Ca 2+ signals in plant immunity

Review13 May 2022Open Access Ca2+ signals in plant immunity Philipp Köster Philipp Köster orcid.org/0000-0002-1359-822X Institute of Plant and Microbial Biology and Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland Contribution: Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Thomas A DeFalco Thomas A DeFalco orcid.org/0000-0003-2897-1485 Institute of Plant and Microbial Biology and Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland Contribution: Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Cyril Zipfel Corresponding Author Cyril Zipfel [email protected] orcid.org/0000-0003-4935-8583 Institute of Plant and Microbial Biology and Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland The Sainsbury Laboratory, University of East Anglia, Norwich, UK Contribution: Funding acquisition, Writing - review & editing Search for more papers by this author Philipp Köster Philipp Köster orcid.org/0000-0002-1359-822X Institute of Plant and Microbial Biology and Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland Contribution: Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Thomas A DeFalco Thomas A DeFalco orcid.org/0000-0003-2897-1485 Institute of Plant and Microbial Biology and Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland Contribution: Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Cyril Zipfel Corresponding Author Cyril Zipfel [email protected] orcid.org/0000-0003-4935-8583 Institute of Plant and Microbial Biology and Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland The Sainsbury Laboratory, University of East Anglia, Norwich, UK Contribution: Funding acquisition, Writing - review & editing Search for more papers by this author Author Information Philipp Köster1, Thomas A DeFalco1 and Cyril Zipfel *,1,2 1Institute of Plant and Microbial Biology and Zürich-Basel Plant Science Center, University of Zürich, Zürich, Switzerland 2The Sainsbury Laboratory, University of East Anglia, Norwich, UK *Corresponding author. Tel: +41 044 63 48222; E-mail: [email protected] The EMBO Journal (2022)41:e110741https://doi.org/10.15252/embj.2022110741 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Calcium ions function as a key second messenger ion in eukaryotes. Spatially and temporally defined cytoplasmic Ca2+ signals are shaped through the concerted activity of ion channels, exchangers, and pumps in response to diverse stimuli; these signals are then decoded through the activity of Ca2+-binding sensor proteins. In plants, Ca2+ signaling is central to both pattern- and effector-triggered immunity, with the generation of characteristic cytoplasmic Ca2+ elevations in response to potential pathogens being common to both. However, despite their importance, and a long history of scientific interest, the transport proteins that shape Ca2+ signals and their integration remain poorly characterized. Here, we discuss recent work that has both shed light on and deepened the mysteries of Ca2+ signaling in plant immunity. The plant immune system All eukaryotes use immune systems to protect themselves against potential pathogens. The plant immune system consists of two characterized perception layers: one that utilizes cell-surface pattern recognition receptors (PRRs) to perceive extracellular immunogenic patterns, and another that relies on intracellular nucleotide-binding leucine-rich repeat (NLR) receptors that recognize pathogenic effectors inside the cell (Jones & Dangl, 2006). In the first layer of the plant immune system, apoplastic immunogenic elicitors such as pathogen-, microbe-, damage-, or herbivore-associated molecular patterns (PAMPs, MAMPs, DAMPs, or HAMPs, respectively) or immune-modulating peptide phytocytokines are recognized by PRRs, which leads to defense responses termed pattern-triggered immunity (PTI) (Boller & Felix, 2009; Yu et al, 2017; DeFalco & Zipfel, 2021). All plant PRRs described to date are receptor kinases (RKs) or receptor proteins (RPs) (Boutrot & Zipfel, 2017; Albert et al, 2020). RKs are characterized by a domain structure reminiscent of metazoan receptor tyrosine kinases (RTKs) (DeFalco & Zipfel, 2021); namely, a ligand-binding extracellular