Mode of action of elicitors: Involvement of plasma membrane functions

International audience

NO-Based Signaling in Plants

In animals, nitric oxide (NO) is an endogenously produced radical involved in cell communication and signal transduction. Its functions in plants are currently being discovered at an unprecedented pace, and insight into NO-derived mechanisms has mainly been gained from research on signal transduction. Numerous studies have firmly placed NO as one component of the signal perception–transduction network that connects plant responses to primary signals, including hormones, elicitors of defence responses or abiotic stresses. Protein kinases and the second messengers Ca 2+, cGMP, and cADPR convey part of the NO signal within cells. Furthermore, NO-based protein modifications are emerging as broa…

Oligosaccharide-triggered plant immunity and plant protection

National audience

β-Aminobutyric Acid Primes an NADPH Oxidase–Dependent Reactive Oxygen Species Production During Grapevine-Triggered Immunity

International audience; The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by β-aminobutyric acid (BABA), a well-known priming-mediated plant resistance inducer. Our results indicate that, in contrast to oligogalacturonides (OG), BABA does not elicit typical defense-related early signaling events nor defense-gene expression in grapevine. However, in OG-elicited cells pretreated with BABA, production of reactive oxygen species (ROS) and expression of the respiratory-burst oxidase homolog RbohD gene were primed. In response to the causal agent of downy mildew Plasmopara viticola, a strong…

Cercospora beticola toxins. Part XVII. The role of the beticolin/Mg2+ complexes in their biological activity Study of plasma membrane H+-ATPase, vacuolar H+-PPase, alkaline and acid phosphatases

Beticolin-1 and beticolin-2, yellow toxins produced by the phytopathogenic fungus Cercospora beticola, inhibit the plasma membrane H(+)-ATPase. Firstly, since beticolins are able to form complexes with Mg2+, the role of the beticolin/Mg2+ complexes in the inhibition of the plasma membrane proton pump has been investigated. Calculations indicate that beticolins could exist under several forms, in the H(+)-ATPase assay mixture, both free or complexed with Mg2+. However, the percentage inhibition of the H(+)-ATPase activity is correlated to the concentration of one single form of beticolin, the dimeric neutral complex Mg2H2B2, which appears to be the active form involved in the H(+)-ATPase inh…

Influence of ATPase activity on PPi dependent H+-transport in tonoplast vesicles of Acer pseudoplatanus

Abstract Tonoplast H + -ATPase and H + -pyrophosphatase (H + -PPase) were previously characterized in Acer pseudoplatanus cells (A. Pugin et al., Plant Sci., 73 (1991) 23–34; A. Fraichard et al., Plant Physiol. Biochem., 31 (1993) 349–359). The present study concerns the relationships between these two enzymes in vitro. ATP and PPi hydrolysis were additive and the inhibition of one did not affect the activity of the second one. ATP and PPi H + -transports were also additive. The H + -PPase inhibition did not change ATP-dependent H + -transport but H + -ATPase inhibition inhibited the PPi dependent H + -transport. Because H + -PPase was reported to transport H + and K + into the vacuole (Dav…

Mechanisms of nitric-oxide-induced increase of free cytosolic Ca2+ concentration in Nicotiana plumbaginifolia cells.

In this study, we investigated a role for nitric oxide (NO) in mediating the elevation of the free cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) in plants using Nicotiana plumbaginifolia cells expressing the Ca(2+) reporter apoaequorin. Hyperosmotic stress induced a fast increase of [Ca(2+)](cyt) which was strongly reduced by pretreating cell suspensions with the NO scavenger carboxy PTIO, indicating that NO mediates [Ca(2+)](cyt) changes in plant cells challenged by abiotic stress. Accordingly, treatment of transgenic N. plumbaginifolia cells with the NO donor diethylamine NONOate was followed by a transient increase of [Ca(2+)](cyt) sensitive to plasma membrane Ca(2+) channel inhibitors …

Calcium signatures and signaling in cytosol and organelles of tobacco cells induced by plant defense elicitors

Calcium signatures induced by two elicitors of plant defense reactions, namely cryptogein and oligogalacturonides, were monitored at the subcellular level, using apoaequorin-transformed Nicotiana tabacum var Xanthi cells, in which the apoaequorin calcium sensor was targeted either to cytosol, mitochondria or chloroplasts. Our study showed that both elicitors induced specific Ca2+ signatures in each compartment, with the most striking difference relying on duration. Common properties also emerged from the analysis of Ca2+ signatures: both elicitors induced a biphasic cytosolic [Ca2+] elevation together with a single mitochondrial [Ca2+] elevation concomitant with the first cytosolic [Ca2+] p…

Ribosomal DNA genes in Fraxinus : Organisation and detection of hybridization beetween F. excelsior l and F. oxyphylla Bieb rDNA spacer probes

National audience

Cloning of two plant cDNAs encoling a beta-type proteasome subunit and a transformer-2-like SR-related protein: Early induction of the corresponding genes in tobacco cells treated with cryptogein

International audience

A β-1,3 Glucan Sulfate Induces Resistance in Grapevine against Plasmopara viticola Through Priming of Defense Responses, Including HR-like Cell Death

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybri…

Nitric oxide: comparative synthesis and signaling in animal and plant cells.

