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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 (NO) production, reactive oxygen species (ROS) production and expression of defense-related genes by OGs. In addition, wild-type Col-0 plants treated with the glutamate-receptor antagonist 6,7-dinitriquinoxaline-2,3-dione (DNQX) had a compromised resistance to Botrytis cinerea and Hyaloperonospora arabidopsidis. Moreover, we provide genetic evidence that AtGLR3.3 is a key component of resistance against H. arabidopsidis. In addition, some OGs-triggered immune events such as defense gene expression, NO and ROS production are also to different extents dependent on AtGLR3.3. Taken together, these data provide evidence for the involvement of GLRs in elicitor/pathogen-mediated plant defense signaling pathways in Arabidopsis thaliana.

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 rapid and transient increase in free cytosolic Ca2þ in Nicotiana plumbaginifolia and tobacco cells, and pre-treatment with a Ca2þ-channel blocker (La3þ) showed that in these cells, MAPK activation was dependent on the cytosolic Ca2þ increase. A partial dependence of MAPK activity on the common Sym pathway could be demonstrated for a cell line of L. japonicus defective for LjSym4 and hence unable to establish an AM symbiosis. Our results show that MAPK activation is triggered by an FE-induced cytosolic Ca2þ transient, and that a Sym genetic determinant acts to modulate the intensity and duration of this activity.

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 accumulated (mainly 5-hydroxyferulic acid). The accumulation of both transcripts and phenolics was calcium-dependent. The transcriptional regulation of phenylpropanoid genes was correlated in a non-linear manner with stimulus intensity and with components of the cryptogein-induced calcium signature. In addition, calmodulin inhibitors increased the sensitivity of cells to low concentrations of cryptogein. These results led us to propose a model of coupling between the cryptogein signal, calcium signalling and the transcriptional response, exerting control of transcription through the coordinated action of two decoding modules exerting opposite effects.

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 stronger ROS production was specifically observed in BABA-treated leaves. This process was correlated with an increased resistance. The NADPH oxidase inhibitor diphenylene iodonium (DPI) abolished this primed ROS production and reduced the BABA-induced resistance (BABA-IR). These results suggest that priming of an NADPH oxidase–dependent ROS production contributes to BABA-IR in the Vitis-Plasmopara pathosystem.

A decade-long investigation of nitric oxide (NO) functions in plants has led to its characterization as a biological mediator involved in key physiological processes. Despite the wealth of information gathered from the analysis of its functions, until recently little was known about the mechanisms by which NO exerts its effects. In the past few years, part of the gap has been bridged. NO modulates the activity of proteins through nitrosylation and probably tyrosine nitration. Furthermore, NO can act as a Ca2+-mobilizing messenger, and researchers are beginning to unravel the mechanisms underlying the cross talk between NO and Ca2+. Nonetheless, progress in this area of research is hindered by our ignorance of the pathways for NO production in plants. This review summarizes the basic concepts of NO signaling in animals and discusses new insights into NO enzymatic sources and molecular signaling in plants.

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 hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.

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). We demonstrate here that the activation of the MBP kinases is calcium dependent and sensitive to staurosporine, a protein kinase inhibitor which annihilates all known responses of tobacco cells to cryptogein. The activation of MBP kinases appeared to be independent of the production of active oxygen species (AOS) and insensitive to calyculin A, a protein phosphatase type 1 and 2A inhibitor. The activation of MAPKs is discussed in relation to the early responses induced by cryptogein.

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 alkalinization but not the formation of active oxygen species and phytoalexins, even in the presence of cryptogein. Staurosporine, a protein kinase inhibitor that was recently reported to suppress cryptogein-induced responses (M.-P. Viard, F. Martin, A. Pugin, P. Ricci, J.-P. Blein [1994] Plant Physiol 104: 1245-1249), inhibited Ca2+ influx induced by cryptogein in a dose-dependent manner. These results suggest that protein phosphorylation followed by Ca2+ influx might be involved in the initial steps of cryptogein signal transduction.

