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Cyclic lipopeptides produced by beneficial bacilli are promising candidates to develop biocontrol strategies in agriculture and reduce the use of conventional chemical pesticides. These biological molecules have shown ability to reduce the disease infection in several pathosystems through two main mechanisms: a direct antagonism against plant pathogens and/or the stimulation of plant immunity. Their activities rely on their amphiphilic properties, enabling them to readily insert into plasma membrane lipids. While the molecular mechanism for antagonistic activities are quite-well understood, involving the alteration of cell plasma membrane integrity and pore formation, little is known about the molecular basis underlying the stimulation of plant immunity through the interaction of lipopeptides with plasma membrane lipids. The present thesis work was therefore dedicated to unraveling how plasma membrane lipids contribute to the detection of lipopeptides by plant cells and subsequently activate plant immunity. The first part of the research used surfactin as a model to study plant immune stimulation by Bacillus lipopeptides, given its well-documented ability to induce systemic resistance in plants. A combination of functional assays in planta and biophysical experiments led to a molecular description of a unique lipid-mediated sensing mechanism of surfactin by plant cells. Compared to the conventional description of plant immunity relying on protein receptors, the surfactin sensing process is based on its interaction with plasma membrane sphingolipids. This interaction induces a disturbance of plasma membrane lipid environment detected via mechanosensitive channels that subsequently trigger a specific immune signature with a low transcriptional change, ultimately leading to the establishment of a systemic resistance in plants. In the second part, fengycin, another lipopeptides produced by beneficial bacilli with potential as biocontrol agent, was compared to surfactin in terms of antifungal and plant eliciting activities, and effects on membrane lipid properties. The two lipopeptides exhibited differences in their biological activities, which were correlated with their distinct effects on membrane lipids. While surfactin primarily influences the packing of membrane lipids, fengycin interacts with membrane lipid through a solubilizing/permeabilizing mechanism, with little effect on the packing of membrane lipids. This led to the actcivation of a plant immune response that is important for surfactin and less pronounced for fengycin. In contrast, fengycin displays direct antifungal properties not observed with surfactin. This section therefore illustrated the pivotal role of the mechanism of interaction with membrane lipids in determining the biological activities of both lipopeptides and, more broadly, amphipathic molecules. The final part of the research focused on the role of the plant cell wall in the mechanosensing of surfactin. This structure is an inherent component of plant cell mechanics and possibly affects plant mechanosensing. By comparing responses to surfactin in root protoplasts fully devoid of cell wall with those of protoplasts with a partially recovered cell wall, the experiments revealed a mitigating impact of the cell wall on cell responses to surfactin. To further investigate the impact of the cell wall on the mechanical disturbance capacity of surfactin, efforts were made to develop a microfluidic device for studying the plant cell deformability. Although the optimal device was not achieved during the thesis, the foundations have beens laid for future research in this area. Altogether, the results of this thesis shed light on a yet underestimated role of plasma membrane lipids in plant immunity. It also opens many research outlooks, aiming to better understand the contribution of the different plant lipid species in the mechanosensing mechanism and to identify markers indicating a state of induced resistance in plants. Ultimately, the knowledge gained from this thesis regarding the lipid-mediated biological activities of amphipathic molecules will contribute to their potential use as novel biocontrol agents for more sustainable strategies in agriculture.

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The plant plasma membrane is a very complex structure, mainly composed of lipids and proteins, that determines cell boundaries and controls the entry and exit of molecules. In plants, the plasma membrane is involved in the perception of elicitors, such as surfactin. This molecule is able to induce the plant defense response which makes it a potential alternative to conventional pesticides.
The molecular mechanism behind this perception process is little-known. To investigate this process, biomimetic models, including lipid monolayers, supported bilayers and liposomes, and biological models, including protoplasts are required. A protoplast is a cell which is delimited by the biological plasma membrane but that does no longer have its cell wall, it is a reliable model because it has a composition similar to the biological membrane.
This master's thesis aimed to develop and optimize the isolation of tomato root protoplasts and to optimize isothermal titration calorimetry (ITC) parameters for measuring the thermodynamics of binding events with protoplasts. After these optimization steps, interaction of protoplasts with surfactin was thermodynamically characterized with ITC and their reactivity in terms of defense response in presence of surfactin was analyzed by reactive oxygen species measurement.
First, to optimize the isolation of tomato root protoplasts, the effect of four factors, including time and speed of centrifugation, the age of roots, the time of incubation with enzymes and the agitation speed during the incubation with enzymes, on protoplast production yield and percentage of viability was investigated. It was shown that all parameters had an influence on protoplast production yield and the protoplast suspension with the highest yield was obtained from 7-day-old roots that were incubated for 17 hours without agitation and that were puri ed with a centrifugation at 600 rcf for 6 minutes. It was also shown that flow cytometry on tomato root protoplasts is complicated due to the high number of debris in protoplast suspension. Its use would require an improvement of the puri fication steps of the protoplast suspension.