Since its identification as an endothelium-derived relaxing factor in the 1980s, nitric oxide has become the source of intensive and exciting research in animals. Nitric oxide is now considered to be a widespread signaling molecule involved in the regulation of an impressive spectrum of mammalian cellular functions. Its diverse effects have been attributed to an ability to chemically react with dioxygen and its redox forms and with specific iron- and thiol-containing proteins. Moreover, the effects of nitric oxide are dependent on the dynamic regulation of its biosynthetic enzyme nitric oxide synthase. Recently, the role of nitric oxide in plants has received much attention. Plants not only…

Involvement of Free Calcium in Action of Cryptogein, a Proteinaceous Elicitor of Hypersensitive Reaction in Tobacco Cells

Treatment of suspension-cultured tobacco (Nicotiana tabacum var Xanthi) cells with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea, induced a great stimulation of Ca2+ influx within the first minutes. Ca2+ influx is essential for the initiation of cryptogein-induced responses, since ethyleneglycol-bis([beta]-amino-ethyl ether)-N,N[prime]-tetraacetic acid or La3+, which block Ca2+ entrance, suppress cryptogein-induced responses such as extracellular alkalinization, active oxygen species, and phytoalexin production. Moreover, once initiated, these responses require sustained Ca2+ influx within the 1st h. A Ca2+ ionophore (A23187) was able to trigger an extracellular alkaliniz…

Stomatal deregulation in Plasmopara viticola-infected grapevine leaves.

International audience; In grapevine, the penetration and sporulation of Plasmopara viticola occur via stomata, suggesting functional relationships between guard cells and the pathogen. This assumption was supported by our first observation that grapevine (Vitis vinifera cv. Marselan) cuttings infected by P. viticola wilted more rapidly than healthy ones when submitted to water starvation. • Here, complementary approaches measuring stomatal conductance and infrared thermographic and microscopic observations were used to investigate stomatal opening/closure in response to infection. • In infected leaves, stomata remained open in darkness and during water stress, leading to increased transpir…

Nitric oxide production in tobacco leaf cells: a generalized stress response?

The function of nitric oxide (NO), a gaseous free radical emitted by many plants, is incompletely understood. In the present study the hypothesis that NO generation, like that of the reactive oxygen species, occurs as a general response to different environmental cues was tested. Leaf peels and mesophyll cell suspensions of Nicotiana tabacum cv. Xanthi were loaded with the NO-specific fluorophore, diaminofluorescein, and subjected to an abiotic stressor. Light stress and mechanical injury had no apparent effect on NO production. In contrast, high temperatures, hyperosmotic stress, salinity and epi-illumination in a microscope all led to rapid surges in NO-induced fluorescence. The fluoresce…

Early changes in gene expression in tobacco cells elicited with cryptogein

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Questioning the role of salicylic acid and cytosolic acidification in mitogen-activated protein kinase activation induced by cryptogein in tobacco cells

Elicitors of plant defence reactions, oligogalacturonides and cryptogein, an elicitin produced by Phytophthora cryptogea, were previously shown to induce a rapid and transient activation of two mitogen-activated protein kinases (MAPKs) in cells of tobacco [ Nicotiana tabacum L. cv. Xanthi; A. Lebrun-Garcia et al. (1998) Plant J 15:773-781]. We verified that these two MAPKs correspond to the salicylic acid-induced protein kinase (SIPK) and the wound-induced protein kinase (WIPK). The involvement of salicylic acid (SA) in cryptogein-induced MAPK activation was investigated using transgenic NahG tobacco cells expressing the salicylate hydroxylase gene and thus unable to accumulate SA. The larg…

Cercospora beticola toxins part 17 . The role of beticolin/Mg2+ complexes in their biological activity study of plasma membrane H+ - ATPase, vacuolar H+ -PPase, alkaline and acid phosphatases

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Plant iNOS: conquest of the Holy Grail

In animals, nitric oxide (NO) is produced by a family of enzymes named nitric oxide synthases (NOSs). Although no NOS-like gene has been found in the Arabidopsis thaliana genome, biochemical studies have suggested that a NOS-like protein is likely to be activated in plants resisting pathogens. This protein has been recently identified as a variant P protein of glycine decarboxylase. This discovery means that studies of nitric oxide signalling functions in plants are now entering a new phase.

Proteinaceous and oligosaccharidic elicitors induce different calcium signatures in the nucleus of tobacco cells.