domain (ECD), a single-span transmembrane helix (TM) and a cytosolic protein kinase domain (Jamieson et al, 2018), while RPs lack a cytoplasmic kinase domain and instead form functional bipartite receptors with adapter RKs (Liebrand et al, 2013; Albert et al, 2015; Postma et al, 2016). Because of their domain architecture, plasma membrane (PM)-localized PRRs (or their complexes) allow extracellular ligand binding to be communicated across the membrane into cytosolic signaling events. The molecular nature of elicitors varies, including proteins, lipids, and carbohydrates, and can be derived from either the potential pathogen or herbivore (e.g., MAMPs, PAMPs, or HAMPs) or the host plant, as in the case of macromolecules released upon cell damage (DAMPs) or secreted peptide phytocytokines (Gust et al, 2017). PRR ECDs are characterized by a variety of subdomains, including leucine-rich repeat (LRR), epidermal growth factor-like (EGF), lectin, and lysin motif (LysM) domains (Boutrot & Zipfel, 2017). The best-studied PRRs to-date are the LRR-RKs FLAGELLIN-SENSING 2 (FLS2) and EF-TU RECEPTOR (EFR), which perceive the bacterial PAMPs flg22 and elf18, respectively (Gómez-Gómez & Boller, 2000; Zipfel et al, 2006). Both FLS2 and EFR form stable ligand-dependent complexes with common LRR-RK co-receptors of the SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK) family, such as BRASSINOSTEROID-INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1, also called SERK3) (Chinchilla et al, 2007; Heese et al, 2007; Roux et al, 2011). Complex formation between PRRs and co-receptors leads to phosphorylation events within the cytoplasmic kinase domains and the activation of receptor-like cytoplasmic kinases (RLCKs), which directly phosphorylate and regulate target proteins in order to activate PTI (Liang & Zhou, 2018; DeFalco & Zipfel, 2021) (Fig 1A). Figure 1. PTI and ETI induce cytoplasmic Ca2+ elevations RKs and RPs are PRRs residing at the PM. They form complexes with co-receptors upon perception of molecular patterns originating from microbes, viruses, herbivores, parasitic plants, or damaged host cells. In turn, RLCKs are activated and released from the complexes to activate downstream signaling to induce pattern-triggered immunity, of which Ca2+ release within few minutes after ligand perception is one facet. Microbes introduce effector proteins into host cells to disturb and overcome immune responses. Cytoplasmic NLRs sense the presence or activity of effectors to induce ETI. To this end, autoinhibition is released, ADP is changed to ATP and oligomerization of NLRs occurs, leading to downstream signaling and finally ETI (A). A significant cytoplasmic Ca2+ increase has been reported to occur in Arabidopsis leaves starting 1.5 h and peaking at about 2 h after infection with avirulent bacteria (B). Schematic Ca2+ signatures of Arabidopsis plants induced by bacterial infection as reported by Grant et al (2000) (B). RK: receptor kinase; co-RK: coreceptor kinase; RP: receptor protein; RLCK: receptor like cytoplasmic kinase; NLR: nucleotide-binding leucine-rich repeat receptor; CC: coiled-coil; TIR: toll/interleukin-related; CNLs: CC-NLRs; TNLs: TIR-NLRs; RNLs: RPW8-NRLs; NBS: nucleotide binding site; LRR: leucine-rich repeats; PTI: pattern-triggered immunity; ETI: effector-triggered immunity, c[Ca2+]: cytoplasmic free Ca2+ concentration. Download figure Download PowerPoint Pathogens introduce effectors into the host cytoplasm that promote pathogenicity, often by disturbing PTI (Jones & Dangl, 2006). To counteract this, plants rely on a second layer of immunity, in which intracellular NLR-type receptors sense effectors and/or their activity, leading to effector-triggered immunity (ETI). Interestingly, plant NLRs share a common architecture with those of animals, featuring a conserved nucleotide-binding domain (NBD) and LRR domain, with variable accessory domains at both N and C termini (DeYoung & Innes, 2006; Jones et al, 2016; Baggs et al, 2017; van Wersch et al, 2020). NLRs are categorized based on their N-terminal domains: coiled-coil (CC)-NLRs (CNLs), toll/interleukin-related (TIR)-NLRs (TNLs), or RPW8-NLRs (RNLs). Of these NLRs, CNLs