Abstract
The sequence information generated through genome and transcriptome analysis from plant tissues has reached unprecedented sizes. Sequence homology-based annotations may provide hints for the possible function and roles of particular plant genes, but the functional annotation remains nonexistent or incomplete for many of them. To discover gene functions, transient expression assays are a valuable tool because they can be done more rapidly and at a higher scale than generating stably transformed tissues. Here, we describe a transient expression assay in protoplasts derived from suspension cells of tobacco (Nicotiana tabacum) for the study of the transactivation capacities of transcription factors. To enhance throughput and reproducibility, this method can be automated, allowing medium-throughput screening of interactions between large compendia of potential transcription factors and gene promoters.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lee TI, Young RA (2000) Transcription of eukaryotic protein-coding genes. Annu Rev Genet 34:77–137
De Sutter V, Vanderhaeghen R, Tilleman S, Lammertyn F, Vanhoutte I, Karimi M, Inzé D, Goossens A, Hilson P (2005) Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J 44:1065–1076
Wehner N, Hartmann L, Ehlert A, Böttner S, Oñate-Sánchez L, Dröge-Laser W (2011) High-throughput protoplast transactivation (PTA) system for the analysis of Arabidopsis transcription factor function. Plant J 68:560–569
Tiwari SB, Wang X-J, Hagen G, Guilfoyle TJ (2001) AUX/IAA proteins are active repressors, and their stability and activity are modulated by auxin. Plant Cell 13:2809–2822
Bharti K, von Koskull-Döring P, Bharti S, Kumar P, Tintschl-Körbitzer A, Treuter E, Nover L (2004) Tomato heat stress transcription factor HsfB1 represents a novel type of general transcription coactivator with a histone-like motif interacting with the plant CREB binding protein ortholog HAC1. Plant Cell 16:1521–1535
Pape S, Thurow C, Gatz C (2010) The Arabidopsis PR-1 promoter contains multiple integration sites for the coactivator NPR1 and the repressor SNI1. Plant Physiol 154:1805–1818
De Boer K, Tilleman S, Pauwels L, Vanden Bossche R, De Sutter V, Vanderhaeghen R, Hilson P, Hamill JD, Goossens A (2011) APETALA2/ETHYLENE RESPONSE FACTOR and basic helix-loop-helix tobacco transcription factors cooperatively mediate jasmonate-elicited nicotine biosynthesis. Plant J 66:1053–1065
Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Vanden Bossche, R., Demedts, B., Vanderhaeghen, R., Goossens, A. (2013). Transient Expression Assays in Tobacco Protoplasts. In: Goossens, A., Pauwels, L. (eds) Jasmonate Signaling. Methods in Molecular Biology, vol 1011. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-414-2_18
Download citation
DOI: https://doi.org/10.1007/978-1-62703-414-2_18
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-413-5
Online ISBN: 978-1-62703-414-2
eBook Packages: Springer Protocols