Skip to main content

Transient Expression Assays in Tobacco Protoplasts

  • Protocol
  • First Online:
Book cover Jasmonate Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1011))

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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Lee TI, Young RA (2000) Transcription of eukaryotic protein-coding genes. Annu Rev Genet 34:77–137

    Article  PubMed  CAS  Google Scholar 

  2. 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

    Article  PubMed  Google Scholar 

  3. 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

    Article  PubMed  CAS  Google Scholar 

  4. 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

    PubMed  CAS  Google Scholar 

  5. 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

    Article  PubMed  CAS  Google Scholar 

  6. 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

    Article  PubMed  CAS  Google Scholar 

  7. 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

    Article  PubMed  Google Scholar 

  8. Karimi M, Inzé D, Depicker A (2002) GATEWAY™ vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints 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

Publish with us

Policies and ethics