Controlled environment agriculture offers a solution to challenges including less available land, water deficits, and consumer demand for pesticide free produce. However, control of soil-borne diseases is a major limiting factor. The goal of this dissertation was to examine predictions of the hypothesis that border cells function to protect plant health by controlling microorganisms associated with plants grown in hydroponic culture. Border cells separate from root tips upon immersion in water, and appear to have important roles in the defense mechanisms of plant roots. The general objectives were (1) to study the delivery of border cells in hydroponics; (2) to evaluate interactions between border cells and microorganisms in hydroponics; and (3) to explore approaches to alter border cell production for improved root disease control. In this study it was confirmed that border cells can be released continuously into the solution of hydroponic culture suggesting that plants grown in this system may use extra energy in the production of new border cells. Free border cells interacted with microorganisms present in the hydroponic solution by secreting an extracellular capsule. Previous studies showed that proteins are a key component of this capsule, including lectins. The interaction of pea lectin and Nectria haematococca spores therefore was explored. Results demonstrated that pea lectin agglutinates fungal spores in a hapten-specific manner, and inhibits their germination. Lectin had no negative effect on root development suggesting that it could be used as a potential control for soil-borne diseases in hydroponics. To control the production of border cells, subsequent studies measured the impact of a transient exposure of root tips to different metabolites secreted by root caps and border cells. Exposure to specific metabolites altered the production of border cells without measurable effects on root growth and development. This is in contrast to results obtained with altered gene expression. For example, gene silencing of a border cell specific gene resulted in altered root growth.

Soilborne plant pathogens are responsible for many of the major crop diseases worldwide. However, plant root tips are generally resistant to pathogen infections. The goal of this dissertation research is to understand the mechanism of this natural resistance by testing the hypothesis that root caps and root border cells control the rhizosphere community through the biological products which they deliver to the soil. Specific objectives of this dissertation project are 1) identifying, isolating, and characterizing the genes important for border cell development and for root exudates delivery, and 2) analyzing the function of extracellular macromolecules in root exudates in root tip-fungal pathogen interaction. The expression of a primary cell wall synthesis gene, PsFut1, encoding Pisum sativum fucosyltransferase, was characterized during border cell production, and the impact of silencing this gene on border cell development was examined. Another gene, BRDgal1, encoding β-galactosidase, was identified and characterized in Pisum sativum during this study. It was shown that this β-galactosidase is specifically produced in and secreted from root border cells. The microarray transcriptional profiling in M. truncatula and mRNA differential display analysis in pea plants were carried out following the induction of border cell production to gain a broader understanding of the genes which potentially influence border cell development. In order to study the commonality of border cell production across different plant species, the expression of rcpme1, the marker gene for border cell production, was compared between the garden pea and a gymnosperm species, the Norway spruce (Picea abies). To accomplish the second objective, the focus of this study was shifted from border cell development to mucilaginous root exudates excreted by border cells and root cap cells. This resulted in a breakthrough in the understanding of the mechanisms of root tip resistance. The presence of extracellular DNA in the root mucilage was discovered and its requirement for root tip resistance to fungal infection was demonstrated. Extracellular proteins in the root mucilage were identified and they were shown to be also required for the root tip resistance to fungal infection. This work provided new insights into understanding plant defense mechanisms.

No description available.

