41 research outputs found
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
Caractérisation des réactions de défense de la vigne et identification d'éliciteurs : l'endopolygalacturonase 1 de Botrytis cinerea, une fonction d'avirulence pour un facteur de virulence.
The fight against grapevine pathogens is mainly carried out by pesticides, the continued use of which is harmful to the environment and the health of users and consumers. The main organizations in charge of viticulture set as a priority the research and use of alternatives to chemical control. However, the genetic improvement of the vine is prohibited in AOC vineyards to preserve the varietal typicity, partly responsible for the quality of the wines. In addition, research undertaken some fifteen years ago reveals that plants have their own immune defenses, which they activate on contact with the microorganisms they recognize via molecules called elicitors. In this context, the objective of this work was both to research and identify active elicitors on grapevine, to characterize the signaling events and the defense reactions activated by these elicitors and finally to verify that the application of elicitors on grapevine could lead to increased resistance to pathogens.We have purified, from the culture filtrates of a strain of Botrytis cinerea with low virulence on grapevine, a protein elicitor which induces a set of defense reactions in cell suspensions of Vitis vinifera cv Gamay: calcium influx, efflux of nitrate, activation of two MAPKs, production of H2O2, activation of defense genes, production of phytoalexins. These results are the first to describe a set of defense reactions in grapevine. They reveal similarities but also differences with the defense mechanisms involved and identified in model plants such as tobacco. Mass spectrometry made it possible to identify this elicitor of Botrytis cinerea; it is the endopolygalacturonase 1 (BcPG1), hitherto considered as a virulence factor. This result is an original contribution to understanding the molecular dialogue between plants and microbes during evolution. Two other elicitors have proven to be effective on grapevine: laminarin, a linear polymer of beta-1,3-glucan extracted from the seaweed Laminaria digitata, and oligogalacturonates (OGs), alpha-1,4 polymers of galacturonic acid in particular produced by hydrolysis of the plant wall by endopolygalacturonases. The qualitative, quantitative and kinetics comparison of events activated by BcPG1 and OG suggested that the eliciting activity of BcPG1 did not come solely from the release of OGs via its enzymatic activity. Desensitization experiments confirmed that BcPG1 and OGs were perceived by grapevine cells as two distinct stimuli. Chemical or physical treatments of the protein made it possible to discriminate between the eliciting and enzymatic activities of BcPG1, thus showing that the eliciting activity of this protein does not come from its enzymatic activity, but probably from structural motifs specific to BcPG1 and recognized by the plant.Different protection tests have been developed to evaluate the protection of grapevine induced by elicitors against two pathogenic agents: Botrytis cinerea (the agent of grey mold) and Plasmopara viticola (the agent of downy mildew). The results show that the development of these pathogens is reduced by more than 50% when grapevine is pre-treated with elicitors. These results are very promising in the perspective of an application in reasoned viticulture.La lutte contre les pathogĂšnes de la vigne sâeffectue principalement par des produits phytosanitaires dont lâutilisation continue est nĂ©faste pour lâenvironnement et la santĂ© des utilisateurs et des consommateurs. Les principaux organismes en charge de la viticulture fixent comme prioritĂ© la recherche et lâutilisation de moyens de lutte alternatifs Ă la lutte chimique. Or, lâamĂ©lioration gĂ©nĂ©tique de la vigne est interdite dans les vignobles dâAOC pour prĂ©server la typicitĂ© variĂ©tale, en partie responsable de la qualitĂ© des crus. Par ailleurs, des recherches engagĂ©es il y a une quinzaine dâannĂ©es rĂ©vĂšlent que les plantes ont des moyens de dĂ©fense propres, quâelles activent au contact des micro-organismes quâelles reconnaissent via des molĂ©cules appelĂ©es Ă©liciteurs. Dans ce contexte, lâobjectif de ce travail Ă©tait Ă la fois de rechercher et dâidentifier des Ă©liciteurs actifs sur la vigne, de caractĂ©riser les Ă©vĂ©nements de signalisation et les