We previously reported elevated cytosolic calcium levels in tobacco cells in response to elicitors [D. Lecourieux, C. Mazars, N. Pauly, R. Ranjeva, A. Pugin, Analysis and effects of cytosolic free calcium elevations in response to elicitors in Nicotiana plumbaginifolia cells, Plant Cell 14 (2002) 2627-2641]. These data suggested that in response to elicitors, Ca2+, as a second messenger, was involved in both systemic acquired resistance (RSA) and/or hypersensitive response (HR) depending on calcium signature. Here, we used transformed tobacco cells with apoaequorin expressed in the nucleus to monitor changes in free nuclear calcium concentrations ([Ca2+](nuc)) in response to elicitors. Two …

Protein phosphorylation is induced in tobacco cells by the elicitor cryptogein

Changes in plasmalemma ion fluxes were observed when tobacco (Nicotiana tabacum) cells were treated with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea. A strong alkalization of the culture medium, accompanied by a leakage of potassium, was induced within a few minutes of treatment. These effects reached a maximum after 30 to 40 min and lasted for several hours. This treatment also resulted in a rapid, but transient, production of activated oxygen species. All these physiological responses were fully sensitive to staurosporine, a known protein kinase inhibitor. Furthermore, a study of protein phosphorylation showed that cryptogein induced a staurosporine-sensitive phosphor…

AM fungal exudates activate MAP kinases in plant cells in dependence from cytosolic Ca2+ increase

International audience; The molecular dialogue occurring prior to direct contact between the fungal and plant partners of arbuscular-mycorrhizal (AM) symbioses begins with the release of fungal elicitors, so far only partially identified chemically, which can activate specific signaling pathways in the host plant. We show here that the activation of MAPK is also induced by exudates of germinating spores of Gigaspora margarita in cultured cells of the non-leguminous species tobacco (Nicotiana tabacum), as well as in those of the model legume Lotus japonicus. MAPK activity peaked about 15 min after the exposure of the host cells to the fungal exudates (FE). FE were also responsible for a rapi…

Cryptogein-Induced Anion Effluxes

Anion effluxes are amongst the earliest reactions of plant cells to elicitors of defence responses. However, their properties and their role in disease resistance remain almost unknown. We previously demonstrated that cryptogein, an elicitor of tobacco defence responses, induces a nitrate (NO(3) (-)) efflux. This efflux is an early prerequisite to the cryptogein-triggered hypersensitive response (HR). Here, we analyzed the electrophysiological properties of the elicitor-mediated NO(3) (-) efflux and clarified the mechanisms through which it contributes to cell death. Application of the discontinuous single electrode voltage-clamp technique in tobacco cells elicited with cryptogein enabled u…

Nitric oxide contributes to cadmium toxicity in Arabidopsis by promoting cadmium accumulation in roots and by up-regulating genes related to iron uptake.

Abstract Nitric oxide (NO) functions as a cell-signaling molecule in plants. In particular, a role for NO in the regulation of iron homeostasis and in the plant response to toxic metals has been proposed. Here, we investigated the synthesis and the role of NO in plants exposed to cadmium (Cd2+), a nonessential and toxic metal. We demonstrate that Cd2+ induces NO synthesis in roots and leaves of Arabidopsis (Arabidopsis thaliana) seedlings. This production, which is sensitive to NO synthase inhibitors, does not involve nitrate reductase and AtNOA1 but requires IRT1, encoding a major plasma membrane transporter for iron but also Cd2+. By analyzing the incidence of NO scavenging or inhibition …

Cercospora beticola toxins. Use of fluorescent cyanine dye to study their effects on tobacco cell suspensions

Abstract The fluorescent dye 3,3′-diethylthiadicarbocyanine iodide [diS-C 2 -(5)] was used to observe plasmalemma transmembrane potential variations of tobacco cells treated with uncoupler (FCCP), respiratory inhibitors (azide and cyanide), and H + -ATPase inhibitors (DCCD and a carbanilate derivative). These chemicals induced an increase in fluorescence, indicating a dissipation of the transmembrane potential. The [diS-C 2 -(5)] was also used to study the effects of two Cercospora beticola toxins on tobacco cells. Changes in fluorescence of [diS-C 2 -(5)] suggested that these two toxins caused a dissipation of the transmembrane potential with a different magnitude whereas kinetics of their…

Calcium homeostasis in plant cell nuclei

International audience; In plant cells, calcium-based signaling pathways are involved in a large array of biological processes, including cell division, polarity, growth, development and adaptation to changing biotic and abiotic environmental conditions. Free calcium changes are known to proceed in a nonstereotypical manner and produce a specific signature, which mirrors the nature, strength and frequency of a stimulus. The temporal aspects of calcium signatures are well documented, but their vectorial aspects also have a profound influence on biological output. Here, we will focus on the regulation of calcium homeostasis in the nucleus. We will discuss data and present hypotheses suggestin…

Activation of MAPK homologues by elicitors in tobacco cells

Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by Phytophthora cryptogea, or oligogalacturonides (OGs)), induced in tobacco cell suspensions (Nicotiana tabacum var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively. These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues. Both are recognized by anti-MAPK antibodies. The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurosporine, lanthanum, EGTA). W…

Changes in carbohydrate metabolism in Plasmopara viticola-infected grapevine leaves.

