Cyclic nucleotide gated channels and related signaling components in plant innate immunity

Abstract

Although plants lack the mobile sentry cells present in animal innate immune systems, plants have developed complex innate immune reactions triggering basal resistance and the hypersensitive response (HR). Cytosolic Ca²⁺ elevation is considered to be an important early event in this pathogen response signal transduction cascade. Plasma membrane (PM)-localized cyclic nucleotide gated channels (CNGCs) contribute to the cytosolic Ca²⁺ rise upon pathogen perception. Recent work suggests that some PM-localized leucine-rich-repeat receptor-like kinases (LRR-RLKs) may be involved in the perception of pathogen associated molecular pattern molecules and triggering some pathogen responses in plants, some of these LRR-RLKs might have cyclic nucleotide cyclase activity. The recognition of pathogens may be connected to cyclic nucleotide generation and the activation of CNGCs, followed by cytosolic Ca²⁺ increase and downstream signaling events (possibly involving nitric oxide, reactive oxygen species (ROS), calmodulin (CaM), CaM-like protein (CML) and protein kinases). Notably, CaM or CML could be the crucial sensor downstream from the early Ca²⁺ signal leading to nitric oxide (NO) production during plant innate immune responses.

Introduction

Of the 57 genes encoding cation-conducting channels in the Arabidopsis genome, 20 are members of the cyclic nucleotide gated channel (CNGC) family.^1,2 Plant CNGCs have been proposed to be involved in multiple plant physiological processes including root growth and gravitropism,³ pollen tube growth,⁴ heavy metal toxicity tolerance,^5,6 sodium stress tolerance,⁷ leaf senescence,⁸ plant disease resistance and innate immunity.^9–14 Importantly, CNGCs are considered as candidates for conducting Ca²⁺ through the plant cell plasma membrane (PM). An increase in cytosolic Ca²⁺ is an essential early step in plant innate immune responses to pathogens.¹⁵ Current models suggest that plants have two innate immune pathogen response systems. One of the systems is basal resistance, which can be activated by the perception of conserved molecules essential to the fitness or integrity of the microbe. The microbial molecules that trigger plant basal pathogen resistance responses are referred to as pathogen-associated molecular patterns (PAMPs). The other system is the gene-to-gene interaction between the translation product of a pathogen avirulence (avr) gene and (if present in the plant), a corresponding plant R (resistance) gene product. The perception of a pathogen at one or both of these levels may cause the rapid and programmed death of cells in the immediate vicinity of the infection; this rapid cell death is known as the hypersensitive response (HR). Nitric oxide (NO)¹⁶ and reactive oxygen species (ROS, including H₂O₂)¹⁷ will be generated during this process which can limit pathogen growth from the infection site. The Arabidopsis cngc2 mutant (‘defense, no death’, or dnd1) displays an impaired HR phenotype¹¹ and lacks a functional CNGC2 cation channel, and does not show a cAMP activated inward Ca²⁺ current.⁹ The impaired HR phenotype to an avirulent pathogen in dnd1 mutant plants can be complemented by the addition of an NO donor.⁹ In the dnd1 mutant, PAMP-induced NO generation in guard cells is impaired as well.⁹ Ali et al.⁹ and Ma et al.¹⁸ also provide evidence showing calmodulin (CaM) or a CaM-like protein (CML), downstream targets of the Ca²⁺ signal, may be involved in PAMP-induced NO synthesis.

