Identification et caractérisation du récepteur à la flagelline (VvFLS2) et recherche du récepteur aux chito-oligosaccharides chez la vigne

Pattern-recognition receptors (PRRs) play a key role in plant immunity by assuring recognition of microbe-associated molecular patterns (MAMPs), signature of microbial presence. MAMP perception constitutes the first layer of pathogen detection and activates defense mechanisms that aim to block the intruder. This study brings an insight into how grapevine (Vitis vinifera) perceives two MAMPs: the flagellin-derived flg22 peptide and chitin, which are conserved motifs occurring over the whole bacterial and fungal classes, respectively. This study analyzed MAMP-triggered early signaling events, defense gene expression and also the efficiency of elicited defense against gray mold and downy mildew diseases. These two MAMPs are active in grapevine suggesting that perception systems exist. So far, no PRR is known for this crop.Given the availability of grapevine genome, we could identify in silico putative grapevine receptors (VvFLS2, VvCERK1-3 and VvCEBiP1-2) that might function as PRRs for flg22 and chitin, respectively. Their functional characterization was firstly achieved by complementation assays in the corresponding A. thaliana mutants and, secondly, by a gene silencing strategy in grapevine.Our results permitted the identification of VvFLS2, the V. vinifera receptor for the bacterial flagellin. The function of VvFLS2 was demonstrated by restoring the flg22 responsiveness in the Arabidopsis fls2 null mutant. Thus, our work provides the first description of an active grapevine PRR-MAMP pair. We further compared VvFLS2 and the Arabidopsis receptor, AtFLS2, in their capability to perceive flagellin-derived flg22 epitopes from endophytic or pathogenic bacteria. Our data clearly show that VvFLS2 differentially recognizes flg22 from different bacteria and suggest that flagellin from the beneficial plant growth-promoting rhizobacteria (PGPR) Burkholderia phytofirmans has evolved to evade grapevine immune recognition system. We also obtained preliminary data on chitin sensing system in grapevine and show that VvCERK3 might be a functional ortholog of AtCERK1 by partly restoring the oxidative burst triggered by chitin in the Arabidopsis cerk1-2 mutant.Les récepteurs PRR (Pattern-recognition receptors) jouent un rôle clé dans l’immunité des plantes en assurant la reconnaissance d’éliciteurs, des motifs moléculaires associés aux microorganismes (MAMP) qui témoigne de leur présence microbienne. La perception de ces MAMPs constitue le premier système de détection d’agents potentiellement pathogène, et déclenche des mécanismes de défense qui ont pour but de bloquer leur développement.Cette étude met en lumière comment la vigne (Vitis vinifera) perçoit deux MAMPs : le peptide flg22 dérivé de la flagelline et la chitine, des motifs conservés existant dans la plupart des espèces bactériennes et fongiques, respectivement. Cette étude analyse les événements précoces de signalisation, l’expression de gènes de défense activés par ces MAMPs et l’efficacité de la résistance induite contre les agents de la pourriture grise et du mildiou de la vigne. Si nos résultats suggèrent que des systèmes de perception pour ces deux MAMPs existent chez la vigne, aucun récepteur PRR n’est actuellement connu pour cette plante cultivée.La disponibilité du génome de la vigne nous a permis d’identifier in silico des récepteurs putatifs (VvFLS2, VvCERK1-3 et VvCEBiP1-2) pouvant fonctionner comme PRR respectif de flg22 et de chitine. Leur analyse fonctionnelle a été réalisée d’une part par complémentation des mutants correspondants d’Arabidopsis et, d’autre part, par une stratégie d’extinction de gène chez la vigne.Nos résultats ont permis d’identifier VvFLS2, le récepteur de la vigne à la flagelline bactérienne. La fonction de VvFLS2 a été démontrée en restaurant la réponse à flg22 du mutant fls2 d’Arabidopsis. Ainsi, nos travaux sont les premiers à décrire un couple PRR-MAMP actif chez la vigne. Nous avons également comparé les capacités de perception de VvFLS2 et du récepteur d’Arabidopsis, AtFLS2, envers des épitopes flg22 provenant de bactéries endophytes ou pathogènes. Nos données montrent clairement que VvFLS2 reconnait différemment les peptides flg22 des différentes bactéries et suggèrent que la flagelline de la bactérie bénéfique Burkholderia phytofirmans a évolué pour échapper au système de reconnaissance immunitaire de la vigne. Nous avons également obtenu des données préliminaires concernant le système de perception de la chitine chez la vigne et montré que VvCERK3 pourrait être un orthologue fonctionnel d’AtCERK1 en restaurant partiellement le burst oxydatif induit par la chitine dans le mutant cerk1-2 d’Arabidopsis

