Variable effects of C-terminal tags on FLS2 function: not all epitope tags are created equal

Receptor-like kinases are the largest family of proteins in plants and are responsible for almost all extracellular perception events, such as sensing pathogen attacks, regulating symbiotic interactions, transducing hormone and peptide signalling, as well as monitoring cell wall status. Despite being of great importance to plant biology very few antibodies are available against receptor-like kinases. This necessitates fusing epitope tags to receptorlike kinases to enable the use of biochemical approaches such as western blotting. Cell biology applications also require the fusion of fluorescent proteins to receptor-like kinases to visualise their dynamics and distribution. As a result of their widespread use epitope tags are often assumed to be benign and have no impact on function. FLS2 is the receptor for bacterial flagellin and is often used as a model for receptor-like kinase function. Previous work implies that C-terminal epitope fusions to FLS2 are functional. However, a detailed complementation analysis of fls2 mutant plants expressing various FLS2 Cterminal epitope fusions shows that the signalling outputs of FLS2-mediated processes in these lines is highly variable and unpredictable. In addition, all but one FLS2 epitope fusion exhibited reduced ability to inhibit plant growth in response to flg22 treatment compared to wild type or control untagged transgenic lines. This raises concerns over the uncritical use of receptor-like kinase epitope tag fusion constructs for functional studies. Many of the subtleties of FLS2 function, and by extension that of other receptor-like kinases, may have been missed or inappropriately interpreted through the use of receptor-like kinase fusions.