Recent bioinformatic analyses resulted in the identification of several leucine-rich-repeat receptor-like kinases (LRR-RLKs), which are postulated to possess a guanylate cyclase (GC) catalytic motif.¹⁹ LRR-RLKs are proposed to be responsible for PAMP perception and initiation of basal defense responses in an infected plant cell.²⁰ In addition, a recently characterized receptor (PEPR1) for a plant (Arabidopsis)-derived ‘elicitor’ (AtPep1, a plant endogenous peptide elicitor) that activates innate immune responses in a fashion similar to pathogen-derived elicitor/PAMPs is a LRR-RLK as well.^21–23 Intriguingly, this PEPR1 receptor is also proposed to have putative GC activity.¹⁹ Thus, pathogen perception signaling could involve translation of pathogen recognition into downstream signaling through cyclic nucleotide synthesis, which in turn will activate CNGCs, causing a rise in cytosolic Ca²⁺ that will trigger downstream signaling events (such as NO and H₂O₂ etc.,) and lead to HR.²⁴

In animal cells, NO synthase (NOS) activation is Ca²⁺/CaM dependent.^25,26 Ca²⁺ or CaM dependent NOS activity has also been reported in tissue of various plant species.^16,27,28 The PAMP lipopolysaccharide (LPS), which is a ubiquitous component of Gram-negative bacteria, has been found to be an elicitor of plant (and animal) innate immune responses.²⁹ Recent work has shown that plant NOS activity is activated by LPS.³⁰ LPS induced NO production is suggested to be dependent on NOS enzyme activity^9,30 and CaM is also proposed to be involved in this regulation process.⁹

CNGCs Facilitate Ca²⁺ Influx into Plant Cells

The expression of CNGCs in heterologous systems and their analysis using electrophysiological techniques have contributed to the characterization of their function in terms of cation uptake.² Previous studies have shown that some CNGCs could be the candidates responsible for the conductance of K⁺ and Na⁺ into the cytosol.^{7,10,13,31–34} In addition to acting as a monovalent cation uptake pathway, increasing evidence indicates that CNGCs participate in Ca²⁺ uptake during essential plant physiological processes. A yeast mutant lacking the CCH1 and MID1 Ca²⁺ transporters shows impaired growth in the presence of α mating factor.³⁵ The expression of CNGC1 in the cch1, mid1 yeast mutant can rescue this phenotype, elucidating the role of CNGC1 in Ca²⁺ permeability.^3,36 The Arabidopsis mutant ‘constitutive expresser of pathogenesis-related (PR) genes22′ (cpr22) was characterized as having a 3 kb fragment deletion in several CNGC genes, which causes the generation of a chimeric gene (CNGC11/12) derived from CNGC11 and CNGC12.¹³ The expression of CNGC11,12, or chimeric CNGC11/12 can complement the phenotype of the yeast cch1, mid1 mutant as well, implying that they have Ca²⁺ transport capability.¹⁴ In addition, the expression of CNGC18 in Escherichia coli (E. coli) leads to increased Ca²⁺ accumulation in E. coli cells, suggesting CNGC18 functions as Ca²⁺ permeable channel.⁴ Electrophysiological results demonstrate that the application of membrane permeable cAMP can elicit an inward Ca²⁺ current into the cytosol of mesophyll and guard cell protoplasts, supporting the existence of functional CNGCs as Ca²⁺ conducting channels in plants.³⁷ Recent electrophysiological studies have indicated that the cngc2 mutant (dnd1) lacks a cAMP-activated inward Ca²⁺ current, further corroborating that CNGCs can act as crucial Ca²⁺ entry pathways into the plant cell.⁹

In animal cells, six genes encode CNGCs, which mediate Ca²⁺ (and/or Na⁺) influx in signal transduction cascades responding to the perception of external cues.³⁸ Plant CNGCs show a broad range of expression profiles in leaves and shoots.³⁹ The evidence provided above indicates that CNGCs act as pathways for Ca²⁺ uptake into the plant cell. In addition to CNGCs, there are other proteins that potentially mediate Ca²⁺ influx into the cytosol, including 20 members of the glutamate receptor (GLR) family of cation channels in plants. In neurons, it is known that members of this family can be activated by extracellular glutamate, functioning as non-selective cation (including Ca²⁺) channels. Plant GLRs are orthologues of animal GLRs and could contribute to Ca²⁺ uptake into plant cells.⁴⁰ Recent work provides evidence indicating that a GLR (i.e., GLR3.3) mediates Ca²⁺ entry into the cytosol.⁴¹ Genetic evidence further demonstrates that the glr3.3 mutant shows impaired glutamate-mediated Ca²⁺ rise in the cytosol. Peiter et al.⁴² reported the characterization of TPC1, a tonoplast-localized non-selective cation channel, and suggested that its major function is to conduct Ca²⁺ current across the tonoplast out of the vacuole, resulting in cytosolic Ca²⁺ rise.

