Analysis of a plant transcriptional regulatory network using transient expression systems

Sara Díaz-Triviño, Yuchen Long, Ben Scheres, Ikram Blilou*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

4 Citations (Scopus)

Abstract

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6). Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR–SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

Original languageEnglish
Title of host publicationPlant gene regulatory networks
Subtitle of host publicationMethods and protocols
EditorsKerstin Kaufmann, Bernd Mueller-Roeber
PublisherHumana Press Inc.
Pages83-103
ISBN (Electronic)9781493971251
ISBN (Print)9781493971244
DOIs
Publication statusPublished - 2017

Publication series

NameMethods in Molecular Biology - Springer Protocols
Volume1629
ISSN (Print)1064-3745

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Keywords

  • Dual luciferase reporter assay
  • Effectors
  • Luciferase
  • Promoter activity
  • Transcriptional regulation
  • Transfection

Cite this

Díaz-Triviño, S., Long, Y., Scheres, B., & Blilou, I. (2017). Analysis of a plant transcriptional regulatory network using transient expression systems. In K. Kaufmann, & B. Mueller-Roeber (Eds.), Plant gene regulatory networks: Methods and protocols (pp. 83-103). (Methods in Molecular Biology - Springer Protocols; Vol. 1629). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7125-1_7