One of the motivating reasons for the development of hydroponics was avoidance of root pathogens. Hydroponics involves growing crops in relatively sterile media, isolated from the underlying soil which may have disease pressure. However, even when hydroponics is coupled with controlled environments such as high tunnels and climate-controlled greenhouses, soil-borne pathogens can enter the growing area and proliferate due to optimal environmental conditions for pathogen growth. Control of root pathogens is difficult and usually achieved through synthetic fungicides since few biocontrol options are available. Compost water extracts (CWE) have recently been gaining the attention of greenhouse growers because they may be a low-cost, environmentally friendly approach to control root disease. CWE are mixtures of compost and water incubated for a defined period of time, either with or without aeration, and with or without additives intended to increase microbial populations, which in turn suppress disease. Much anecdotal, but very little scientific, evidence exists describing CWE effect on suppressing soil-borne pathogens. The present study 1) examined the effect of an aerated CWE on disease suppression at the laboratory scale and in container studies using different soilless substrates, 2) investigated a phenotypic change at the root level caused by CWE that may be associated with disease suppression, and 3) isolated some factors in the production of CWE that affect the ability of a CWE to suppress disease. The common model pathogen-host system of Fusarium solani f.sp. pisi and pea was used to examine CWE-induced disease suppression, with information then being translatable to similar patho-systems involved in greenhouse crop production. In the first study, laboratory-based root growth and infection assays resulted in 100% suppression of F. solani when roots were drenched in CWE. These protected seedlings were then taken to a greenhouse and transplanted into fine coconut coir, watered with hydroponic nutrient solution, and grown for five weeks. At the end of the experiment, 23% of the shoots of the pathogen-inoculated, CWE-drenched seedlings remained healthy while only 2% of the inoculated seedlings without CWE drench remained healthy. All of the roots of the inoculated seedlings developed lesions, even those drenched in CWE. However, 29% of the CWE drenched roots were able to recover from disease, growing white healthy roots past the lesion, while only 2% recovered naturally. A shorter-term container study was conducted in the laboratory to determine the effects of CWE-induced suppression when peas were grown in different substrates and to determine if the hydroponic nutrient solution had an effect on the suppression. Peas were grown in sterilized fine and coarse coconut coir fiber and sand irrigated with water, with a second set of fine coir irrigated with hydroponic nutrient solution. Pea seeds with 20-25mm radicles were inoculated with pathogen and sown directly into CWE-drenched substrate and grown for three weeks. At the end of the experiment, 80%, 60%, 90%, and 50% of the shoots of the inoculated, CWE-drenched seedlings remained healthy when grown in fine coir, coarse coir, sand, and fine coir irrigated with hydroponic nutrient solution, respectively. Nearly 100% of the roots grown in coconut coir substrates again developed necrotic lesions but 83%, 87%, 100%, and 87% grew healthy roots beyond the disease region. The hydroponic nutrient solution had a negative effect on suppression, with a reduction of at least 30 percentage points. Sand demonstrated a natural ability to suppress F. solani. Only 23% of inoculated seedlings had dead or dying shoots by the end of the experiment (compared to 77-80% in coir substrates) and although all but one of the roots developed lesions, all were able to recover on their own with CWE. CWE further increased shoot health and also prevented 57% of the roots from developing lesions. In a second study, two different CWE were used to examine the effect on root border cell dispersion and dynamics in pea, maize, cotton, and cucumber and its relation to disease suppression. Dispersal of border cells after immersion of roots into water or CWE was measured by direct observation over time using a compound microscope and stereoscope. Pictures were taken and the number of border cells released into suspension were enumerated by counting the total number of cells in aliquots taken from the suspension. Border cells formed a mass surrounding root tips within seconds after exposure to water, and most cells dispersed into suspension spontaneously. In CWE, >90% of the border cell population instead remained appressed to the root surface, even after vigorous agitation. This altered border cell phenomena was consistent for pea, maize, and cotton and for both CWE tested. For most cucumber roots (n=86/95), inhibition of border cell dispersal in both CWE was similar to that observed in pea, maize, and cotton. However, some individual cucumber roots (8±5%) exhibited a distinct phenotype. For example, border cells of one root immersed into CWE remained tightly adhered to the root tip even after 30 minutes while border cells of another root immersed at the same time in the same sample of CWE expanded significantly within 5 minutes and continued to expand over time. In a previous study, sheath development over time in growth pouches also was distinct in cucumber compared with pea, with detachment of the sheaths over time, and root infection was reduced by only 38% in cucumber compared with 100% protection in pea (Curlango-Rivera et al. 2013). Further research is needed to evaluate whether this difference in retention of border cell sheaths plays a role in the observed difference in inhibition of root infection. In the third study, a series of investigations were conducted to isolate different factors that contribute to the suppression ability of a CWE by changing incrementally changing some aspect of the CWE production process. The basic aerated CWE recipe (with molasses, kelp, humic acid, rock phosphate, and silica) provided 100% protection of pea from root disease while the non-aerated basic recipe CWE provided 72% protection. Aerated CWE made of only compost and water resulted in 58% protection. It was found that molasses did not contribute to the suppression ability of the ACWE, while kelp contributed strongly. When soluble kelp was added by itself to the compost and water, the CWE provided 80% suppression. However, when all additives were included except molasses and kelp, suppression remained high (93%) indicating that humic acids, rock phosphate, and/or silica were also major contributors toward the suppression effect. Optimal fermentation time for ACWE was 24 hr to achieve 100% suppression, with increased time resulting in inconsistent suppression results. Optimal fermentation time for NCWE was 3 days or 8 days. These studies are important contributions to understanding the differences that might be expected in CWE suppression when growing in different substrates, some of the factors in the production of CWE that affects the ability of a CWE to suppress disease, and the phenotypic effect CWE has on the root zone of plants and the possible relationship between that effect and disease suppression.

