PAMP recognition and the plant-pathogen arms race

Plants have evolved systems analogous to animal innate immunity that recognise pathogen-associated molecular patterns (PAMPs). PAMP detection is an important component of non-host resistance in plants and serves as an early warning system for the presence of potential pathogens. Binding of a PAMP to the appropriate pattern recognition receptor leads to downstream signalling events and, ultimately, to the induction of basal defence systems. To overcome non-host resistance, pathogens have evolved effectors that target specific regulatory components of the basal defence system. In turn, this has led to the evolution in plants of cultivar-specific resistance mediated by R proteins, which guard the targets of effectors against pathogen manipulation; the arms race continues.     

CNGCs: prime targets of plant cyclic nucleotide signalling?

Cyclic nucleotide-gated channels (CNGCs) are a recently identified family of plant ion channels. They show a high degree of similarity to Shaker-type voltage-gated channels and contain a C-terminal cyclic nucleotide-binding domain with an overlapping calmodulin-binding domain. Heterologously expressed plant CNGCs show activation by cyclic nucleotides and permeability to monovalent and divalent cations. In plants, downstream effectors of cyclic nucleotide signals have so far remained obscure, and CNGCs might be their prime targets. The unique position of CNGCs as ligand-gated Ca(2+)-permeable channels suggests that they function at key sites where cyclic nucleotide and Ca(2+) signalling pathways interact. Such processes include plant defence responses, and two recently characterized Arabidopsis mutants in CNGC genes indeed show altered pathogen responses.     

Disruption of PAMP-induced MAP kinase cascade by a Pseudomonas syringae effector activates plant immunity mediated by the NB-LRR protein SUMM2

Pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) serves as a primary plant defense response against microbial pathogens, with MEKK1, MKK1/MKK2, and MPK4 functioning as a MAP kinase cascade downstream of PAMP receptors. Plant Resistance (R) proteins sense specific pathogen effectors to initiate a second defense mechanism, termed effector-triggered immunity (ETI). In a screen for suppressors of the mkk1 mkk2 autoimmune phenotype, we identify the nucleotide-binding leucine-rich repeat (NB-LRR) protein SUMM2 and find that the MEKK1-MKK1/MKK2-MPK4 cascade negatively regulates SUMM2-mediated immunity. Further, the MEKK1-MKK1/MKK2-MPK4 cascade positively regulates basal defense targeted by the Pseudomonas syringae pathogenic effector HopAI1, which inhibits MPK4 kinase activity. Inactivation of MPK4 by HopAI1 results in activation of SUMM2-mediated defense responses. Our data suggest that SUMM2 is an R protein that becomes active when the MEKK1-MKK1/MKK2-MPK4 cascade is disrupted by pathogens, supporting the hypothesis that R proteins evolved to protect plants when microbial effectors suppress basal resistance.     

Systemic acquired resistance is induced by R gene-mediated responses independent of cell death

On infection by pathogens, plants initiate defence responses that are able to curtail infection locally. These responses are mediated either by receptor-like proteins that recognize pathogen-associated molecular patterns or by the protein products of disease resistance ( R ) genes. At the same time, primary defence responses often result in the generation of signals that induce what is known as systemic acquired resistance (SAR), such that defence responses are enhanced on secondary pathogen challenge in distal tissues. R protein-mediated SAR induction is normally accompanied by a type of programmed cell death known as the hypersensitive response (HR) and, in some instances, cell death alone has been implicated in the induction of SAR. This has raised the question of whether R protein-mediated signalling per se induces SAR or whether SAR is an indirect result of the induction of HR. Using the Rx gene of potato, which confers resistance to Potato Virus X in the absence of cell death, we have shown that the HR is dispensable for R protein-mediated induction of SAR and that Rx-induced SAR is mediated by the same salicylate-dependent pathway induced by other R proteins.     

