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.
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References
Brunkard JO, Runkel AM, Zambryski PC (2015) The cytosol must flow: intercellular transport through plasmodesmata. Curr Opin Cell Biol 35:13–20
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
Reagan BC, Burch-Smith TM (2020) Viruses reveal the secrets of plasmodesmal cell biology. Mol Plant-Microbe Interact 33:26–39
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
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
Cheval C, Faulkner C (2018) Plasmodesmal regulation during plant–pathogen interactions. New Phytol 217:62–67
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
Raven J (2005) Evolution of plasmodesmata. In: Oparka KJ (ed) Plasmodesmata. Blackwell, Oxford, pp 33–52
Niklas KJ (2014) The evolutionary-developmental origins of multicellularity. Am J Bot 101:6–25
Crawford KM, Zambryski PC (2000) Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport. Curr Biol 10:1032–1040
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
Waigmann E, Zambryski P (1995) Tobacco mosaic virus movement protein-mediated protein transport between trichome cells. Plant Cell 7:2069–2079
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
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
Gallagher KL, Paquette AJ, Nakajima K, Benfey PN (2004) Mechanisms regulating SHORT-ROOT intercellular movement. Curr Biol 14:1847–1851
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
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
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
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
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
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
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
Brunkard JO, Zambryski P (2019) Plant cell-cell transport via plasmodesmata is regulated by light and the circadian CLOCK. Plant Physiol 181:1459–1467
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
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
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
Brunkard JO, Runkel AM, Zambryski PC (2013) Plasmodesmata dynamics are coordinated by intracellular signaling pathways. Curr Opin Plant Biol 16:614–620
Brunkard JO, Burch-Smith TM (2018) Ties that bind: the integration of plastid signalling pathways in plant cell metabolism. Essays Biochem 62:95–107
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
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
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
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
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
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
Horner W, Brunkard JO (2021) Cytokinins stimulate plasmodesmatal transport in leaves. Front Plant Sci 12:674128
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
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
Burch-Smith TM, Schiff M, Liu Y, Dinesh-Kumar SP (2006) Efficient virus-induced gene silencing in Arabidopsis. Plant Physiol 142:21–27
Hellens R, Mullineaux P, Klee H (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451
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
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
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
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.
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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
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DOI: https://doi.org/10.1007/978-1-0716-2132-5_19
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