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 possible involvement of phospholipase D and phospholipase A in the metabolism of vacuolar membrane lipids. The phospholipase activities were studied by following the hydrolysis of added sn-2-[14C]linoleyl-PC to tonoplast vesicles. Tonoplast was obtained by osmotic lysis of pure preparations of vacuoles isolated from protoplasts derived from Acer pseudoplatanus cells [4], This present work demonstrated clearly the presence of phospholipase D and phospholipase A1 activities associated with the tonoplast of Acer. The phospholipase A1 showed an optimal activity at pH about 6–6.5, did not necessarily require divalent cations, but was stimulated by Mg2+ and particularly by Ca2+. This work presents the first evidence for the presence of phospholipases A1 in plant cells.

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) activity and modifications in the starch degradation pathway, especially an increased α-amylase activity. Together with these alterations in starch metabolism, we have observed an accumulation of hexoses, an increase in invertase activity, and a reduction of photosynthesis, indicating a source-to-sink transition in infected leaf tissue. Additionally, we have measured an accumulation of the disaccharide trehalose correlated to an increased trehalase gene expression and enzyme activity. Altogether, these results highlight a dramatic alteration of carbohydrate metabolism correlated with later stages of P. viticola development in leaves.

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 depends on ergosterol concentration in the range 0.002e10 mM. Among sterols, this calcium mobilization was specific for ergosterol and, ergosterolinduced pH and [Ca2þ]cyt changes were specifically desensitized after two subsequent applications of ergosterol. Specific modulators allowed elucidating some events in the signalling pathway triggered by ergosterol. The action of BAPTA, LaCl3, nifedipine, verapamil, neomycin, U73122 and ruthenium red suggested that the first phase was linked to calcium influx from external medium which subsequently triggered the second phase linked to calcium release from internal stores. The calcium influx and the [Ca2þ]cyt increase depended on upstream protein phosphorylation. The extracellular alkalinization and ROS production depended on calcium influx but, the ergosterol-induced MAPK activation was calciumindependent. ROS were not involved in cytosolic calcium rise as described in other models, indicating that ROS do not systematically participate in the amplification of calcium signalling. Interestingly, ergosterol-induced ROS production is not linked to cell death and ergosterol does not induce any calcium elevation in the nucleus.

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 to cryptogein, a well studied elicitor of defence response, and partly responsible for cryptogein-induced NO production. However, other cryptogein-induced calcium-dependent events including anion efflux, H2O2 production, MAPK activation and hypersensitive response (HR) did not depend on iGluRs indicating that different calcium channels regulate different processes at the cell level. We have also demonstrated that cryptogein induces efflux of glutamate in the apoplast by exocytosis. Taken together, our results demonstrate for the first time, an involvement of a putative iGluR in plant defence signaling and NO production, by mechanisms that show homology with glutamate mode of action in mammals.

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 of its synthesis during Cd2+ treatment, we demonstrated that NO contributes to Cd2+-triggered inhibition of root growth. To understand the mechanisms underlying this process, a microarray analysis was performed in order to identify NO-modulated root genes up- and down-regulated during Cd2+ treatment. Forty-three genes were identified encoding proteins related to iron homeostasis, proteolysis, nitrogen assimilation/metabolism, and root growth. These genes include IRT1. Investigation of the metal and ion contents in Cd2+-treated roots in which NO synthesis was impaired indicates that IRT1 up-regulation by NO was consistently correlated to NO's ability to promote Cd2+ accumulation in roots. This analysis also highlights that NO is responsible for Cd2+-induced inhibition of root Ca2+ accumulation. Taken together, our results suggest that NO contributes to Cd2+ toxicity by favoring Cd2+ versus Ca2+ uptake and by initiating a cellular pathway resembling those activated upon iron deprivation.

