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Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems

Part of the Methods in Molecular Biology book series (MIMB,volume 1629)

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.

Key words

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

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Acknowledgments

We are grateful to Dr Erin Spark, Dr Wenkun Zhou, and Jorge Zamora Zaragoza for their helpful comments on this chapter.

pEGB-35S:Renilla:Tnos vector for constitutive rLUC expression was kindly provided by Prof. Smeekens Laboratory, Utrecht University, the Netherlands. pGreen vectors containing fLUC for promoters assays and constitutive rLUC were kindly provided by Dr. Rumyana Karlova, Wageningen University and Research Centre, the Netherlands. Plasmid for constitutive rLUC expression p-CMV-Renilla and mammalian protocol was kindly provided by Dr. Isabel Sanchez Perez Department of Biochemistry; School of Medicine; Biomedical Research Institute of Madrid CSIC/UAM; Madrid, Spain.

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Correspondence to Ikram Blilou .

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Díaz-Triviño, S., Long, Y., Scheres, B., Blilou, I. (2017). Analysis of a Plant Transcriptional Regulatory Network Using Transient Expression Systems. In: Kaufmann, K., Mueller-Roeber, B. (eds) Plant Gene Regulatory Networks. Methods in Molecular Biology, vol 1629. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7125-1_7

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  • DOI: https://doi.org/10.1007/978-1-4939-7125-1_7

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