Interestingly, the elevation of cytosolic Ca²⁺ above homeostatic levels can activate the TPC1 channel and enhance the Ca²⁺ influx current due to the EF hands (Ca²⁺ binding) of the TPC1 channel; it is a Ca²⁺-activated Ca²⁺-conducting channel.⁴² TPC1-dependent Ca²⁺ flux into the cytosol is involved in plant hormone mediated Ca²⁺ signaling cascades. However, a recent study found that TPC1 does not play an important role in mediating the Ca²⁺ signal induced by different types of stresses, including PAMP-dependent cytosolic Ca²⁺ elevation and other aspects of innate immune signaling.⁴³ Ranf et al.⁴³ suggest TPC1 is not the main vacuolar cation channel influencing Ca²⁺ homeostasis under physiological conditions. Thus the role of the pool of vacuolar Ca²⁺ (a major component of total intracellular Ca²⁺ and a potential source of Ca²⁺ for cytosolic Ca²⁺ signals), as well as the molecular identity of the channel (possibly) mediating vacuolar Ca²⁺ efflux during plant innate immune signaling need to be explored in the future.

Signaling Molecule Involvement in Plant Innate Immunity

During their life cycle, plants are under constant challenge from different types of microorganisms. The successful invasion by a plant pathogen can cause many fatal problems to their host. Thus, plants must respond to the threat of invasion from numerous types of pathogenic microorganisms. The HR is a mechanism used by plants to prevent the spread of infection by microbial pathogens; it is characterized by the rapid death of cells in the local region surrounding an infection.⁴⁴

Recently, many excellent reviews have described various aspects of plant innate immunity.^{15,20,45–50} At present, the current model (see cited reviews above) supports the aforementioned two protective systems (basal response and R gene mediated resistance). It is well illustrated that PAMPs, essential components of the pathogen, can activate plant basal defenses. Perception of PAMPs requires the presence of pattern recognition receptor (PRR) proteins within the PM, which trigger the internal responses to the pathogen/PAMPs. Basal response follows, leading to PAMP-triggered immunity (PTI), including metabolic and transcriptional changes during this process.^45,48

Cytosolic Ca²⁺ elevation is considered a critical step in the signal transduction pathways in plant innate immunity.¹⁵ Animal and plant cells have some similarity in terms of the use of Ca²⁺ as a key molecule (secondary messenger) involved in numerous signaling responses to abiotic and biotic stresses. If the invaded plant has an R gene which encodes a protein that directly or indirectly interacts with the microbial effector, activating the gene-for-gene interaction, the plant immune system can induce another reaction called effector-triggered immunity (ETI). Current models distinguish the PTI and ETI as two overlapping but mechanistically distinct systems in that HR has been classically considered to be pathogen avr gene product related.²⁰ In addition, other recent work suggests a close relationship between ETI and PTI in plants in terms of sharing similar downstream targets and signal transduction components.⁵¹ It has been reported that PAMPs, such as bacterial flagellin, can elicit basal defense responses and R gene-mediated signal transduction pathways, illustrating the intimate linkage between the ETI and PTI.⁵²

