Benoît Poinssot

Early Signaling Events Induced by Elicitors of Plant Defenses

Plant pathogen attacks are perceived through pathogen-issued 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.

Laminarin elicits defense responses in grapevine and induces protection against Botrytis cinerea and Plasmopara viticola

Grapevine (Vitis 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 P. 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.

The Endopolygalacturonase 1 from Botrytis cinerea Activates Grapevine Defense Reactions Unrelated to Its Enzymatic 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 plant cell walls. Nevertheless, the intensity and kinetics of events triggered by OGA were very different when compared with T4BcPG1 effects. Moreover, chemical treatments of T4BcPG1 and desensitization assays have allowed us to discriminate enzymatic and elicitor activities, indicating that elicitor activity was not due to released oligogalacturonides. Thus, BcPG1 should be considered as both an avirulence and a virulence factor. The role of the secreted BcPG1 in the pathogenicity of Botrytis cinerea is discussed.

Integrated Signaling Network Involving Calcium, Nitric Oxide, and 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 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.

Carbohydrates in plant immunity and plant protection: roles and potential application as foliar sprays

Increasing interest is devoted to carbohydrates for their roles in plant immunity. Some of them are elicitors of plant defenses whereas other ones act as signaling molecules in a manner similar to phytohormones. This review first describes the main classes of carbohydrates associated to plant immunity, their role and mode of action. More precisely, the state of the art about perception of “PAMP, MAMP, and DAMP (Pathogen-, Microbe-, Damage-Associated Molecular Patterns) type” oligosaccharides is presented and examples of induced defense events are provided. A particular attention is paid to the structure/activity relationships of these compounds. The role of sugars as signaling molecules, especially in plant microbe interactions, is also presented. Secondly, the potentialities and limits of foliar sprays of carbohydrates to stimulate plant immunity for crop protection against diseases are discussed, with focus on the roles of the leaf cuticle and phyllosphere microflora.

Nonspecific Lipid-Transfer Protein Genes Expression in Grape (Vitis sp.) Cells in Response to Fungal Elicitor Treatments

Nonspecific lipid transfer proteins (nsLTPs) are small, basic cystein-rich proteins believed to be involved in plant defense mechanisms. Three cDNAs coding nsLTPs from grape (Vitis vinifera sp.) were cloned by reverse-transcriptase-polymerase chain reaction (RT-PCR) and PCR. The expression of nsLTP genes was investigated in 41B-rootstock grape cell suspension, in response to various defense-related signal molecules. Ergosterol (a fungi-specific sterol) and a proteinaceous elicitor purified from Botrytis cinerea strongly and rapidly induced the accumulation of nsLTP mRNAs. Jasmonic acid, cholesterol, and sitosterol also promoted nsLTPs mRNA accumulation, although to a lesser extent, whereas salicylic acid had no effect. High performance liquid chromatography analysis indicated that the amounts of three LTP isoforms (previously named P1, P2, and P4) were increased by ergosterol. None of the four isoforms displayed any significant antifungal properties, with the exception of the P4 isoform, which reduced Botrytis mycelium growth in vitro, but only in calcium-free medium. The results are discussed in the context of plant-pathogen interactions.

Glutathione deficiency of the Arabidopsis mutant pad2-1 affects oxidative stress-related events, defense gene expression, and the hypersensitive response.

