Fluorescent Protein Tagging of Arabidopsis MAPKs for In Vivo Localization Studies
Mitogen-activated protein kinases (MAPK) are key regulatory elements in many processes. They are highly conserved throughout eukaryotes. In plants, MAPKs are involved in biotic and abiotic stress responses; they regulate cell division, cell growth, and also programmed cell death. In vivo visualization of MAPKs is crucial for understanding of their spatiotemporal organization. Cloning of MAPK–fluorescent protein fusions might present difficulties related to the preservation of protein–protein interactions essential for MAPK localization, interactions with upstream and downstream regulators, and finally substrate targeting. In this chapter we describe cloning of MAPKs in the flexible MultiSite Gateway® cloning system followed by easy and quick testing of binary vectors by transient assays in Arabidopsis thaliana and Nicotiana benthamiana.
Key wordsMitogen activated protein kinase Multisite Gateway® cloning FAST transient transformation Green fluorescent protein GFP-Trap® immunoprecipitation Native promoter Floral dipping
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