International audience; The oomycete Plasmopara viticola is responsible for downy mildew, a severe grapevine disease. In infected grapevine leaves, we have observed an abnormal starch accumulation at the end of the dark period, suggesting modifications in starch metabolism. Therefore, several complementary approaches, including transcriptomic analyses, measurements of enzyme activities, and sugar quantification, were performed in order to investigate and to understand the effects of P. viticola infection on leaf starch and-to a larger extent-carbohydrate metabolism. Our results indicate that starch accumulation is associated with an increase in ADP-glucose pyrophosphorylase (AGPase) activit…

Colonization of flax roots and early physiological responses of flax cells inoculated with pathogenic and nonpathogenic strains of Fusarium oxysporum

ABSTRACT Fusarium oxysporum includes nonpathogenic strains and pathogenic strains that can induce necrosis or tracheomycosis in plants. The objective of this study was to compare the abilities of a pathogenic strain (Foln3) and a nonpathogenic strain (Fo47) to colonize flax roots and to induce early physiological responses in flax cell culture suspensions. Both strains colonized the outer cortex of the root; however, plant defense reactions, i.e., the presence of wall appositions, osmiophilic material, and collapsed cells, were less frequent and less intense in a root colonized by Foln3 than by Fo47. Early physiological responses were measured in flax cell suspensions confronted with germin…

Phosphoproteins Involved in the Signal Transduction of Cryptogein, an Elicitor of Defense Reactions in Tobacco

We previously reported that the signal transduction of cryptogein, an elicitor of defense reactions in Nicotiana tabacum cells, involves upstream protein phosphorylation. In the present study, induction of these early physiological events was further investigated with inhibitors of protein phosphatase (PP), okadaïc acid, and calyculin A. Calyculin A mimicked the effects of cryptogein, inducing an influx of calcium, an extracellular alkalinization, and the production of active oxygen species (AOS), suggesting that during cryptogein signal transduction the balance between specific protein kinase (PK) and PP activities was modified. To identify the phosphorylated proteins that could be involv…

Priming: getting ready for battle

International audience; Infection of plants by necrotizing pathogens or colonization of plant roots with certain beneficial microbes causes the induction of a unique physiological state called “priming.” The primed state can also be induced by treatment of plants with various natural and synthetic compounds. Primed plants display either faster, stronger, or both activation of the various cellular defense responses that are induced following attack by either pathogens or insects or in response to abiotic stress. Although the phenomenon has been known for decades, most progress in our understanding of priming has been made over the past few years. Here, we summarize the current knowledge of p…

ß-1,4 cellodextrins, ß-1,3 glucans and a-1,4 oligogalacturonides are potent elicitors of various defense-related responses in grapevine

International audience; Plant cell-wall degradation products can be considered as ‘microbe-induced molecular patterns’ (MIMPs) recognized through plant receptors as ‘pathogen-induced modified self’. So cell-wall oligosaccharides originating from plants can play an important role in the perception of the invading pathogen by the plant. Cellodextrins (CD) are the end-products from cellulose degradation in plant cell walls, consisting og a linear ß-(1,4) linked glucose backbone. The role of these oligosaccharides in triggering plant defense reactions has not yet been established. Here, we investigated the elicitor activity of CD oligomers in grapevine cells. We show that CD triggered induction…

Intracellular compartmentation and plant cell signalling

Compartmentation is an essential feature of eukaryotic cells, and is crucial for the regulation of cell metabolism. Recent progress has significantly improved the understanding of signal transduction pathways in plants, including the activation of light-signalling networks and the tightly controlled generation of the calcium message. Cell compartmentation is important for the regulation and proliferation of these signalling processes.

Cercospora beticola toxins. IX. Relationship between structure of beticolins, inhibition of plasma membrane H+ -ATPase and partition in lipid membranes

Beticolins are yellow toxins produced by the fungus Cercospora beticola. The effect of one of them, beticolin-1, has been investigated on corn root plasma membrane H + -ATPase (EC 3.6.1.35) at different purification levels (plasma membrane fraction. partially, or highly purified enzyme). The results obtained demonstrated that (1) the purified proton pump was inhibited directly by low amounts of the toxin (I 50 =1.62 ± 0.18 μM), (2) the biological effects of beticolin-1 were similar to those of CBT (Cercospora beticola toxin). Furthermore, it was established that the efficiency of the different beticolins was clearly related to their ability to interact with the lipid bilayers, determined by…

Activite auxinolytique du Phialophora cinerescens (Wr.) van Beyma

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Cytosolic calcium rises and related events in ergosterol-treated Nicotiana cells

International audience; The typical fungal membrane component ergosterol was previously shown to trigger defence responses and protect plants against pathogens. Most of the elicitors mobilize the second messenger calcium, to trigger plant defences. We checked the involvement of calcium in response to ergosterol using Nicotiana plumbaginifolia and Nicotiana tabacum cv Xanthi cells expressing apoaequorin in the cytosol. First, it was verified if ergosterol was efficient in these cells inducing modifications of proton fluxes and increased expression of defence-related genes. Then, it was shown that ergosterol induced a rapid and transient biphasic increase of free [Ca2þ]cyt which intensity dep…

Nuclear protein kinases: still enigmatic components in plant cell signalling

International audience; Plants constantly face changing conditions in their environment. Unravelling the transduction mechanisms from signal perception at the plasma membrane level down to gene expression in the nucleus is a fascinating challenge. Protein phosphorylation, catalysed by protein kinases, is one of the major posttranslational modifications involved in the specificity, kinetic(s) and intensity of a signal transduction pathway. Although commonly assumed, the involvement of nuclear protein kinases in signal transduction is often poorly characterized. In particular, both their regulation and mode of action remain to be elucidated and may lead to the unveiling of new original mechan…

