Identification and characterization of grapevine Shaker-type K+ channels involved in the control of the berry acidity

La vigne (Vitis vinifera L.) est une espèce fruitière à fort impact économique, largement cultivée pour la production de raisin de cuve et donc de vin. Cette espèce est également considérée comme une plante modèle pour étudier la physiologie des plantes à fruits acides. Chez la vigne, comme pour toutes les plantes, le K+ joue un rôle crucial dans de nombreux mécanismes physiologiques. Lors de la maturation du raisin, qui est initiée par une étape clé appelée véraison, le K+ va être fortement accumulé dans la baie. Pendant la maturation de la baie, cet ion initie et contrôle les flux massifs de sucre et de nutriments nécessaires au chargement de la baie. En revanche, une accumulation trop importante de K+ dans la baie de raisin entraîne une diminution de l’acidité des moûts conduisant à l’obtention de vins de faibles qualité organoleptiques et un potentiel de vieillissement altéré. Dans ce contexte, la compréhension des flux K+ intervenant lors de la maturation de la baie semble cruciale. Mon travail s’est focalisé sur la caractérisation de canaux potassiques appartenant à la famille des Shaker. Les différents membres de cette famille sont connus pour dominer la conductance potassique à la membrane plasmique des cellules végétales. Cette famille composée de 9 membres divisée en 5 sous-familles a été étudiée de façon exhaustive chez la plante modèle A. thaliana. Chez la vigne, 9 membres ont aussi été identifiés. Mais de façon surprenante, nous ne retrouvons pas la même répartition des sous-unités dans les différentes sous-familles. Par exemple, la sous-famille des sous-unités sortantes comprend 2 membres chez A. thaliana, alors que 4 membres ont été identifiés chez la vigne. Durant ma thèse, j’ai étudié ces 4 sous-unités pour comprendre leur implication dans le transport du K+ chez la vigne et le chargement de la baie pendant sa maturation. Dans ce cadre, ma thèse se divise en 3 axes. Le premier axe expose la caractérisation de VvK5.1, une sous-unité Shaker sortante typique mais avec un territoire d’expression élargi suggérant fortement l’implication fonctionnelle de canaux Shaker de type sortants dans de nouvelles fonctions. Le deuxième axe présente la caractérisation de VvK5.2. Cette sous-unité dotée d’une particularité structurale semble complètement adaptée aux besoins physiologiques de la baie de raisin lors de sa maturation. Pour finir, le dernier axe de recherche regroupe les deux sous-unités appelées VvK5.3 et VvK5.4 qui partagent 95% d’identité au niveau peptidique mais curieusement diffèrent par la présence d’une mutation au niveau du motif de sélectivité au K+.Grapevine (Vitis vinifera L.) is a fruit species with high economic impact largely cultivated for wine production. This species is also considered as a model plant for studying the physiology of acidic fruit development. In grapevine, as for all plants, K+ plays a crucial role in many physiological mechanisms. During berry ripening, which is initiated by a key step called veraison, the K+ is strongly accumulated in berry cells. During this maturation phase, K+ also initiates and controls the massive fluxes of sugar and nutrients loaded in berry. However, an excessive accumulation of K+ in the fruit causes a decrease of acidity of the must resulting to the production of low quality wines with a poor potential of aging. In this context, understanding K+ fluxes occurring during the maturation of berries is crucial. My work focused on the characterization of K+ channels belonging to the Shaker family that is known to dominate the K+ conductance in plant cell plasma membrane. This family with nine sub-units divided into five subfamilies has been largely studied in the A. thaliana model plant. Similarly, 9 shaker sub-units have also been identified in grapevine genome but, surprisingly, the numbers of members within each group are not strictly conserved between the two species. Indeed, the sub-family of outward sub-units contains 2 members in A. thaliana while 4 members have been identified in vine. During my thesis, I studied the 4 VvK5s outward subunits from grapevine to understand their involvement in the transport of K+ during the ripening phase of the berry. In this context, my thesis is divided into 3 axes. The first axis is a characterization of VvK5.1, a typical outward rectifying K+ Shaker channel with an extended territory of expression strongly suggesting his involvement in new functions for outward subunits. The second axis is the characterization of VvK5.2, a subunit with structural and functional features completely adapted to the specific physiological needs for a complete nutrient loading of grape berry. Finally, the last research axis focus on the two subunits VvK5.3 and VvK5.4 that share 95% of identity at the peptidic level but curiously differ by a mutation that changes one aminoacid in the K+ selectivity motif

Recherche de récepteurs de l’immunité des plantes et étude de l’impact de biostimulants sur leur expression et la résistance induite chez des organes sensibles