La migration cellulaire concerne des cellules individuelles ou bien des groupes de cellules migrant de manière collective et coordonnée. De nombreux processus physiologiques, notamment au cours du développement embryonnaire, ainsi que pathologiques, notamment lors de maladies inflammatoires ou de la formation de métastases nécessitent des mouvements cellulaire collectifs. Au cours de l'ovogénèse chez la Drosophile, un groupe de cellules, appelés cellules de bordure, migrent entre les cellules nourricières, collectivement, au sein du follicule ovarien. Ces cellules de bordure constituent un modèle de choix pour étudier les mécanismes régulant la migration collective in vivo. La migration de ce groupe de cellules est divisée en deux phases. Lors de la première moitié de migration, du début de la migration à la moitié du parcours, les cellules de bordure adoptent un mouvement linéaire, au cours duquel chaque cellule maintient sa position au sein de l'entité, et une seule et même cellule conduit le groupe vers l'avant. Ensuite, à mi-chemin, ces groupes commencent à effectuer des mouvements de rotation sur eux-mêmes pour aller atteindre l'ovocyte, permettant à n'importe quelle cellule de pouvoir mener la migration. L'objectif de ma thèse a été d'élucider les mécanismes régulant le choix entre ces deux modes de migration (linéaire et rotationnel). Le cytosquelette d'acto-myosine est un des acteurs principaux régulant la contraction cellulaire nécessaire à la motilité des cellules. Au cours de ma thèse, nous avons mis en évidence le rôle de la myosine non musculaire de type II dans le contrôle du passage d'un mouvement linéaire à rotationnel. Nos travaux démontrent que l'apparition des mouvements de rotation effectués par les cohortes de cellules de bordure est corrélée à une augmentation de l'activité de la myosine non musculaire de type II. De plus, nous avons montré que l'activité de la myosine non musculaire de type II pouvait être régulée de manière antagoniste par les récepteurs de guidance. En conclusion, mes travaux de thèse nous ont permis de démontré le rôle clé de la myosine non musculaire de type II dans l'adaptation du mode de migration au cours de mouvements collectifs des cellules de bordure. De plus nous avons identifié les facteurs régulant l'activité de la myosine non musculaire de type II. En effet, cette dernière est régulée positivement par EGFR. / In many biological processes, cells can move individually or in a coordinated and collective manner. Collective migrations are necessary during several embryo developmental processes, and pathologies such as inflammatory diseases or metastasis formation. During Drosophila oogenesis, border cells, a group of 6-10 cells, migrate in between nurse cell until the oocyte, within the egg chamber and provide a good model to study collective cell migration in vivo. Border cell migration is divided in to two phases. From the anterior pole of the egg chamber to the half of migrated distance, border cell adopt a linear movement, in which each cell maintain its position within the cluster and one leader cell drive the migration. Midway of the migration path, border cell clusters rotate to reach the oocyte. During this second phase, any cell can take the lead of the migration. The aim of my PhD research works was to identify mechanisms regulating the choice between linear and rotational movements. Acto-myosin cytoskeleton is one of the main regulators of cell contraction necessary for cell motility. Through our research, we demonstrated that non-muscle myosin II (NMII) regulate the switch between linear and rotational behaviour. These results led us to identify mechanisms regulating NMII activity during border cell migration. Border cells express two guidance receptors: PVR (Platelet-derived growth factor receptor (PDGFR) and Vascular endothelial growth factor receptor (VEGFR) receptor Related) and EGFR (Epidermal Growth Factor Receptor). Recent studies shown that PVR play a crucial role in the first phase and EGFR predominantly regulate the second phase of migration. Our data shows that NMII is antagonistically regulated by PVR and EGFR. Indeed, the inhibition of NMII in border cell over expressing EGFR completely blocks the rotational movement To conclude, my PhD works allow us to demonstrate the key role of NMII for the regulation of border cell migration. Moreover, we found that EGFR positively regulates NMII activity.

