Skip to main content
SpringerLink
Log in

Quantifying Plasmodesmatal Transport with an Improved GFP Movement Assay

  • Protocol
  • First Online:
Plasmodesmata

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2457))

Abstract

Plasmodesmata (PD) are membrane-lined channels that cross the cell wall to connect the cytosol of adjacent plant cells, permitting diverse cytosolic molecules to move between cells. PD are essential for plant multicellularity, and the regulation of PD transport contributes to metabolism, developmental patterning, abiotic stress responses, and pathogen defenses, which has sparked broad interest in PD among diverse plant biologists. Here, we present a straightforward method to reproducibly quantify changes in the rate of PD transport in leaves. Individual cells are transformed with Agrobacterium to express fluorescent proteins, which then move beyond the transformed cell via PD. Forty-eight to 72 h later, the extent of GFP movement is monitored by confocal fluorescence microscopy. This assay is versatile and may be combined with transient gene overexpression, virus-induced gene silencing, physiological treatments, or pharmaceutical treatments to test how PD transport responds to specific conditions. We expect that this improved method for monitoring PD transport in leaves will be broadly useful for plant biologists working in diverse fields.

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 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
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. Brunkard JO, Runkel AM, Zambryski PC (2015) The cytosol must flow: intercellular transport through plasmodesmata. Curr Opin Cell Biol 35:13–20

    Article  CAS  PubMed  Google Scholar 

  2. Paultre DSG, Gustin MP, Molnar A, Oparka KJ (2016) Lost in transit: long-distance trafficking and phloem unloading of protein signals in arabidopsis homografts. Plant Cell 28:2016–2025

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Reagan BC, Burch-Smith TM (2020) Viruses reveal the secrets of plasmodesmal cell biology. Mol Plant-Microbe Interact 33:26–39

    Article  CAS  PubMed  Google Scholar 

  4. Khang CH, Berruyer R, Giraldo MC et al (2010) Translocation of Magnaporthe oryzae effectors into rice cells and their subsequent cell-to-cell movement. Plant Cell 22:1388–1403

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Brunkard JO, Zambryski PC (2017) Plasmodesmata enable multicellularity: new insights into their evolution, biogenesis, and functions in development and immunity. Curr Opin Plant Biol 35:76–83

    Article  PubMed  Google Scholar 

  6. Cheval C, Faulkner C (2018) Plasmodesmal regulation during plant–pathogen interactions. New Phytol 217:62–67

    Article  PubMed  Google Scholar 

  7. Aung K, Kim P, Li Z et al (2020) Pathogenic bacteria target plant plasmodesmata to colonize and invade surrounding tissues. Plant Cell 32:595–611

    Article  CAS  PubMed  Google Scholar 

  8. Raven J (2005) Evolution of plasmodesmata. In: Oparka KJ (ed) Plasmodesmata. Blackwell, Oxford, pp 33–52

    Chapter  Google Scholar 

  9. Niklas KJ (2014) The evolutionary-developmental origins of multicellularity. Am J Bot 101:6–25

    Article  CAS  PubMed  Google Scholar 

  10. Crawford KM, Zambryski PC (2000) Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport. Curr Biol 10:1032–1040

    Article  CAS  PubMed  Google Scholar 

  11. Crawford KM, Zambryski PC (2001) Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states. Plant Physiol 125:1802–1812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Waigmann E, Zambryski P (1995) Tobacco mosaic virus movement protein-mediated protein transport between trichome cells. Plant Cell 7:2069–2079

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Atkins D, Hull R, Wells B et al (1991) The tobacco mosaic virus 30K movement protein in transgenic tobacco plants is localized to plasmodesmata. J Gen Virol 72:209–211