National audienceLa stimulation des défenses immunitaires des plantes représente une stratégie durable deprotection des cultures qui pourrait permettre de réduire l’utilisation de pesticides chimiquesencore trop répandue en viticulture. Son principe réside en l’application, au contact des cellulesvégétales, de molécules élicitrices détectées comme des signaux de danger par la plante. Cessignaux de dangers peuvent être de différente nature et origine comme par exemple la chitine,un chito-oligosaccharide (COS) retrouvé dans les parois fongiques. Leur détection par desrécepteurs de l’immunité entraine une cascade de signalisation complexe, conduisant àl’activation de réactions de défense et à terme à une protection accrue face aux pathogènes.Malgré des résultats encourageants obtenus avec des éliciteurs oligosaccharidiques enconditions contrôlées, l’efficacité de cette méthode de protection reste encore insuffisante surle terrain. Dans ce cadre, notre premier objectif consistera à caractériser les récepteursparticipant à la perception de ces éliciteurs COS. Pour cela, une double approche decomplémentation de mutants d’Arabidopsis et d’édition génomique CRISPR-Cas9 sur vignepermettra d’évaluer l’impact d’une perte/gain de fonction. Pour tenter de comprendre ladifférence d’efficacité observée entre les conditions naturelles et expérimentales, un secondobjectif sera d’analyser l’expression de ces récepteurs en fonction des organes et des stades dedéveloppement de la plante. Enfin, nous vérifierons si l’utilisation de produits de biostimulationpour améliorer l’état physiologique de la plante peut moduler l’expression des récepteurs enquestion et ainsi permettre d’améliorer la résistance induite en réponse à ce type d’éliciteurs

Grapevine Potassium Nutrition and Fruit Quality in the Context of Climate Change

International audiencePotassium (K(+)) nutrition is of relevant interest for winegrowers because it influences grapevine growth, berry composition, as well as must and wine quality. Indeed, wine quality strongly depends on berry composition at harvest. However, K(+) content of grape berries increased steadily over the last decades, in part due to climate change. Currently, the properties and qualities of many fruits are also impacted by environment. In grapevine, this disturbs berry properties resulting in unbalanced wines with poor organoleptic quality and low acidity. This requires a better understanding of the molecular basis of K(+) accumulation and its control along grape berry development. This mini-review summarizes our current knowledge on K(+) nutrition in relation with fruit quality in the context of a changing environment

Unique features of the grapevine VvK5.1 channel support novel functions for outward K+ channels in plants.

Grapevine (Vitisvinifera L.), one of the most important fruit crops, is a model plant for studying the physiology of fleshy fruits. Here, we report on the characterization of a new grapevine Shaker-type K+ channel, VvK5.1. Phylogenetic analysis revealed that VvK5.1 belongs to the SKOR-like subfamily. Our functional characterization of VvK5.1 in Xenopus oocytes confirms that it is an outwardly rectifying K+ channel that displays strict K+ selectivity. Gene expression level analyses by RT-qPCR showed that the VvK5.1 expression was detected in berries, roots, and flowers. In contrast to its Arabidopsis thaliana counterpart that is involved in K+ secretion in the root pericycle, allowing root-to-shoot K+ translocation, VvK5.1 expression territory is strongly enlarged. We showed by in situ hybridization that VvK5.1 is expressed in the phloem and perivascular cells of berries and in flower pistil. In the root, in addition to be expressed in the root pericycle like AtSKOR, a strong expression of VvK5.1 is detected in small cells facing the xylem that are involved in lateral root formation. This fine and selective expression pattern of VvK5.1 at early stage of lateral root primordia supports a suggested role for outward channel as switch on cell division initiation

Cell-specific expression and roles of the grapevine outwardly rectifying K+ channel VvK5.1

International audienc

Study of the stress-related signalling of endoplasmic reticulum in grapevine immunity associated to LysM receptor kinases (VvLYKs)

National audienceEnhancing our understanding of plant immunity is a critical task to develop more sustainableplant health protection methods. An interesting strategy is to study how plants, and in particulargrapevine, perceives and responds to different microorganisms. Microorganisms are notably recognized by 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 unfoldedproteins accumulate in the endoplasmic reticulum, is also involved during the plant immune response. It’s 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 downregulation of the plant immunity during the establishment of mycorrhizal symbiosis. In addition, we also intend to characterize the UPR signalling pathways in grapevine which is still unknown and finally how different biotic factors are involved in the stress-related signalling of endoplasmic reticulum

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.It’s 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

The grapevine LysM receptor-like kinase VvLYK5-1 mediates chitin-triggered immunity

International audienceThe establishment of defense reactions to protect plants against invading pathogens first requiresthe recognition of Microbe-Associated Molecular Patterns (MAMPs), detected by plasmamembrane-bound Pattern Recognition Receptors (PRRs). These MAMPs, also termed elicitors, areused in several biocontrol products that are gradually developing to reduce the use of chemicalpesticides in agriculture. Chitin, the main component of fungal cell walls, as well as its deacetylatedderivative, chitosan, are two chitooligosaccharides (COS) that can be found in some of theseproducts. Unfortunately, the mechanism allowing the perception of these molecules is still poorlyunderstood in Vitis vinifera, sometimes hampering the improvement and the generalization ofthese emerging crop protection tools. On the contrary, chitin perception in the model plantArabidopsis thaliana is well described and relies on a tripartite complex formed by threemembrane-bound LysM Receptor-Like Kinases named AtLYK1/CERK1, AtLYK4 & AtLYK5, thelatter having the strongest affinity to chitin. In grapevine, COS perception has for the moment onlybeen demonstrated to rely on VvLYK1-1 & VvLYK1-2, two constitutively expressed genes that didnot specifically respond to pathogens according to genes expression analysis. In this context, weperformed experiments to complement the Arabidopsis lyk5 simple mutant and the lyk4/5 doublemutant, which showed altered response to COS, by overexpressing their grapevine orthologousgenes (VvLYK5-1/2). Our results revealed that MAPK activation and defense gene expression werepartially restored after chitin treatment by the complementation with VvLYK5-1 but not withVvLYK5-2. These preliminary results seem to indicate that VvLYK5-1 participates in the chitinrecognition in grapevine, together with VvLYK1-1 previously identified. Furthermore, as theperception of chitosan does not seem altered in the Atlyk5 mutant, it would also appear that theperception of chitin and chitosan does not involve the same receptors. FRET-FLIM experimentsinitiated with these co-receptors will soon provide additional information to better decipher themechanism of COS perception in grapevin