La migration cellulaire joue un rôle essentiel dans le développement des organismes multicellulaires et dans certaines pathologies comme le cancer, où elle permet la formation de métastases. Le trafic vésiculaire est un régulateur clé de la migration cellulaire, notamment en contrôlant la localisation de protéines impliquées dans la migration telles que les intégrines, les cadhérines et les récepteurs transmembranaires. En particulier, notre laboratoire a montré que l'endocytose contrôle l'orientation et la communication cellulaire durant la migration cellulaire collective. Notre hypothèse est que d'autres événements du trafic vésiculaire pourraient aussi être impliqués dans ce type de migration. Ainsi, le but de cette thèse a été de déterminer la fonction des petites GTPases Arf, importantes pour la formation de vésicules et le tri de cargo dans ces vésicules et de leurs régulateurs dans la migration cellulaire collective. Un modèle pour étudier la migration cellulaire collective est les chambres d’œufs de Drosophila melanogaster. En effet, lors de l’ovogénèse, des cellules folliculaires appelées cellules de bordure migrent à travers les cellules nourricières pour atteindre l’ovocyte. Conformément à notre hypothèse, un fort défaut de migration est observé lorsque les Arfs sont déplétées spécifiquement dans les cellules de bordure. De plus, un constat similaire est observé après la déplétion de certains régulateurs des Arfs (ArfGAPs et ArfGEFs). Notamment, nous avons démontré que l’ArfGAP Drongo et sa fonction d'activation de l’activité GTPase sont essentielles pour le détachement initial des cellules de bordure du tissu folliculaire. Drongo promeut le détachement en contrôlant la localisation de la myosine phosphatase afin de réguler l’activité de la myosine II à l’arrière des cellules. De plus, nous avons montré que Drongo agit sur l’Arf de classe III (Arf51F) de manière antagoniste à l’ArfGEF Steppke pour déplacer la myosine phosphatase de l’arrière du groupe de cellules. D’un autre côté, nous avons aussi démontré qu’une autre GAP, ArfGAP1, contrôle la directionnalité de migration. Cette ArfGAP agit potentiellement en régulant la localisation de certains déterminants de la migration tels que l’E-cadhérine et les récepteurs tyrosine kinase. Ainsi, nos recherches ont démontré un rôle essentiel des Arfs ainsi que des rôles spécifiques de deux ArfGAPs dans la migration cellulaire collective. / Cell migration is implicated in various important biological processes, notably it is central for the dissemination of cancer cells. Vesicular trafficking is a key regulator of cell migration, notably by controlling the localisation of proteins involved in migration such as integrins, cadherins and transmembrane receptors. In particular, our laboratory has shown that endocytosis controls orientation and cellular communication during collective cell migration. Our hypothesis is that other events of vesicular trafficking might be implicated in collective cell migration. Thus, the purpose of this thesis was to assess the function of small GTPases Arf, important for vesicle formation and cargo sorting into those vesicles, and their regulators in collective cell migration. A powerful model to study collective cell migration is the migration of follicular cells named border cells during oogenesis in Drosophila melanogaster. Border cells (BCs) detach from the follicle epithelium surrounding the egg chambers and form a small cluster of six to ten cells that migrates invasively between the giant nurse cells that compose the center of the egg chamber, toward the oocyte. Accordingly to our hypothesis, a strong migration defect is observed when the Arfs are depleted specifically in the border cells. Moreover, a similar finding is observed after depletion of some Arfs regulators (ArfGAPs and ArfGEFs). In particular, the ArfGAP Drongo and its GTPase-activating function are essential for the initial detachment of the border cell cluster from the basal lamina. We demonstrated through protein localization and genetic interactions that Drongo controls the localisation of the myosin phosphatase in order to regulate myosin II activity at the back of the cluster and promote border cells detachment. Moreover, we showed that Drongo acts on the class III Arf (Arf51F) antagonistically to the guanine exchange factor Steppke to displace myosin phosphatase from the back of the cluster. On the other hand, we have also demonstrated that the GAP ArfGAP1 controls the directionality of migration. This ArfGAP potentially acts by regulating the localization of certain determinants of migration such as E-cadherin and receptors tyrosine kinase. Thus, our research has demonstrated an essential role for Arfs in collective cell migration and specific contributions of two ArfGAPs in this migration process.