The grapevine (Vitis vinifera) LysM receptor kinases VvLYK1-1 and VvLYK1-2 mediate chitooligosaccharide-triggered immunity

Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator

Identification and characterization of the grapevine flagelline eceptor (VvFLS2) and search for the receptor to chito oligosaccharides in grapevine

Les récepteurs PRR (Pattern-recognition receptors) jouent un rôle clé dans l’immunité des plantes en assurant la reconnaissance d’éliciteurs, des motifs moléculaires associés aux microorganismes (MAMP) qui témoigne de leur présence microbienne. La perception de ces MAMPs constitue le premier système de détection d’agents potentiellement pathogène, et déclenche des mécanismes de défense qui ont pour but de bloquer leur développement.Cette étude met en lumière comment la vigne (Vitis vinifera) perçoit deux MAMPs : le peptide flg22 dérivé de la flagelline et la chitine, des motifs conservés existant dans la plupart des espèces bactériennes et fongiques, respectivement. Cette étude analyse les événements précoces de signalisation, l’expression de gènes de défense activés par ces MAMPs et l’efficacité de la résistance induite contre les agents de la pourriture grise et du mildiou de la vigne. Si nos résultats suggèrent que des systèmes de perception pour ces deux MAMPs existent chez la vigne, aucun récepteur PRR n’est actuellement connu pour cette plante cultivée.La disponibilité du génome de la vigne nous a permis d’identifier in silico des récepteurs putatifs (VvFLS2, VvCERK1-3 et VvCEBiP1-2) pouvant fonctionner comme PRR respectif de flg22 et de chitine. Leur analyse fonctionnelle a été réalisée d’une part par complémentation des mutants correspondants d’Arabidopsis et, d’autre part, par une stratégie d’extinction de gène chez la vigne.Nos résultats ont permis d’identifier VvFLS2, le récepteur de la vigne à la flagelline bactérienne. La fonction de VvFLS2 a été démontrée en restaurant la réponse à flg22 du mutant fls2 d’Arabidopsis. Ainsi, nos travaux sont les premiers à décrire un couple PRR-MAMP actif chez la vigne. Nous avons également comparé les capacités de perception de VvFLS2 et du récepteur d’Arabidopsis, AtFLS2, envers des épitopes flg22 provenant de bactéries endophytes ou pathogènes. Nos données montrent clairement que VvFLS2 reconnait différemment les peptides flg22 des différentes bactéries et suggèrent que la flagelline de la bactérie bénéfique Burkholderia phytofirmans a évolué pour échapper au système de reconnaissance immunitaire de la vigne. Nous avons également obtenu des données préliminaires concernant le système de perception de la chitine chez la vigne et montré que VvCERK3 pourrait être un orthologue fonctionnel d’AtCERK1 en restaurant partiellement le burst oxydatif induit par la chitine dans le mutant cerk1-2 d’Arabidopsis.Pattern-recognition receptors (PRRs) play a key role in plant immunity by assuring recognition of microbe-associated molecular patterns (MAMPs), signature of microbial presence. MAMP perception constitutes the first layer of pathogen detection and activates defense mechanisms that aim to block the intruder. This study brings an insight into how grapevine (Vitis vinifera) perceives two MAMPs: the flagellin-derived flg22 peptide and chitin, which are conserved motifs occurring over the whole bacterial and fungal classes, respectively. This study analyzed MAMP-triggered early signaling events, defense gene expression and also the efficiency of elicited defense against gray mold and downy mildew diseases. These two MAMPs are active in grapevine suggesting that perception systems exist. So far, no PRR is known for this crop.Given the availability of grapevine genome, we could identify in silico putative grapevine receptors (VvFLS2, VvCERK1-3 and VvCEBiP1-2) that might function as PRRs for flg22 and chitin, respectively. Their functional characterization was firstly achieved by complementation assays in the corresponding A. thaliana mutants and, secondly, by a gene silencing strategy in grapevine.Our results permitted the identification of VvFLS2, the V. vinifera receptor for the bacterial flagellin. The function of VvFLS2 was demonstrated by restoring the flg22 responsiveness in the Arabidopsis fls2 null mutant. Thus, our work provides the first description of an active grapevine PRR-MAMP pair. We further compared VvFLS2 and the Arabidopsis receptor, AtFLS2, in their capability to perceive flagellin-derived flg22 epitopes from endophytic or pathogenic bacteria. Our data clearly show that VvFLS2 differentially recognizes flg22 from different bacteria and suggest that flagellin from the beneficial plant growth-promoting rhizobacteria (PGPR) Burkholderia phytofirmans has evolved to evade grapevine immune recognition system. We also obtained preliminary data on chitin sensing system in grapevine and show that VvCERK3 might be a functional ortholog of AtCERK1 by partly restoring the oxidative burst triggered by chitin in the Arabidopsis cerk1-2 mutant