Death don't have no mercy and neither does calcium: Arabidopsis CYCLIC NUCLEOTIDE GATED CHANNEL2 and innate immunity

Plant innate immune response to pathogen infection includes an elegant signaling pathway leading to reactive oxygen species generation and resulting hypersensitive response (HR); localized programmed cell death in tissue surrounding the initial infection site limits pathogen spread. A veritable symphony of cytosolic signaling molecules (including Ca(2+), nitric oxide [NO], cyclic nucleotides, and calmodulin) have been suggested as early components of HR signaling. However, specific interactions among these cytosolic secondary messengers and their roles in the signal cascade are still unclear. Here, we report some aspects of how plants translate perception of a pathogen into a signal cascade leading to an innate immune response. We show that Arabidopsis thaliana CYCLIC NUCLEOTIDE GATED CHANNEL2 (CNGC2/DND1) conducts Ca(2+) into cells and provide a model linking this Ca(2+) current to downstream NO production. NO is a critical signaling molecule invoking plant innate immune response to pathogens. Plants without functional CNGC2 lack this cell membrane Ca(2+) current and do not display HR; providing the mutant with NO complements this phenotype. The bacterial pathogen-associated molecular pattern elicitor lipopolysaccharide activates a CNGC Ca(2+) current, which may be linked to NO generation due to buildup of cytosolic Ca(2+)/calmodulin.     

The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis

Plants use pattern recognition receptors (PRRs) to perceive pathogen-associated molecular pattern (PAMPs) and initiate defence responses. PAMP-triggered immunity (PTI) plays an important role in general resistance, and constrains the growth of most microbes on plants. Despite the importance of PRRs in plant immunity, the vast majority of them remain to be identified. We recently showed that the Arabidopsis LysM receptor kinase CERK1 is required not only for chitin signalling and fungal resistance, but plays an essential role in restricting bacterial growth on plants. We proposed that CERK1 may mediate the perception of a bacterial PAMP, or an endogenous plant cell wall component released during infection, through its extracellular carbohydrate-binding LysM-motifs. Here we report reduced activation of a PAMP-induced defence response on plants lacking the CERK1 gene after treatment with crude bacterial extracts. This demonstrates that CERK1 mediates perception of an unknown bacterial PAMP in Arabidopsis.Key words: PAMP, PRR, PTI, LysM, chitin, bacteria, carbohydrate     

Heterotrimeric G-protein regulation of ROS signalling and calcium currents in Arabidopsis guard cells

Heterotrimeric G proteins composed of Gα, Gβ, and Gγ subunits are important signalling agents in both animals and plants. In plants, G proteins modulate numerous responses, including abscisic acid (ABA) and pathogen-associated molecular pattern (PAMP) regulation of guard cell ion channels and stomatal apertures. Previous analyses of mutants deficient in the sole canonical Arabidopsis Gα subunit, GPA1, have shown that Gα-deficient guard cells are impaired in ABA inhibition of K(+) influx channels, and in pH-independent activation of anion efflux channels. ABA-induced Ca(2+) uptake through ROS-activated Ca(2+)-permeable channels in the plasma membrane is another key component of ABA signal transduction in guard cells, but the question of whether these channels are also dependent on Gα for their ABA response has not been evaluated previously. We used two independent Arabidopsis T-DNA null mutant lines, gpa1-3 and gpa1-4, to investigate this issue. We observed that gpa1 mutants are disrupted both in ABA-induced Ca(2+)-channel activation, and in production of reactive oxygen species (ROS) in response to ABA. However, in response to exogenous H(2)O(2) application, I(Ca) channels are activated normally in gpa1 guard cells. In addition, H(2)O(2) inhibition of stomatal opening and promotion of stomatal closure are not disrupted in gpa1 mutant guard cells. These data indicate that absence of GPA1 interrupts ABA signalling between ABA reception and ROS production, with a consequent impairment in Ca(2+)-channel activation.     

Interplay between calcium signalling and early signalling elements during defence responses to microbe- or damage-associated molecular patterns

While diverse microbe- or damage-associated molecular patterns (MAMPs/DAMPs) typically trigger a common set of intracellular signalling events, comparative analysis between the MAMPs flg22 and elf18 revealed MAMP-specific differences in Ca(2+) signalling, defence gene expression and MAMP-mediated growth arrest in Arabidopsis thaliana. Such MAMP-specific differences are, in part, controlled by BAK1, a kinase associated with several receptors. Whereas defence gene expression and growth inhibition mediated by flg22 were reduced in bak1 mutants, BAK1 had no or minor effects on the same responses elicited by elf18. As the residual Ca(2+) elevations induced by diverse MAMPs/DAMPs (flg22, elf18 and Pep1) were virtually identical in bak1 mutants, a differential BAK1-mediated signal amplification to attain MAMP/DAMP-specific Ca(2+) amplitudes in wild-type plants may be hypothesized. Furthermore, abrogation of reactive oxygen species (ROS) accumulation, either in the rbohD mutant or through inhibitor application, led to loss of a second Ca(2+) peak, demonstrating a feedback effect of ROS on Ca(2+) signalling. Conversely, mpk3 mutants showed a prolonged accumulation of ROS but this did not significantly impinge on the overall Ca(2+) response. Thus, fine-tuning of MAMP/DAMP responses involves interplay between diverse signalling elements functioning both up- or downstream of Ca(2+) signalling.     

Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation.

The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis.     

Cyclic lipopeptides from Bacillus subtilis activate distinct patterns of defence responses in grapevine

Non‐self‐recognition of microorganisms partly relies on the perception of microbe‐associated molecular patterns (MAMPs) and leads to the activation of an innate immune response. Bacillus subtilis produces three main families of cyclic lipopeptides (LPs), namely surfactins, iturins and fengycins. Although LPs are involved in induced systemic resistance (ISR) activation, little is known about defence responses induced by these molecules and their involvement in local resistance to fungi. Here, we showed that purified surfactin, mycosubtilin (iturin family) and plipastatin (fengycin family) are perceived by grapevine plant cells. Although surfactin and mycosubtilin stimulated grapevine innate immune responses, they differentially activated early signalling pathways and defence gene expression. By contrast, plipastatin perception by grapevine cells only resulted in early signalling activation. Gene expression analysis suggested that mycosubtilin activated salicylic acid (SA) and jasmonic acid (JA) signalling pathways, whereas surfactin mainly induced an SA‐regulated response. Although mycosubtilin and plipastatin displayed direct antifungal activity, only surfactin and mycosubtilin treatments resulted in a local long‐lasting enhanced tolerance to the necrotrophic fungus Botrytis cinerea in grapevine leaves. Moreover, challenge with specific strains overproducing surfactin and mycosubtilin led to a slightly enhanced stimulation of the defence response compared with the LP‐non‐producing strain of B. subtilis. Altogether, our results provide the first comprehensive view of the involvement of LPs from B. subtilis in grapevine plant defence and local resistance against the necrotrophic pathogen Bo. cinerea. Moreover, this work is the first to highlight the ability of mycosubtilin to trigger an immune response in plants.     

The cyclic nucleotide-gated channels AtCNGC11 and 12 are involved in multiple Ca²⁺-dependent physiological responses and act in a synergistic manner

Arabidopsis cyclic nucleotide-gated ion channels (AtCNGCs) form a large family consisting of 20 members. These channels have so far been reported to be involved in a diverse range of physiological phenomena. For example, AtCNGC18 was reported to play an important role in pollen tube growth, while AtCNGC2, 4, 11, and 12 were implicated in mediating pathogen defence. To identify additional functions for AtCNGC11 and 12, various physiological aspects were analysed using both AtCNGC11 and 12 single knockout mutants as well as a double mutant. Although AtCNGC11 and 12 can function as K(+) and Ca(2+) channels in yeast, it was found that the loss of AtCNGC11 and 12 in Arabidopsis caused increased sensitivity to Ca(2+) but not K(+), indicating a specific function for these genes in Ca(2+) signalling in planta. However, they did not show an alteration in Ca(2+) accumulation, suggesting that AtCNGC11 and 12 are not involved in general Ca(2+) homeostasis but rather in the endogenous movement of Ca(2+) and/or Ca(2+) signalling. Furthermore, these channels synergistically contribute to the generation of a Ca(2+) signal that leads to gravitropic bending. Finally, AtCNGC11 and 12 gene expression was induced during dark-induced senescence and AtCNGC11 and 12 knockout mutants displayed enhanced chlorophyll loss, which was even more pronounced in the double mutant, also indicating synergistic roles in senescence. The findings indicate that (i) some CNGC family members have multiple physiological functions and (ii) some plant CNGCs share the same biological function and work in a synergistic manner.     