    Article  CAS  PubMed  Google Scholar 

  14. Yuan C, Lazarowitz SG, Citovsky V (2016) Identification of a functional plasmodesmal localization signal in a plant viral cell-to-cell-movement protein. MBio 7:e02052–e02015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Gallagher KL, Paquette AJ, Nakajima K, Benfey PN (2004) Mechanisms regulating SHORT-ROOT intercellular movement. Curr Biol 14:1847–1851

    Article  CAS  PubMed  Google Scholar 

  16. Koizumi K, Wu S, MacRae-Crerar A, Gallagher KL (2011) An essential protein that interacts with endosomes and promotes movement of the SHORT-ROOT transcription factor. Curr Biol 21:1559–1564

    Article  CAS  PubMed  Google Scholar 

  17. Koizumi K, Hayashi T, Wu S, Gallagher KL (2012) The SHORT-ROOT protein acts as a mobile, dose-dependent signal in patterning the ground tissue. Proc Natl Acad Sci U S A 109:13010–13015

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Tucker JE, Mauzerall D, Tucker EB (1989) Symplastic transport of carboxyfluorescein in staminal hairs of Setcreasea purpurea is diffusive and includes loss to the vacuole. Plant Physiol 90:1143–1147

    Article  CAS  PubMed  Google Scholar 

  19. Wright KM, Oparka KJ (1996) The fluorescent probe HPTS as a phloem-mobile, symplastic tracer: an evaluation using confocal laser scanning microscopy. J Exp Bot 47:439–445

    Article  CAS  Google Scholar 

  20. Faulkner C, Petutschnig E, Benitez-Alfonso Y et al (2013) LYM2-dependent chitin perception limits molecular flux via plasmodesmata. Proc Natl Acad Sci U S A 110:9166–9170

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Maule AJ, Gaudioso-Pedraza R, Benitez-Alfonso Y (2013) Callose deposition and symplastic connectivity are regulated prior to lateral root emergence. Commun Integr Biol 6:e26531

    Article  Google Scholar 

  22. Wang X, Sager R, Cui W et al (2013) Salicylic acid regulates plasmodesmata closure during innate immune responses in Arabidopsis. Plant Cell 25:2315–2329

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Brunkard JO, Zambryski P (2019) Plant cell-cell transport via plasmodesmata is regulated by light and the circadian CLOCK. Plant Physiol 181:1459–1467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Benitez-Alfonso Y, Cilia M, San Roman A et al (2009) Control of Arabidopsis meristem development by thioredoxin-dependent regulation of intercellular transport. Proc Natl Acad Sci U S A 106:3615–3620

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Stonebloom S, Brunkard JO, Cheung AC et al (2012) Redox states of plastids and mitochondria differentially regulate intercellular transport via plasmodesmata. Plant Physiol 158:190–199

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Burch-Smith TM, Brunkard JO, Choi YG, Zambryski PC (2011) Organelle-nucleus cross-talk regulates plant intercellular communication via plasmodesmata. Proc Natl Acad Sci U S A 108:E1451–E1460

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Brunkard JO, Runkel AM, Zambryski PC (2013) Plasmodesmata dynamics are coordinated by intracellular signaling pathways. Curr Opin Plant Biol 16:614–620

    Article  CAS  PubMed  Google Scholar 

  28. Brunkard JO, Burch-Smith TM (2018) Ties that bind: the integration of plastid signalling pathways in plant cell metabolism. Essays Biochem 62:95–107

    Article  PubMed  PubMed Central  Google Scholar 

  29. Ganusova EE, Reagan BC, Fernandez JC et al (2020) Chloroplast-to-nucleus retrograde signalling controls intercellular trafficking via plasmodesmata formation. Philos Trans R Soc B Biol Sci 375:20190408

    Article  CAS  Google Scholar 

  30. Bobik K, Fernandez JC, Hardin SR et al (2019) The essential chloroplast ribosomal protein uL15c interacts with the chloroplast RNA helicase ISE2 and affects intercellular trafficking through plasmodesmata. New Phytol 221:850–865