Identification of the VvFLS2 grapevine flagellin receptor by a functional genomics strategy

National audienc

Induced resistance in grapevine: from concept to vineyard application

SPE IPM Chapitre 11International audienc

Cytokinin Metabolism of Pathogenic Fungus Leptosphaeria maculans Involves Isopentenyltransferase, Adenosine Kinase and Cytokinin Oxidase/Dehydrogenase

Among phytohormones, cytokinins (CKs) play an important role in controlling crucial aspects of plant development. Not only plants but also diverse microorganisms are able to produce phytohormones, including CKs, though knowledge concerning their biosynthesis and metabolism is still limited. In this work we demonstrate that the fungus Leptosphaeria maculans, a hemi-biotrophic pathogen of oilseed rape (Brassica napus), causing one of the most damaging diseases of this crop, is able to modify the CK profile in infected B. napus tissues, as well as produce a wide range of CKs in vitro, with the cis-zeatin derivatives predominating. The endogenous CK spectrum of L. maculans in vitro consists mainly of free CK bases, as opposed to plants, where other CK forms are mostly more abundant. Using functional genomics, enzymatic and feeding assays with CK bases supplied to culture media, we show that L. maculans contains a functional: (i) isopentenyltransferase (IPT) involved in cZ production; (ii) adenosine kinase (AK) involved in phosphorylation of CK ribosides to nucleotides; and (iii) CK-degradation enzyme cytokinin oxidase/dehydrogenase (CKX). Our data further indicate the presence of cis–trans isomerase, zeatin O-glucosyltransferase(s) and N6-(Δ2-isopentenyl)adenine hydroxylating enzyme. Besides, we report on a crucial role of LmAK for L. maculans fitness and virulence. Altogether, in this study we characterize in detail the CK metabolism of the filamentous fungi L. maculans and report its two novel components, the CKX and CK-related AK activities, according to our knowledge for the first time in the fungal kingdom. Based on these findings, we propose a model illustrating CK metabolism pathways in L. maculans

Identification of the VvFLS2 grapevine flagellin receptor by a functional genomics strategy

National audiencePattern recognition receptors (PRRs) emerge as important components of plant disease resistance in plants. PRRs mediate detection of potential pathogens via the perception of pathogen associated molecular patterns (PAMPs). A well-studied PRR is the Arabidopsis leucine-rich repeat receptor kinase (LRR-RK) FLS2 that recognizes bacterial flagellin. Until now, functional FLS2 orthologues have been characterized in Nicotiana benthamiana, rice and tomato. We have shown that flg22 triggers a battery of defense responses and induces partial resistance to a necrotrophic fungus Botrytis cinerea. The putative grapevine FLS2 orthologue, designated as VvFLS2, was identified by an in silico approach and its functionality was demonstrated by complementation of the Arabidopsis fls2c null mutant. In parallel, a silencing approach in grapevine was developed to obtain stable transgenic lines silenced in VvFLS2

Identification and characterization of the grapevine flagellin receptor vvfls2

International audienc

Recognition of Elicitors in Grapevine: From MAMP and DAMP Perception to Induced Resistance

International audienceIn a context of a sustainable viticulture, the implementation of innovative eco-friendly strategies, such as elicitor-triggered immunity, requires a deep knowledge of the molecular mechanisms underlying grapevine defense activation, from pathogen perception to resistance induction. During plant-pathogen interaction, the first step of plant defense activation is ensured by the recognition of microbe-associated molecular patterns, which are elicitors directly derived from pathogenic or beneficial microbes. Vitis vinifera, like other plants, can perceive elicitors of different nature, including proteins, amphiphilic glycolipid, and lipopeptide molecules as well as polysaccharides, thanks to their cognate pattern recognition receptors, the discovery of which recently began in this plant species. Furthermore, damage-associated molecular patterns are another class of elicitors perceived by V. vinifera as an invader's hallmark. They are mainly polysaccharides derived from the plant cell wall and are generally released through the activity of cell wall-degrading enzymes secreted by microbes. Elicitor perception and subsequent activation of grapevine immunity end in some cases in efficient grapevine resistance against pathogens. Using complementary approaches, several molecular markers have been identified as hallmarks of this induced resistance stage. This review thus focuses on the recognition of elicitors by Vitis vinifera describing the molecular mechanisms triggered from the elicitor perception to the activation of immune responses. Finally, we discuss the fact that the link between elicitation and induced resistance is not so obvious and that the formulation of resistance inducers remains a key step before their application in vineyards