Au cours de l’ovogenèse chez la mouche du vinaigre: Drosophila melanogaster, un groupe de cellules folliculaires appelées cellules de bord, migrent à travers les cellules nourricières pour atteindre l’ovocyte. Cet événement, nécessitant la transition épithélio- mésenchymateuse (TEM), la réorientation, puis l’arrêt, ressemble à la formation de métastases. L’endocytose est un régulateur clé de plusieurs événements polarisés, y compris la migration cellulaire. En effet, différentes protéines impliquées dans la migration, comme les intégrines et les E-cadhérines (cadhérines épithéliales), sont régulées par transport à travers les endosomes. De même, l’endocytose restreint au front de migration l’activité des récepteurs tyrosine kinases (RTKs) qui guident les cellules de bord dans leur mouvement. Cependant les mécanismes moléculaires de cette restriction spatiale de l’activité des RTKs demeurent largement inconnus. Nous avons testé l’implication du trafic vésiculaire à travers la machinerie d’endocytose, dans la migration dirigée des cellules de bord, car ce système est facilement accessible pour l’expression de protéines et l’analyse de mutants. Nous avons commencé par confirmer une observation précédente du rôle de l’endosome précoce dans la migration des cellules de bord. Ensuite, nous avons identifié l’endosome de recyclage (ER) comme un régulateur clé de cette migration. En effet, nous avons démontré que l’expression dans les cellules de bord d’une forme dominante négative de Rab11, la petite GTPase régulant le transport vésiculaire à travers l’ER, bloque la migration ou entraîne de sévères défauts de migration dans environ 80% des chambres d’œufs examinées. De plus, nous observons par immunofluorescence une relocalisation de l’activité des RTKs alors que d’autres protéines de migration ne sont pas affectées par Rab11 dominant négatif. Ce résultat a été par la suite confirmé par une interaction génétique entre Rab11 et les RTKs. D’autre part, nous avons montré que le complexe exocyste, un effecteur de Rab11, est impliqué dans la migration des cellules de bord. Nous avons trouvé par microscopie confocale en tissu fixé et par microscopie en temps réel que Sec15, un composant de ce complexe, est polarisé, de façon Rab11- dépendante, dans des vésicules qui s’accumulent au front de migration tout au long du mouvement des cellules de bord. De plus, la perte de l’activité de Sec15 perturbe à son tour la migration. Ainsi, toutes ces données démontrent le rôle fondamental d’un cycle d’endo- exocytose dans le maintien des RTKs actifs au niveau du front de migration des cellules de bord le long de leur mouvement. / During Drosophila melanogaster’s oogenesis, a cluster of folllicle cells, called border cells, perform an invasive migration through the surrounding nurse cells to reach the oocyte. This event resembles metastasis formation since it requires epithelial- mesenchymal transition, reorientation and arrest. Endocytosis plays a fundamental role in many polarized processes, including cell migration, since different migration proteins, like integrins and E-cadherins traffic through the endocytic pathway. Furthermore, receptor tyrosine kinases (RTKs) that guide border cells during their migration are regulated by endocytosis, although the mechanisms involved are largely unknown. We tested the implication of vesicular trafficking through the endocytic machinery, in border cells’ directed migration, because this system is easily accessible for protein expression and mutant analysis. We first confirmed previous observation that trafficking through the early endosome is necessary for border cells migration, and then we identified the recycling endosome as a key compartment for this migration. Indeed, we showed that overexpression in border cells of a dominant negative form of Rab11, the small GTPase regulating vesicular trafficking through the recycling endosome, blocks migration or leads to severe migration defects in about 80% of examined egg chambers. Furthermore, using immunofluorescence, we observed a relocalization of RTKs activity, whereas other migration proteins were not redistributed upon dominant negative Rab11 expression. This result was further confirmed by a genetic interaction between Rab11 and RTKs. Moreover, we showed that the exocyst complex, an effector of Rab11, is also involved in border cells migration. We found by using confocal microscopy of fixed tissues and time-lapse microscopy of living egg chambers, that Sec15, a member of this complex, is distributed in vesicles which are polarized, in a Rab11- dependent manner, throughout border cells migration. In addition, loss of Sec15 also impairs migration. Together these data demonstrate a fundamental role for an endo- exocytic cycle in the maintenance of active RTKs at the leading edge of border cells during their migration.