rĂ©actions de dĂ©fense activĂ©s par ces Ă©liciteurs et enfin de vĂ©rifier que lâapplication dâĂ©liciteurs sur la vigne pouvait conduire Ă une rĂ©sistance accrue aux pathogĂšnes.Nous avons purifiĂ©, Ă partir des filtrats de culture dâune souche de Botrytis cinerea peu virulente sur la vigne, un Ă©liciteur protĂ©ique qui induit un ensemble de rĂ©actions de dĂ©fense dans des suspensions cellulaires de Vitis vinifera cv Gamay : influx de calcium, efflux de nitrate, activation de deux MAPK, production dâH2O2, activation de gĂšnes de dĂ©fense, production de phytoalexines. Ces rĂ©sultats sont les premiers Ă dĂ©crire un ensemble de rĂ©actions de dĂ©fense chez la vigne. Ils rĂ©vĂšlent des similitudes mais aussi des diffĂ©rences avec les mĂ©canismes de dĂ©fense mis en jeu et identifiĂ©s dans des plantes modĂšles comme le tabac. La spectromĂ©trie de masse a permis dâidentifier cet Ă©liciteur de Botrytis cinerea ; il sâagit de lâendopolygalacturonase 1 (BcPG1), jusque lĂ considĂ©rĂ©e comme un facteur de virulence. Ce rĂ©sultat est une contribution originale Ă la comprĂ©hension du dialogue molĂ©culaire entre les plantes et les microbes au cours de lâĂ©volution. Deux autres Ă©liciteurs se sont rĂ©vĂ©lĂ©s efficaces sur la vigne : la laminarine, polymĂšre linĂ©aire de beta-1,3-glucane extrait de lâalgue Laminaria digitata, et les oligogalacturonates (OG), polymĂšres alpha-1,4 dâacide galacturonique notamment produits par lâhydrolyse de la paroi vĂ©gĂ©tale par les endopolygalacturonases. La comparaison au plan qualitatif, quantitatif et cinĂ©tique des Ă©vĂ©nements activĂ©s par BcPG1 et les OG laissaient supposer que lâactivitĂ© Ă©licitrice de BcPG1 ne provenait pas uniquement de la libĂ©ration dâOG via son activitĂ© enzymatique. Des expĂ©riences de dĂ©sensibilisation ont confirmĂ© que BcPG1 et les OG Ă©taient perçus par les cellules de vigne comme deux stimuli distincts. Des traitements chimiques ou physiques de la protĂ©ine ont permis de discriminer les activitĂ©s Ă©licitrice et enzymatique de BcPG1, montrant ainsi que lâactivitĂ© Ă©licitrice de cette protĂ©ine ne provient pas de son activitĂ© enzymatique, mais vraisemblablement de motifs structuraux propres Ă BcPG1 et reconnus par la plante.DiffĂ©rents tests de protection ont Ă©tĂ© dĂ©veloppĂ©s pour Ă©valuer la protection de la vigne induite par des Ă©liciteurs vis Ă vis de deux agents pathogĂšnes : Botrytis cinerea (lâagent de la pourriture grise) et Plasmopara viticola (lâagent du mildiou). Les rĂ©sultats montrent que le dĂ©veloppement de ces pathogĂšnes est rĂ©duit de plus de 50% lorsque la vigne est prĂ©-traitĂ©e par des Ă©liciteurs. Ces rĂ©sultats sont trĂšs prometteurs dans la perspective dâune application en viticulture raisonnĂ©e
Caractérisation des réactions de défense de la vigne et identification d'éliciteurs : l'endopolygalacturonase 1 de Botrytis cinerea, une fonction d'avirulence pour un facteur de virulence.
The fight against grapevine pathogens is mainly carried out by pesticides, the continued use of which is harmful to the environment and the health of users and consumers. The main organizations in charge of viticulture set as a priority the research and use of alternatives to chemical control. However, the genetic improvement of the vine is prohibited in AOC vineyards to preserve the varietal typicity, partly responsible for the quality of the wines. In addition, research undertaken some fifteen years ago reveals that plants have their own immune defenses, which they activate on contact with the microorganisms they recognize via molecules called elicitors. In this context, the objective of this work was both to research and identify active elicitors on grapevine, to characterize the signaling events and the defense reactions activated by these elicitors and finally to verify that the application of elicitors on grapevine could lead to increased resistance to pathogens.We have purified, from the culture filtrates of a strain of Botrytis cinerea with low virulence on grapevine, a protein elicitor which induces a set of defense reactions in cell suspensions of Vitis vinifera cv Gamay: calcium influx, efflux of nitrate, activation of two MAPKs, production of H2O2, activation of defense genes, production of phytoalexins. These results are the first to describe a set of defense reactions in grapevine. They reveal similarities but also differences with the defense mechanisms involved and identified in model plants such as tobacco. Mass spectrometry made it possible to identify this