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 events leading from perception of rhamnolipids to the induction of plant defences that include expression of a wide range of defence genes and a hypersensitive response (HR) -like response. In addition, rhamnolipids potentiated defence responses induced by the chitosan elicitor and by the culture filtrate of B. cinerea. We also demonstrated that rhamnolipids have direct antifungal properties by inhibiting spore germination and mycelium growth of B. cinerea. Ultimately, rhamnolipids efficiently protected grapevine against the fungus. We propose that rhamnolipids are acting as microbe-associated molecular patterns (MAMPs) in grapevine and that the combination of rhamnolipid effects could participate in grapevine protection against grey mould disease.

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 and elicitors share common signaling events, it led us to question whether stomatal movements and H2O2 production in guard cells could play a key role in elicitor-induced protection against pathogens that use stomata for infection. This study was performed using the grapevine–Plasmopara viticola pathosystem. Using epidermal peels, we showed that, as for ABA, the elicitor-induced stomatal closure is mediated by reactive oxygen species (ROS) production in guard cells. In plants, we observed that the protection against downy mildew induced by some elicitors is probably not due only to effects on stomatal movements or to a guard-cell-specific activation of ROS production.

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 suggesting that, while interacting with other organelles, the nucleus has the potential to generate and regulate calcium signals on its own.

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.g. MAPK (mitogen-activated protein kinase), are known to be translocated into the nucleus. In the present study, we investigated the ability of cryptogein, a proteinaceous elicitor of tobacco defence reactions, to induce different nuclear protein kinase activities. We found that at least four nuclear protein kinases are activated in response to cryptogein treatment in a time-dependent manner, some of them exhibiting Ca2+-dependent activity. The present study focused on one 47 kDa protein kinase with a Ca2+-independent activity, closely related to the MAPK family. After purification and microsequencing, this protein kinase was formally identified as SIPK (salicyclic acid-induced protein kinase), a biotic and abiotic stress-activated MAPK of tobacco. We also showed that cytosolic activation of SIPK is not sufficient to promote a nuclear SIPK activity, the latter being correlated with cell death. In that way, the present study provides evidence of a functional nuclear MAPK activity involved in response to an elicitor treatment.

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 be accounted for by the limited specificity of the pharmacological agents used to suppress NO synthesis but also by the reduced content of L-arginine as well as the inactivity of nitrate-permeable anion channels in nitrate reductase- and/or nitrate/nitrite-deficient plants. Another unresolved issue concerns the molecular mechanisms underlying NO effects in plants. Here, we provide evidence that the second messenger Ca21, as well as protein kinases including MAPK and SnRK2, are very plausible mediators of the NO signals. These findings open new perspectives about NO-based signaling in plants.

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. Furthermore, protein kinases regulated through NO-dependent mechanisms are being discovered, offering fresh perspective on processes such as stress tolerance.

Colonization of roots by selected strains of fluorescent Pseudomonas spp. can trigger induced systemic resistance (ISR) against foliar pathogens in a plant species-specific manner. It has been suggested that early responses in cell suspension cultures in response to rhizobacterial elicitors, such as generation of active oxygen species (AOS) and extracellular medium alkalinization (MA), are linked to the development of ISR in whole plants. Perception of flagellin was demonstrated to elicit ISR in Arabidopsis, and bacterial lipopolysaccharides (LPS) have been shown to elicit several defense responses and to act as bacterial determinants of ISR in various plant species. In the present study, the LPS-containing cell walls, the pyoverdine siderophores, and the flagella of Pseudomonas putida WCS358, P. fluorescens WCS374, and P. fluorescens WCS417, which are all known to act as elicitors of ISR in selected plant species, were tested for their effects on the production of AOS, MA, elevation of cytoplasmic Ca2+ ([Ca2+]cyt), and defense-related gene expression in tobacco suspension cells. The LPS of all three strains, the siderophore of WCS374, and the flagella of WCS358 induced a single, transient, early burst of AOS, whereas the siderophores of WCS358 and WCS417 and the flagella of WCS374 and WCS417 did not. None of the compounds caused cell death. Once stimulated by the active compounds, the cells became refractory to further stimulation by any of the active elicitors, but not to the elicitor cryptogein from the oomycete Phytophthora cryptogea, indicating that signaling upon perception of the different rhizobacterial compounds rapidly converges into a common response pathway. Of all compounds tested, only the siderophores of WCS358 and WCS417 did not induce MA; the flagella of WCS374 and WCS417, although not active as elicitors of AOS, did induce MA. These results were corroborated by using preparations from relevant bacterial mutants. The active rhizobacterial elicitors led to a rapid increase in [Ca2+]cyt, peaking at 6 min, whereas the inactive siderophores of WCS358 and WCS417 elicited a single spike at 1 min. Elicitation of the cells by cell-wall LPS of WCS358 or the siderophore of WCS374 induced a weak, transient expression of several defense-related genes, including PAL and GST. The spectrum of early responses of the suspension cells was not matched by the expression of ISR in whole tobacco plants against Erwinia carotovora pv. carotovora. Of the live bacterial strains, only WCS358 elicited significant ISR, but application of the LPS or the siderophore of all three strains also elicited ISR. Notably, the absence of elicitation of AOS and MA in suspension-cultured cells but induction of ISR in whole plants by the siderophore of WCS358, which was lost upon treatment with the siderophore-minus mutant of WCS358, indicates that the early responses in suspension cells are not predictive of the ability to induce ISR in whole plants. Possible explanations for these discrepancies are discussed.