Store-operated Ca2+ influx causes Ca2+ release from the intracellular Ca2+ channels that is required for T cell activation

The precise control of many T cell functions relies on cytosolic Ca(2+) dynamics that is shaped by the Ca(2+) release from the intracellular store and extracellular Ca(2+) influx. The Ca(2+) influx activated following T cell receptor (TCR)-mediated store depletion is considered to be a major mechanism for sustained elevation in cytosolic Ca(2+) concentration ([Ca(2+)](i)) necessary for T cell activation, whereas the role of intracellular Ca(2+) release channels is believed to be minor. We found, however, that in Jurkat T cells [Ca(2+)](i) elevation observed upon activation of the store-operated Ca(2+) entry (SOCE) by passive store depletion with cyclopiazonic acid, a reversible blocker of sarco-endoplasmic reticulum Ca(2+)-ATPase, inversely correlated with store refilling. This indicated that intracellular Ca(2+) release channels were activated in parallel with SOCE and contributed to global [Ca(2+)](i) elevation. Pretreating cells with (-)-xestospongin C (10 microM) or ryanodine (400 microM), the antagonists of inositol 1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR), respectively, facilitated store refilling and significantly reduced [Ca(2+)](i) elevation evoked by the passive store depletion or TCR ligation. Although the Ca(2+) release from the IP3R can be activated by TCR stimulation, the Ca(2+) release from the RyR was not inducible via TCR engagement and was exclusively activated by the SOCE. We also established that inhibition of IP3R or RyR down-regulated T cell proliferation and T-cell growth factor interleukin 2 production. These studies revealed a new aspect of [Ca(2+)](i) signaling in T cells, that is SOCE-dependent Ca(2+) release via IP3R and/or RyR, and identified the IP3R and RyR as potential targets for manipulation of Ca(2+)-dependent functions of T lymphocytes.     

Ethylene activates a plasma membrane Ca(2+)-permeable channel in tobacco suspension cells

Here, the effects of the ethylene-releasing compound, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currents across plasma membranes and on the cytosolic Ca(2+) activity ([Ca(2+)](c)) of tobacco (Nicotiana tabacum) suspension cells were characterized using a patch-clamp technique and confocal laser scanning microscopy. Exposure of tobacco protoplasts to ethephon and ACC led to activation of a plasma membrane cation channel that was permeable to Ba(2+), Mg(2+) and Ca(2+), and inhibited by La(3+), Gd(3+) and Al(3+). The ethephon- and ACC-induced Ca(2+)-permeable channel was abolished by the antagonist of ethylene perception (1-metycyclopropene) and by the inhibitor of ACC synthase (aminovinylglycin), indicating that activation of the Ca(2+)-permeable channels results from ethylene. Ethephon elicited an increase in the [Ca(2+)](c) of tobacco suspension cells, as visualized by the Ca(2+)-sensitive probe Fluo-3 and confocal microscopy. The ethephon-induced elevation of [Ca(2+)](c) was markedly inhibited by Gd(3+) and BAPTA, suggesting that an influx of Ca(2+) underlies the elevation of [Ca(2+)](c). These results indicate that an elevation of [Ca(2+)](c), resulting from activation of the plasma membrane Ca(2+)-permeable channels by ethylene, is an essential component in ethylene signaling in plants.     

The multiple mechanisms of Ca2+ signalling by listeriolysin O, the cholesterol-dependent cytolysin of Listeria monocytogenes

Cholesterol-dependent cytolysins (CDCs) represent a large family of conserved pore-forming toxins produced by several Gram-positive bacteria such as Listeria monocytogenes, Streptococcus pyrogenes and Bacillus anthracis. These toxins trigger a broad range of cellular responses that greatly influence pathogenesis. Using mast cells, we demonstrate that listeriolysin O (LLO), a prototype of CDCs produced by L. monocytogenes, triggers cellular responses such as degranulation and cytokine synthesis in a Ca(2+)-dependent manner. Ca(2+) signalling by LLO is due to Ca(2+) influx from extracellular milieu and release of from intracellular stores. We show that LLO-induced release of Ca(2+) from intracellular stores occurs via at least two mechanisms: (i) activation of intracellular Ca(2+) channels and (ii) a Ca(2+) channels independent mechanism. The former involves PLC-IP(3)R operated Ca(2+) channels activated via G-proteins and protein tyrosine kinases. For the latter, we propose a novel mechanism of intracellular Ca(2+) release involving injury of intracellular Ca(2+) stores such as the endoplasmic reticulum. In addition to Ca(2+) signalling, the discovery that LLO causes damage to an intracellular organelle provides a new perspective in our understanding of how CDCs affect target cells during infection by the respective bacterial pathogens.