Plants must adapt to environmental constraints. For this, they are able to perceive several types of stress in isolation or in combination manner. At the cellular level, after the perception of stress, cell signaling is set up to allow the establishment of the specific response. The calcium ion is known to be one of the ubiquitous second messengers which is involved in most of the stresses perceived by the plant. Changes of free cytosolic calcium but also in other cellular compartments are able to activate or inactivate several mechanisms involved in the cell to cope with the changes of environmental conditions. Several calcium reporters have been intensively used to visualize calcium signals in different conditions. In this chapter, we will present only genetically encoded fluorescent reporters for calcium imaging in living plant tissues to measure variations in calcium at several scales. The FRET (fluorescence resonance energy transfer) YC3.60 and the intensiometric GCamP3 sensors will be used in this method chapter. The image analyses will be also detailed for fluorescence quantification of calcium variation.

Upon pathogen recognition, a transient rise in cytoplasmic calcium levels is one of the earliest events in plants and a prerequisite for defense initiation and signal propagation from a local site to systemic plant tissues. However, it is unclear if calcium signaling differs in the context of priming: Do plants exposed to a first pathogen stimulus and have consequently established systemic acquired resistance (SAR) display altered calcium responses to a second pathogen stimulus? Several calcium indicator systems including aequorin, YC3.6 or R-GECO1 have been used to document local calcium responses to the bacterial flg22 peptide but systemic calcium imaging within a single plant remains a technical challenge. Here, we report on an experimental approach to monitor flg22-induced calcium responses in systemic leaves of primed plants. The calcium-dependent protein kinase CPK5 is a key calcium sensor and regulator of the NADPH oxidase RBOHD and plays a role in the systemic calcium-ROS signal propagation. We therefore compared flg22-induced cytoplasmic calcium changes in Arabidopsis wild-type, cpk5 mutant and CPK5-overexpressing plants (exhibiting constitutive priming) by introgressing the calcium indicator R-GECO1-mTurquoise that allows internal normalization through mTurquoise fluorescence. Aequorin-based analyses were included for comparison. Based on the R-GECO1-mTurquoise data, CPK5-OE appears to reinforce an \"oscillatory-like\" Ca2+ signature in flg22-treated local tissues. However, no change was observed in the flg22-induced calcium response in the systemic tissues of plants that had been pre-challenged by a priming stimulus - neither in wild-type nor in cpk5 or CPK5-OE-lines. These data indicate that the mechanistic manifestation of a plant immune memory in distal plant parts required for enhanced pathogen resistance does not include changes in rapid calcium signaling upstream of CPK5 but rather relies on downstream defense responses.

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Genetically encoded Ca2+ indicators (GECIs) enable long-term monitoring of cellular and subcellular dynamics of this second messenger in response to environmental and developmental cues without relying on exogenous dyes. Continued development and optimization in GECIs, combined with advances in gene manipulation, offer new opportunities for investigating the mechanism of Ca2+ signaling in fungi, ranging from documenting Ca2+ signatures under diverse conditions and genetic backgrounds to evaluating how changes in Ca2+ signature impact calcium-binding proteins and subsequent cellular changes. Here, we attempted to express multi-color (green, yellow, blue, cyan, and red) circularly permuted fluorescent protein (FP)-based Ca2+ indicators driven by multiple fungal promoters in Fusarium oxysporum, F. graminearum, and Neurospora crassa. Several variants were successfully expressed, with GCaMP5G driven by the Magnaporthe oryzae ribosomal protein 27 and F. verticillioides elongation factor-1α gene promoters being optimal for F. graminearum and F. oxysporum, respectively. Transformants expressing GCaMP5G were compared with those expressing YC3.60, a ratiometric Cameleon Ca2+ indicator. Wild-type and three Ca2+ signaling mutants of F. graminearum expressing GCaMP5G exhibited improved signal-to-noise and increased temporal and spatial resolution and are also more amenable to studies involving multiple FPs compared to strains expressing YC3.60.

