Whole-Seedling-Based Chemical Genetic Screens in Arabidopsis
Forward genetics has been extremely powerful for dissecting biological pathways in various model organisms. However, it is limited by the fact that redundant gene families and essential genes cannot be readily uncovered through such methods. Chemical gene
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Introduction Humanity has been benefiting from small molecules, such as natural products, for millennia. Although high-throughput screening (HTS) of small molecules is widely used in the pharmaceutical and biotechnology industry for drug discovery, its implementation for functional studies is more recent. Chemical genetic screens exploit small molecules to perturb gene functions in specific biological pathways [1]. Forward chemical genetics (phenotype-based screening) searches for compounds from diverse small-molecule libraries that can affect the phenotype of interest. Further identification of the molecular target of the specific molecule is usually necessary for revealing the mechanism of the chemical. In reverse chemical genetics (target-based screening), the target of interest (e.g., a protein) is often known. By screening for compounds that can bind to the target, the consequences and phenotypes caused by the small molecules can be determined (Fig. 1).
Glenn R. Hicks and Chunhua Zhang (eds.), Plant Chemical Genomics: Methods and Protocols, Methods in Molecular Biology, vol. 2213, https://doi.org/10.1007/978-1-0716-0954-5_3, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Shuai Huang and Xin Li
Fig. 1 Comparison of forward and reverse chemical genetics. (a) Phenotypebased forward chemical genetics. (b) Target-based reverse chemical genetics
Compared with classical genetics, chemical genetics enables antagonists with low selectivity to inhibit the function of redundant gene families. Chemicals targeting essential genes can also be used at lower concentrations to avoid lethality. In addition, chemicals can be added or removed at will, and HTS methods enable a large quantity of chemicals to be tested in a relatively short time period with the availability of liquid handling robots. Over the decades,
Whole Seedling Screens
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chemical genetics has shown its power in illustrating various biological processes in plants [2–5]. Using chemical genetics to study plant–microbe interactions and dissect plant immune pathways has also emerged in recent years [6–9]. Multiple chemical genetic screening protocols have been previously described in Arabidopsis with either reporter lines or cell cultures to study plant signaling pathways [6, 7, 10, 11]. Here, we describe a whole-seedling-based high-throughput chemical genetic screening method simply exploiting plant growth and developmental phenotype as the readout, which greatly simplifies and accelerates the general screening procedure. Using this approach, we successfully identified a small molecule involved in resistance protein-mediated plant immune responses in Arabidopsis [12]. Our protocol is easy to establish and should also be applicable to other plant biology fields.
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Materials 1. Murashige and Skoog (MS) medium, premixed. 2. 96-well culture plates. 3. Small-molecule libraries commercially available from different suppliers. 4. Ethyl methanesulfonate (EMS). 5. Agar. 6. Phytagel. 7. Sucrose. 8. Commercial bleach. 9. Tween-20.
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