The grapevine LysM receptor-like kinase VvLYK5-1 mediates chitin-triggered immunity

International audienceThe establishment of defense reactions to protect plants against invading pathogens first requiresthe recognition of Microbe-Associated Molecular Patterns (MAMPs), detected by plasmamembrane-bound Pattern Recognition Receptors (PRRs). These MAMPs, also termed elicitors, areused in several biocontrol products that are gradually developing to reduce the use of chemicalpesticides in agriculture. Chitin, the main component of fungal cell walls, as well as its deacetylatedderivative, chitosan, are two chitooligosaccharides (COS) that can be found in some of theseproducts. Unfortunately, the mechanism allowing the perception of these molecules is still poorlyunderstood in Vitis vinifera, sometimes hampering the improvement and the generalization ofthese emerging crop protection tools. On the contrary, chitin perception in the model plantArabidopsis thaliana is well described and relies on a tripartite complex formed by threemembrane-bound LysM Receptor-Like Kinases named AtLYK1/CERK1, AtLYK4 & AtLYK5, thelatter having the strongest affinity to chitin. In grapevine, COS perception has for the moment onlybeen demonstrated to rely on VvLYK1-1 & VvLYK1-2, two constitutively expressed genes that didnot specifically respond to pathogens according to genes expression analysis. In this context, weperformed experiments to complement the Arabidopsis lyk5 simple mutant and the lyk4/5 doublemutant, which showed altered response to COS, by overexpressing their grapevine orthologousgenes (VvLYK5-1/2). Our results revealed that MAPK activation and defense gene expression werepartially restored after chitin treatment by the complementation with VvLYK5-1 but not withVvLYK5-2. These preliminary results seem to indicate that VvLYK5-1 participates in the chitinrecognition in grapevine, together with VvLYK1-1 previously identified. Furthermore, as theperception of chitosan does not seem altered in the Atlyk5 mutant, it would also appear that theperception of chitin and chitosan does not involve the same receptors. FRET-FLIM experimentsinitiated with these co-receptors will soon provide additional information to better decipher themechanism of COS perception in grapevin

DataSheet_1_Increasing vineyard sustainability: innovating a targeted chitosan-derived biocontrol solution to induce grapevine resistance against downy and powdery mildews.pdf

The European Green Deal aims to reduce the pesticide use, notably by developing biocontrol products to protect crops from diseases. Indeed, the use of significant amounts of chemicals negatively impact the environment such as soil microbial biodiversity or groundwater quality, and human health. Grapevine (Vitis vinifera) was selected as one of the first targeted crop due to its economic importance and its dependence on fungicides to control the main damaging diseases worldwide: grey mold, downy and powdery mildews. Chitosan, a biopolymer extracted from crustacean exoskeletons, has been used as a biocontrol agent in many plant species, including grapevine, against a variety of cryptogamic diseases such as downy mildew (Plasmopara viticola), powdery mildew (Erysiphe necator) and grey mold (Botrytis cinerea). However, the precise molecular mechanisms underlying its mode of action remain unclear: is it a direct biopesticide effect or an indirect elicitation activity, or both? In this study, we investigated six chitosans with diverse degrees of polymerization (DP) ranging from low to high DP (12, 25, 33, 44, 100, and 470). We scrutinized their biological activities by evaluating both their antifungal properties and their abilities to induce grapevine immune responses. To investigate their elicitor activity, we analyzed their ability to induce MAPKs phosphorylation, the activation of defense genes and metabolite changes in grapevine. Our results indicate that the chitosans with a low DP are more effective in inducing grapevine defenses and possess the strongest biopesticide effect against B. cinerea and P. viticola. We identified chitosan with DP12 as the most efficient resistance inducer. Then, chitosan DP12 has been tested against downy and powdery mildews in the vineyard trials performed during the last three years. Results obtained indicated that a chitosan-based biocontrol product could be sufficiently efficient when the amount of pathogen inoculum is quite low and could be combined with only two fungicide treatments during whole season programs to obtain a good protection efficiency. On the whole, a chitosan-based biocontrol product could become an interesting alternative to meet the chemicals reduction targeted in sustainable viticulture.</p