Nitrate-induced Ca2+ signaling is crucial for the primary nitrate response in plants. However, the molecular mechanism underlying the generation of the nitrate-specific calcium signature remains unknown. We report here that a cyclic nucleotide-gated channel (CNGC) protein, CNGC15, and the nitrate transceptor (NRT1.1) constitute a molecular switch that controls calcium influx depending on nitrate levels. The expression of CNGC15 is induced by nitrate, and its protein is localized at the plasma membrane after establishment of young seedlings. We found that disruption of CNGC15 results in the loss of the nitrate-induced Ca2+ signature (primary nitrate response) and retards root growth, reminiscent of the phenotype observed in the nrt1.1 mutant. We further showed that CNGC15 is an active Ca2+-permeable channel that physically interacts with the NRT1.1 protein in the plasma membrane. Importantly, we discovered that CNGC15-NRT1.1 interaction silences the channel activity of the heterocomplex, which dissociates upon a rise in nitrate levels, leading to reactivation of the CNGC15 channel. The dynamic interactions between CNGC15 and NRT1.1 therefore control the channel activity and Ca2+ influx in a nitrate-dependent manner. Our study reveals a new nutrient-sensing mechanism that utilizes a nutrient transceptor-channel complex assembly to couple nutrient status to a specific Ca2+ signature.

Calcium (Ca2+) is a universal second messenger involved in various cellular processes, leading to plant development and to biotic and abiotic stress responses. Intracellular variation in free Ca2+ concentration is among the earliest events following the plant perception of environmental change. These Ca2+ variations differ in their spatio-temporal properties according to the nature, strength and duration of the stimulus. However, their conversion into biological responses requires Ca2+ sensors for decoding and relaying. The occurrence in plants of calmodulin (CaM) but also of other sets of plant-specific Ca2+ sensors such as calmodulin-like proteins (CMLs), Ca2+-dependent protein kinases (CDPKs) and calcineurin B-like proteins (CBLs) indicate that plants possess specific tools and machineries to convert Ca2+ signals into appropriate responses. Here, we focus on recent progress made in monitoring the generation of Ca2+ signals at the whole plant or cell level and their long distance propagation during biotic interactions. The contribution of CaM/CMLs and CDPKs in plant immune responses mounted against bacteria, fungi, viruses and insects are also presented.

As calcium sensor relays in plants, calcineurin B-like (CBL) proteins provide an important contribution to decoding Ca2+ signatures elicited by a variety of abiotic stresses. Currently, it is well known that CBLs perceive and transmit the Ca2+ signals mainly to a group of serine/threonine protein kinases called CBL-interacting protein kinases (CIPKs). In this study, we report that the CBL10 member of this family has a novel interaction partner besides the CIPK proteins. Yeast two-hybrid screening with CBL10 as bait identified an Arabidopsis cDNA clone encoding a TOC34 protein, which is a member of the TOC (Translocon of the Outer membrane of the Chloroplasts) complex and possesses the GTPase activity. Further analyses showed that in addition to CBL10, CBL7 also interacts with TOC34 at much lower strength in the yeast two-hybrid system. However, the rest of the CBL family members failed to interact with TOC34. Bimolecular fluorescence complementation (BiFC) analysis verified that the CBL10-TOC34 interaction occurs at the outer membrane of chloroplasts in vivo. In addition, we also demonstrated that CBL10 physically associates with TOC34 in vitro, resulting in a significant decrease in the GTPase activity of the TOC34 protein. Taken together, our findings clearly indicate that a member of the CBL family, CBL10, can modulate not only the CIPK members but also TOC34, allowing the CBL family to relay the Ca2+ signals in more diverse ways than currently known.

Spatial and temporal changes of cytoplasmic calcium ions ([Ca(2+)]c), caused by external stimuli, are known as the Ca(2+) signature and presumably control cellular and developmental responses. Multiple types of ion channels, pumps, and transporters on plasma and organellar membranes modulate influx and efflux of Ca(2+) to and from the extracellular environment and internal Ca(2+) stores to form Ca(2+) signatures. Expression of a fluorescent protein-based Ca(2+) probe, Cameleon YC3.60, in Fusarium oxysporum enabled us to study how disruption of three Ca(2+) channel genes, including FoCCH1, FoMID1 and FoYVC1, affects Ca(2+) signature formation at polarized hyphal tips and whether specific changes in the Ca(2+) signature caused by these mutations are related to growth-related phenotypes. Resulting mutants displayed altered amplitude, interval, and duration of Ca(2+) pulses under various external Ca(2+) concentrations as well as changes in sporulation and growth. Loss of FoMID1 and FoCCH1, genes encoding putative plasma membrane channel proteins, had a major impact on Ca(2+) signatures and growth, while disruption of FoYVC1, which encodes a vacuolar channel, only subtly affected both traits. Results from our study provide new insights into the underpinning of Ca(2+) signaling in fungi and its role in controlling growth and also raise several new questions.