Intracellular Ca2+ 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 Ca2+ signals is limited. Investigation of cell- and tissue-specific properties of Ca2+-dependent signaling processes requires versatile Ca2+ 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 Ca2+ signaling in living cells. In this study, we compared two fluorescence-based Ca2+ 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 Ca2+ signals on a cellular scale, which allowed identification of defined [Ca2+]cyt oscillations in epidermal and guard cells in response to the fungal elicitor chitin. Moreover, we discovered that flg22- and chitin-induced Ca2+ signals in the root initiate from the elongation zone.

Summary Pattern recognition receptors (PRRs) sense microbial patterns and activate innate immunity against attempted microbial invasions. The leucine‐rich repeat receptor kinases (LRR‐RK) FLS2 and EFR, and the LRR receptor protein (LRR‐RP) receptors RLP23 and RLP42, respectively, represent prototypical members of these two prominent and closely related PRR families. We conducted a survey of Arabidopsis thaliana immune signaling mediated by these receptors to address the question of commonalities and differences between LRR‐RK and LRR‐RP signaling. Quantitative differences in timing and amplitude were observed for several early immune responses, with RP‐mediated responses typically being slower and more prolonged than those mediated by RKs. Activation of RLP23, but not FLS2, induced the production of camalexin. Transcriptomic analysis revealed that RLP23‐regulated genes represent only a fraction of those genes differentially expressed upon FLS2 activation. Several positive and negative regulators of FLS2‐signaling play similar roles in RLP23 signaling. Intriguingly, the cytoplasmic receptor kinase BIK1, a positive regulator of RK signaling, acts as a negative regulator of RP‐type immune receptors in a manner dependent on BIK1 kinase activity. Our study unveiled unexpected differences in two closely related receptor systems and reports a new negative role of BIK1 in plant immunity.

Roots grow in highly dynamic and heterogeneous environments. Biological activity as well as uneven nutrient availability or localized stress factors result in diverse microenvironments. Plants adapt their root morphology in response to changing environmental conditions, yet it remains largely unknown to what extent developmental adaptations are based on systemic or cell-autonomous responses. We present the dual-flow-RootChip, a microfluidic platform for asymmetric perfusion of Arabidopsis roots to investigate root-environment interactions under simulated environmental heterogeneity. Applications range from investigating physiology, root hair development and calcium signalling upon selective exposure to environmental stresses to tracing molecular uptake, performing selective drug treatments and localized inoculations with microbes. Using the dual-flow-RootChip, we revealed cell-autonomous adaption of root hair development under asymmetric phosphate (Pi) perfusion, with unexpected repression in root hair growth on the side exposed to low Pi and rapid tip-growth upregulation when Pi concentrations increased. The asymmetric root environment further resulted in an asymmetric gene expression of RSL4, a key transcriptional regulator of root hair growth. Our findings demonstrate that roots possess the capability to locally adapt to heterogeneous conditions in their environment at the physiological and transcriptional levels. Being able to generate asymmetric microenvironments for roots will help further elucidate decision-making processes in root-environment interactions.

This protocol provides a detailed description of how to fabricate and use the dual-flow-RootChip (dfRootChip), a novel microfluidic platform for investigating root nutrition, root-microbe interactions and signaling and development in controlled asymmetric conditions. The dfRootChip was developed primarily to investigate how plants roots interact with their environment by simulating environmental heterogeneity. The goal of this protocol is to provide a detailed resource for researchers in the biological sciences wishing to employ the dfRootChip in particular, or microfluidic devices in general, in their laboratory.

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