The endopolygalacturonase 1 from botrytis cinerea activates grapevine defense reactions unrelated to its enzyumatic activity

A purified glycoprotein from Botrytis cinerea(strain T4), identified as endopolygalacturonase 1 (T4BcPG1) by mass spectrometry analysis, has been shown to activate defense reactions in grapevine (Vitis vinifera cv. Gamay). These reactions include calcium influx, production of active oxygen species, activation of two mitogen-activated protein kinases, defense gene transcript accumulation, and phytoalexin production. Most of these defense reactions were also activated in grapevine in response to purified oligogalacturonides (OGA) with a degree of polymerization of 9 to 20. In vivo, these active OGA might be a part of the released products resulting from endopolygalacturonase activity on plan…

Allosteric regulation by Mg2+ of the vacuolar H(+)-PPase from Acer pseudoplatanus cells. Ca2+/Mg2+ interactions.

The tonoplast H(+)-PPase was previously characterized in Acer pseudoplatanus cells (Pugin et al (1991) Plant Sci 73, 23-34; Fraichard et al (1993) Plant Physiol Biochem 31, 349-359). Tonoplast vesicles were obtained from vacuoles isolated from protoplasts of A pseudoplatanus suspension cultures and used to study kinetic effects of Mg2+ and Ca2+ on PPi hydrolysis. The concentrations of ionic species (free Mg2+, free PPi, and MgPPi complexes) were calculated with apparent dissociation constants of 55.3 microM for MgPPi and 59.6 microM for CaPPi. Our results indicated that the substrate of the tonoplast PPase was a MgPPi complex and that free Mg2+ was essential for PPi hydrolysis. With fixed f…

The Tonoplast H+ -ATPase of Acer pseudoplatanus is a vacuolar-type ATPase that operates with a phosphoenzyme intermediate

The tonoplast H+-ATPase of Acer pseudoplatanus has been purified from isolated vacuoles. After solubilization, the purification procedure included size-exclusion and ion-exchange chromatography. The H+-ATPase consists of at least eight subunits, of 95, 66, 56, 54, 40, 38, 31, and 16 kD, that did not cross-react with polyclonal antibodies raised to the plasmalemma ATPase of Arabidopsis thaliana. The 66-kD polypeptide cross-reacted with monoclonal antibodies raised to the 70-kD subunit of the vacuolar H+-ATPase of oat roots. The functional molecular size of the tonoplast H+-ATPase, analyzed in situ by radiation inactivation, was found to be around 400 kD. The 66-kD subunit of the tonoplast H+…

The Elicitor Cryptogein Blocks Glucose Transport in Tobacco Cells

Abstract Cryptogein is a 10-kD protein secreted by the oomycetePhytophthora cryptogea that induces a hypersensitive response on tobacco (Nicotiana tabacum var. Xanthi) plants and a systemic acquired resistance against various pathogens. The mode of action of this elicitor has been studied using tobacco cell suspensions. Our previous data indicated that within minutes, cryptogein signaling involves various events including changes in ion fluxes, protein phosphorylation, sugar metabolism, and, eventually, cell death. These results suggested that transport of sugars could be affected and, thus, involved in the complex relationships between plant and microorganisms via elicitors. This led us to…

Nitric oxide signalling in plants: interplays with Ca2+ and protein kinase

International audience; Much attention has been paid to nitric oxide (NO)research since its discovery as a physiological mediator of plant defence responses. In recent years, newer roles have been attributed to NO, ranging from root development to stomatal closure. The molecular mechanisms underlying NO action in plants are just begun to emerge. The currently available data illustrate that NO can directly influence the activity of target proteins through nitrosylation and has the capacity to act as a Ca2+-mobilizing intracellular messenger. The interplay between NO and Ca2+ has important functional implications, expanding and enriching the possibilities for modulating transduction processes…

Involvement of the glutamate receptor AtGLR3.3 in plant defense signaling and resistance toHyaloperonospora arabidopsidis

Like their animal counterparts, plant glutamate receptor-like (GLR) homologs are intimately associated with Ca(2+) influx through plasma membrane and participate in various physiological processes. In pathogen-associated molecular patterns (PAMP)-/elicitor-mediated resistance, Ca(2+) fluxes are necessary for activating downstream signaling events related to plant defense. In this study, oligogalacturonides (OGs), which are endogenous elicitors derived from cell wall degradation, were used to investigate the role of Arabidopsis GLRs in defense signaling. Pharmacological investigations indicated that GLRs are partly involved in free cytosolic [Ca(2+)] ([Ca(2+)]cyt) variations, nitric oxide (N…

Calcium in plant defence‐signalling pathways

In plant cells, the calcium ion is a ubiquitous intracellular second messenger involved in numerous signalling pathways. Variations in the cytosolic concentration of Ca2+ ([Ca2+]cyt) couple a large array of signals and responses. Here we concentrate on calcium signalling in plant defence responses, particularly on the generation of the calcium signal and downstream calcium-dependent events participating in the establishment of defence responses with special reference to calcium-binding proteins.