Secondly, it was determined that the best con gfiuration for the ITC measurements is to put the protoplasts in the measuring cell with a low agitation. In this case there was no decrease of the protoplast concentration and of their percentage of viability during a measurement. In presence of surfactin, a binding event was observed. However, optimization of the concentration of protoplasts is still required in order to obtain an optimal pro le of heat flow for the determination of the thermodynamic parameters.
Production of reactive oxygen species (ROS) was also observed in presence of surfactin con firming
the good reactivity of protoplasts in terms of defense response.
In conclusion, this thesis paves the way to produce and use root protoplasts in biophysical experiments
such as ITC to better understand the perception of elicitors in plants. La membrane plasmique végétale est une structure très complexe, principalement composée de lipides et de protéines, qui détermine les limites de la cellule et qui contrôle l’entrée et la sortie de molécules. Chez les plantes, la membrane plasmique est impliquée dans la perception d’éliciteurs, tel que la surfactine, qui est capable d’induire la réponse de défense dans la plante et qui en fait une alternative potentielle aux pesticides conventionnels. Le mécanisme moléculaire à l’origine de ce processus de perception est peu connu. Pour étudier ce processus, des modèles biomimétiques, tels que les monocouches lipidiques, les bicouches lipidiques supportées et les liposomes, et des modèles biologiques, tel que les protoplastes, sont nécessaires. Un protoplaste est une cellule qui est délimitée par la membrane plasmique biologique mais qui ne dispose plus de sa paroi cellulaire. C’est un modèle fiable parce qu’il a une composition similaire à celle de la membrane biologique.
Cette thèse a pour but de développer et d’optimiser l’isolation des protoplastes de racines de tomates ainsi que d’optimiser les paramètres de titrage calorimétrique isotherme (ITC) pour mesurer la thermodynamique des événements dûs à une interaction avec les protoplastes. Après ces étapes d’optimisation, l’interaction des protoplastes avec la surfactine a été caractérisée thermodynamiquement avec l’ITC et leur réactivité en termes de réponse de défense en présence de surfactine a été analysée par la mesure des espèces réactives de l’oxygène.
Premièrement, pour optimiser l’isolation des protoplastes de racines de tomates, l’effet de quatre facteurs, dont le temps et la vitesse de centrifugation, l’âge des racines, le temps d’incubation avec les enzymes et la vitesse d’agitation pendant l’incubation avec les enzymes, sur le rendement en protoplastes et le pourcentage de viabilité a été´etudié. Il a été démontré que tous les paramètres avaient une influence sur le rendement en protoplastes et la suspension de protoplastes ayant le rendement le plus élevé a été obtenue à partir de racines de 7 jours qui ont été incubées pendant 17 heures sans agitation et qui ont été purifiées par une centrifugation à 600 rcf pendant 6 minutes. L’utilisation de la cytométrie de flux s’est révélée difficile suite à la présence d’un nombre importants de débris racinaires dans la suspension de protoplastes. Pour pouvoir employer la cytométrie, une amélioration des étapes de purification de la suspension de protoplastes est requise.
Ensuite, il a été déterminé que la meilleure configuration pour les mesures à l’aide de l’ITC est de placer les protoplastes dans la cellule de mesure avec une faible agitation. Dans ce cas, il n’y a eu aucune diminution de la concentration en protoplastes et de leur pourcentage de viabilité au
cours de la mesure. En présence de surfactine, un événement dû à une interaction a été observé.
Cependant, l’optimisation de la concentration en protoplastes est toujours nécessaire afin d’obtenir un profil optimal du flux thermique pour la détermination des paramètres thermodynamiques.
La production d’espèces réactives de l’oxygène (ROS) a également été observée en présence de surfactine, ce qui a permis de confirmer que les protoplastes ont une bonne réactivité en termes de
réponse de défense.
En conclusion, ce mémoire pose les bases pour l’obtention et l’utilisation de protoplastes de racines dans des expériences biophysiques tel que l’ITC, afin de mieux comprendre la perception des éliciteurs chez les plantes.

Root protoplasts --- Optimization --- Isothermal Titration Calorimetry --- Surfactin --- Reactive Oxygen Species --- Protoplaste racinaire --- Optimisation --- Titrage Calorimétrique Isotherme --- Surfactine --- Espèces Réactives de l'Oxygène --- Physique, chimie, mathématiques & sciences de la terre > Chimie --- Sciences du vivant > Biochimie, biophysique & biologie moléculaire --- Sciences du vivant > Biologie végétale (sciences végétales, sylviculture, mycologie...)