Abstract
In plants, stable expression is arguably the method of choice to test transgene function but it is a slow and labor-intensive process. This bottleneck generally limits the number of transgenes that can be tested, and as such hinders construct optimization. In the face of this challenge, transient expression in Nicotiana benthamiana leaves has emerged as a powerful screening platform to test gene expression, as well as subcellular distribution and function of many proteins within a week. This system relies on the infiltration of Agrobacterium tumefaciens (Agrobacterium) carrying DNA of interest into the leaf air spaces of N. benthamiana plants. Agrobacterium rapidly transforms the plant cells and the leaves can be analyzed within a few days. Investigating the subcellular localization of a protein of interest often relies on its fusion to a fluorescent tag. While the amount of accumulation of such fusion proteins can often be gauged by observing the fluorescence of the tag at the whole-leaf level, subcellular protein distribution is best determined in protoplasts extracted from transformed leaves. Here I present a simple and effective method to transform N. benthamiana leaves with Agrobacterium and to prepare protoplasts from these leaves to characterize the subcellular localization of proteins of interest.
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Acknowledgments
I would like to thank Rosemary White, Rob Allen and Sarah Covshoff for useful comments on the manuscript. This research was supported by Bill and Melinda Gates Foundation grant OPP1060461, titled “RIPE—Realizing increased photosynthetic efficiency for sustainable increases in crop yield”, sub-objective on “CCMs into chloroplasts” held by MR Badger and GD Price.
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Rolland, V. (2018). Determining the Subcellular Localization of Fluorescently Tagged Proteins Using Protoplasts Extracted from Transiently Transformed Nicotiana benthamiana Leaves. In: Covshoff, S. (eds) Photosynthesis. Methods in Molecular Biology, vol 1770. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7786-4_16
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DOI: https://doi.org/10.1007/978-1-4939-7786-4_16
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