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 intensity of some reactions induced by CD was different when compared with betaGlu and OGA effects. Moreover, desensitization assays using H2O2 production showed that cells treated with CD remained fully responsive to a second application of OGA, suggesting a different mode of perception of these oligosaccharides by grape cells. None of CD, betaGlu, or OGA induced HSR gene expression nor did they induce cell death. In accordance with elicitor activity in grapevine cells, CD-incubated leaves challenged with Botrytis cinerea also resulted in a significant reduction of the disease. Data suggest that CD could operate via other distinct reaction pathways than betaGlu and OGA. They also highlight the requirement of a specific DP for each oligosaccharide to induce the defence response.

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 transpiration. This deregulation was restricted to the colonized area, was not systemic and occurred before the appearance of symptoms. Cytological observations indicated that stomatal lock-open was not related to mechanical forces resulting from the presence of the pathogen in the substomatal cavity. In contrast to healthy leaves, stomatal closure in excised infected leaves could not be induced by a water deficit or abscisic acid (ABA)treatment. However, ABA induced stomatal closure in epidermal peels from infected leaves, indicating that guard cells remained functional. • These data indicate that the oomycete deregulates guard cell functioning, causing significant water losses. This effect could be attributed to a nonsystemic compound, produced by the oomycete or by the infected plant, which inhibits stomatal closure or induces stomatal opening; or a reduction of the back-pressure exerted by surrounding epidermal cells. Both hypotheses are under investigation.

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 variation, iii) the activation of active oxygen species (AOS) production, and iv) defense gene expression, including phenylalanine ammonia lyase and stilbene synthase, which encode enzymes responsible for phytoalexin biosynthesis. Interestingly enough, mitogen-activated protein kinase (MAPK) activation is independent of this regulation pathway that closely connects Ca2+, NO, and AOS.

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 elicitor transduction pathway can use a combination or a partial combination of such events which can differ in kinetics and intensity depending on the stimulus. The links between the signaling events allow amplification of the signal transduction and ensure specificity to get appropriate plant defense reactions. This review first describes the early events induced by cryptogein, an elicitor of tobacco defense reactions, in order to give a general scheme for signal transduction that will be use as a thread to review signaling events monitored in different elicitor or plant models.

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 germinated microconidia of both strains. Both pathogenic (Foln3) and nonpathogenic strains (Fo47) triggered transient H 2 O 2 production in the first few minutes of the interaction, but the nonpathogenic strain also induced a second burst 3 h postinoculation. Ca 2+ influx was more intense in cells inoculated with Fo47 than in cells inoculated with Foln3. Similarly, alkalinization of the extracellular medium was higher with Fo47 than with Foln3. Inoculation of the fungi into flax cell suspensions induced cell death 10 to 20 h postinoculation, with a higher percentage of dead cells observed with Fo47 than with Foln3 beginning at 14 h. This is the first report showing that early physiological responses of flax cells can be used to distinguish pathogenic and nonpathogenic strains of the soil-borne fungus F. oxysporum .