elicitor of Botrytis cinerea; it is the endopolygalacturonase 1 (BcPG1), hitherto considered as a virulence factor. This result is an original contribution to understanding the molecular dialogue between plants and microbes during evolution. Two other elicitors have proven to be effective on grapevine: laminarin, a linear polymer of beta-1,3-glucan extracted from the seaweed Laminaria digitata, and oligogalacturonates (OGs), alpha-1,4 polymers of galacturonic acid in particular produced by hydrolysis of the plant wall by endopolygalacturonases. The qualitative, quantitative and kinetics comparison of events activated by BcPG1 and OG suggested that the eliciting activity of BcPG1 did not come solely from the release of OGs via its enzymatic activity. Desensitization experiments confirmed that BcPG1 and OGs were perceived by grapevine cells as two distinct stimuli. Chemical or physical treatments of the protein made it possible to discriminate between the eliciting and enzymatic activities of BcPG1, thus showing that the eliciting activity of this protein does not come from its enzymatic activity, but probably from structural motifs specific to BcPG1 and recognized by the plant.Different protection tests have been developed to evaluate the protection of grapevine induced by elicitors against two pathogenic agents: Botrytis cinerea (the agent of grey mold) and Plasmopara viticola (the agent of downy mildew). The results show that the development of these pathogens is reduced by more than 50% when grapevine is pre-treated with elicitors. These results are very promising in the perspective of an application in reasoned viticulture.La lutte contre les pathogĂšnes de la vigne sâeffectue principalement par des produits phytosanitaires dont lâutilisation continue est nĂ©faste pour lâenvironnement et la santĂ© des utilisateurs et des consommateurs. Les principaux organismes en charge de la viticulture fixent comme prioritĂ© la recherche et lâutilisation de moyens de lutte alternatifs Ă la lutte chimique. Or, lâamĂ©lioration gĂ©nĂ©tique de la vigne est interdite dans les vignobles dâAOC pour prĂ©server la typicitĂ© variĂ©tale, en partie responsable de la qualitĂ© des crus. Par ailleurs, des recherches engagĂ©es il y a une quinzaine dâannĂ©es rĂ©vĂšlent que les plantes ont des moyens de dĂ©fense propres, quâelles activent au contact des micro-organismes quâelles reconnaissent via des molĂ©cules appelĂ©es Ă©liciteurs. Dans ce contexte, lâobjectif de ce travail Ă©tait Ă la fois de rechercher et dâidentifier des Ă©liciteurs actifs sur la vigne, de caractĂ©riser les Ă©vĂ©nements de signalisation et les rĂ©actions de dĂ©fense activĂ©s par ces Ă©liciteurs et enfin de vĂ©rifier que lâapplication dâĂ©liciteurs sur la vigne pouvait conduire Ă une rĂ©sistance accrue aux pathogĂšnes.Nous avons purifiĂ©, Ă partir des filtrats de culture dâune souche de Botrytis cinerea peu virulente sur la vigne, un Ă©liciteur protĂ©ique qui induit un ensemble de rĂ©actions de dĂ©fense dans des suspensions cellulaires de Vitis vinifera cv Gamay : influx de calcium, efflux de nitrate, activation de deux MAPK, production dâH2O2, activation de gĂšnes de dĂ©fense, production de phytoalexines. Ces rĂ©sultats sont les premiers Ă dĂ©crire un ensemble de rĂ©actions de dĂ©fense chez la vigne. Ils rĂ©vĂšlent des similitudes mais aussi des diffĂ©rences avec les mĂ©canismes de dĂ©fense mis en jeu et identifiĂ©s dans des plantes modĂšles comme le tabac. La spectromĂ©trie de masse a permis dâidentifier cet Ă©liciteur de Botrytis cinerea ; il sâagit de lâendopolygalacturonase 1 (BcPG1), jusque lĂ considĂ©rĂ©e comme un facteur de virulence. Ce rĂ©sultat est une contribution originale Ă la comprĂ©hension du dialogue molĂ©culaire entre les plantes et les microbes au cours de lâĂ©volution. Deux autres Ă©liciteurs se sont rĂ©vĂ©lĂ©s efficaces sur la vigne : la laminarine, polymĂšre linĂ©aire de beta-1,3-glucane extrait de lâalgue Laminaria digitata, et les oligogalacturonates (OG), polymĂšres alpha-1,4 dâacide galacturonique notamment produits par lâhydrolyse de la paroi vĂ©gĂ©tale par les endopolygalacturonases. La comparaison au plan qualitatif, quantitatif et cinĂ©tique des Ă©vĂ©nements activĂ©s par BcPG1 et les OG laissaient supposer que lâactivitĂ© Ă©licitrice de BcPG1 ne provenait pas uniquement de la libĂ©ration dâOG via son activitĂ© enzymatique. Des expĂ©riences de dĂ©sensibilisation ont confirmĂ© que BcPG1 et les OG Ă©taient perçus par les cellules de vigne comme deux stimuli