Two lysine motif receptor-like kinases (VvLYKs) participate in chitin-triggered immunity in grapevine

International audienceIn nature, plants are constantly exposed to potentially pathogenic microbes such as bacteria, fungi, oomycetes or viruses. However, plants have developed effective immune systems triggering various defence reactions against invading pathogens upon the perception of pathogen-associated molecular patterns (PAMPs; Dodds and Rathjen, 2010). The recognition of these conserved microbial signatures is ensured by Pattern Recognition Receptors (PRRs) which also detect plant endogenous molecules released during pathogen invasion, called damage-associated molecular patterns (DAMPs; Boller and Felix, 2009). Chitin, a fungal cell wall component, is a well-known PAMP that triggers defence responses in many mammal and plant species. The aim of the study was to determine the effects of chito-oligosaccharides on grapevine’s immunity and identify the receptor(s) involved in the perception of chito-oligosaccharides in grapevine. Material and methods Grapevine cells (Vitis vinifera cv Gamay) were cultivated as described in Gauthier et al. (2014). Arabidopsis thaliana plants from wild-type (WT) Columbia (Col-0), mutant and transgenic lines were grown in vitro for two weeks in controlled conditions for defence responses or in jiffy peat pellets in a controlled growth chamber for four weeks for protection assays. Grapevine cells or Arabidopsis plants were treated with water, chitin, chitosan (Elicityl, 0.1 g/l for cells and 1 g/l for plants) or flagelline (10 μM) taken as a positive control. ROS production and cytosolic Ca2+ variations ([Ca2+]cyt) in grapevine cells were performed according to Dubreuil-Maurizi et al. (2010) after elicitor treatments, by measuring the chemiluminescence of luminol for H2O2 production and using apoaequorin expressing cells to detect variations of [Ca2+]cyt. Protein extraction, SDS-PAGE and western blotting for MAPK phosphorylation analysis were carried out as previously described (Trdà et al., 2014). RNA extraction and quantitative real-time PCR were performed using primers for the Highlights • Two Pattern Recognition Receptors (PRRs) VvLYK1-1 and VvLYK1-2 participate in the signaling of chito-oligosaccharides in grapevine • VvLYK1-1 is involved in powdery mildew resistance amplification of defence marker genes (CHIT4C, STS1-2, PAL, RBOHD, and FRK1). Two days after elicitor treatment, Botrytis cinerea and Plasmopara viticola infections were performed on grapevine plants. For protection assays to Erysiphe necator, leaves were infected, maintained on agar medium in the incubator and then sampled at 0, 4, 8, 12 and 24 hours post inoculation. Results and discussion In grapevine cells, chitin treatment induced a rapid and transient increase in free [Ca2+]cyt that peaked at 2 min but not chitosan, even if the basal level remained higher during the whole experiment. Both chito-oligosaccharides did not trigger any H2O2 production contrary to the flagelline epitope flg-22. Chitin and chitosan treatment induced the phosphorylation of two MAPKs with relative molecular masses of 45 and 49 kDa in grapevine cells but chitosan activated the phosphorylation of these two MAPKs longer than the chitin treatment. The expression of defence marker genes activated by different elicitors was then followed by qPCR. Among them, both chito-oligosaccharides induced the expression of four grapevine defence genes encoding an acidic chitinase (CHIT4C), a stilbene synthase (STS1-2), a phenylalanine ammonia lyase (PAL) and a respiratory burst oxidase homolog D (RBOHD), 1 hour post-treatment (hpt). The efficacy of chitin- and chitosan-induced immunity was investigated in Vitis vinifera leaf discs infected by the necrotrophic fungus B. cinerea or with the biotrophic oomycete P. viticola, the causal agents of gray mold and downy mildew, respectively. If chitin pretreatment induced a low but significant resistance against these pathogens, chitosan reduced very significantly the B. cinerea lesion diameter and the P. viticola sporulation. Taken toghether, these results demonstrate that grapevine perceives chitin and chitosan suggesting that at least one PRR for chito-oligosaccharides perception exists. To identify the receptor of chito-oligosaccharides in grapevine, the grapevine family of LysM receptor like kinases was characterised and three proteins, respectively named VvLYK1-1, VvLYK1-2 and VvLYK1-3, showed a close relation to Arabidopsis CERK1/LYK1 (Chitin-Elicitor Receptor Kinase 1) and the rice ortholog CERK1. By functional complementation of the Arabidopsis cerk1/lyk1 mutant, impaired in chitin perception and signalling, we demonstrated that VvLYK1-1 and VvLYK1-2 are involved in the signalling of chito-oligosaccharides in Vitis vinifera. Moreover, VvLYK1-1 plays a key role in basal resistance against the grapevine powdery mildew causal agent E. necator