Au cours de l’ovogenèse chez la mouche du vinaigre: Drosophila melanogaster, un groupe de cellules folliculaires appelées cellules de bord, migrent à travers les cellules nourricières pour atteindre l’ovocyte. Cet événement, nécessitant la transition épithélio- mésenchymateuse (TEM), la réorientation, puis l’arrêt, ressemble à la formation de métastases. L’endocytose est un régulateur clé de plusieurs événements polarisés, y compris la migration cellulaire. En effet, différentes protéines impliquées dans la migration, comme les intégrines et les E-cadhérines (cadhérines épithéliales), sont régulées par transport à travers les endosomes. De même, l’endocytose restreint au front de migration l’activité des récepteurs tyrosine kinases (RTKs) qui guident les cellules de bord dans leur mouvement. Cependant les mécanismes moléculaires de cette restriction spatiale de l’activité des RTKs demeurent largement inconnus. Nous avons testé l’implication du trafic vésiculaire à travers la machinerie d’endocytose, dans la migration dirigée des cellules de bord, car ce système est facilement accessible pour l’expression de protéines et l’analyse de mutants. Nous avons commencé par confirmer une observation précédente du rôle de l’endosome précoce dans la migration des cellules de bord. Ensuite, nous avons identifié l’endosome de recyclage (ER) comme un régulateur clé de cette migration. En effet, nous avons démontré que l’expression dans les cellules de bord d’une forme dominante négative de Rab11, la petite GTPase régulant le transport vésiculaire à travers l’ER, bloque la migration ou entraîne de sévères défauts de migration dans environ 80% des chambres d’œufs examinées. De plus, nous observons par immunofluorescence une relocalisation de l’activité des RTKs alors que d’autres protéines de migration ne sont pas affectées par Rab11 dominant négatif. Ce résultat a été par la suite confirmé par une interaction génétique entre Rab11 et les RTKs. D’autre part, nous avons montré que le complexe exocyste, un effecteur de Rab11, est impliqué dans la migration des cellules de bord. Nous avons trouvé par microscopie confocale en tissu fixé et par microscopie en temps réel que Sec15, un composant de ce complexe, est polarisé, de façon Rab11- dépendante, dans des vésicules qui s’accumulent au front de migration tout au long du mouvement des cellules de bord. De plus, la perte de l’activité de Sec15 perturbe à son tour la migration. Ainsi, toutes ces données démontrent le rôle fondamental d’un cycle d’endo- exocytose dans le maintien des RTKs actifs au niveau du front de migration des cellules de bord le long de leur mouvement. / During Drosophila melanogaster’s oogenesis, a cluster of folllicle cells, called border cells, perform an invasive migration through the surrounding nurse cells to reach the oocyte. This event resembles metastasis formation since it requires epithelial- mesenchymal transition, reorientation and arrest. Endocytosis plays a fundamental role in many polarized processes, including cell migration, since different migration proteins, like integrins and E-cadherins traffic through the endocytic pathway. Furthermore, receptor tyrosine kinases (RTKs) that guide border cells during their migration are regulated by endocytosis, although the mechanisms involved are largely unknown. We tested the implication of vesicular trafficking through the endocytic machinery, in border cells’ directed migration, because this system is easily accessible for protein expression and mutant analysis. We first confirmed previous observation that trafficking through the early endosome is necessary for border cells migration, and then we identified the recycling endosome as a key compartment for this migration. Indeed, we showed that overexpression in border cells of a dominant negative form of Rab11, the small GTPase regulating