and TNLs function as sensors while RNLs function as helpers downstream of TNLs (Baggs et al, 2017; Wu et al, 2017; Jubic et al, 2019; Feehan et al, 2020). NLRs can be present in an inactive state, in which the LRR domain is likely autoinhibitory, and adenosine diphosphate (ADP) is bound to their NBD (Williams et al, 2011; Bernoux et al, 2016). Upon activation, ADP is exchanged to adenosine triphosphate (ATP) and autoinhibition is released (Fig 1A). In animals, NLR activation often leads to oligomerization via N-terminal domains and the formation of large multimeric structures (Danot et al, 2009). A similar oligomerization mechanism has been long hypothesized for plant NLRs, but has only been recently corroborated by structural data that are discussed in detail below. PTI and ETI have traditionally been viewed as independent pathways; however, at least some signaling components are shared by both layers of immunity (Thomma et al, 2011). Activation of either layer of the immune system triggers numerous overlapping cell signaling events, including Ca2+ fluxes, production of apoplastic reactive oxygen species (ROS), mitogen-activated protein kinase (MAPK) cascades, transcriptional reprogramming, and phytohormone biosynthesis (Cui et al, 2015; Yu et al, 2017; Zhou & Zhang, 2020; DeFalco & Zipfel, 2021). ETI is generally also accompanied by a form of programmed cell death termed the hypersensitive response (HR) at the site of infection (DeYoung & Innes, 2006; Jones & Dangl, 2006), although HR-like cell death is also induced by some forms of PTI (Wang et al, 2020). Recent work has further demonstrated that PTI and ETI are linked at transcriptional and/or molecular levels (Ngou et al, 2021; Pruitt et al, 2021; Tian et al, 2021; Yuan et al, 2021); however, the exact mechanisms governing linkage of these immune pathways remains to be elucidated fully. As changes in intracelluar Ca2+ levels have been well documented downstream of both PRR and NLR activation, Ca2+ signaling is thought to be key to both layers of the plant immune system (Seybold et al, 2014; Moeder et al, 2019). Ca2+ in immunity Ca2+ is a universal second messenger in eukaryotes (Clapham, 2007). Owing to its cytotoxicity, cytosolic Ca2+ levels must be maintained at low (~10−8 to 10−7 M) levels in living cells, and thus Ca2+ is sequestered in intracellular stores (in plants, primarily the vacuole and the endoplasmatic reticulum, but also the vesicular compartments, the chloroplasts and mitochondria) or the apoplast via active transport, generating enormous electrochemical potential gradients across membranes (Clapham, 2007; Edel et al, 2017; Costa et al, 2018). Ca2+-permeable channels can therefore generate rapid, transient increases in Ca2+ concentrations, which are in turn interpreted by a large suite of Ca2+-binding sensor proteins that regulate diverse cellular processes (DeFalco et al, 2010). Ca2+ signaling is thus summarized in three steps: encoding (via stimulus-triggered Ca2+ fluxes), decoding (via Ca2+ sensor proteins), and responses (via regulation of downstream cellular processes). In plants, Ca2+ signaling is involved in all aspects of life, including growth regulation, development, abiotic stress responses, and reproduction (Kudla et al, 2018), as well as the establishment of beneficial plant-microbe interactions (Tian et al, 2020). In this review, we focus on how cytoplasmic Ca2+ signals are encoded via transport across the PM during immune signaling. Ca2+ influx and the oxidative burst (Doke, 1983, 1985; Apostol et al, 1989; Keppler et al, 1989) were among the first cellular responses to pathogen infection or elicitor treatment to be described (Atkinson et al, 1996; Levine et al, 1996; Zimmermann et al, 1997; Lecourieux et al, 2002). ROS production during the oxidative burst was eventually attributed to the activity of PM-localized NADPH oxidases of the RESPIRATORY BURST OXIDASE HOMOLOGUE (RBOH) family (Torres et al, 2002); in the model plant Arabidopsis thaliana (hereafter, Arabidopsis), a single member, RBOHD, is responsible for ROS production in response to elicitors (Nühse et al, 2007; Zhang et al, 2007). In contrast, the molecular nature of the Ca2+ channel(s) involved in plant immunity remained comparably elusive for many years (Seybold et al, 2014). Cytosolic Ca2+ signals evoked by treatment with various immunogenic elicitors were first measured in plant cell culture using Ca2+ radioisotopes, Ca2+-sensitive dyes, or electrophysiological approaches (Atkinson et al, 1996; Levine et al, 1996; Gelli et al, 1997; Zimmermann et al, 1997). The development of genetically encoded Ca2+ indicators (GECIs) greatly expanded the possibilities for real-time, kinetic analysis of Ca2+ fluxes in intact tissues upon infection or elicitor treatment. The first GECI deployed in plants was aequorin (AEQ) from Aequoria victoria (Knight et al, 1991), which forms a holo-enzyme with its cofactor coelenterazine and emits light upon Ca2+-binding. When challenged with either virulent or avirulent strains of the pathogenic bacterium Pseudomonas syringae, Arabidopsis plants expressing AEQ showed a first Ca2+ signal peak after ~10 min. A second, stronger, more persistent Ca2+ signal was seen after 1.5–2 h only with avirulent, ETI-activating P. syringae (Grant et al, 2000; Kang et al, 2010; Hung et al, 2014). The similar kinetics of early Ca2+ elevation induced by P. syringae and that triggered by elicitors (Blume et al, 2000; Lecourieux et al, 2002) and the biphasic nature of the response to ETI-inducing bacteria suggested that PTI and ETI may induce distinct Ca2+ signals (Fig 1B). Subsequent analyses of AEQ-expressing Arabidopsis plants have shown perception of diverse elicitors, including PAMPs, DAMPs, and phytocytokines, to be sufficient to elicit rapid Ca2+ signals (Ranf et al, 2008, 2011; Vadassery et al, 2009; Krol et al, 2010). Such PTI Ca2+ signaling requires functional PRRs and downstream signaling components, including RLCKs such as the RLCK-VII/ AVRPPHB SUSCEPTIBLE 1 (PBS1)-LIKE (PBL) family members BOTRYTIS-INDUCED KINASE 1 (BIK1) and PBL1 (Li et al, 2014; Ranf et al, 2014; Monaghan et al, 2015). More recently, the deployment of fluorescent GECIs in plants has allowed for the analysis of elicitor-induced Ca2+ signals at the cellular level. Such fluorescent GECIs include ratiometric (e.g., yellow cameleons) and intensiometric (e.g., GCaMPs and GECOs) sensors (Grenzi et al, 2021b; Waadt et al, 2021). Flourescent GECIs have been utilized to show that elicitor-induced Ca2+ signals in leaves are oscillatory at the single-cell level (Thor & Peiter, 2014; Keinath et al, 2015) and that in roots both elicitor application and laser ablation-induced cell damage lead to the formation of Ca2+ transients (Keinath et al, 2015; Marhavý et al, 2019; Waadt et al, 2020). ROS and Ca2+—tightly linked second messengers There is extensive interplay between Ca2+ and ROS signaling (Gilroy et al, 2016); however, the initial PTI-related Ca2+ signal triggered by P. syringae was shown to be only mildly reduced by treatment with the NADPH oxidase inhibitor DPI or catalase, while there was no effect on the longer-term, effector-triggered signal (Grant et al, 2000). Similarly, rbohd mutants showed a slight, quantitative defect in elicitor-triggered Ca2+ signals when measured in seedlings (Ranf et al, 2011). In contrast, elicitor-induced ROS production can be severely attenuated by treatment with Ca2+ channel blockers (Ranf et al, 2011). Elicitor perception can directly activate RBOHD via phosphorylation by BIK1 (Kadota et al, 2014; Li et al, 2014), suggesting a complex relationship between Ca2+ and ROS in immune signaling and a model wherein, upon elicitor perception, initial activation of RBOHD through PRR-mediated phosphorylation primes the system for subsequent activation through Ca2+ signaling (Kadota et al, 2015) (Fig 2). Ca2+ not only activates RBOHD directly via its cytoplasmic Ca2+-binding EF-hand domains but also indirectly via Ca2+-regulated kinase-mediated RBOHD phosphorylation (Ogasawara et al, 2008; Dubiella et al, 2013). Interestingly, BIK1 and CALCIUM DEPENDENT PROTEIN KINASE 5 (CPK5) activate RBOHD through phosphorylation at distinct sites (Dubiella et al, 2013; Kadota et al, 2014; Li et al, 2014). While target residues have been described to be strictly required for PTI-induced ROS bursts (Nühse et al, 