and Gurley, W.B. (1999). Plant heat shock transcription factors: divergence in structure and function, Biotechnologia 3:125-142.
Pan, S., Sehnke, P.C., Ferl, R.J. and Gurley, W.B. (1999). Specific interactions with TBP and TFIIB in vitro suggest that 14-3-3 proteins may participate in the regulation of transcription when part of a DNA binding complex, Plant Cell 11:1591-1602.
Nagao, R. and Gurley, W. B. (1999) Use of heat shock promoters to control gene expression in plants, In: Inducible Gene Expression in Plants, P. H. S. Reynolds (ed.), CAB International, pp. 97-126.
Czarnecka-Verner, E., Yuan, C. –X., Nover, L., Scharf, K. –D., English,G. and Gurley, W. B. (1998) Plant heat shock transcription factors: positiveand negative aspects of regulation. Acta Physiologiae Plantarum 19: 529-537.
Yuan, C. -X., Czarnecka-Verner, E. and Gurley, W. B. (1997). Expression of human heat shock transcription factors 1 and 2 in HeLa cells and yeast. CellStress and Chaperones 2:263-275.
Baldwin, D. A. and Gurley, W. B. (1996) Isolation and Characterization of cDNAs encoding transcription factor IIB from Arabidopsis andsoybean. Plant J. 10: 561-568.
Czarnecka-Verner, E., Yuan, C. -X., Fox, P. C. and Gurley, W. B. (1995) Isolation and characterization of six heat shock transcription factor cDNA clones from soybean. Plant Mol. Biol. 29: 37-51.