distincts. Des traitements chimiques ou physiques de la protĂ©ine ont permis de discriminer les activitĂ©s Ă©licitrice et enzymatique de BcPG1, montrant ainsi que lâactivitĂ© Ă©licitrice de cette protĂ©ine ne provient pas de son activitĂ© enzymatique, mais vraisemblablement de motifs structuraux propres Ă BcPG1 et reconnus par la plante.DiffĂ©rents tests de protection ont Ă©tĂ© dĂ©veloppĂ©s pour Ă©valuer la protection de la vigne induite par des Ă©liciteurs vis Ă vis de deux agents pathogĂšnes : Botrytis cinerea (lâagent de la pourriture grise) et Plasmopara viticola (lâagent du mildiou). Les rĂ©sultats montrent que le dĂ©veloppement de ces pathogĂšnes est rĂ©duit de plus de 50% lorsque la vigne est prĂ©-traitĂ©e par des Ă©liciteurs. Ces rĂ©sultats sont trĂšs prometteurs dans la perspective dâune application en viticulture raisonnĂ©e
Ătude de la signalisation liĂ©e au stress du rĂ©ticulum endoplasmique dans lâimmunitĂ© de la vigne associĂ©e aux rĂ©cepteurs kinases Ă motifs LysM (VvLYKs)
National audienceDans le contexte de rĂ©duction des produits phytosanitaires en agriculture, lâamĂ©lioration desconnaissances sur lâimmunitĂ© et la symbiose des plantes est un enjeu important dans larecherche de nouveaux modes de protection des cultures. Ce projet de thĂšse vise Ă amĂ©liorernos connaissances sur le rĂŽle des rĂ©cepteurs kinases Ă domaine LysM (LYKs) dans la balanceentre immunitĂ© et symbiose chez la vigne ainsi quâĂ dĂ©terminer le rĂŽle de la rĂ©ponse au stressdu rĂ©ticulum endoplasmique (Unfolded Protein Response; UPR) dans ces voies. Des travauxantĂ©rieurs ont identifiĂ© 15 rĂ©cepteurs VvLYKs chez la vigne et permis de caractĂ©riser le rĂŽle deVvLYK1-1 et VvLYK1-2 dans la perception de la chitine. Dans ce contexte, le premier objectifde la thĂšse est de caractĂ©riser la fonction de nouveaux VvLYKs impliquĂ©s dans la perceptionde facteurs Myc qui permettent le contournement de lâimmunitĂ© lors de la mise en place dessymbioses mycorhiziennes. Le second objectif est de caractĂ©riser les voies de signalisation delâUPR chez la vigne. En effet, mĂȘme si le rĂŽle de lâUPR dans lâimmunitĂ© des plantes nâest plusĂ dĂ©monter, notamment via les facteurs de transcriptions bZIP17, bZIP28 et bZIP60, nosconnaissances restent limitĂ©es quant Ă son fonctionnement chez de nombreuses plantes dâintĂ©rĂȘtagronomique, dont la vigne. Pour ce faire, une approche de gĂ©nomique fonctionnelle par Ă©ditiongĂ©nomique CRISPR/Cas9 chez la vigne permettra de valider le rĂŽle des rĂ©cepteurs VvLYKs etdes VvbZIPs dans lâimmunitĂ© et la symbiose mutualiste
Identification of PAD2 as a Îł-glutamylcysteine synthetase highlights the importance of glutathione in disease resistance of Arabidopsis
The Arabidopsis pad2-1 mutant belongs to a series of non-allelic camalexin-deficient mutants. It was originally described as showing enhanced susceptibility to virulent strains of Pseudomonas syringae and was later shown to be hyper-susceptible to the oomycete pathogen Phytophthora brassicae (formerly P. porri). Surprisingly, in both pathosystems, the disease susceptibility of pad2-1 was not caused by the camalexin deficiency, suggesting additional roles of PAD2 in disease resistance. The susceptibility of pad2-1 to P. brassicae was used to map the mutation to the gene At4g23100, which encodes Îł-glutamylcysteine synthetase (Îł-ECS, GSH1). GSH1 catalyzes the first committed step of glutathione (GSH) biosynthesis. The pad2-1 mutation caused an S to N transition at amino acid position 298 close to the active center. The conclusion that PAD2 encodes GSH1 is supported by several lines of evidence: (i) pad2-1 mutants contained only about 22% of wild-type amounts of GSH, (ii) genetic complementation of pad2-1 with wild-type GSH1 cDNA restored GSH production, accumulation of camalexin in response to P. syringae and resistance to P. brassicae and P. syringae, (iii) another GSH1 mutant, cad2-1, showed pad2-like phenotypes, and (iv) feeding of GSH to excised leaves of pad2-1 restored camalexin production and resistance to P. brassicae. Inoculation of Col-0 with P. brassicae caused a coordinated increase in the transcript abundance of GSH1 and GSH2, the gene encoding the second enzyme in GSH biosynthesis, and resulted in enhanced foliar GSH accumulation. The pad2-1 mutant showed enhanced susceptibility to additional pathogens, suggesting an important general role of GSH in disease resistance of Arabidopsis
The xyloglucans : are they new elicitors of Arabidopsis thaliana immunity ?