2007), individual contribution from other phosphorylation sites and the impact of certain phosphorylation patterns remain to be uncovered. Figure 2. Ca2+ and ROS signals are tightly interconnected Upon activation of PRR complexes during PTI, RLCKs activate Ca2+ channels leading to cytoplasmic Ca2+ signals. Ca2+ ions can directly activate the NADPH-oxidase RBOHD through binding to its N-terminal EF-hands, but also induce the activity of Ca2+-regulated kinases that phosphorylate the cytoplasmic N terminus of RBOHD (indicated by grey arrows targeting RBOHD p-sites). In addition, RLCKs directly phosphorylate the N terminus and thereby activate RBOHD (indicated by black arrows targeting RBOHD p-sites). Reactive oxygen species derived from RBOHD activity can be perceived by cysteine pairs of the RK HPCA1/CARD1. This is required for H2O2 induced Ca2+ signals in Arabidopsis, the signaling pathway downstream of HPCA1 activation is not known. Download figure Download PowerPoint A recent AEQ-based screen for impaired H2O2-induced Ca2+ signaling identified an LRR-RK, HYDROGEN PEROXIDE INDUCED Ca2+ INCREASE 1 (HPCA1), as a putative ROS sensor (Wu et al, 2020a). Interestingly, HPCA1 was independently identified as CANNOT RESPOND TO DMBQ 1 (CARD1), which showed a loss of response to the quinone compound 2,6-dimethoxy-1,4-benzoquinone (DMBQ), which regulates interactions with parasitic plants and also triggers HPCA1/CARD1-dependent Ca2+ signaling (Laohavisit et al, 2020). Both the nature of the channel(s) that are regulated by HPCA1/CARD1, as well as the exact role of ROS in regulating Ca2+ signaling via such sensor(s) remain unclear. Interestingly, AEQ-measured calcium signals in response to H2O2 were reduced in cngc2 and cngc4 mutants (Tian et al, 2019), suggesting that these channels may function downstream of ROS perception. Shaping immune signals via Ca2+ efflux Ca2+ signals are generated via the coordinated action of channels and active transporters and involve influx from the apoplast and release from intracellular stores (Spalding & Harper, 2011; Edel et al, 2017; Resentini et al, 2021). In addition, plants possess three major families of proteins that mediate active Ca2+ transport out of the cytosol: Ca2+/H+ exchangers (CAXs), autoinhibited Ca2+-ATPases (ACAs) and ER Ca2+-ATPases (Geisler et al, 2000; Shigaki & Hirschi, 2000; García Bossi et al, 2020). ACA autoinhibition can be relieved by Ca2+/CaM-binding, which allows for rapid feedback regulation of Ca2+ signals (Geisler et al, 2000). The PM-localized ACA8 and its homolog ACA10 were identified as interactors of FLS2, and aca8 aca10 mutants displayed quantitative defects in flg22-induced calcium signals and compromised resistance to P. syringae infection (Frei dit Frey et al, 2012), as well as disturbed stomatal closure upon PAMP perception (Yang et al, 2017), suggesting that Ca2+ efflux across the PM to the apoplast shapes Ca2+ signaling during PTI. Two tonoplast-localized ACAs, ACA4 and ACA11, have also been implicated in immunity, as aca4 aca11 mutants display autoimmune phenotypes and spontaneous cell death (Boursiac et al, 2010). Although aca4 aca11 mutants have wildtype total calcium content (Boursiac et al, 2010), subsequent work has revealed that basal cytosolic calcium levels are elevated in aca4 aca11 (Hilleary et al, 2020). Elicitor-induced calcium signals also show elevated peaks in aca4 aca11 mutants (Fig 3), which can be rescued by mis-localization of PM ACAs to the tonoplast (Hilleary et al, 2020), indicating that transport of Ca2+ into the vacuole is critical to maintain Ca2+ homeostasis and modulate signaling during PTI. Figure 3. Disturbance of the Ca2+ efflux machinery impairs plant immunity Ca2+ exchangers (CAX) and autoinhibited Ca2+-ATPase (ACAs) reside at the PM or tonoplast and establish low cytoplasmic Ca2+ concentrations and rapid termination of Ca2+ signals through export of the Ca2+ ions into the apoplast or vacuolar lumen (A). This function is disturbed in Arabidopsis aca4 aca11 mutants, which consequently show an autoimmune phenotype (B). PTI induced Ca2+ signatures are compromised in those lines, with slower onset of the signal, and higher peak concentration and retarded reduction of the Ca2+ signals. Schematic Ca2+ signatures as reported by Hilleary et al (2020) (C). Download figure Download PowerPoint Plasma membrane-localized Ca2+ channels involved in immunity Extensive work has demonstrated that elicitor-induced Ca2+ signals strictly require PM-localized, Ca2+-permeable channels, as treatment with blockers such as Gd3+ or La3+ abolishes such signals (Blume et al, 2000; Grant et al, 2000; Lecourieux et al, 2002; Kwaaitaal et al, 2011; Ranf et al, 2011; Maintz et al, 2014; DeFalco et al, 2017). While such studies clearly implicate Ca2+-permeable channels as components of immune signaling, their nature has remained hidden. However, recent work has started to decipher how Ca2+ signals are generated upon immune activation, and the defense-related roles of several classes of plant Ca2+ channels have begun to be characterized. Below, we discuss immunity-related channel candidates by their phylogenetic groups rather than following a chronological order of identification or a strict PTI/ETI dichotomy. CNGCs—from strong phenotypes to complex regulation One of the first families of potential Ca2+ channels identified in plants were the tetrameric cyclic nucleotide-gated channels (CNGCs) (Köhler & Neuhaus, 1998). Plant CNGCs comprise large gene families (e.g., 20 members in Arabidopsis) (Mäser et al, 2001) and are named for their topology and domain organization, which are reminiscent of mammalian cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-modulated (HCN) families (Kaupp & Seifert, 2002; Matulef & Zagotta, 2003). Individual CNGCs have six transmembrane helices and cytosolic N and C termini, with the cyclic nucleotide-binding domain (CNBD) located within the CNGC C terminus (Kaplan et al, 2007). While previous reports have indicated that the CNBDs of plant CNGCs may bind cyclic nucleotides (Baxter et al, 2008), and some electrophysiological analyses have indicated that application of cAMP or cGMP can promote CNGC activity (Leng et al, 2002; Zhang et al, 2007; Gao et al, 2014, 2016; Meena et al, 2019), it remains unclear whether cyclic nucleotides are bona fide agonists for plant CNGCs in planta. Furthermore, the existence of guanylate and adenylate cyclases (GCs and ACs) in plant proteomes is still under debate and will not be discussed in detail here. Indeed, while studies suggest multiple plant proteins, including RKs, to display GC activity (Qi et al, 2010; Turek & Irving, 2021), the low determined in vitro activities of the putative GCs and the position of their putative active sites within the kinase domains of RKs argues against a physiological relevance for such potential GC activity (Ashton, 2011; Bojar et al, 2014). Nevertheless, extensive electrophysiological work over the past two decades has shown that at least some CNGCs form Ca2+-permeable, non-selective cation channels (Jarratt-Barnham et al, 2021). CNGCs are directly regulated by the conserved Ca2+ sensor calmodulin (CaM), with one or more CaM-binding domains (CaMBDs) found within the cytosolic C termini of all CNGCs examined to date (Arazi et al, 1999; Köhler & Neuhaus, 2000; Hua et al, 2003; Fischer et al, 2013, 2017; DeFalco et al, 2016a) as well as the N terminus of some CNGC isoforms (DeFalco et al, 2016a). Ca2+/CaM regulation of CNGCs is complex (DeFalco et al, 2016b) as a Ca2+-independent IQ motif CaMBD at the C-terminal end of the channel is essential for CNGC function (DeFalco et al, 2016a; Pan et al, 2019), with additional Ca2+-dependent CaMBDs providing negative (feedback) regulation (DeFalco et al, 2016a; Pan et al, 2019; Tian et al, 2019). Plant CNGCs are divided into four subfamilies based on phylogeny, with group IV CNGCs further divided into groups IVa and IVb (Mäser et al, 2001). The best-studied CNGCs to-date are the two Arabidopsis group IVb members, CNGC2 and CNGC4, which were first isolated as the defense, no death (dnd) or HR-like lesion mimic (hlm) mutants dnd1 and dnd2/hlm1 (null mutants of CNGC2 and CNGC4, respectively) (Clough et al, 2000; Balagué et al, 2003; Jurkowski et al, 2004). The dnd mutants were initially described to be defective in the induction of HR, despite still being able to