As discussed above, candidates for facilitating Ca²⁺ influx into the cytosol are CNGCs, GLRs and TPC1. In addition, plant HR also includes the generation of NO¹⁶ and ROS.¹⁷ Furthermore, PAMPs can induce NO generation in the plant cell; NO is another signaling molecule crucial to plant innate immunity as well.^9,30,53 Disrupting the CNGC2 cation channel causes a lack of HR in the dnd1 mutant.¹¹ Recent work indicates that CNGC2 participates in plant innate immunity, acting as a PM Ca²⁺ channel.⁹ In this study, the bacterial PAMP LPS could elicit NO generation in leaf guard cells and facilitate Ca²⁺ influx into the cytosol. The addition of the Ca²⁺ channel blocker Gd³⁺ or the Ca²⁺ chelator EGTA abolished LPS induced NO synthesis, indicating that cytosolic Ca²⁺ elevation acts upstream from LPS-induced NO generation. Importantly, this LPS mediated NO induction is impaired in dnd1 guard cells. In addition, calmodulin (CaM), an important target that can bind to Ca²⁺, is involved in the plant innate immune response. CaM antagonists block LPS-induced NO generation in leaf guard cells and avirulent pathogen mediated HR.^9,18 These results underlie the hypothesis that the application of a CaM antagonist can disrupt the CaM-mediated innate immunity signal transduction pathway since CaM can regulate NO generation and NO is a pivotal molecule in this plant innate immunity system. However, it should be noted that Ca²⁺-dependent protein kinases (CDPKs) might be inhibited as well by the application of CaM antagonists. Two CDPKs were recently reported to play an important role in H₂O₂ generation in response to a pathogen.⁵⁴ Thus, block of HR by a CaM antagonist could be due to effects on several different proteins downstream from cytosolic Ca²⁺ elevation. However, recent work from this lab suggests that effects of the CaM antagonist on occur downstream from the rise in cytosolic Ca²⁺ during pathogen response signaling.¹⁸

At present a wealth of evidence supports the essential role of NO in the plant HR response to avirulent pathogens.^29,55–59 NO generation contributing to HR formation is due to (arginine-dependent) NOS activity.^16,60,61 Maximal activity of all three NOS isoforms in animal cells requires Ca²⁺/CaM as a cofactor.^{25,26,62–64} Importantly, animal NOS contains a CaM binding domain.⁶⁴ Although indirect evidence suggests that plant NOS activity depends on Ca²⁺/CaM,^16,18,27,28 plant NOS has not been identified.^66–68 Thus, this conclusion remains speculative.

In addition to the plant innate immune signaling transduction pathway steps mentioned above, we briefly add the point that ROS is another crucial signaling component during plant-pathogen interactions. ROS, including H₂O₂, superoxide and hydroxyl radicals are involved in signal cascades and the activation of HR during plant-pathogen interactions.¹⁷ ROS generation is also considered to be a crucial event downstream of cytosolic Ca²⁺ elevation.¹⁵ ROS and NO have the ability to act independently or synergistically to alter gene expression during HR^{17,20,29,56,68–71} and the ratio of NO to H₂O₂ is an important factor influencing HR.⁷²

Possible Signaling Molecules Upstream from Cytosolic Ca²⁺ Rise

In the previous sections of this review, we have briefly illustrated the significance of cytosolic Ca²⁺ elevation and Ca²⁺ regulated downstream steps in plant innate immunity. Not much is currently known about the signaling events that occur upstream from cytosolic Ca²⁺ influx in the plant innate immune response to pathogens. Cyclic nucleotides can activate CNGC channels and initiate Ca²⁺ entry into the cytosol.^9,37 This activation is impaired in the dnd1 mutant.⁹ The evidence that LPS can activate Ca²⁺ influx⁹ raises some interesting questions: how does plant cell perception of the PAMP LPS (or a pathogen) lead to the activation of inward Ca²⁺ current? What regulator or intermediate plays a key role in this signal transduction pathway? One possibility is that PAMP/pathogen-PRR recognition elicits the synthesis of cyclic nucleotide (either cAMP or cGMP rise). Therefore, we can speculate that cyclic nucleotide level changes might occur during pathogen/PAMP interaction, upstream from activating CNGCs.⁷³

