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Wall-associated kinases (WAKs) are pectin receptors found in the plasma membranes of angiosperms. Pectin binding by WAKs is required for cell growth and expansion, as WAKs regulate turgor by inducing the transcription of vacuolar invertases. However WAKs also bind pectin fragments generated when the cell wall is damaged, and these pectin fragments, oligogalacturonic acids (OGs), elicit a stress response in the cell that is characterized by the generation of reactive oxygen species and the induction of transcription of genes involved in a pathogen response. This study attempts to characterize components of these WAK signaling pathways. This was accomplished by creating transgenic Arabidopsis plants expressing epitope-tagged versions of proteins previously shown to have increased phosphorylation in response to OGs. These proteins include WAK1, putative WAK co-receptors WAKL7 and WAKL16, a cytoplasmic kinase ROG1, and the putative scaffold proteins REM1.2 and REM1.3. The proteins were then investigated by visualizing subcellular localization via fluorescence confocal microscopy, investigating in vitro kinase activity, and performing immunoprecipitation of epitope-tagged proteins from plant tissues to identify protein-protein interactions. Transgenic plants were successfully generated expressing WAKL16-GFP, REM1.3-GFP, ROG1-GFP, and ROG1-His. In vitro localization experiments using transformed protoplasts suggested compartment or vesicle localization of WAK1-YFP, WAKL16-GFP, and REM1.3-GFP. The kinase activity of ROG1-His was investigated, but ROG1-His did not phosphorylate the artificial substrate myelin basic protein. Finally, immunoprecipitation revealed successful purification of REM1.3-GFP and WAKL16-GFP, and several unknown proteins co-precipitating with ROG1-His and WAKL16-GFP.
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