Bi-directional Dual-flow-RootChip for Physiological Analysis of Plant Primary Roots Under Asymmetric Perfusion of Stress Treatments.

Due to technical limitations, research to date has mainly focused on the role of abiotic and biotic stress-signalling molecules in the aerial organs of plants, including the whole shoot, stem, and leaves. Novel experimental platforms including the dual-flow-RootChip (dfRC), PlantChip, and RootArray have since expanded this to plant-root cell analysis. Based on microfluidic platforms for flow stream shaping and force sensing on tip-growing organisms, the dfRC has further been expanded into a bi-directional dual-flow-RootChip (bi-dfRC), incorporating a second adjacent pair of inlets/outlet, enabling bi-directional asymmetric perfusion of treatments towards plant roots (shoot-to-root or root-to-shoot). This protocol outlines, in detail, the design and use of the bi-dfRC platform. Plant culture on chip is combined with guided root growth and controlled exposure of the primary root to solute changes. The impact of surface treatment on root growth and defence signals can be tracked in response to abiotic and biotic stress or the combinatory effect of both. In particular, this protocol highlights the ability of the platform to culture a variety of plants, such as Arabidopsis thaliana, Nicotiana benthamiana, and Solanum lycopersicum, on chip. It demonstrates that by simply altering the dimensions of the bi-dfRC, a broad application basis to study desired plant species with varying primary root sizes under microfluidics is achieved. Key features Expansion of the method developed by Stanley et al. (2018a) to study the directionality of defence signals responding to localised treatments. Description of a microfluidic platform allowing culture of plants with primary roots up to 40 mm length, 550 µm width, and 500 µm height. Treatment with polyvinylpyrrolidone (PVP) to permanently retain the hydrophilicity of partially hydrophobic bi-dfRC microchannels, enabling use with surface-sensitive plant lines. Description of novel tubing array setup equipped with rotatable valves for switching treatment reagent and orientation, while live-imaging on the bi-dfRC. Graphical overview Graphical overview of bi-dfRC fabrication, plantlet culture, and setup for root physiological analysis.(a) Schematic diagram depicting photolithography and replica molding, to produce a PDMS device. (b) Schematic diagram depicting seed culture off chip, followed by sub-culture of 4-day-old plantlets on chip. (c) Schematic diagram depicting microscopy and imaging setup, equipped with a media delivery system for asymmetric treatment introduction into the bi-dfRC microchannel root physiological analysis under varying conditions.

Encoding, transmission, decoding, and specificity of calcium signals in plants.

Calcium acts as a signal and transmits information in all eukaryotes. Encoding machinery consisting of calcium channels, stores, buffers, and pumps can generate a variety of calcium transients in response to external stimuli, thus shaping the calcium signature. Mechanisms for the transmission of calcium signals have been described, and a large repertoire of calcium binding proteins exist that can decode calcium signatures into specific responses. Whilst straightforward as a concept, mysteries remain as to exactly how such information processing is biochemically implemented. Novel developments in imaging technology and genetically encoded sensors (such as calcium indicators), in particular for multi-signal detection, are delivering exciting new insights into intra- and intercellular calcium signaling. Here, we review recent advances in characterizing the encoding, transmission, and decoding mechanisms, with a focus on long-distance calcium signaling. We present technological advances and computational frameworks for studying the specificity of calcium signaling, highlight current gaps in our understanding and propose techniques and approaches for unravelling the underlying mechanisms.

A dual-flow RootChip enables quantification of bi-directional calcium signaling in primary roots.

One sentence summary: Bi-directional-dual-flow-RootChip to track calcium signatures in Arabidopsis primary roots responding to osmotic stress. Plant growth and survival is fundamentally linked with the ability to detect and respond to abiotic and biotic factors. Cytosolic free calcium (Ca2+) is a key messenger in signal transduction pathways associated with a variety of stresses, including mechanical, osmotic stress and the plants' innate immune system. These stresses trigger an increase in cytosolic Ca2+ and thus initiate a signal transduction cascade, contributing to plant stress adaptation. Here we combine fluorescent G-CaMP3 Arabidopsis thaliana sensor lines to visualise Ca2+ signals in the primary root of 9-day old plants with an optimised dual-flow RootChip (dfRC). The enhanced polydimethylsiloxane (PDMS) bi-directional-dual-flow-RootChip (bi-dfRC) reported here adds two adjacent inlet channels at the base of the observation chamber, allowing independent or asymmetric chemical stimulation at either the root differentiation zone or tip. Observations confirm distinct early spatio-temporal patterns of salinity (sodium chloride, NaCl) and drought (polyethylene glycol, PEG)-induced Ca2+ signals throughout different cell types dependent on the first contact site. Furthermore, we show that the primary signal always dissociates away from initially stimulated cells. The observed early signaling events induced by NaCl and PEG are surprisingly complex and differ from long-term changes in cytosolic Ca2+ reported in roots. Bi-dfRC microfluidic devices will provide a novel approach to challenge plant roots with different conditions simultaneously, while observing bi-directionality of signals. Future applications include combining the bi-dfRC with H2O2 and redox sensor lines to test root systemic signaling responses to biotic and abiotic factors.

Interior design: how plant pathogens optimize their living conditions.

Pathogens use effectors to suppress host defence mechanisms, promote the derivation of nutrients, and facilitate infection within the host plant. Much is now known about effectors that target biotic pathways, particularly those that interfere with plant innate immunity. By contrast, an understanding of how effectors manipulate nonimmunity pathways is only beginning to emerge. Here, we focus on exciting new insights into effectors that target abiotic stress adaptation pathways, tampering with key functions within the plant to promote colonization. We critically assess the role of various signalling agents in linking different pathways upon perturbation by pathogen effectors. Additionally, this review provides a summary of currently known bacterial, fungal, and oomycete pathogen effectors that induce biotic and abiotic stress responses in the plant, as a first step towards establishing a comprehensive picture for linking effector targets to pathogenic lifestyles.