At present, a gene encoding a plant (Arabidopsis) adenylate cyclase (AC) (cAMP synthesis) has not been identified⁷⁴ (but see Moutinho et al.⁷⁵), neither has a plant cyclic nucleotide phosphodiesterase (PDE) (i.e., facilitating catalysis of cyclic nucleotide) been cloned.⁷⁶ Thus, it is still unknown how pathogen perception is translated to cAMP (or cGMP) elevation in the plant cell, but much evidence indicates a cAMP rise could increase the cytosolic Ca²⁺ level. Pathogen elicitors have been reported to cause a transient cytosolic cAMP elevation in several plant species.^77–80 In French bean cells (Phaseolus vulgaris L.), Bindschedler et al.⁸¹ reported that a cytosolic cAMP rise enhanced Ca²⁺ dependent ROS production induced by elicitor. Moreover, current studies in our lab (Ma W, Smigel A, Walker RK, Verma R and Berkowitz GA, unpublished data) indicate that the application of AC inhibitor can: (i) impair avirulent pathogen induced cytosolic cAMP elevation; (ii) inhibit avirulent pathogen induced cytosolic Ca²⁺ elevation; (iii) abolish synthesis of the downstream pathogen signaling molecules (NO and ROS); and (iv) block plant HR to an avirulent pathogen. Correspondingly, the application of PDE inhibitor can: (i) hasten HR and cAMP rise induced by an avirulent pathogen; (ii) increase avirulent pathogen induced cytosolic Ca²⁺ elevation; and (iii) lead to NO synthesis in the absence of plant PAMP in leaf guard cells. The information above supports the speculation that pathogen induced cAMP rise (through either activation of AC or inhibition of PDE) could open CNGCs and lead to Ca²⁺ influx through PM as the early signal in plant innate immune response.

Recently, Kwezi et al. reported the identification of 26 putative plant membrane-bound and soluble guanylate cyclases (GCs).¹⁹ In animals, soluble GCs can bind to and be activated by NO.⁸² Several studies have identified cGMP acting as a messenger molecule downstream from NO in plant pathogen response signal transduction cascades.^71,83 In Arabidopsis, a soluble GC (AtGC1) had been identified in earlier work; it does not show a response to NO.⁸⁴ Although it is still unknown whether the other putative GCs could be responsive to the NO signal and activate cGMP synthesis, this discovery sheds light on a new connection between plant innate immunity and CNGCs. Kwezi et al.¹⁹ show that AtBRI1 (a receptor for brassinosteriods) functions as a GC in vitro. Among the other 25 putative GCs that Kwezi et al.¹⁹ identified, some encode LRR-RLKs. It has been demonstrated that some of the 233 known Arabidopsis LRR-RLKs can recognize PAMPs and elicit basal defense responses in plant cells.²⁰ Moreover, recent studies from Ryan and his colleagues identified a family of plant endogenous peptide elicitors, AtPep1-6, these peptides are thought to be involved in activating plant innate immune responses in a fashion similar to PAMPs.²² The AtPep receptor (PEPR1) is localized to the PM; PEPR1 is also a LRR-RLK.²³ Importantly, this PEPR1 receptor is one of the LRR-RLKs identified by Kwezi et al.¹⁹ to have a cytoplasmically-localized putative GC domain similar to AtBRI1. Therefore, the possible GC activity of these receptors leads us to speculate that the perception of pathogen can increase cGMP synthesis and activate CNGCs, cytosolic Ca²⁺ rise and downstream signaling.²⁴

CaM or CML could be the Candidate Linking Ca²⁺ Signaling and Downstream NO Production

Cytosolic Ca²⁺ rise is linked to downstream NO production in the plant immune response.^9,15 However, little is known about the molecules and/or mechanisms that link Ca²⁺ elevation in the cytosol to downstream NO synthesis. It is known that CaMs or CMLs are involved in plant signaling in response to pathogens. The overexpression of soybean CaM (SCaM-4 or SCaM-5) in tobacco can increase resistance to pathogens.⁸⁵ The silencing of the tobacco CaM NtCaM13 enhances the susceptibility of the plant to virulent bacteria or fungi.⁸⁶ Reducing the expression of CML APR134 in tomato diminishes HR while constitutive expression of AtCML43 (the Arabidopsis ortholog of CML APR134) in Arabidopsis accelerates HR development.^87,88 Recently, Ali et al.⁹ found that the application of a CaM antagonist can abolish LPS-induced NO generation in plant cells. NO generation in response to application of PAMPs such as LPS to plant cells occurs through an arginine-dependent pathway; this suggests involvement of a NOS type enzyme in pathogen-induced NO production.^9,30 Other work has shown that the NO production occurring during HR depends on NOS activity.^16,60,61 Recent work from this lab suggested a Ca²⁺/CaM dependence of NOS activity and that CaM or a CML may act either directly or indirectly on NOS to transmit the early signal of cytosolic Ca²⁺ elevation to NO generation during pathogen response signaling cascades leading to HR.¹⁸