International audience; Grapevine (Vittis vinifera L.) is susceptible to many pathogens, such as Botrytis cinerea, Plasmopara viticola, Uncinula necator, and Eutypa lata. Phytochemicals are used intensively in vineyards to limit pathogen infections, but the appearance of pesticide-resistant pathogen strains and a desire to protect the environment require that alternative strategies be found. In the present study, the, beta-1,3-glucan laminarin derived from the brown algae Laminaria digitata was shown both to be an efficient elicitor of defense responses in grapevine cells and plants and to effectively reduce B. cinerea and P viticola development on infected grapevine plants. Defense reactions elicited by laminarin in grapevine cells include calcium influx, alkalinization of the extracellular medium, an oxidative burst, activation of two mitogen-activated protein kinases, expression of 10 defense-related genes with, different kinetics and intensities, increases in chitinase and beta-1,3-glucanase activities, and the production of two phytoalexins (resveratrol and epsilon-viniferin). Several of these effects were checked and confirmed in whole plants. Laminarin did not induce cell death. When applied to grapevine plants, laminarin reduced infection by B. cinerea and R viticola by approximately 55 and 75%, respectively. Our data describing a large set of defense reactions in grapevine indicate that the activation of defense responses using elicitors could be a valuable strategy to protect plants against pathogens.

There is much interest in the transduction pathways by which avirulent pathogens or derived elicitors activate plant defense responses. However, little is known about anion channel functions in this process. The aim of this study was to reveal the contribution of anion channels in the defense response triggered in tobacco by the elicitor cryptogein. Cryptogein induced a fast nitrate (NO(3)(-)) efflux that was sensitive to anion channel blockers and regulated by phosphorylation events and Ca(2+) influx. Using a pharmacological approach, we provide evidence that NO(3)(-) efflux acts upstream of the cryptogein-induced oxidative burst and a 40-kD protein kinase whose activation seems to be controlled by the duration and intensity of anion efflux. Moreover, NO(3)(-) efflux inhibitors reduced and delayed the hypersensitive cell death triggered by cryptogein in tobacco plants. This was accompanied by a delay or a complete suppression of the induction of several defense-related genes, including hsr203J, a gene whose expression is correlated strongly with programmed cell death in plants. Our results indicate that anion channels are involved intimately in mediating defense responses and hypersensitive cell death.

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 respond to atmospheric nitric oxide, but also possess the capacity to produce nitric oxide enzymatically. Initial investigations into nitric oxide functions suggested that plants use nitric oxide as a signaling molecule via pathways remarkably similar to those found in mammals. These findings complement an emerging body of evidence indicating that many signal transduction pathways are shared between plants and animals.

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 for binding sites, suggesting that they interact with the same receptor. The four elicitins induced Ca2+ influx, extracellular medium alkalinization, and the production of active oxygen species in tobacco cell suspensions, but the intensity and kinetics of these effects were different from one elicitin to another. As a general observation the concentrations that induce similar levels of biological activities were lower for basic elicitins (with the exception of cinnamomin-induced Ca2+ uptake). The qualitative similarity of early events induced by elicitins indicates a common transduction scheme, whereas fine signal transduction tuning is different in each elicitin.

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 show that tobacco cells treated with OGs or cryptogein differed in their ability to respond to a subsequent stimulation: OGs-treated cells were refractory to the same stimulus but remained responsive to cryptogein whereas cryptogein-treated cells still responded weakly to a second application of cryptogein or OGs. Desensitization induced by OGs and cryptogein could correspond, respectively, to some forms of homologous or heterologous desensitization described in animal systems. Alternatively, cryptogein-activated pathways via large calcium influx could promote important cellular modifications leading to a reduction of response in general.

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.