Damaged-Associated Molecular Patterns (DAMPs) are endogenous molecules released from the plant cell wall after wounding by pathogens. DAMPs are recognized by Pattern- Recognition Receptors (PRRs) that play a key role in plant immunity by mediating defense responses. The plant cell wall-derived oligogalacturonides (OG) are well characterized DAMPs that elicit plant immune responses such as MAPK activation, [Ca2+]cyt variations, H2O2 production, defense-related gene expression and enhanced resistance against Botrytis cinerea. Our study focused on a new polysaccharide component of the plant cell wall called xyloglucans (Xh) and compared the immune events triggered by OG and Xh in Arabidopsis thaliana. Our results indicated that Xh can be considered as new elicitors as they induced MAPK activation, the expression of defense-related genes, callose deposition and triggered immunity against Botrytis cinerea. By using a genetic approach, our data indicated that the Xh-triggered immunity against B. cinerea requires the phytoalexin and jasmonic aciddependent pathways
Study of the stress-related signalling of endoplasmic reticulum in grapevine immunity associated to LysM receptor kinases (VvLYKs)
National audienceIn the actual situation of decreasing the use of chemicals in agriculture, enhancing our understanding of plant immunity is a critical task to develop more sustainable plant health protection methods. An interesting strategy is to study how plants, and in particular grapevine, perceives and responds to different microorganisms. Microorganisms are notably recognizedby LysM Receptor-like Kinase (LYKs) and previous works have identified 16 LYKs encoded by the grapevine genome (VvLYKs) (Roudaire et al. 2023). Among them, VvLYK1-1, VvLYK1-2 and VvLYK5-1 are involved in chitin perception and thus play a role in the plant immunity. Interestingly, immune responses are also involved during symbiotic interaction but the receptors involved in this process are still unknown. In addition, it has been demonstrated that the Unfolded Protein Response (UPR), which is activated when unfolded proteins accumulate in the endoplasmic reticulum, is also involved during the plant immune response.Itâs therefore important to improve our knowledge about the role of the different grapevine LYK receptors involved in the balance between immunity and symbiosis and in addition to determine the role of stress-related signalling of endoplasmic reticulum and the consequent activation of the UPR in these two different pathways.In this context, we aim to characterize new VvLYKs involved in the perception of Myc-factors which allow the down-regulation of the plant immunity during the establishment of mycorrhizal symbiosis. In addition we also intend to characterize the UPR signalling pathways in grapevinewhich is still unknown and finally ho
UTILISATION D'OLIGOMĂRES D'ALGINATES POUR AMĂLIORER LA PROTECTION DES PLANTES CONTRE LES PATHOGĂNES
The invention concerns the use of alginate oligomers in sequence α(1-4)Guluronane and/or Ă(1-4)Mannuronane having a DP greater than 30, as an active ingredient of biocontrol products in plants, and that may be combined with another molecules such as phytohormones, against cryptogamic diseases of plants, in particular of vine (Vitis vinifera), or to protect other species against aggressors such as pathogenic microbes or insects.L'invention concerne l'utilisation d'oligomĂšres d'alginate dans la sĂ©quence α(1-4)Guluronane et/ou Ă(1-4)Mannuronane ayant un DP supĂ©rieur Ă 30, en tant que principe actif de produits de lutte biologique dans des plantes, et qui peuvent ĂȘtre combinĂ©es avec d'autres molĂ©cules telles que des phytohormones, contre des maladies cryptogamiques de plantes, en particulier de la vigne (Vitis vinifera), ou pour protĂ©ger d'autres espĂšces contre des agressions telles que des microbes pathogĂšnes ou des insectes