Cercospora beticola toxins. X Inhibition of plasma membrane H+ -ATPase by beticolin-1

International audience

Are grapevine stomata involved in the elicitor-induced protection against downy mildew?

Stomata, natural pores bordered by guard cells, regulate transpiration and gas exchanges between plant leaves and the atmosphere. These natural openings also constitute a way of penetration for microorganisms. In plants, the perception of potentially pathogenic microorganisms or elicitors of defense reactions induces a cascade of events, including H2O2 production, that allows the activation of defense genes, leading to defense reactions. Similar signaling events occur in guard cells in response to the perception of abscisic acid (ABA), leading to stomatal closure. Moreover, few elicitors were reported to induce stomatal closure in Arabidopsis and Vicia faba leaves. Because responses to ABA…

Phospholipase activities associated with the tonoplast from Acer pseudoplatanus cells: identification of a phospholipase A1 activity

In higher plants, the lipolytic enzymes and their physiological functions are not well characterized [1]. Most reports demonstrated that phospholipid catabolism in plants is achieved by the concerted actions of membrane-bound enzymes including phospholipase D, phosphatidate phosphatase, lipolytic acyl hydrolases and lipoxygenases [1,2]. With the exception of the phospholipase D, the literature on plant phospholipases is still very limited. We previously reported that tonoplast from Acer pseudoplatanus cells contains small amounts of phosphatidc acid and lysophospholipids, which were produced together with free fatty acids, particularly after addition of Ca2+[3]. These data suggested the pos…

Cryptogein, a fungal elicitor, remodels the phenylpropanoid metabolism of tobacco cell suspension cultures in a calcium-dependent manner

Plant cells use calcium-based signalling pathways to transduce biotic and/or abiotic stimuli into adaptive responses. However, little is known about the coupling between calcium signalling, transcriptional regulation and the downstream biochemical processes. To understand these relationships better, we challenged tobacco BY-2 cells with cryptogein and evaluated how calcium transients (monitored through the calcium sensor aequorin) impact (1) transcript levels of phenylpropanoid genes (assessed by RT-qPCR); and (2) derived-phenolic compounds (analysed by mass spectrometry). Most genes of the phenylpropanoid pathway were up-regulated by cryptogein and cell wall-bound phenolic compounds accumu…

Integrated signaling network involving calcium, nitric oxide, active oxygen species but not mitogen-activated protein kinases in BcPG1-elicited grapevine defenses.

We have already reported the identification of the endopolygalacturonase 1 (BcPG1) from Botrytis cinerea as a potent elicitor of defense responses in grapevine, independently of its enzymatic activity. The aim of the present study is the analysis of the signaling pathways triggered by BcPG1 in grapevine cells. Our data indicate that BcPG1 induces a Ca2+ entry from the apoplasm, which triggers a phosphorylation-dependent nitric oxide (NO) production via an enzyme probably related to a NO synthase. Then NO is involved in i) cytosolic calcium homeostasis, by activating Ca2+ release from internal stores and regulating Ca2+ fluxes across the plasma membrane, ii) plasma membrane potential variat…

Comparison of the effects of cryptogein and oligogalacturonides on tabacco cells and evidence of different forms of desensitization induced by these elicitors

Abstract The effects of cryptogein and oligogalacturonides (OGs) were compared on tobacco cells by measuring calcium influx and calcium-dependent responses including extracellular alkalinization and H 2 O 2 production. The main difference is the higher calcium influx and the sustained H 2 O 2 production induced by cryptogein compared to OGs. Amplitude and duration of calcium signalling triggered by cryptogein or OGs may explain the necrotic effect of cryptogein, and the absence of necrosis in tobacco plants treated with OGs. We used induction of alkalinization and H 2 O 2 production to investigate cryptogein effects after a first treatment with cryptogein or OGs, and reciprocally. Results s…

Calcium signaling in plant cell organelles delimited by a double membrane.

AbstractIncreases in the concentration of free calcium in the cytosol are one of the general events that relay an external stimulus to the internal cellular machinery and allow eukaryotic organisms, including plants, to mount a specific biological response. Different lines of evidence have shown that other intracellular organelles contribute to the regulation of free calcium homeostasis in the cytosol. The vacuoles, the endoplasmic reticulum and the cell wall constitute storage compartments for mobilizable calcium. In contrast, the role of organelles surrounded by a double membrane (e.g. mitochondria, chloroplasts and nuclei) is more complex. Here, we review experimental data showing that t…

Early events induced by elicitor cryptogein in tobacco cells: Involvement of a plasma membrane NADPH oxidase and activation of glycolysis and the pentose phosphate pathway

International audience

Signalisation calcique cytosolique et nucléaire et réponses des plantes aux stimulus biotiques et abiotiques

Bacterial rhamnolipids are novel MAMPs conferring resistance to Botrytis cinerea in grapevine

International audience; Rhamnolipids produced by the bacteria Pseudomonas aeruginosa are known as very efficient biosurfactant molecules. They are used for a wide range of industrial applications, especially in food, cosmetics and pharmaceutical formulations as well as in bioremediation of pollutants. In this paper, the role of rhamnolipids as novel molecules triggering defence responses and protection against the fungus Botrytis cinerea in grapevine is presented. The effect of rhamnolipids was assessed in grapevine using cell suspension cultures and vitro-plantlets. Ca2+ influx, mitogen-activated protein kinase activation and reactive oxygen species production form part of early signalling…

Evidence for specific, high-affinity binding sites for a proteinaceous elicitor in tobacco plasma membrane

Abstract Binding of cryptogein, a proteinaceous elicitor, was studied on tobacco plasma membrane. The binding of the [125I]cryptogein was saturable, reversible and specific with an apparent Kd of 2 nM. A single class of cryptogein binding sites was found with a sharp optimum pH for binding at about pH 7.0. The high-affinity correlates with cryptogein concentrations required for biological activity in vivo.