The Arabidopsis (Arabidopsis thaliana) phytoalexin-deficient mutant pad2-1 displays enhanced susceptibility to a broad range of pathogens and herbivorous insects that correlates with deficiencies in the production of camalexin, indole glucosinolates, and salicylic acid (SA). The pad2-1 mutation is localized in the GLUTAMATE-CYSTEINE LIGASE (GCL) gene encoding the first enzyme of glutathione biosynthesis. While pad2-1 glutathione deficiency is not caused by a decrease in GCL transcripts, analysis of GCL protein level revealed that pad2-1 plants contained only 48% of the wild-type protein amount. In contrast to the wild type, the oxidized form of GCL was dominant in pad2-1, suggesting a distinct redox environment. This finding was corroborated by the expression of GRX1-roGFP2, showing that the cytosolic glutathione redox potential was significantly less negative in pad2-1. Analysis of oxidative stress-related gene expression showed a higher transcript accumulation in pad2-1 of GLUTATHIONE REDUCTASE, GLUTATHIONE-S-TRANSFERASE, and RESPIRATORY BURST OXIDASE HOMOLOG D in response to the oomycete Phytophthora brassicae. Interestingly, oligogalacturonide elicitation in pad2-1 revealed a lower plasma membrane depolarization that was found to act upstream of an impaired hydrogen peroxide production. This impaired hydrogen peroxide production was also observed during pathogen infection and correlated with a reduced hypersensitive response in pad2-1. In addition, a lack of pathogen-triggered expression of the ISOCHORISMATE SYNTHASE1 gene, coding for the SA-biosynthetic enzyme isochorismate synthase, was identified as the cause of the SA deficiency in pad2-1. Together, our results indicate that the pad2-1 mutation is related to a decrease in GCL protein and that the resulting glutathione deficiency negatively affects important processes of disease resistance.

The grapevine flagellin receptor VvFLS2 differentially recognizes flagellin‐derived epitopes from the endophytic growth‐promoting bacterium Burkholderia phytofirmans and plant pathogenic bacteria

• The role of flagellin perception in the context of plant beneficial bacteria still remains unclear. Here, we characterized the flagellin sensing system flg22-FLAGELLIN SENSING 2 (FLS2) in grapevine, and analyzed the flagellin perception in the interaction with the endophytic plant growth-promoting rhizobacterium (PGPR) Burkholderia phytofirmans. • The functionality of the grapevine FLS2 receptor, VvFLS2, was demonstrated by complementation assays in the Arabidopsis thaliana fls2 mutant, which restored flg22-induced H₂O₂ production and growth inhibition. Using synthetic flg22 peptides from different bacterial origins, we compared recognition specificities between VvFLS2 and AtFLS2. • In grapevine, flg22-triggered immune responses are conserved and led to partial resistance against Botrytis cinerea. Unlike flg22 peptides derived from Pseudomonas aeruginosa or Xanthomonas campestris, flg22 peptide derived from B. phytofirmans triggered only a small oxidative burst, weak and transient defense gene induction and no growth inhibition in grapevine. Although, in Arabidopsis, all the flg22 epitopes exhibited similar biological activities, the expression of VvFLS2 into the fls2 background conferred differential flg22 responses characteristic for grapevine. • These results demonstrate that VvFLS2 differentially recognizes flg22 from different bacteria, and suggest that flagellin from the beneficial PGPR B. phytofirmans has evolved to evade this grapevine immune recognition system.

β-Aminobutyric acid primes an NADPH oxidase-dependent reactive oxygen species production during grapevine-triggered immunity.

The molecular mechanisms underlying the process of priming are poorly understood. In the present study, we investigated the early signaling events triggered by beta-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.

Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis

The recent publication of the grapevine genome sequence facilitates the use of qRT-PCR to study gene expression changes. For this approach, reference genes are commonly used to normalize data and their stability of expression should be systematically validated. Among grapevine defenses is the production of the antimicrobial stilbenic phytoalexins, notably the highly fungitoxic pterostilbene, which plays a crucial role in grapevine interaction with Plasmopara viticola and Botrytis cinerea. As a resveratrol O-methyltransferase (ROMT) gene involved in pterostilbene synthesis was recently identified, we investigated the accumulation of the corresponding transcripts to those of two other stilbene biosynthesis related genes phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) in response to pathogen infection. Using three computer-based statistical methods and Ct values or LRE method generated values as input data, we have first identified two reference genes (VATP16 and 60SRP) suitable for normalization of qPCR expression data obtained in grapevine leaves and berries infected by P. viticola and B. cinerea, respectively. Next, we have highlighted that the expression of ROMT is induced in P. viticola-infected leaves and also in B. cinerea-infected berries, confirming the involvement of pterostilbene in grapevine defenses.