carry out ETI to avirulent pathogens (Yu et al, 1998). These dnd mutants display numerous phenotypic defects, including dwarf morphology, delayed flowering, elevated concentrations of the phytohormone salicylic acid (SA), spontaneous cell death, and dis-regulated auxin signaling (Clough et al, 2000; Balagué et al, 2003; Chan et al, 2003; Jurkowski et al, 2004; Chin et al, 2013; Chakraborty et al, 2021). In keeping with the immune-related phenotypes of dnd1/cngc2 mutants, CNGC2 was also suggested to be a mediator of Ca2+ fluxes in plant immunity, as production of the signaling molecule nitric oxide (NO) was reported to be reduced in cngc2 mutants compared to WT plants after treatment with the PAMP lipopolysaccharide (LPS) (Ali et al, 2007). The same study used pharmacological inhibitors to implicate CaM, Ca2+ channels, and a NO synthase (NOS)-type protein to be required for this process. Given the lack of mammalian-type NOS enzymes in land plants (Santolini et al, 2017) and the myriad functions of CaM (DeFalco et al, 2010), results from such pharmacological studies must however be interpreted cautiously. Subsequent work using AEQ reporter lines suggested that CNGC2 is required for full Ca2+ signals in response to some but not all elicitors (Ma et al, 2012). Given the convergence of signaling downstream of diverse PRRs (Couto & Zipfel, 2016; Bjornson et al, 2021), it remains unclear how such specificity may be achieved. Interestingly, virus-induced gene silencing (VIGS) of IVb isoforms in tomato compromised ROS production in response to flg22, further suggesting that these CNGCs may positively regulate PTI (Saand et al, 2015). Recently, loss-of-function cngc2 and cngc4 mutants were each isolated in an AEQ-based forward genetic screen for compromised Ca2+ signaling upon flg22 treatment (Tian et al, 2019). Both mutants displayed defects in Ca2+ influx and ROS production after treatment with flg22 and exhibited compromised resistance to P. syringae. Remarkably, these phenotypes were however strictly dependent on high Ca2+ concentrations in the growth media, as cngc2 and cngc4 responses under low Ca2+ growth were indistinguishable from those of WT plants. Interestingly, PRR signaling mutants, such as bik1, do not display such conditional phenotypes (Li et al, 2014; Ranf et al, 2014; Monaghan et al, 2015). Detailed electrophysiological characterization of the heterologously expressed channels in Xenopus laevis oocytes found the single subunits to be inactive, while CNGC2-CNGC4 heteromers produce strong currents (Tian et al, 2019), in keeping with a model wherein these channel subunits function together (Chin et al, 2013). CNGC2-CNGC4 currents were inhibited by CaM; further experiments suggested that phosphorylation of the CNGC4 C terminus by BIK1 can partially release this negative regulation (Tian et al, 2019) (Fig 4A). This work further highlights the complex regulation to which CNGCs are likely subject, including by CaM, phosphorylation, and, potentially, ligand-binding (Jarratt-Barnham et al, 2021). Figure 4. CNGCs fulfil diverse roles in plant immune signaling CNGCs form homo- or heterotertramers at the PM. Arabidopsis CNGC2 and CNGC4 homotetramers are inactive, but heterotetramers allow cation fluxes into the cytosol. Ca2+-bound Calmodulin (CAM) inhibits those channels, generating a negative feedback loop. Upon initiation of PTI, activated BIK1 phosphorylates CNGC4 to release CAM-mediated inhibition and to induce Ca2+ influx (A). In rice, PRR complexes activate RLCK185 upon ligand perception, which phosphorylates and thereby activates OsCNGC9. If the OsCNGC9 containing tetramer is homomeric or heteromeric is not known (B). In Arabidopsis, CNGC activity can lead to the induction of cell death via to date not resolved signaling pathways. CNGC19 and CNGC20 form complexes at the PM, and are phosphorylated by BAK1, which initiates degradation of the channels. In bak1/bkk1 coRK mutants, accumulation of CNGC19/CNGC20 channels leads to Ca2+ influx, ultimately causing cell death. Download figure Download PowerPoint Both cngc2 and cngc4 mutants are hypersensitive to Ca2+ concentration in growth media (Chan et al, 2003; Chin et al, 2013), an