Complexity of Signaling Steps in Plant Innate Immunity

The model we develop in this review of early steps in plant innate immune signaling includes the speculation that perception of a pathogen elicits cyclic nucleotide generation, causing activation of CNGCs. This in turn will initiate a rise in cytosolic Ca²⁺; resulting Ca²⁺ binding to CaM (or a CML) transmits the pathogen perception signal further, leading to NO synthesis. NO is required for HR and it can also interact with H₂O₂ to potentiate HR during the plant innate immune response.^16,72 However, it should be noted that some studies raise the possibility that these downstream components in the pathogen response signal transduction pathway could be upstream from cytosolic Ca²⁺ elevation as well. As mentioned above, NO generation could be upstream from cGMP synthesis.⁸³ In animal cells, NO is a diffusible messenger molecule that can bind and activate soluble GC.⁵⁸ It has also been reported that NO synthesis during plant pathogen signaling cascades can also cause cytosolic Ca²⁺ rise.^89–91

During plant innate immune signaling, Ca²⁺ conductance across the tonoplast as well as the PM could contribute to cytosolic Ca²⁺ elevation. As pointed out in recent reviews, cyclic ADP ribose (cADPR) is thought to activate tonoplast ryanodine receptors, which provide a pathway for Ca²⁺ release from the vacuole and/or other intracellular pools of Ca²⁺ in the plant cell.^27,91 As suggested in these reviews, in this manner, some evidence^83,92 indicates cADPR may act as a mediator between NO and cytosolic Ca²⁺ elevation, thus provides mechanism of Ca²⁺ release to cytosol downstream from NO generation. Furthermore, recent work suggests that S-nitrosylation and phosphorylation may be involved in NO-mediated signaling. These post-translational modifications have been suggested to mediate NO activation of proteins, including Ca²⁺ conducting channels involved in pathogen response signaling.^27,91 In addition, previous work suggests that H₂O₂ induced by pathogen perception could activate PM Ca²⁺-conducting channels.⁹³ Therefore, the whole pathway should be viewed as a complex interactive network. It could be possible that the initial Ca²⁺ influx through CNGCs induces downstream molecules (either NO or H₂O₂) that can diffuse to neighboring cells and activate new Ca²⁺ signals, propagating the HR. Furthermore, it could also be possible that downstream molecules induced by additional Ca²⁺ influx contribute to amplifying the pathogen perception signal.⁷³ In order to understand this complicated plant innate immune signaling transduction cascade, the missing pieces of this puzzle need further study.

Abbreviations

AC

adenylate cyclase

avr

avirulence

cADPR

cyclic ADP ribose

CaM

calmodulin

CDPKs

Ca²⁺ -dependent protein kinases

CMLs

CaM-like proteins

CNGC

cyclic nucleotide gated channel

E. coli

Escherichia coli

ETI

effector-triggered immunity

GC

guanylate cyclase

GLR

glutamate receptor

HR

hypersensitive response

LPS

lipopolysaccharide

LRR-RLK

leucine-rich-repeat receptor-like kinase

NO

nitric oxide

NOS

nitric oxide synthase

PAMP

pathogen associated molecular pattern

PDE

phosphodiesterase

PM

plasma membrane

PRR

pattern recognition receptor

PTI

PAMP-triggered immunity

ROS

reactive oxygen species