Comparison of binding properties and early biological effects of elicitins in tobacco cells

Abstract Elicitins are a family of small proteins secreted by Phytophthora species that have a high degree of homology and elicit defense reactions in tobacco (Nicotiana tabacum). They display acidic or basic characteristics, the acidic elicitins being less efficient in inducing plant necrosis. In this study we compared the binding properties of four elicitins (two basic and two acidic) and early-induced signal transduction events (Ca2+ influx, extracellular medium alkalinization, and active oxygen species production). The affinity for tobacco plasma membrane-binding sites and the number of binding sites were similar for all four elicitins. Furthermore, elicitins compete with one another fo…

Relationship between active oxygen species, lipid peroxidation, necrosis, and phytoalexin production induced by elicitins in Nicotiana 1

International audience

Cercospora beticola toxins. VII. Fluorometric study of their interactions with biological membranes

International audience

Early signaling events induced by elicitors of plant defenses

International audience; Plant pathogen attacks are perceived through pathogenissued compounds or plant-derived molecules that elicit defense reactions. Despite the large variety of elicitors, general schemes for cellular elicitor signaling leading to plant resistance can be drawn. In this article, we review early signaling events that happen after elicitor perception, including reversible protein phosphorylations, changes in the activities of plasma membrane proteins, variations in free calcium concentrations in cytosol and nucleus, and production of nitric oxide and active oxygen species. These events occur within the first minutes to a few hours after elicitor perception. One specific eli…

Changes in lipid composition in tobacco cells treated with cryptogein , an elicitor from Phytophthora cryptogea

Abstract Changes in lipid composition occurred when tobacco cells (Nicotiana tabacum var. Xanthi) were treated with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea. The most striking change was an increase in acylated steryl glycosides and steryl esters levels, certainly resulting from the glycosylation and/or esterification of free sterols. Moreover, in vivo pulse-labelling experiments with [14C]acetate also showed that a progressive decline in the incorporation rate of [14C]acetate into free sterols started with the induction of sesquiterpenoid synthesis and lasted when sesquiterpenoid synthesis stops. This phenomenon was accompanied by a significant increase in the synth…

Involvement of putative glutamate receptors in plant defence signaling and NO production

International audience; Ionotropic glutamate receptors (iGluRs) are non-selective cation channels permeable to calcium, present in animals and plants. In mammals, glutamate is a well-known neurotransmitter and recently has been recognized as an immunomodulator. As animals and plants share common mechanisms that govern innate immunity with calcium playing a key role in plant defence activation, we have checked the involvement of putative iGluRs in plant defence signaling. Using tobacco cells, we first provide evidence supporting the activity of iGluRs as calcium channels and their involvement in NO production as reported in animals. Thereafter, iGluRs were shown to be activated in response t…

Elicitor and resistance-inducing activities of -1,4 cellodextrins in grapevine, comparison with -1,3 glucans and -1,4 oligogalacturonides

Cellodextrins (CD), water-soluble derivatives of cellulose composed of beta-1,4 glucoside residues, have been shown to induce a variety of defence responses in grapevine (Vitis vinifera L.) cells. The larger oligomers of CD rapidly induced transient generation of H2O2 and elevation in free cytosolic calcium, followed by a differential expression of genes encoding key enzymes of the phenylpropanoid pathway and pathogenesis-related (PR) proteins as well as stimulation of chitinase and beta-1,3 glucanase activities. Most of these defence reactions were also induced by linear beta-1,3 glucans (betaGlu) and alpha-1,4 oligogalacturonides (OGA) of different degree of polymerization (DP), but the i…

Characterization of a new, nonpathogenic mutant of Botrytis cinerea with impaired plant colonization capacity.

