Abstract
Several plant proteins function as intercellular messenger to specify cell fate and coordinate plant development. Such intercellular communication can be achieved by direct, selective, or nonselective (diffusion-based) trafficking through plasmodesmata (PD), the symplasmic membrane-lined nanochannels adjoining two cells. A trichome rescue trafficking assay was reported to allow the detection of protein movement in Arabidopsis leaf tissue using transgenic gene expression. Here, we provide a protocol to dissect the mode of intercellular protein movement in Arabidopsis root. This assay system involves a root ground tissue-specific GAL4/UAS transactivation expression system in combination with fluorescent reporter proteins. In this system, mCherry, a red fluorescent protein, can move cell to cell via diffusion, while mCherry-H2B is tightly cell autonomous. Thus, a protein fused to mCherry-H2B that can move out from the site of synthesis likely contains a selective trafficking signal to impart a cell-to-cell gain-of-trafficking function to the cell-autonomous mCherry-H2B. This approach can be adapted to investigate the cell-to-cell trafficking properties of any protein of interest.
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Acknowledgments
We thank Roger Tsien for providing the mCherry construct and Jim Haseloff for seeds of the J0571 plant line. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2013R1A1A2007230) and by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant PJ009495), Rural Development Administration, Republic of Korea.
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Kumar, D., Chen, H., Rim, Y., Kim, JY. (2015). GAL4 Transactivation-Based Assay for the Detection of Selective Intercellular Protein Movement. In: Heinlein, M. (eds) Plasmodesmata. Methods in Molecular Biology, vol 1217. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1523-1_15
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DOI: https://doi.org/10.1007/978-1-4939-1523-1_15
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