Abstract
Receptor-like kinases (RLKs) are the largest family of proteins in plants and are responsible for perceiving the vast majority of extracellular stimuli. Thus, RLKs function in diverse processes, including sensing pathogen attacks, regulating symbiotic interactions, transducing hormone and peptide signals, and monitoring cell wall status. However, despite their fundamental role in plant biology, very few antibodies are available against RLKs, which necessitates the use of epitope tags and fluorescent protein fusions in biochemical analyses such as immunoblot analysis and intracellular visualization. Epitope tags are widely used and are typically assumed to be benign, with no influence on protein function. FLAGELLIN SENSITIVE2 (FLS2) is the receptor for bacterial flagellin and often is used as a model for RLK function. Previous work implies that carboxyl-terminal epitope fusions to FLS2 maintain protein function. Here, a detailed complementation analysis of Arabidopsis (Arabidopsis thaliana) fls2 mutant plants expressing various FLS2 C-terminal epitope fusions revealed highly variable and unpredictable FLS2-mediated signaling outputs. In addition, only one out of four FLS2 epitope fusions maintained the ability to inhibit plant growth in response to flg22 treatment comparable to that in the wild type or control untagged transgenic lines. These results raise concerns over the widespread use of RLK epitope tag fusions for functional studies. Many of the subtleties of FLS2 function, and by extension those of other RLKs, may have been overlooked or inappropriately interpreted through the use of RLK epitope tag fusions.
Highlights
Receptor-like kinases (RLKs) are the largest family of proteins in plants and are responsible for perceiving the vast majority of extracellular stimuli
During work to characterize the flg22-induced responses of various forms of FLAGELLIN SENSITIVE2 (FLS2), we consistently observed that all lines expressing FLS2-mGFP6 fusions exhibited greatly reduced MAPK activation compared with that in wildtype Columbia-0 (Col-0) plants
All generated epitope-tagged FLS2 transgenic lines were tested for FLS2 expression, and those lines showing a range of mRNA expression to control for expression level effects were selected for further study. fls2/FLS2pro:FLS2 control lines were selected to cover the range of mRNA expression observed between the tagged lines under investigation and Col-0 to control for expression-level effects (Fig. 1; Supplemental Fig. S3)
Summary
Receptor-like kinases (RLKs) are the largest family of proteins in plants and are responsible for perceiving the vast majority of extracellular stimuli. Receptor-like kinases (RLKs) form the largest gene family in plants (Shiu and Bleecker, 2001) and are the principal sensing mechanism for physical extracellular signals Their roles in governing processes such as plant-microbe interactions (Macho and Zipfel, 2014), cell wall integrity (Voxeur and Höfte, 2016), hormonal status (Belkhadir et al, 2014; Xu et al, 2014), peptide signaling (Hara et al, 2007), and developmental processes (Clark et al, 1997; Fisher and Turner, 2007) are well studied. We present complementation assays for FLS2 C-terminal fusions as a resource for the community and identify the best constructs to use in future work
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