Skip to main content
SpringerLink
Log in

Transient Assays for the Analysis of miRNA Processing and Function

  • Protocol
  • First Online:
Plant MicroRNAs

Part of the book series: Methods in Molecular Biology ((MIMB,volume 592))

Abstract

Transient assays provide a convenient alternative to stable transformation. For small RNA analysis in plants, the most widely used method, commonly named agroinfiltration, makes use of Agrobacterium tumefaciens to deliver transgenes into leaf cells of Nicotiana benthamiana. Compared to the generation of stably transformed plants, agroinfiltration is more rapid, and samples can be analyzed a few days after inoculation. Agroinfiltration has been used successfully in many different applications, including the analysis of small RNAs. We describe here a protocol for analysis of miRNA processing using agroinfiltration of N. benthamiana leaves.

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

Access this chapter

Log in via an institution
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 139.00
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. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  2. Chung MH, Chen MK, Pan SM (2000) Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Res 9:471–476

    Article  CAS  PubMed  Google Scholar 

  3. Himber C, Dunoyer P, Moissiard G, Ritzenthaler C, Voinnet O (2003) Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J 22:4523–4533

    Article  CAS  PubMed  Google Scholar 

  4. Usharani KS, Periasamy M, Malathi VG (2006) Studies on the activity of a bidirectional promoter of Mungbean yellow mosaic India virus by agroinfiltration. Virus Res 119:154–162

    Article  CAS  PubMed  Google Scholar 

  5. Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551

    Article  CAS  PubMed  Google Scholar 

  6. Baumberger N, Tsai CH, Lie M, Havecker E, Baulcombe DC (2007) The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation. Curr Biol 17(18):1609–1614

    Article  CAS  PubMed  Google Scholar 

  7. Pazhouhandeh M, Dieterle M, Marrocco K, Lechner E, Berry B, Brault V, Hemmer O, Kretsch T, Richards KE, Genschik P, Ziegler-Graff V (2006) F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function. Proc Natl Acad Sci U S A 103:1994–1999

    Article  CAS  PubMed  Google Scholar 

  8. Hoffmann T, Kalinowski G, Schwab W (2006) RNAi-induced silencing of gene expression in strawberry fruit (Fragaria × ananassa) by agroinfiltration: a rapid assay for gene function analysis. Plant J 48:818–826

    Article  CAS  PubMed  Google Scholar 

  9. Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato and Arabidopsis. Plant Biotechnol J 3:259–273

    Article  CAS  PubMed  Google Scholar 

  10. Llave C, Kasschau KD, Carrington JC (2000) Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway. Proc Natl Acad Sci U S A 97:13401–13406

    Article  CAS  PubMed  Google Scholar 

  11. Wydro M, Kozubek E, Lehmann P (2006) Optimization of transient Agrobacterium-mediated gene expression system in leaves of Nicotiana benthamiana. Acta Biochim Pol 53:289–298

    CAS  PubMed  Google Scholar 

  12. Tzfira T, Citovsky V (2006) Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Curr Opin Biotechnol 17:147–154

    CAS  PubMed  Google Scholar 

  13. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev 67:16–37 table of contents

    Article  CAS  PubMed  Google Scholar 

  14. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42:819–832

    Article  CAS  PubMed  Google Scholar 

  15. Hartley JL, Temple GF, Brasch MA (2000) DNA cloning using in vitro site-specific recombination. Genome Res 10:1788–1795

    Article  CAS  PubMed  Google Scholar 

  16. Sambrook J, Russell DW (eds) (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  17. Palatnik JF, Allen E, Wu X, Schommer C, Schwab R, Carrington JC, Weigel D (2003) Control of leaf morphogenesis by microRNAs. Nature 425:257–263

    Article  CAS  PubMed  Google Scholar 

  18. Weigel D, Glazebrook J (eds) (2002) Arabid­opsis: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA

    Google Scholar 

  19. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A 74:5463–5467

    Article  CAS  PubMed  Google Scholar 

  20. Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Gleave AP, Laing WA (2005) Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1:13

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. Jia-Wei Wang for discussion and Heike Wollmann for discussion and suggestions in the preparation of this chapter. FFF is supported by DAAD. Work on small RNAs in the Weigel laboratory is supported by European Community FP6 IP SIROCCO (contract LSHG-CT-2006-037900) and by the Max Planck Society.

Author information

Authors and Affiliations

  1. Max Planck Institute for Developmental Biology, Tübingen, Germany

    Felipe F. de Felippes & Detlef Weigel

Authors
  1. Felipe F. de Felippes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Detlef Weigel
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Delaware Biotechnology Institute, University of Delaware, Innovation Way 15, Newark, 19711, U.S.A.

    Blake C. Meyers

  2. Delaware Biotechnology Institute, University of Delaware, Innovation Way 15, Newark, 19711, U.S.A.

    Pamela J. Green

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

de Felippes, F.F., Weigel, D. (2010). Transient Assays for the Analysis of miRNA Processing and Function. In: Meyers, B., Green, P. (eds) Plant MicroRNAs. Methods in Molecular Biology, vol 592. Humana Press. https://doi.org/10.1007/978-1-60327-005-2_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-005-2_17

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60327-004-5

  • Online ISBN: 978-1-60327-005-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation