The grateful dead: calcium and cell death in plant innate immunity

Authors


*E-mail gerald.berkowitz@uconn.edu; Tel. (+1) 860 486 1945; Fax (+1) 860 486 0534.

Summary

Plant cells sensing pathogenic microorganisms evoke defence systems that can confer resistance to infection. This innate immune reaction can include triggering of basal defence responses as well as programmed cell death, or hypersensitive response (HR). In both cases (basal defence and HR), pathogen perception is translated into elevated cytosolic Ca2+ (mediated by plasma membrane and intracellular channels) as an early step in a signalling cascade. Cyclic nucleotide-gated channels contribute to this influx of Ca2+ into the cell. The molecular nature of other transport proteins contributing to the Ca2+ elevation is unclear. Pathogen recognition occurs at two levels: the perception of pathogen-associated molecular pattern (PAMP) molecules widely present in microorganisms, and an interaction between pathogen avirulence gene products (if present) and corresponding plant R (resistance) gene products. The Ca2+ elevation occurring in response to PAMP perception or R gene interactions could occur due to phosphorylation events, G-protein signalling and/or an increase in cyclic nucleotides. Downstream from the initial Ca2+ rise, the signalling cascade includes: activation of calmodulin and protein kinases, and nitric oxide and reactive oxygen species generation. Some of these downstream events amplify the Ca2+ signal by further activation of Ca2+ transporters.

Introduction

During their life cycle, plants are exposed to invading microorganisms, including bacteria, true fungi, filamentous protist oomycetes, and viruses that are potentially pathogenic, in that impairment of host plant growth, development, survival and/or reproduction could result from successful infection. As sessile organisms without mobile sentry cells, plants must respond to such potentially injurious microbe invasion using defence strategies that preclude chemotaxis-facilitated phagocytosis (a cornerstone of innate immunity in animals). In addition to the absence of a mobile self/non-self-surveillance system, plants lack the powerful recombinatorial-based adaptive immunity system of mammals. Nonetheless, plants have a complex multilayered system of host survival-enhancing innate immune responses to pathogens that can include cellular-level apoptotic programmed cell death (PCD).

Plants share with animals the use of Ca2+ as a cytosolic secondary messenger molecule involved in numerous cell-signalling cascades responding to abiotic and biotic stimuli. Perturbations in cytosolic Ca2+ homeostasis are known to be an essential early step in pathogen perception and subsequent innate immune response of plant cells (Dangl et al., 1996). This review will focus on our understanding of the role Ca2+ plays in the signal transduction pathway leading to innate immune-associated host cell apoptosis in plants; unless specifically noted, information pertains to leaf (as opposed to root) tissue and cells. Recent advancements in, as well as outstanding questions related to, (i) the cellular-level molecular events linking pathogen perception/recognition to altered cytosolic Ca2+, (ii) the cell and intracellular Ca2+ transporters potentially involved in microbe-associated perturbations in Ca2+ homeostasis, and (iii) the downstream events (in the signalling and pathogen response systems) affected by altered cytosolic Ca2+ levels will be highlighted.

Innate immunity in plants involves two interrelated and interconnected defence responses: (i) a basal reaction to microbe invasion that is a general non-self-recognition phenomenon, and (ii) disease-resistance (R) gene, or gene-for-gene mediated reaction to potential pathogens (Jones and Dangl, 2006; He et al., 2007; Hofius et al., 2007). In addition to innate immune responses displayed by cells directly challenged by pathogen attack and cells neighbouring the site of infection, plant response to pathogens includes systemic acquired resistance, which involves long-distance transport of plant-derived signalling molecules and potentiates plant defences (localized to the site of infection as well as in distal tissues) against current and subsequent pathogen attack (Grant and Lamb, 2006).

Systemic acquired resistance does not involve PCD as a plant-protective response to pathogen attack, while plant innate immunity can be thought of as an integrated system that can lead to apoptosis in the cells at the site of infection. This PCD at the infection site [also referred to as the hypersensitive response (HR)] limits the spread of pathogens within the plant; it is one of many plant defensive strategies that limit and/or ameliorate disease. As is the case with animal PCD, in addition to Ca2+ as a cytosolic signalling molecule, reactive oxygen species (ROS), including H2O2, superoxide (O2), hydroxyl radicals (OH) and nitric oxide (NO), are involved in the induction, signal cascade and execution of PCD during plant HR to pathogens.

Excellent reviews have been recently published on various aspects of plant innate immunity, Ca2+ transport and signalling, and Ca2+ involvement in pathogen responses (Sanders et al., 2002; Hetherington and Brownlee, 2004; Nürnberger et al., 2004; Zipfel and Felix, 2005; Garcia-Brugger et al., 2006; Grün et al., 2006; Jones and Dangl, 2006; Lecourieux et al., 2006; He et al., 2007; Hofius et al., 2007). Here, we summarize and update the relevant information in this fast-changing field. Most of the studies cited in this review were performed with Arabidopsis thaliana plants/cells; important exceptions are noted as discussed below.

Plant innate immunity overview

Current models (see reviews cited above) of plant innate immune defence against potentially pathogenic microbes conceptualize the aforementioned two protective systems: basal responses and R gene-mediated resistance, as different ‘layers’ of a single plant defence network reacting to (in the case of basal resistance) non-self antigens and (with R gene resistance) pathogen-associated virulence proteins that typically inactivate some component of the basal defence system. Plant basal defences are activated by perception of evolutionarily conserved microbial components essential to the proper functioning of the potential pathogen: microbial (or pathogen)-associated molecular patterns (MAMPS or PAMPS, subsequently referred to here as PAMPs). PAMPs can be essential components of non-pathogenic microbes, hence the dual name. In addition to PAMPs, activators of basal resistance include compounds released from the plant or invading microbe cell wall by hydrolytic enzymes of the plant or pathogen, i.e. breakdown products of an initial host–pathogen assault/defence ‘skirmish’. Recent studies (Lecourieux et al., 2006; Qutob et al., 2006) indicate that microbial toxins as well can act as PAMPs in broad classes of plants. PAMP perception occurs at the plant cell plasma membrane (PM), facilitated by a multiplicity of pattern recognition receptor (PRR) proteins that transduce PAMP/pathogen recognition into convergent signalling within the host cell under attack (Jones and Dangl, 2006; He et al., 2007). This basal response includes metabolic and transcriptional changes in the host cell, leading to PAMP-triggered immunity (PTI). Protein effectors produced by (specific races of) invading microbes can interfere with host resistance afforded by PTI. These specific gene products can enhance the virulence of the invading microbe, thus conveying effector-triggered susceptibility (ETS) to the invaded host cells under attack. ‘Layered’ on top of host cell basal resistance (PTI) is a secondary network of responses to the pathogen, in this case species- and/or cultivar-specific. This secondary response network is induced by interaction of a translation product from a plant-encoded (cultivar- and/or species-specific) R gene either directly or indirectly with the specific protein effector produced by the pathogen; hence such microbial effector proteins can also be thought of as elicitors (i.e. of ETS). If the plant under attack has an R gene that encodes a protein that specifically interacts with the microbial effector, this gene-for-gene interaction can provide a signal that induces another network of immune responses in the plant; effector-triggered immunity (ETI). ETI conveys disease resistance to the host plant and typically includes, among the network of plant defence responses providing disease resistance, a HR to the specific race of microbe (Jones and Dangl, 2006). If ETI imparts effective resistance to the host plant through this gene-for-gene microbe:plant interaction, the microbial gene encoding the effector protein (which inactivates the PTI basal defence network of the plant) triggering this plant R gene-based immunity ironically ends up as an avirulence (avr) gene, and the pathogen/plant host interaction is termed ‘incompatible’.

It should be noted that even current reviews distinguish PTI and ETI as perhaps overlapping but mechanistically distinct resistance systems, with HR classically thought of as solely an ETI response to pathogen avr gene products (Hofius et al., 2007). However, some current work (i) refers to PAMP responses (PTI) and avr gene product elicitation of ETI in plants as more intimately related, (ii) notes that they share many similar signal transduction components as well as downstream targets, and (iii) presents plant immune response to pathogens harbouring avr genes as invoking an ‘accelerated’ immune response (e.g. Mészáros et al., 2006). Some PAMPs, such as the bacterial flagellin protein, have been shown to activate key regulators of R gene-mediated signalling pathways as well as basal defence networks, thus suggesting anintimate overlap and linkage of the two pathogen defence responses of plants (Robatzek et al., 2006). Providing further texture to the characterization of plant innate immunity is the recent demonstration that the host plant itself can generate specific gene products upon pathogen recognition that elicit many of the ETI immune responses evoked by microbial avr gene products (Huffaker et al., 2006; Yamaguchi et al., 2006).

Ca2+ rise in the cytosol is an early signalling event in plant immune responses to both pathogen PAMPs and avr genes; i.e. PTI and ETI. The plant HR response to avirulent pathogens, PCD occurring during ETI, is one of the most actively studied systems of apoptosis in plants. Here, we develop a model linking pathogen-associated Ca2+ elevation in the cytosol with HR, focusing on how pathogen perception could be linked to this Ca2+ rise, what specific Ca2+ transporters contribute to the rise, and recent work filling in some of the substantial gaps in our current understanding of the steps linking this Ca2+ rise to HR in plant cells responding to pathogen infection. The cytosolic Ca2+ elevation that is a critical step in plant innate immunity is mediated by an increase in Ca2+ influx rather than a decrease in Ca2+ efflux from the cytosol (see References in Takabatake et al., 2007). Therefore, focus here will be on Ca2+ influx systems. Presumably, as is the case with many Ca2+-signalling pathways, continued function of the PM Ca2+ ATPase and the vacuolar H+/Ca2+ antiporter (both of these transport systems are pathways for Ca2+ movement out of the cytosol) would return cytosolic Ca2+ to homeostatic levels after pathogen-associated elevations.

Mechanisms facilitating Ca2+ influx into the plant cell cytosol

There are some notable differences between plant leaf and animal cell morphology and Ca2+ transport that are relevant to Ca2+ signalling during plant innate immunity. Plant cells are surrounded by a pectin-containing wall that provides a reservoir of Ca2+ in the apoplast outside the plant cell PM that can be on the order of ∼1–10 mM (White and Broadley, 2003). In studies of plant cell signalling in response to pathogens, it is important to consider that the cell wall could act as a source of chleated Ca2+ juxtaposed next to the PM in planta, and could supply Ca2+ to an incubation medium (when no exogenous Ca2+ is added) and thus provide a source for Ca2+ uptake to facilitate Ca2+-dependent mechanisms for cell-level studies in the absence of added Ca2+. Another morphological difference between plant and animal cells is that the vacuole could represent 90% of the symplastic volume delimited by the plant cell PM and may also contain mM Ca2+ (Lecourieux et al., 2006). Animal cells lack such an internal organelle and potential Ca2+ source contributing to cytosolic Ca2+ changes, although the endoplasmic reticulum can also be an internal source of Ca2+ in both animal and plant cells. Ca2+ levels in the plant cell mitochondria and chloroplasts are regulated differently compared with the cytosol; these pools of Ca2+ can also affect Ca2+-based signalling in the plant cell (Xiong et al., 2006). As in animal cells, [Ca2+] in the plant cell cytosol is tightly regulated and maintained under homeostasis at ∼0.1–0.3 μM (McAinsh et al., 1992).

Due to the steep Ca2+ concentration gradient (inside low) between the apoplast and the cytosol, as well as the PM electrical potential gradient (inside approximately −150 mV), it would be expected that passive Ca2+ conduction through channels would dominate the overall Ca2+ uptake pathway into the plant cell, and this appears to be the case (White et al., 2002). The plant genome does not encode any channel proteins with deduced sequences corresponding to Ca2+-selective channels of animals. Patch clamp studies indicate that the major inward Ca2+ current across the PM occurs through non-selective weakly voltage-gated cation channels (Demidchik et al., 2002). Only recently have specific gene products been associated with Ca2+ channel currents recorded from the plant cell PM and tonoplast (the limiting membrane of the vacuole).

Peiter et al. (2005) recently characterized TPC1 (a non-selective cation channel) and demonstrated that it is responsible for the major conductance of Ca2+ across the tonoplast from the vacuole to the cytosol. TPC1 conductance is strongly gated by both voltage and Ca2+ the channel has (Ca2+ binding) EF hands on an intramembrane loop extending into the cytosol and may be activated by Ca2+ through direct binding. Ca2+ rise from homeostatic levels in the cytosol could dramatically activate this Ca2+-activated Ca2+-conducting channel. Thus this tonoplast protein provides a mechanism for amplification of an initial pathogen-induced rise in cytosolic Ca2+ into an even greater (either larger and/or more sustained) Ca2+ signal in the cytosol. Peiter et al. (2005) have demonstrated that TPC1-dependent Ca2+ flux into the cytosol is involved in (hormone-based) Ca2+-signalling pathways, and that tpc1 loss-of-function mutant plants lack the ability to translate a cytosolic Ca2+ increase arising from influx across the PM into a Ca2+-dependent physiological response.

There are up to 57 cation-conducting channels encoded in the plant genome (see Mäser et al., 2001). Aside from K+-selective channels and the tonoplast-localized TPC1, 40 of these cation channels belong to two large protein families about which little is known. There are 20 members each in the glutamate receptor (GLR) channel and cyclic nucleotide-gated channel (CNGC) families of proteins. Plant GLRs are orthologues of the non-selective cation (including Ca2+) conducting ligand-gated channels found in neurones of the mammalian central nervous system; members of this channel family are activated by extracellular glutamate. Many of the plant GLRs are targeted to the cell secretory pathway and expressed in leaves/above ground organs (Davenport, 2002); thus they can be considered as candidates to contribute to channel-mediated Ca2+ influx into plant cells. Recently, Qi et al. (2006) undertook the first characterization of a plant GLR (i.e. GLR3.3) that includes genetic evidence that the native channel can provide a Ca2+ uptake pathway into the plant cell cytosol. Ca2+ conduction was activated by a number of (extracellular) amino acids including glutamate, and, importantly, a glr3.3 loss-of-function mutant lacked a glutamate-dependent rise in cytosolic Ca2+.

Animals have six CNGC genes; the translation products of these genes are involved in signal transduction cascades, allowing for perception of external cues to facilitate downstream influx of Ca2+ or Na+ into the responding cell cytosol (Kaupp and Seifert, 2002). Of the 20 members of the plant CNGC family (i.e. sharing sequence similarity and structural homology to animal CNGCs), publicly available expression profiling databases indicate that only one has a deduced organelle-targeting transit peptide and most are expressed in leaves and plant shoots (Talke et al., 2003). Cation conduction across the PM by these channels is activated by cytosolic cAMP and (in some cases) cGMP (Leng et al., 1999); however, CNGCs are voltage-gated (hyperpolarizing membrane potentials increase conductance probably due to increased channel opening) as well (Leng et al., 1999; 2002; Lemtiri-Chlieh and Berkowitz, 2004). Expression in heterologous systems has demonstrated that several members of this family (including CNGC2) can form homomeric channels and conduct Ca2+ across the PM (Leng et al., 1999; Ma et al., 2006). Application of cAMP to the plant cell cytosol activates inward Ca2+ currents across the plant cell PM, demonstrating the presence of functional CNGCs in plants (Lemtiri-Chlieh and Berkowitz, 2004). Recent work (Ali et al., 2007) has shown that cells of a cngc2 mutant (dnd1) lack a cAMP-activated inward PM Ca2+ current, confirming that CNGC translation products are components of a PM channel that allows for Ca2+ entry into the plant cell. Although the possibility exists that other types of ligand- and voltage-gated channels or transporters could contribute to pathogen-associated influx of Ca2+ into the plant cell cytosol (Sanders et al., 2002), the only specific gene products currently known that are potential candidates for facilitating this influx are TPC1, GLRs and CNGCs.

Pathogen-associated molecular patterns (PAMPs), pathogen perception and cytosolic Ca2+ rise

Although Ca2+ influx into the cytosol during plant cell response to pathogen perception likely occurs from the apoplast as well as from internal Ca2+ stores, much evidence (reviewed in Lecourieux et al., 2006) is consistent with the initial Ca2+ elevation requiring influx into the cell across the PM. Early patch clamp studies (Gelli and Blumwald, 1997; Zimmerman et al., 1997) provided evidence that PAMPs, as well as race-specific pathogen virulence proteins (i.e. avr gene products if the interaction is an incompatible one with the host), increase the open probability of PM inwardly conducting Ca2+-permeable channels in plant cell (parsley and tomato) protoplasts. Most likely, the cytosolic Ca2+ rise, referred to as an early signal induced upon pathogen perception, occurs through such channels as a transient elevation initiated within minutes or even seconds after pathogen perception/recognition (Lecourieux et al., 2002; Hu et al., 2004). Although the amplitude and/or frequency of repeated cytosolic Ca2+ oscillations or spikes can impart specificity to a plant cell responding to a specific external signal, most evidence indicates that Ca2+ signalling in response to pathogen recognition is manifest by a transient cytosolic elevation rather than repeated Ca2+ oscillations. Lecourieux et al. (2002, 2005), Moscatiello et al. (2006) and Hu et al. (2004), among others, evaluated numerous examples of PAMP (referred to by these authors as elicitor)-induced cytosolic Ca2+ elevations in cells, cell cultures and intact plants. The Ca2+-dependent luminescence of recombinant cytosolic-localized jellyfish aequorin (see Grant et al., 2000; Plieth, 2006) was used to characterize this transient Ca2+ elevation as rising from homeostatic levels to 1–3 μM typically in a biphasic fashion. The initial peak can occur within minutes, or even seconds. It should be noted that Grant et al. (2000) concluded that such a biphasic Ca2+ elevation occurred in aequorin-expressing Arabidopsis plants only in the presence of a pathogen with an avr gene; they concluded that the major, second rise in cytosolic Ca2+ of the biphasic pattern was specifically induced during avr gene product elicitation of ETI. Pike et al. (2005) provided electrophysiological evidence that a pathogen-induced Ca2+ influx occurs due to R gene-mediated signalling as well. However, recent patch clamp data from our laboratory (Ali et al., 2007) indicate that a PAMP alone is capable of activating inward PM Ca2+ currents in plant cells. There is sufficient evidence available (mentioned above) to support the conclusion that PAMPs (which would be present in pathogens in the absence of avr gene products), as well as pathogen avr gene products that initiate ETI during an incompatible interaction with a plant host harbouring a corresponding R gene, and of course the pathogen itself are all capable of activating inward Ca2+ currents across the PM as an early plant cell response to pathogen perception.

At present, it is unclear how binding of a PAMP to a PM integral protein such as a PRR leads to activation (increased open probability) of a Ca2+-conducting channel. Current models (Jones and Dangl, 2006; He et al., 2007; Hofius et al., 2007) speculate that plant PRRs may include many of the 233 members of the leucine-rich-repeat receptor-like kinase (LRR-RLK) family of plant PM integral proteins. For some PAMPs, such as the bacterial proteins flagellin and elongation factor EF-Tu, the specific PRR protein has been identified (FLS2 and EFR, respectively), allowing for insights into some signalling steps downstream from pathogen perception (Zipfel et al., 2004; 2006). Importantly, FLS2 and EFR are LRR-RLKs, and perception of the PAMP flagellin by FLS2 results in downstream protein phosphorylation (Peck et al., 2001). Protein kinase (PK)-dependent phosphorylation could be a step in signalling cascades induced by PRR perception of a pathogen (PAMP), either upstream or downstream from the initial PAMP-induced cytosolic Ca2+ elevation. Effects of the PAMPs cryptogein and lipopolysaccharide (LPS) involve altered phosphorylation of plant proteins, downstream from an initial cytosolic Ca2+ elevation (Lecourieux-Ouaked et al., 2000; Gerber and Dubery, 2004). Some evidence also suggests that after perception of the PAMP cryptogein, the cytosolic Ca2+ elevation is dependent on kinase activity (Lecourieux et al., 2002). However, kinase inhibitors did not block the cytosolic Ca2+ elevation caused by the elicitor endopolygalacturonase I (Vandelle et al., 2006). The early work of Gelli et al. (1997) indicated that the avr gene product activation of a plant PM Ca2+-conducting channel is mediated by phosphorylation, although not necessarily of the channel protein itself. Phosphorylation (again, not necessarily of the channel itself) events are involved upstream from activation of inwardly conducting Ca2+-permeable channels involved in other signalling pathways (Stoelzle et al., 2003).

Candidates for the pathogen-associated PM Ca2+ uptake pathway

Members of a transient receptor potential channel subfamily appear to mediate Ca2+ influx into phagocytes and other animal cells capable of invoking innate immune responses (including Ca2+-associated PCD) to pathogens (Massullo et al., 2006); however, transient receptor potential channel orthologues are not present in plants (Mäser et al., 2001). As mentioned above, CNGC and GLR channels conduct Ca2+ into plant cells; they are potential candidates for the pathogen/PAMP/elicitor (of ETI)-activated Ca2+ influx pathway. The structure:function aspects of ionotropic GLRs make them intriguing possible pathogen-responsive proteins. Qi et al. (2006) have recently shown that a number of amino acids can activate GLR-mediated Ca2+ influx across the plant PM. The precise role of GLRs in plant leaf cells is unclear; Qi et al. (2006) speculate that GLRs in roots could be activated by the amino acids present in the rhizosphere. Could an increase in apoplast amino acids arising from cell (microbe and/or host cell) necrosis during initial plant-pathogen skirmishes lead to activation of GLRs, thus initiating the associated Ca2+-signalling cascade? There is little evidence at present supporting this speculation. One report does provide a loose association between GLR-mediated Ca2+ uptake and altered plant response to pathogens (Kang et al., 2006); in this case, GLR-mediated response to the pathogen did not involve HR. Another possible cytosolic metabolite that could accumulate in the leaf apoplast due to cell injury is ATP (or ADP). Extracellular ATP/ADP may bind to an as-yet-unidentified PM receptor and initiate Ca2+ influx into plant cells that has been associated with NADPH oxidase-dependent ROS production (Jeter et al., 2004; Song et al., 2006). However, other reports indicate that extracellular ATP is required in the plant leaf apoplast in order to prevent signalling, leading to PCD (Chivasa et al., 2005).

In contrast to GLRs, there is direct evidence supporting a role for CNGCs in PAMP/pathogen-induced Ca2+ entry into plant cells, and initiation of a signalling cascade leading to HR. Expression of some CNGCs is enhanced in bean leaves inoculated with an avirulent pathogen during HR (Ali et al., 2003). Loss-of-function mutations in CNGC2 and CNGC4 prevent plant HR to avirulent pathogens (Clough et al., 2000; Balaguéet al., 2003; Jurkowski et al., 2004). CNGC11 and CNGC12 are also involved in plant defence response to avirulent pathogens; they do not, however, appear to mediate HR (Yoshioka et al., 2006). Although the subunit composition of native plant CNGC channel complexes is not known, it appears that they are tetrameric (Leng et al., 2002), as is the case with animal CNGCs. All animal CNGCs are heterotetramers, composed of at least two CNGC gene translation products (Zheng and Zagotta, 2004). Perhaps this is also the case with plant CNGCs (for example, CNGC2 and CNGC4 may be subunits of the same channel complex); this could explain why loss of either CNGC2 or CNGC4 could prevent the same downstream Ca2+-dependent pathogen signalling leading to HR. Application of the PAMP LPS activates an inward Ca2+ current in leaf protoplasts that likely occurs through a CNGC (Ali et al., 2007).

Inward Ca2+ current into plant leaf cells occurs upon cAMP activation of PM channels (Lemtiri-Chlieh and Berkowitz, 2004); earlier work showed that cAMP increases cytosolic Ca2+ (from extracellular Ca2+) in tobacco cell cultures (Volotovski et al., 1998). Pathogen elicitors cause a transient rise in cytosolic cAMP in a number of plant species (Bolwell, 1992; Kurosaki and Nishi, 1993; Cooke et al., 1994; Jiang et al., 2005). Increasing cytosolic cAMP enhanced Ca2+-dependent elicitor-induced ROS production in bean cell cultures (Bindschedler et al., 2001); some similar effects were found with Arabidopsis cell cultures (Davies et al., 2006). Preliminary studies from our laboratory (W. Ma et al., unpublished data) indicate that inhibitors of adenylate cyclase (AC) can: (i) reduce the early component (i.e. in the biphasic elevation) of avirulent pathogen induced cytosolic Ca2+ elevation monitored in cells of aequorin-transformed plants; (ii) block downstream pathogen-signalling pathways, leading to NO and ROS generation (monitored in guard cells of epidermal peels); and (iii) delay plant HR response to an avirulent pathogen.

A gene encoding the plant leaf AC (i.e. generating cAMP) has not yet been identified or cloned (but see Moutinho et al., 2001), nor has a cyclic nucleotide phosphodiesterase (PDE) (i.e. breaking down cyclic nucleotides) been cloned (Newton and Smith, 2004). Therefore, it is unclear how the enzymes responsible for cAMP homeostasis are regulated, or how pathogen perception could be translated into a rise in cAMP. However, the aforementioned pathogen-induced rise in plant cell cytosolic cAMP (mediated by either activation of AC or inhibition of PDE) could open CNGC channels, leading to the primary PM Ca2+ influx and the initial component of the biphasic PAMP/pathogen-associated Ca2+ rise.

As mentioned above, pathogen perception by PRRs could involve an initial phosphorylation event at the plant cell PM, and there is some evidence that inward Ca2+ conduction into the plant cell during pathogen signalling could involve phosphorylation of the Ca2+ channel involved. Thus, PRR-mediated phosphorylation of CNGCs (either directly or indirectly) could be a mechanism by which CNGC-mediated Ca2+ currents are activated in the signalling pathway. Although phosphorylation/dephosphorylation is one of many ways in which animal CNGC currents are modulated, evidence is lacking about possible phosphorylation of plant CNGCs. A possible mechanism for translation of pathogen perception by the plant into cAMP rise could be through G-protein signalling. Pharmacological and G-protein loss-of-function mutant studies suggest that G proteins mediate (i) PM Ca2+ currents, (ii) elicitor-mediated cytosolic Ca2+ rise, and (iii) downstream generation of ROS (Thuleau et al., 1998; Bindschedler et al., 2001; Ortega et al., 2002; Suharsono et al., 2002; Assmann, 2005). Although the evidence is lacking for plants, some G-protein signalling pathways in animals involve G-protein α-subunit interaction with, and activation of, AC and resulting cAMP rise (Simonds, 1999). The hypothesized interaction between a G protein (acting downstream from a PRR) and AC could provide a mechanism leading to activation of CNGCs and increased conductance of Ca2+ across the PM. An alternative possibility (also speculative at this point) is that in plants, G proteins could inhibit PDE, thus leading to a rise in cytosolic cAMP (by reducing its breakdown) and activation of CNGCs.

It is interesting to note that in the guard cells of the stomatal complex, perception of PAMPs and bacterial pathogens leads to a signal transduction pathway resulting in stomatal closure, which blocks bacterial pathogen entry into the leaf (Melotto et al., 2006). The plant hormone abscisic acid (ABA) also initiates signalling in the guard cell that leads to stomatal closure, and the two pathways share at least some steps (Klüsener et al., 2002; Melotto et al., 2006). Important work by Liu et al. (2007) has recently identified the ABA binding protein in the guard cell PM as a G-protein-coupled receptor. G-protein signalling, perhaps, could be involved in pathogen- and ABA hormone perception-signalling pathways in the guard cell, both of which involve cytosolic Ca2+ elevations (Klüsener et al., 2002; Sokolovski et al., 2005; Ali et al., 2007). Mori et al. (2006) provided genetic evidence that Ca2+-dependent kinases (CDPKs) are involved in ABA-activated inward Ca2+ conduction in guard cells during stomatal closure signalling. Hence, CDPK regulation (directly or indirectly) could also be a common component of the two signalling pathways in guard cells.

Contribution of Ca2+ release from internal stores to pathogen signalling

Conductance from both extracellular and intracellular Ca2+ pools contributes to pathogen-associated cytosolic Ca2+ elevation (Lamotte et al., 2004; Lecourieux et al., 2006). Candidates for contributing to the pathway for movement of Ca2+ into the cytosol from intracellular stores are TPC1 (clearly located in the tonoplast; Peiter et al., 2005), as well as inositol 1,4,5-triphosphate (IP3)- and cyclic ADP ribose (cADPR)-mediated Ca2+ release systems found in intracellular plant membranes. No genes have yet been associated with either IP3- or cADPR-mediated Ca2+ release into the plant cell cytosol, so little is known about the specific mechanisms regulating these Ca2+ transport pathways; in fact, there is some controversy regarding the precise intracellular membrane localization (although they are typically associated with the tonoplast and/or endoplasmic reticulum) of these Ca2+ transporters (Sanders et al., 2002; Pottosin and Schönknecht, 2007).

The aforementioned G-protein involvement in pathogen signalling could, in addition to modulating PM Ca2+ conductance, also play a role in release of Ca2+ from intracellular stores. Apone et al. (2003) provided genetic evidence that G-protein signalling in plants is required for phosphatidylinositol-specific phospholipase C (PI-PLC)-dependent IP3 production. PI-PLC inhibitor studies (reviewed by Lecourieux et al., 2006) indicate that cytosolic Ca2+ elevation in response to a number of PAMPs involves, in part, IP3-mediated Ca2+ flux. PLC activation downstream from elicitor (avr gene product) perception plays a role in generation of ROS (de Jong et al., 2004). Aside from IP3-dependent rise in cytosolic Ca2+, cADPR also modulates release of Ca2+ from the vacuole and/or endoplasmic reticulum. Some evidence suggests that cADPR may be involved in pathogen signalling as well as pathogen-induced Ca2+ release from internal stores in plant cells (Durner et al., 1998; Blume et al., 2000). Not much is currently known about steps upstream from cADPR signalling in plants, but some components of ABA-signalling pathways could be involved (Klüsener et al., 2002; Sanchez et al., 2004). In animal cells, NO is thought to activate Ca2+ release from intracellular stores through cADPR (Wendehenne et al., 2001).

Ca2+-activated Ca2+ currents through the channel TPC1 dominate Ca2+ efflux from the vacuole; this channel is expressed at high density in the tonoplast (Peiter et al., 2005; Pottosin and Schönknecht, 2007). Importantly, TPC1 is involved in Ca2+-mediated effects of several PAMPs, shown in tobacco and rice (Kadota et al., 2004; Kurusu et al., 2005). Because cytosolic Ca2+ elevation has a great effect on TPC1 conductance (Peiter et al., 2005), future work could reveal TPC1 as an important ‘amplifier’ of initial cytosolic Ca2+ influx across the PM during pathogen signalling.