International audience; Botrytis cinerea is a necrotrophic pathogen that attacks more than 200 plant species.Here, the nonpathogenic mutant A336, obtained via insertional mutagenesis, was characterized.Mutant A336 was nonpathogenic on leaves and fruits, on intact and wounded tissue, while still able to penetrate the host plant. It grew normally in vitro on rich media but its conidiation pattern was altered. The mutant did not produce oxalic acid and exhibited a modified regulation of the production of some secreted proteins (acid protease 1 and endopolygalacturonase 1). Culture filtrates of the mutant triggered an important oxidative burst in grapevine ( Vitis vinifera ) suspension cells, a…

Caractérisation du mode d’action des rhamnolipides dans la défense des plantes et l’induction d’une résistance contre des agents phytopathogènes biotrophes et nécrotrophes

National audience; Les rhamnolipides (RL) sont des glycolipides amphiphiles produits par différentes bactéries notamment du genre Pseudomonas et Burkholderia. Les RL comprennent généralement une chaîne d’acides gras couplée à un ou plusieurs rhamnoses. Il sont impliqués dans la formation de biofilms, dans la mobilité bactérienne et constituent, pour certaines bactéries, des facteurs de virulence (Vatsa et al. 2010). Nous avons montré que les RL sont perçus en tant qu’éliciteurs par les cellules de vigne et que cette reconnaissance provoque un influx de Ca2+, l’activation de plusieurs MAPK (Mitogen-Activated Protein Kinase), la production de formes actives de l’oxygène ainsi que l’expression…

Activation of a nuclear-localized SIPK in tobacco cells challenged by cryptogein, an elicitor of plant defence reactions.

When a plant cell is challenged by a well-defined stimulus, complex signal transduction pathways are activated to promote the modulation of specific sets of genes and eventually to develop adaptive responses. In this context, protein phosphorylation plays a fundamental role through the activation of multiple protein kinase families. Although the involvement of protein kinases at the plasma membrane and cytosolic levels are now well-documented, their nuclear counterparts are still poorly investigated. In the field of plant defence reactions, no known study has yet reported the activation of a nuclear protein kinase and/or its nuclear activity in plant cells, although some protein kinases, e.…

Involvement of plasma membrane proteins in plant defense responses. Analysis of the cryptogein signal transduction in tobacco

International audience; Cryptogein, a 98 amino acid protein secreted by the fungus Phytophthora cryptogea, induces a hypersensitive response and systemic acquired resistance in tobacco plants (Nicotiana tabacum var Xanthi). The mode of action of cryptogein has been studied using tobacco cell suspensions. The recognition of this elicitor by a plasma membrane receptor leads to a cascade of events including protein phosphorylation, calcium influx, potassium and chloride effluxes, plasma membrane depolarization, activation of a NADPH oxidase responsible for active oxygen species (AOS) production and cytosol acidification, activation of the pentose phosphate pathway, and activation of two mitoge…

Signal perception and transduction, secondary messengers and gene activation in elicitin-triggered HR and SAR in tobacco

National audience

Nitric Oxide in Plants: Production and Cross-talk with Ca2+ Signaling

International audience; Nitric oxide (NO) is a diatomic gas that performs crucial functions in a wide array of physiological processes in animals. The past several years have revealed much about its roles in plants. It is well established that NO is synthesized from nitrite by nitrate reductase (NR) and via chemical pathways. There is increasing evidence for the occurrence of an alternative pathway in which NO production is catalysed from L-arginine by a so far non-identified enzyme. Contradictory results have been reported regarding the respective involvement of these enzymes in specific physiological conditions. Although much remains to be proved, we assume that these inconsistencies can …

Activation of MAPK homologues by elicitors in tobacco cells

International audience; Elicitors of plant defence reactions (such as cryptogein, an elicitin produced by Phytophthora cryptogea, or oligogalacturonides (OGs)), induced in tobacco cell suspensions (Nicotiana tabacum var Xanthi) a rapid and transient activation of two protein kinases (PKs) with apparent molecular masses of 50 and 46 kDa, respectively. These PKs activated and phosphorylated at tyrosine residues, phosphorylated myelin basic protein (MBP) at serine/threonine residues. Both are recognized by anti-MAPK antibodies. The two MBP kinases possessed the same kinetics of activation, and their activation depended, to the same extent, on different exogenously applied compounds (staurospor…

Transbilayer distribution of phosphatidycholine and phosphatidyl-ethanolamine in the vacuolar menbrane of Acer pseudoplatanus cells

International audience

Nitric oxide: Chemistry and bioactivity in animal and plant cells

Abstract In mammals, nitric oxide (NO) is a reactive free radical involved in diverse physiological functions. NO and its redox-related forms NO + and NO − react with di(oxygen) and its derivatives, with metalloproteins and thiol-containing proteins. NO-mediated nitrosation of proteins represents an important cellular regulatory mechanism. Biosynthesis of NOis catalysed by nitric oxide synthase (NOS). Three isoenzymes representing distinct gene products have been identified: the inducible NOS isoform, the constitutive neuronal and endothelial isoforms. Inducible and constitutive NOSs have the same structural features, but their activities differ in their dependence to calcium and the rate o…

Early signaling network in tobacco cells elicited with methyl jasmonate and cyclodextrins.

We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmonate (MJ) and cyclodextrins (CDs) in tobacco (Nicotiana tabacum) cell cultures, paying particular attention to changes in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)), the production of hydrogen peroxide (H(2)O(2)) and nitric oxide (NO), and late events like the induction of capsidiol. Our data indicate that MJ and CDs trigger a [Ca(2+)](cyt) rise promoted by Ca(2+) influx through Ca(2+)-permeable channels. The joint presence of MJ and CDs provokes a first increase in [Ca(2+)](cyt) similar to that observed in MJ-treated cells, followed by a second peak similar to that found in the presence…