vesicular trafficking through the recycling endosome, blocks migration or leads to severe migration defects in about 80% of examined egg chambers. Furthermore, using immunofluorescence, we observed a relocalization of RTKs activity, whereas other migration proteins were not redistributed upon dominant negative Rab11 expression. This result was further confirmed by a genetic interaction between Rab11 and RTKs. Moreover, we showed that the exocyst complex, an effector of Rab11, is also involved in border cells migration. We found by using confocal microscopy of fixed tissues and time-lapse microscopy of living egg chambers, that Sec15, a member of this complex, is distributed in vesicles which are polarized, in a Rab11- dependent manner, throughout border cells migration. In addition, loss of Sec15 also impairs migration. Together these data demonstrate a fundamental role for an endo- exocytic cycle in the maintenance of active RTKs at the leading edge of border cells during their migration.

La coiffe racinaire est cruciale à la croissance et survie du méristème subapical de racine. Elle libère des cellules frontières (CFs) qui assurent la protection de l’apex racinaire. Les CFs associées à leur mucilage forment le piège extracellaire de racine (RET). La caractérisation du RET et des exsudats racinaires de trois essences ligneuses sahéliennes à partir de plantules cultivées in vitro a été réalisée. B. aegyptiaca et A. raddiana prospèrent dans les zones semi-arides, à l’opposé de T. indica. La morphologie des CFs et l’organisation du RET ont été déterminées par microscopie. La compostion en glycopolymères et la détection des arabinogalactanes proteines (AGPs) dans le RET et les exsudats racinaires ont été déterminées par des analyses biochimiques. L’effet des exsudats racinaires sur la croissance d’Azospirillum brasilense, une bactérie bénéfique pour la plante a été évalué. B. aegyptiaca produit des CFs de type border cells (BCs) alors que les autres Fabaceae produisent des BCs et des border-like cells. Les BCs sont entourées d’un dense mucilage riche en polymères de paroi. Le RET et les exsudats racinaires issus de B. aegyptiaca et A. raddiana sont plus riches en AGPs que ceux provenant T. indica. Les AGPs pourraient contribuer à la survie des plantules dans un contexte semiaride. Ce travail ouvre de nouvelles perspectives de recherche concernant l'implication du RET dans la survie des plantes à l'aridité. / The root cap is primordial for seedling growth and supports root apical meristem integrity. The root cap releases root border cells (RBCs) that surround the root tip and ensure seedling protection against numerous stresses. RBCs and their associated mucilage form the root extracellular trap (RET). Here, RET and root exudate characterization of three Sahelian woody seedlings are performed. In contrast to B. aegyptiaca and A. raddiana which thrive in semi-arid areas, T. indica is more sensitive to drought. B. aegyptiaca, A. raddiana and T. indica seedlings were sub-cultured in vitro. RBC morphologies and RET organization were determined using microscopic approaches. The polysaccharide composition and arabinogalactan protein (AGP) content were determined by biochemical approaches in the RET and the root exudates. Moreover, the effect of root exudates on the growth of Azospirillum brasilense a plant benefical bacteria has been performed. While B. aegyptiaca produces only border cell (BC) type, the two Fabaceae seedlings release both BCs and border-like cells (BLCs). BCs are enclosed in a dense mucilage enriched in cell wall polymers. Compared to T. indica, RET and root exudates of B. aegyptiaca and A. raddiana include more abundant AGPs. In this context, AGPs could contribute to woody seedling survival. This work opens new research perspectives regarding involvement of RET in plant survival to aridity.