Downstream effects of cytosolic Ca2+ elevation during pathogen signalling

Cytosolic Ca2+ elevation leads to a network of signalling events during plant innate immune response to pathogens; as mentioned above, one aspect of this resistance response that limits infection is HR, involving PCD (Hofius et al., 2007). Downstream events from Ca2+ elevation in this signalling cascade (i.e. their effect is Ca2+-dependent) which have been demonstrated to positively regulate PCD are: activation of CDPKs, mitogen-activated protein kinase (MAPK) cascades, NO and H2O2 (ROS) generation, and increase in Ca2+ bound to calmodulin (CaM). With several of these signal cascade events, it is clear that one way they facilitate HR is through effects on gene expression. ROS and NO can act independently and together to alter gene expression related to PCD during HR, and through actions independent of gene expression (Clarke et al., 2000; Neill et al., 2002; Romero-Puertas et al., 2004; Wendehenne et al., 2004; Delledonne, 2005; Torres et al., 2006; Van Breusegem and Dat, 2006; Zaninotto et al., 2006; Hofius et al., 2007). The ratio of NO to H2O2 is an important factor affecting HR (Delledonne et al., 2001). Possible mechanisms mediating Ca2+ elevation effects on NO and ROS generation will be discussed below.

Mitogen-activated protein kinase (MAPK)-signalling cascades (including the salicylic acid- and wound-induced MAPKs) downstream from Ca2+ elevation during pathogen response signalling activate plant pathogen defence-related gene expression, including some transcription factors related to HR (Kim and Zhang, 2004; Pedley and Martin, 2005; Ren et al., 2006; Hofius et al., 2007; Stulemeijer et al., 2007; Suarez-Rodriguez et al., 2007). It is unclear how cytosolic Ca2+ elevation activates MAPK cascades, but Ca2+ dependency of the initial phosphorylation suggests that Ca2+-dependent PKs may be involved (Lecourieux-Ouaked et al., 2000; Garcia-Brugger et al., 2006).

Plasma membrane localized NADPH oxidase generates ROS (O2) that causes the oxidative burst leading to HR. The O2 is converted to H2O2 by superoxide dismutase (SOD) (Lecourieux et al., 2006). In addition to possible effects on MAPK cascades and gene expression (Garcia-Brugger et al., 2006), recent work (Kobayashi et al., 2007) provides strong genetic evidence identifying two CDPKs as phosphorylating NADPH oxidase and thereby upregulating ROS production during pathogen signalling. In this recent work, however, prevention of NADPH oxidase activation in CDPK loss-of-function mutants was not investigated, leaving open the possibility that other mechanisms may be involved as well. NADPH oxidase has its own Ca2+ binding domains, and other work has shown that Ca2+ is required for maximal NADPH oxidase activity in vitro (Sagi and Fluhr, 2006), suggesting that Ca2+ elevations during pathogen signalling could directly activate ROS production. Another possible mechanism facilitating NADPH oxidase activation during pathogen signalling is suggested by a recent report (Elmayan et al., 2007) demonstrating a physical and functional interaction of a 14-3-3 protein with this ROS-generating enzyme.

Calmodulins (6–13 genes; Zielinski, 1998; Takabatake et al., 2007) and CaM-like proteins (CMLs) (about 50 genes; Bouchéet al., 2005) are a large family of Ca2+ binding, signalling proteins in plants; many isoforms have overlapping expression patterns. Increases in cytosolic Ca2+ lead to complexing of CaMs and CMLs with Ca2+, altering the structure of these Ca2+ binding proteins in a manner that allows them to interact with other plant proteins. CaMs (and CMLs) are involved in plant innate immunity, and in this context, can transmit the initial signal of cytosolic Ca2+ elevation (upon pathogen perception) to downstream targets in this signal transduction cascade. Pathogen infection results in the induction and/or suppression of different plant CaM isoforms (Garcia-Brugger et al., 2006). Manipulating plant CaM expression affects (non-HR related) basal resistance to a range of pathogens (Heo et al., 1999; Takabatake et al., 2007). In addition, altered expression of CaM/CMLs has been associated with modulation of HR; reducing expression of the CML APR134 in tomato depressed HR, and increasing CML 43 expression (an Arabidopsis orthologue of APR134) enhanced HR to an avirulent pathogen (Bouchéet al., 2005; Chiasson et al., 2005). The mechanismmediating these phenotypes has not yet been identified. At this point, identification of a specific mechanism allowing for CaM to translate a cytosolic Ca2+ elevation into a positive effect on HR can only be speculative, due to the lack of strong evidence clearly identifying CaM/CML-regulated proteins in vivo (Bouchéet al., 2005; Garcia-Brugger et al., 2006). One possibility is CaM activation of NAD kinase, which would increase the concentration of NADPH, the substrate for the oxidase responsible for generating ROS during the HR oxidative burst (Harding et al., 1997; Harding and Roberts, 1998).

Ca2+, CNGCs and nitric oxide synthase; interactions during pathogen signalling

Nitric oxide, perhaps one of the most intensively studied molecules in biology over the past two decades, is clearly involved in plant innate immunity (Wendehenne et al., 2004; Delledonne, 2005), and has been referred to as the ‘concert master’ of plant HR to pathogens (Dangl, 1998). In animals (and likely in plants as well), NO is generated in such signalling cascades through the action of nitric oxide synthase (NOS), which catalyses NO production from the substrate arginine and requires Ca2+/CaM activation (Crawford and Guo, 2005; Crawford, 2006). Inhibitors of arginine-dependent NO generation block pathogen- and/or PAMP-associated NO generation in plants and plant cells and impair HR (Delledonne et al., 1998; Zhang et al., 2003; Zeidler et al., 2004), but a plant NOS enzyme has not yet been identified (Guo et al., 2003; Crawford et al., 2006; Zemojtel et al., 2006).

