Direct visualization of protein interactions in plant cells

Subramaniam, Rajagopal; Desveaux, Darrell; Spickler, Catherine; Michnick, Stephen W.; Brisson, Normand

doi:10.1038/90831

Cited by 131 publications

(77 citation statements)

References 24 publications

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“…This finding was consistent with the results of a recent study using a transient expression system in tobacco and tomato protoplasts (Subramaniam et al, 2001). The mechanism by which SA enhances NPR1-TGA2 complex formation is unknown.…”

Section: Discussionsupporting

confidence: 82%

In Vivo Interaction between NPR1 and Transcription Factor TGA2 Leads to Salicylic Acid–Mediated Gene Activation in Arabidopsis

2002

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The Arabidopsis NPR1 protein is a key regulator of salicylic acid (SA)-mediated gene expression in systemic acquired resistance. Based on yeast two-hybrid analysis, NPR1 has been suggested to interact with members of the TGA family of transcription factors, including TGA2 (AHBP-1b). However, genetic evidence demonstrating that the NPR1-TGA interaction occurs in planta is still lacking, and the role of this interaction in SA-mediated gene activation has yet to be determined. In this study, we expressed a truncated form of TGA2 in Arabidopsis and found that the resulting transgenic lines displayed phenotypes similar to those of npr1 mutants. This dominant-negative effect of the TGA2 mutant shows that TGA2 and NPR1 interact in planta. We also present biochemical evidence indicating that this interaction is specific and enhanced by SA treatment. Moreover, using a chimera reporter system, we found that a chimeric TGA2GAL4 transcription factor activated a UAS GAL :: GUS reporter gene in response to SA and that this activation was abolished in the npr1 mutant. NPR1 is required for the DNA binding activity of the transcription factor. These genetic data clearly demonstrate that TGA2 is a SA-responsive and NPR1-dependent transcription activator.

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Section: Discussionsupporting

confidence: 82%

In Vivo Interaction between NPR1 and Transcription Factor TGA2 Leads to Salicylic Acid–Mediated Gene Activation in Arabidopsis

2002

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“…NPR1 is localized in both the cytoplasm and the nucleus of unstimulated cells, but concentrates in the nucleus in response to SA and, in fact, nuclear localization of NPR1 is required for PR gene expression (Kinkema et al, 2000). Furthermore, using an in vivo protein fragment complementation assay, based on association of reconstituted murine dihydrofolate reductase (mDHFR) with a fluorescent probe to detect protein-protein interactions, it has been shown that the interaction between NPR1 and the bZIP factor TGA2 is itself induced by SA and localized predominantly in the nucleus (Subramaniam et al, 2001). Thus, the interaction of transcription factor(s) with NPR1 provides a link between an SAR-inducing agent, SA, and the changes in gene expression that are associated with SAR.…”

mentioning

confidence: 99%

Transcriptional Regulation: a Genomic Overview

Riechmann

2002

The Arabidopsis Book

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The availability of the Arabidopsis thaliana genome sequence allows a comprehensive analysis of transcriptional regulation in plants using novel genomic approaches and methodologies. Such a genomic view of transcription first necessitates the compilation of lists of elements. Transcription factors are the most numerous of the different types of proteins involved in transcription in eukaryotes, and the Arabidopsis genome codes for more than 1,500 of them, or approximately 6% of its total number of genes. A genome-wide comparison of transcription factors across the three eukaryotic kingdoms reveals the evolutionary generation of diversity in the components of the regulatory machinery of transcription. However, as illustrated by Arabidopsis, transcription in plants follows similar basic principles and logic to those in animals and fungi. A global view and understanding of transcription at a cellular and organismal level requires the characterization of the Arabidopsis transcriptome and promoterome, as well as of the interactome, the localizome, and the phenome of the proteins involved in transcription. Introduction. The Arabidopsis Book ©2002 American Society of Plant BiologistsFirst published on April 4, 2002 doi: 10.1199/tab.0085 : e0085. The Arabidopsis Book 2 of 46In addition, gene expression profiling technologies, such as DNA microarrays, allow monitoring transcription factor activity at a genome-wide level. These studies should eventually lead to an understanding of the interplay of the transcription factors with the genome whose expression they control.This chapter intends to provide a genomic perspective on transcriptional regulation in Arabidopsis. The first section briefly reviews the different types of proteins directly involved in transcription in eukaryotes, and our current understanding on how they function. The following sections consist of a description of the Arabidopsis complement of genes and proteins involved in transcriptional control, in particular sequence-specific DNA-binding transcription factors and chromatin-related proteins. Transcriptional regulators often act in a combinatorial fashion, and this mode of action is reviewed in the context of Arabidopsis promoters and cis-regulatory sequences, and of protein-protein interactions. Finally, genome-wide functional analyses of transcription factors, the characterization of the Arabidopsis promoterome, and of the transcriptome by gene expression profiling experiments, are considered. The availability of the genome sequence of different prokaryotic and eukaryotic organisms has provided for new ways of searching for unity and diversity among biological systems, and given birth to the field of comparative genomics. Although the subject of this book is Arabidopsis, reference is made in this chapter to other eukaryotic organisms, in order to situate the Arabidopsis genome information in a broader biological context. Transcription machinery: concepts, components, and mechanismsIn eukaryotic organisms, regulation of gene expression proceeds ...

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“…Further, such analysis is not limited to a specific cell type; we have already demonstrated the utility of PCA strategies in bacteria and mammalian cells and more recently in plant cells (Pelletier et al, 1998;Michnick, 1999, 2001;Subramaniam et al, 2001). We have demonstrated that pharmacological perturbations of interactions can be observed, even if the site of action of the perturbant is distant from the interaction being studied.…”

Section: Summary and Perspectivesmentioning

confidence: 97%

Dynamic visualization of expressed gene networks

Remy

Michnick

2003

Journal Cellular Physiology

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Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. While our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact living cells. The experimental strategy is based on protein fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, etc). In this review, we describe how we go from descriptive to quantitative representations of biochemical networks at an individual to whole genome level and how our approach will lead ultimately to better descriptions of the biochemical machineries that underlie living processes.

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Direct visualization of protein interactions in plant cells

Cited by 131 publications

References 24 publications

In Vivo Interaction between NPR1 and Transcription Factor TGA2 Leads to Salicylic Acid–Mediated Gene Activation in Arabidopsis

In Vivo Interaction between NPR1 and Transcription Factor TGA2 Leads to Salicylic Acid–Mediated Gene Activation in Arabidopsis

Transcriptional Regulation: a Genomic Overview

Dynamic visualization of expressed gene networks

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