Abstract
The protoplast transient expression system has become a powerful and popular tool for studying molecular mechanisms underlying various plant signal transduction pathways. Arabidopsis mesophyll protoplasts display intact and active physiological responses and are easy to isolate and transfect, which facilitate high-throughput screening and systematic and genome-wide characterization of gene functions. The system is suitable for most Arabidopsis accessions and mutant plants. Genetic complementation of mutant defective in sensor functions, gene expression, enzymatic activities, protein interactions, and protein trafficking can be easily designed and explored in cell-based assays. Here, we describe the detailed protocols for protoplast isolation, polyethylene glycol-calcium transfection, and different assays for quantifying the output of various signaling pathways.
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Acknowledgments
We thank the present and past members of the Sheen Laboratory for their efforts to set up and improve the Arabidopsis mesophyll protoplasts transient expression system. We gratefully acknowledge Dr. Guillaume Tena, Dr. Kun-Hsiang Liu, Dr. Yan Xiong, Dr. Joonyup Kim, and Dr. Matthew Ramon for their technical help. We also would like to thank our greenhouse manager Jenifer Bush for providing plant material. This work was supported by NSF IOS 0618292, NIH R01 GM070567, and MGH-CCIB fund.
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Niu, Y., Sheen, J. (2011). Transient Expression Assays for Quantifying Signaling Output. In: Wang, ZY., Yang, Z. (eds) Plant Signalling Networks. Methods in Molecular Biology, vol 876. Humana Press. https://doi.org/10.1007/978-1-61779-809-2_16
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DOI: https://doi.org/10.1007/978-1-61779-809-2_16
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