In a recent characterization of the signalling steps linking CNGC2 loss-of-function with the prevention of HR in the dnd1 mutant, work from this laboratory (Ali et al., 2007) has shown that exogenous application of an NO donor to mutant plants restores HR; this work, along with that of Delledonne et al. (1998), indicates that NO is required for signalling downstream from Ca2+ influx to result in HR. Using the guard cell in epidermal peels as a model cell system, Ali et al. found that application of a PAMP (LPS)-induced (arginine-dependent, i.e. suggesting involvement of a ‘NOS’-like enzyme) NO generation in wild-type cells that was downstream from an influx of extracellular Ca2+ in dnd1 mutant cells, PAMP induction of NO generation was impaired. This linkage of a pathogen/PAMP-activated PM inward Ca2+ current with NOS-dependent NO generation expanded on prior studies of Lamotte et al. (2004). A new step in the signalling pathway linking CNGC-mediated Ca2+ currents to downstream NO generation (as well as HR in the case of plants responding to an avirulent pathogen) during pathogen signalling possibly identified by Ali et al. is the involvement of CaM (or CML) upstream from NO. The CaM antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) blocked both NO generation (in cells) and HR in plants. The effect of W7 was downstream from the Ca2+ channel, as PAMP activation of the channel was demonstrated in the presence of W7. It is not known whether the still unidentified plant NOS requires CaM/Ca2+ for function as is the case with animal NOS enzymes (although some studies suggest this possibility, see Corpas et al., 2004), so use of a CaM inhibitor should be interpreted with caution. In addition, W7 would also bind to plant proteins such as CDPKs that have similar EF hand motifs as CaM, so the specificity of this agent is also problematic. However, this work provides evidence suggesting that a cytosolic Ca2+ elevation could lead to NO generation by the concomitant rise in CaM/Ca2+ activating NOS. No alternatives for a mechanism linking Ca2+ to downstream NO generation in pathogen signalling have yet been proposed.

It should be noted that CNGCs are inactivated by a rise in cytosolic Ca2+/CaM (Hua et al., 2003a; Li et al., 2005; Ali et al., 2006). The rise of Ca2+/CaM that would occur, then, upon initial pathogen perception and early signalling could feed back to inhibit continued CNGC function during the signalling cascade.

Up to this point, CNGC function has been discussed here within the context of cAMP as an activating ligand with regard to early events upstream from PM Ca2+ channel opening upon pathogen perception by PRRs. At least in the case of some plant CNGC gene products (including CNGC2 and CNGC4, both of which are involved in HR) expressed (in isolation) in heterologous systems, cGMP as well as cAMP activates currents (Leng et al., 1999; Balaguéet al., 2003; Borsics et al., 2007). The specificity of cyclic nucleotide activation of CNGCs in native plant membranes may be different (Lemtiri-Chlieh and Berkowitz, 2004). Modelling of the cyclic nucleotide binding domains of plant CNGCs suggests that some differences are present in amino acid residues at positions that may impact relative affinity for cAMP versus cGMP (Hua et al., 2003b; also R.W. Mercier and G.A. Berkowitz, unpublished data). Thus, the possibility exists that various native CNGC channel complexes in plant membranes (i.e. composed of different members of the 20 gene family) may have differing affinities (i.e. activation profiles) for cAMP and cGMP. Such differences in cAMP versus cGMP activation are known to be present in animal CNGCs (Kaupp and Seifert, 2002).

This point about plant CNGCs and cGMP as an activating ligand is raised here because several studies have identified cGMP as a messenger molecule involved downstream from NO generation in pathogen-signalling cascades leading to HR (Durner et al., 1998; Clarke et al., 2000). It has also been established that (in addition to cytosolic Ca2+ elevation leading to NO generation during pathogen-signalling cascades) NO can trigger a downstream influx of Ca2+ into the cytosol in response to PAMP and elicitor application (Lamotte et al., 2004; 2005;Vandelle et al., 2006). The mechanism transducing NO generation to downstream cytosolic Ca2+ elevation is not known. One possibility is a direct regulatory effect of NO on ion channels; nitrosylation of cysteine residues of some (cell membrane as well as intracellular localized) Ca2+-conducting animal channels alters their structure and associated opening probability (Xu et al., 1998; Yoshida et al., 2006). The role cGMP plays downstream from NO in plant innate immunity is also unclear, but CNGC involvement seems a logical possibility.

Animals have two forms of guanylate cyclase (GC), the enzyme that synthesizes cGMP from GTP; a membrane bound isozyme and a soluble variant (Denninger and Marletta, 1999). In animals, soluble GC binds to, and is activated by, NO (Boon et al., 2005). Perhaps NO activates a plant GC isozyme in a similar fashion. If so, then NO generation during a pathogen response-signalling cascade could lead to cGMP generation in plants, and this rise in activating ligand could increase open probability of cGMP-responsive CNGCs. NO is a diffusible molecule, and therefore could be an intercellular signal (Shapiro, 2005). There is some evidence that NO plays a significant role in spreading an initial pathogen perception signal from a cell at the site of infection to neighbouring cells (Zhang et al., 2003). This cell-to-cell signalling cascade involving diffused NO activating GC and leading to Ca2+ influx through CNGCs could provide a mechanistic basis for how cells neighbouring an infection site undergo PCD during HR.

The aforementioned speculation concerning NO and downstream increase of cGMP (occurring perhaps in either the cell at the site of infection and/or neighbouring cells) presupposes the presence in the plant of a GC that is activated by NO binding. No such enzyme has yet been identified. One soluble GC has been cloned from Arabidopsis (Ludidi and Gehring, 2003), but it was specifically found to be unresponsive to NO in this study. More recently, the same group identified a new family of 27 putative membrane-bound and soluble GCs (Kwezi et al., 2007). In one case, the translation product of these genes was demonstrated to have GC activity. None of them have been checked for NO sensitivity, and their deduced amino acid sequences do not contain an NO binding motif, which involves a haem iron associated with the enzyme in the case of soluble GCs of animals (Wendehenne et al., 2001). Although the identification of 27 putative GCs in this work has not yet led to a mechanistic connection between NO generation and downstream cGMP synthesis, it does lead to an intriguing insight into a possible new connection between pathogen response signalling in plants and CNGCs. One of the putative GCs (At1g73080) is a NB-LRR that acts as a receptor for a plant-derived ‘elicitor’ that activates HR and innate immune responses in a fashion similar to pathogen-derived elicitor/PAMPs (Huffaker et al., 2006; Yamaguchi et al., 2006). In addition, some of the other putative GCs identified by Kwezi et al. (2007) are LRR-RLKs. As discussed above, some of the 233 known (Arabidopsis) LRR-RLKs are thought to be as-yet-unidentified PRRs responsible for PAMP perception and initiation of basal defence responses in an infected plant cell (Hofius et al., 2007). Thus, the identification of an elicitor receptor involved in pathogen signalling as a GC, and the possibility that some LRR-RLKs thought to be PRRs could have a GC function, raise the possibility that pathogen perception could directly lead to increase in cGMP, and activate CNGCs, cytosolic Ca2+ elevation and downstream signalling.

