Live Cell Imaging with R-GECO1 Sheds Light on flg22- and Chitin-Induced Transient [Ca(2+)]cyt Patterns in Arabidopsis

Intracellular Ca(2+) transients are an integral part of the signaling cascade during pathogen-associated molecular pattern (PAMP)-triggered immunity in plants. Yet, our knowledge about the spatial distribution of PAMP-induced Ca(2+) signals is limited. Investigation of cell- and tissue-specific properties of Ca(2+)-dependent signaling processes requires versatile Ca(2+) reporters that are able to extract spatial information from cellular and subcellular structures, as well as from whole tissues over time periods from seconds to hours. Fluorescence-based reporters cover both a broad spatial and temporal range, which makes them ideally suited to study Ca(2+) signaling in living cells. In this study, we compared two fluorescence-based Ca(2+) sensors: the Förster resonance energy transfer (FRET)-based reporter yellow cameleon NES-YC3.6 and the intensity-based sensor R-GECO1. We demonstrate that R-GECO1 exhibits a significantly increased signal change compared with ratiometric NES-YC3.6 in response to several stimuli. Due to its superior sensitivity, R-GECO1 is able to report flg22- and chitin-induced Ca(2+) signals on a cellular scale, which allowed identification of defined [Ca(2+)]cyt oscillations in epidermal and guard cells in response to the fungal elicitor chitin. Moreover, we discovered that flg22- and chitin-induced Ca(2+) signals in the root initiate from the elongation zone.