    Article  CAS  PubMed  Google Scholar 

  31. Azim MF, Burch-Smith TM (2020) Organelles-nucleus-plasmodesmata signaling (ONPS): an update on its roles in plant physiology, metabolism and stress responses. Curr Opin Plant Biol 58:48–59

    Article  CAS  PubMed  Google Scholar 

  32. Brunkard JO, Xu M, Regina Scarpin M et al (2020) TOR dynamically regulates plant cell-cell transport. Proc Natl Acad Sci U S A 117:5049–5058

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kumagai MH, Donson J, Della-Cioppa G et al (1995) Cytoplasmic inhibition of carotenoid biosynthesis with virus-derived RNA. Proc Natl Acad Sci U S A 92:1679–1683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Ratcliff F, Martin-Hernandez AM, Baulcombe DC (2001) Tobacco rattle virus as a vector for analysis of gene function by silencing. Plant J 25:237–245

    Article  CAS  PubMed  Google Scholar 

  35. Horner W, Brunkard JO (2021) Cytokinins stimulate plasmodesmatal transport in leaves. Front Plant Sci 12:674128

    Article  PubMed  PubMed Central  Google Scholar 

  36. Bombarely A, Rosli HG, Vrebalov J et al (2012) A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research. Mol Plant-Microbe Interact 25:1523–1530

    Article  CAS  PubMed  Google Scholar 

  37. Zipfel C, Kunze G, Chinchilla D et al (2006) Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation. Cell 125:749–760

    Article  CAS  PubMed  Google Scholar 

  38. Burch-Smith TM, Schiff M, Liu Y, Dinesh-Kumar SP (2006) Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol 142:21–27

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hellens R, Mullineaux P, Klee H (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451

    Article  CAS  PubMed  Google Scholar 

  40. Liu Y, Schiff M, Marathe R, Dinesh-Kumar SP (2002) Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus. Plant J 30:415–429

    Article  CAS  PubMed  Google Scholar 

  41. Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629

    Article  Google Scholar 

  42. Brunkard JO, Burch-Smith TM, Runkel AM, Zambryski P (2015) Investigating plasmodesmata genetics with virus-induced gene silencing and an agrobacterium-mediated GFP movement assay. In: Heinlein M (ed) Plasmodesmata, Methods in molecular biology (Methods and protocols), vol 1217. Humana Press, New York

    Google Scholar 

Download references

Acknowledgments

This work was supported by NIH grant DP5-OD023072 to J.O.B. We thank Dr. Tessa M. Burch-Smith, Dr. Anne M. Runkel, and Dr. Patricia Zambryski for their contributions to a previous version of this method [42] and Snigdha Chatterjee for constructive comments on the manuscript.

Author information

Authors and Affiliations

  1. Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI, USA

    Wilson Horner & Jacob O. Brunkard

  2. Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, CA, USA

    Wilson Horner & Jacob O. Brunkard

  3. Plant Gene Expression Center, U.S. Department of Agriculture, Agricultural Research Service, Albany, CA, USA

    Wilson Horner & Jacob O. Brunkard

Authors
  1. Wilson Horner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jacob O. Brunkard
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jacob O. Brunkard .

Editor information

Editors and Affiliations

  1. Centre for Plant Sciences, School of Biology, University of Leeds, Leeds, UK

    Yoselin Benitez-Alfonso

  2. Institut de Biologie Moléculaire des Plantes, CNRS, Université de Strasbourg, Strasbourg, France

    Manfred Heinlein

Rights and permissions

Reprints and permissions

Copyright information

© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Horner, W., Brunkard, J.O. (2022). Quantifying Plasmodesmatal Transport with an Improved GFP Movement Assay. In: Benitez-Alfonso, Y., Heinlein, M. (eds) Plasmodesmata. Methods in Molecular Biology, vol 2457. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2132-5_19

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-2132-5_19

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2131-8

  • Online ISBN: 978-1-0716-2132-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation