Abstract
Transient expression in Arabidopsis thaliana seedlings allows fast expression of fluorescent markers for different subcellular compartments. This protocol describes a transient transformation assay with five-day-old seedlings using Agrobacterium tumefaciens-mediated vacuum infiltration. Three days after infiltration of the Agrobacterium containing an expression vector for a fluorescent marker of interest, cotyledon cells expressing the fluorescent protein can be imaged in a confocal microscope. This assay allows high-throughput screening of new constructs and the study of the localization of a large number of subcellular markers in Arabidopsis seedlings including wild-type, stable over-expressing and mutant lines.
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References
Hwang H-H, Yu M, Lai E-M (2017) Agrobacterium-mediated plant transformation: biology and applications. The Arabidopsis Book 15:e0186
Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure JD (2008) Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J 56:169–179
Li J-F, Park E, von Arnim AG, Nebenfüh A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6. https://doi.org/10.1186/1746-4811-5-6
Wu H-Y, Liu K-H, Wang Y-C, Wu J-F, Chiu W-L, Chen C-Y, Wu S-H, Sheen J, Lai E-M (2014) AGROBEST: an efficient Agrobacterium-mediated transient expression method for versatile gene function analyses in Arabidopsis seedlings. Plant Methods 10:19. https://doi.org/10.1186/1746-4811-10-19
Wang Y-C, Yu M, Shih P-Y, Wu H-Y, Lai E-M (2018) Stable pH suppresses defense signaling and is the key to enhance Agrobacterium-mediated transient expression in Arabidopsis seedlings. Sci Rep 8:1–9
Koncz C, Schell J (1986) The promoter of TL-DNA gene 5 controls the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396
Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature Protocols 1 (4):2019–2025
Montesinos JC, Langhans M, Sturm S, Hillmer S, Aniento F, Robinson DG, Marcote MJ (2013) Putative p24 complexes in Arabidopsis contain members of the delta and beta subfamilies and cycle in the early secretory pathway. J Exp Bot 64:3147–3167
Martinière A, Lavagi I, Nageswaran G et al (2012) Cell wall constrains lateral diffusion of plant plasma-membrane proteins. PNAS 109:12805–12810
Pereira C, Pereira S, Satiat-Jeunemaitre B, Pissarra J (2013) Cardosin A contains two vacuolar sorting signals using different vacuolar routes in tobacco epidermal cells. Plant J 76:87–100
Subramoni S, Nathoo N, Klimov E, Yuan Z-C (2014) Agrobacterium tumefaciens responses to plant-derived signaling molecules. Front Plant Sci 5:322. https://doi.org/10.3389/fpls.2014.00322
Acknowledgments
We thank the microscopy sections of Serveis Centrals de Suport a la Investigació Experimental, University of Valencia (SCSIE). F.A. was supported by the Ministerio de Economía y Competitividad (grant no BFU2016-76607P) and Generalitat Valenciana (ISIC/2013/004, GVACOMP2014-202). V.V. was supported by a Nigel Groome/Oxford Brookes University 150th Anniversary Studentship. C.B.-S. and J.S.-S. are recipients of a fellowship from the Ministerio de Ciencia (FPU program) and an EMBO short-term fellowship.
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Bernat-Silvestre, C., De Sousa Vieira, V., Sánchez-Simarro, J., Aniento, F., Marcote, M.J. (2021). Transient Transformation of A. thaliana Seedlings by Vacuum Infiltration. In: Sanchez-Serrano, J.J., Salinas, J. (eds) Arabidopsis Protocols . Methods in Molecular Biology, vol 2200. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0880-7_6
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DOI: https://doi.org/10.1007/978-1-0716-0880-7_6
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