Chez les plantes, le RET (Root Extracellular Trap) est une structure cellulo-moléculaire jouant un rôle central dans la défense racinaire face aux stress abiotiques et biotiques. De nombreuses similitudes de composition ont été observées entre le RET et le NET (Neutrophil Extracellular Trap) du système immunitaire des mammifères, connu pour capturer et tuer certains microorganismes bactériens et fongiques. Le RET est composé de cellules frontières et de leurs sécrétions (composés de haut et de bas poids moléculaire) comprenant des polysaccharides de la paroi cellulaire, des protéoglycannes et des métabolites secondaires. Il contient également des protéines antimicrobiennes et de l'ADN extracellulaire, tout comme le NET. Dans le cadre de mon projet de thèse, nous avons caractérisé la composition moléculaire et la structuration de cette entité de défense chez deux légumineuses, le soja (Glycine max (Merr) L.) et le pois (Pisum sativum L.), par des approches d’imagerie cellulaire photonique et électronique. Nous avons également étudié l’impact du RET du soja sur des pathogènes telluriques, à savoir Phytophthora parasitica et Aphanomyces euteiches. Nous avons ainsi pu mettre en évidence la présence de différents morphotypes de cellules frontières et de mucilage au sein du RET de soja et de pois. Pour la première fois, nous avons montré la présence d’hétéromannanes, de xyloglucane et de cellulose dans le RET, formant une ossature stabilisant le mucilage et reliant les cellules frontières entre elles. Ces polysaccharides structuraux semblent être essentiels à l’intégrité structurale et fonctionnelle du RET. Enfin, nos résultats ont montré que le RET de soja était impliqué dans la défense précoce de la racine contre P. parasitica. Cette étude apporte de nouvelles connaissances relatives à la composition moléculaire et la structure du RET, nous amenant ainsi à comparer le RET à d’autres modèles que le NET des mammifères, tels que les biofilms bactériens et les mucilages de graines. En effet, de nombreuses similitudes existent entre ces différents complexes en termes de composition et de fonctionnement, qui méritent d’être explorer plus en détail dans l’avenir. / In higher plants, the RET (Root Extracellular Trap) is a complex made up of border cells and secretions, released by root tips and believed to play a central role in biotic and abiotic stress tolerance. This structure is quite similar to the Neutrophil Extracellular Trap (NET) known as one of the first lines of defense in mammals, able to trap and kill microbial pathogens. RET secretions consist of high and low-molecular weight compounds including cell wall polysaccharides, proteoglycans and secondary metabolites. They also contain a variety of anti-microbial proteins and extracellular DNA much like the NET. During my thesis work, we investigated the release and morphology of root border cells in soybean (Glycine max (Merr) L.) using light and scanning electron microscopy. The molecular composition of the mucilage was also investigated using immunocytochemistry, anti-cell wall glycan antibodies and confocal microscopy. Immunocytochemistry was also applied to pea (Pisum sativum L.) border cells and secretions to examine the occurrence of specific polysaccharides. We also studied the impact of soybean RET on the soilborne pathogens, Phytophthora parasitica and Aphanomyces euteiches. Our findings showed that root tips of soybean released three border cell morphotypes all of which secreted substantial amounts of mucilage. Immunocytochemical data showed that mucilage was enriched in pectin and the two hemicellulosic polysaccharides xyloglucan and heteromannan. Mucilage also contained cellulose, histone and extracellular DNA. Interestingly, the structural polysaccharides formed a fibrous network surrounding the cells and holding them together, supporting their role in maintaining mucilage architecture and integrity. In addition, we found that xyloglucan and cellulose were also secreted into the mucilage of pea, connecting border cells together. Finally, our findings revealed that RET prevented P. parasitica zoospores from colonizing soybean root tip, by stopping their penetration and inducing their death. Overall the study revealed novel insights into the composition, structure and function of plant RETs. Currently, the RET is much less studied than its mammal counterpart, the NET, but structural and functional similarities exist between these two traps. Interestingly, similarities do also exist between the RET and other important biological complexes, including bacterial biofilms and seed mucilage, that deserve to be further investigated and compared in the context of immunity.