We still cannot be sure whether a NO-activated GC is present in plants, and whether diffusible NO acts as a signal through effects on such a target protein. An alternative to NO as a diffusible extracellular signal that could initiate pathogen-responsive cascades in cells neighbouring the site of pathogen perception is H2O2, which has been shown to activate PM Ca2+ channels and lead to cytosolic Ca2+ elevations involved in HR (Levine et al., 1996; Pei et al., 2000). Lemtiri-Chlieh and Berkowitz (2004) have noted that the CNGC Ca2+ currents recorded from native mesophyll and guard cell protoplasts appear similar to the H2O2-activated Ca2+ currents recorded by Pei et al. (2000) and others. Thus, NO and H2O2, which are downstream messenger/PCD activator molecules generated from initial Ca2+ influx into a cell upon pathogen perception, could also act to generate subsequent Ca2+ influx either in the original cell under infection, so as to amplify the Ca2+-mediated HR signal, or spread the primary pathogen perception signal to neighbouring cells.

Conclusion: Ca2+-mediated death in plant innate immunity – a complex signalling network

This review identified a number of possible mechanisms linking pathogen perception to activation of PM and intracellular Ca2+ channels/transporters. Candidate channels involved in pathogen-induced Ca2+ conduction were presented. The available information does not necessarily point to one specific signal step in each of these cases. Perhaps pathogen perception is translated to opening of Ca2+ channels through a number of different transduction systems, and more than one type of Ca2+ transporter may be involved. Further, it is clear that the signalling cascade downstream from Ca2+ channel function involves numerous pathways that may overlap and interact. For these reasons, we are left with a complex signalling network as a possible pathogen response pathway that translates pathogen perception to Ca2+-mediated death during innate immune responses. An attempt to integrate the informational flow and signalling steps in this plant response network is presented as a model in Fig. 1.

Figure 1.

Model of possible mechanisms involved with pathogen-associated Ca2+ rise in a plant cell, and signal transduction pathways leading to the hypersensitive response and plant cell death. In all cases, arrows imply activation unless they are shown with the notation ‘inhibition’. Some arrows are shown with broken lines for clarity. Membrane protein Ca2+ transporters are shown in red, proteins involved in the signalling pathway are shown in blue, signalling molecules are shown in orange, and defence response events evoked by the signalling cascade are shown in green. ABA, abscisic acid; avr, avirulence; cADPR, cyclic ADP ribose; CaM, calmodulin; CDPKs, Ca2+-dependent kinases; CMLs, CaM-like proteins; CNGC, cyclic nucleotide gated channel; ETI, effector-triggered immunity; ETS, effector-triggered susceptibility; GC, guanylate cyclase; GLR, glutamate receptor; GPCR1, G protein coupled receptor; HR, hypersensitive response; IP3, inositol 1, 4, 5-triphosphate; LPS, lipopolysaccharide; LRR-RLK, leucine-rich-repeat receptor like kinase; MAPK, mitogen-activated protein kinase; NO, nitric oxide; NOS, nitric oxide synthase; O2-, superoxide; OH-, hydroxyl radicals; PAMP, pathogen associated molecular pattern; PCD, programmed cell death; PDE, phosphodiesterase; PI-PLC, phosphatidylinositol-specific phospholipase C; PK, protein kinase; PM, plasma membrane; PRR, pattern recognition receptor; PTI, PAMP-triggered immunity; ROS, reactive oxygen species; SOD, superoxide dismutase; W7, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

The signalling network shown in Fig. 1 has the following steps. Pathogen/PAMP perception occurs due to PAMP binding to pathogen response receptors (LRR-RLKs), which initiates a pathogen perception-signalling cascade. The signalling cascade leads to activation of PM inwardly conducting Ca2+ channels and/or transporters. The perception signal could be transmitted to the PM channels (directly or indirectly) through kinase activity (RLK or PK), or indirectly through altered cyclic nucleotide levels. Perception of a pathogen avr gene translation product in the cytosol occurs through direct (or indirect) binding and recognition of the avr gene product to a plant R gene translation product, which could be either a soluble or membrane-bound recognition protein. This R geneinteraction also initiates a signalling cascade involving PM Ca2+ channel activation as a primary signal. Candidates for facilitating influx of Ca2+ across the PM are CNGCs, GLRs or the ATP/ADP-dependent transporter. In the case of this transporter or GLRs, pathogen perception could occur due to the extracellular presence of amino acids or adenosine phosphates (from dead plant or microbe cells). PAMP/pathogen perception could also involve G-protein signalling (including a G-protein-coupled receptor 1 and membrane-bound G protein), which could alter cytosolic cyclic nucleotides (affecting CNGCs) and, in addition, lead to release of intracellular Ca2+ stores through PLC activation and IP3 generation. The initial influx of Ca2+ into the cytosol can lead to the following downstream effects. Ca2+ could directly activate NADPH oxidase [leading to increased ROS (H2O2)], and the tonoplast Ca2+-activated Ca2+ release channel TPC1. CDPKs and CaMs are activated through Ca2+ binding. CaM/Ca2+ can increase NADPH oxidase generation of ROS (by increasing NADPH substrate level), increase NOS generation of NO, and inhibit CNGCs. CDPKs could activate NADPH oxidase as well. Cytosolic Ca2+ elevation also leads to activation of MAPK-signalling cascades that result in defence gene activation, including HR/PCD-related genes. H2O2 can work independently, and in a synergistic fashion together with NO, to induce HR. In addition, NO and ROS (including H2O2) can induce pathogen response defence gene activation, including transcription related to HR/PCD. NO could also cause cADPR elevation, activating cADPR-responsive intracellular Ca2+ release channels. NO (indirectly, through GC generation of cGMP) and H2O2 (directly) can activate PM Ca2+ channels. NO and/or H2O2 can diffuse to neighbouring cells to initiate PM Ca2+ channels and a new signalling cascade.

Acknowledgements

This work was supported by National Science Foundation Award MCB-0344141 to G.A.B., and is dedicated to the memory of Dr Martin Gibbs (Brandies Univ., National Academy of Sciences), an insightful scientist and inspirational teacher.

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