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 free Mg2+ concentrations, PPase activity showed Michaelis-Menten kinetics with respect to MgPPi. Moreover, free Mg2+ acted as an allosteric activator with a Hill coefficient of 2.4, indicating at least two Mg2+ binding sites on the enzyme. The Mg-imidodiphosphate complex was a competitive inhibitor of the substrate MgPPi but did not change significantly the allosteric activation by free Mg2+. This result confirmed the presence of Mg2+ regulatory sites. Ca2+ acted as an uncompetitive inhibitor of MgPPi hydrolysis. Furthermore, the sensitivity of the H(+)-PPase to Ca2+ increased with decrease in free Mg2+ concentration. Therefore, Ca2+ and Mg2+ may compete for a common binding site. Taken together, our results confirm that activation by free Mg2+ and inhibition by Ca2+ could be involved in the regulation of the PPase activity in vivo.

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 fluorometric studies : the toxins that exhibited the lower I 50 (50% inhibitory concentrations) values were the molecules that had the lowest partition coefficient to liposomes.

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 association with membranes were comparable.

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.

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 synthesis rate of phosphatidylethanolamine occuring after a period of 12 h (80% of [14C]incorporated into lipids was found in PE). These pulse-labelling experiments also indicated a transient neosynthesis of high levels of acylated steryl glycosides and steryl esters in elicited cells. These results demonstrated that glycosylation and/or esterification of sterols (preexistent or neosynthetised) is an initial event among plant cell responses associated with the hypersensitive reaction.

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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+-ATPase was rapidly phosphorylated by [[gamma]-32P]ATP in vitro. The complete loss of radio-activity in the 66-kD subunit after a short pulse-chase experiment with unlabeled ATP reflected a rapid turnover, which characterizes a phosphorylated intermediate. Phosphoenzyme formed from ATP is an acylphosphate-type compound as shown by its sensitivity to hydroxylamine and alkaline pH. These results lead us to suggest that the tonoplast H+-ATPase of A. pseudoplatanus is a vacuolar-type ATPase that could operate with a plasmalemma-type ATPase catalytic mechanism.

The interactions of two beticolins, Cercospora beticola toxins, and of their magnesium complexes with liposomes or plasma membrane were studied. The fluorometric pH titration curves of beticolins in liposomes and in plasma membranes reveal the presence of the dissociated form of beticolins. The concentration of the magnesium complex in these membranes increases at high pH. The partition coefficient of beticolin-1 on liposomes is 3-fold higher than that of beticolin-2 and the fluorescence of both compounds on liposomes is similar. The addition of magnesium to liposomes causes a 40-fold and 20-fold increase in the partition coefficient of beticolin-1 and -2, respectively, as a result of the interactions between membrane, magnesium and beticolins. Beticolins react to a ΔpH across the liposome membrane but the formation of the magnesium complex completely abolishes this effect.

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 (Davies et al., Proc. Natl. Acad. Sci. USA, 89 (1992) 11701–11705), these results led us to suggest that the inhibition of the H + -ATPase activity could modify the H + /K + stoichiometry for the benefit of K + -transport.

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 phosphorylation of several polypeptides. These data suggest that phosphorylated proteins may be essential for the transduction of elicitor signals.

When tobacco cells were treated with cryptogein, a proteinaceous elicitor from Phytophthora cryptogea, changes in plasmalemma fluxes were noticed. A strong alkalinization of the culture medium, accompagnied with a leakage of potassium, were induced few minutes after treatment, and reached a plateau within 30 min. Then, they remained at the same level for several hours. This treatment also induced a rapid, but transient production of activated oxygen species (20–80 min). All these physiological responses were fully sensitive to staurosporine, a known protein kinase inhibitor. A simultaneous treatment with cryptogein and staurosporin stimulated the accumulation of capsidiol, a sesquiterpenoid phytoalexin. These different results are in agreement with those reported by Felix et al and Conrath et al and led us to study the time course of protein phosphorylation during the first min of the elicitation process. It appeared that cryptogein induced a staurosporine-sensitive phosphorylation of microsomal proteins. Therefore, these data suggest that phosphorylated proteins were essential for transduction of elicitor signals.

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 priming in various induced-resistance phenomena in plants.