Thèse Régulation de l'Architecture de la Paroi Cellulaire des Cordons d'Infection à Croissance en Pointe Inversée Lors de la Symbiose Légumineuses-Rhizobiums. H/F - Doctorat.Gouv.Fr

Établissement : Université de Toulouse École doctorale : SEVAB - Sciences Ecologiques, Vétérinaires, Agronomiques et Bioingenieries Laboratoire de recherche : LIPME - Laboratoire des Interactions Plantes-Microbes-Environnement Direction de la thèse : Fernanda DE CARVALHO-NIEBEL ORCID 0000000255969420 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-06-01T23:59:59 Les légumineuses, qui englobent des espèces cultivées à haute valeur nutritionnelle pour l'alimentation humaine et animale, peuvent établir des symbioses avec des bactéries rhizobiennes fixatrices d'azote, hébergées à l'intérieur des nodules racinaires spécialisés. Le transport des rhizobiums de la surface racinaire vers le nodule en développement est crucial dans ce processus, au cours duquel l'hôte végétale guide l'entrée sélective du partenaire bactérien via des compartiments spécialisés, appelés cordons d'infection. De découvertes pionnières ont mis en évidence la nécessité d'orchestrer la sécrétion ciblée de certaines protéines pariétales végétales vers la paroi du cordon d'infection, afin de soutenir la croissance polarisée des rhizobia à l'intérieur. Cependant, les mécanismes moléculaires sous-jacents à cette sécrétion polarisée et à l'organisation structurale de la paroi du cordon d'infection pour une croissance polarisée, demeurent peu connus. Ce projet de thèse vise à comprendre comment des protéines pariétales végétales façonnent le paysage physico-chimique de la paroi afin de faciliter la colonisation symbiotique de la plante hôte, en utilisant la symbiose Medicago truncatula-Sinorhizobium meliloti comme système modèle. En combinant des approches de génomique fonctionnelle, de biochimie et d'imagerie cellulaire in vivo, ce projet cherchera à élucider le rôle d'une classe spécifiques de glycoprotéines symbiotiques, ainsi que leur interaction fonctionnelle avec d'autres composants de la paroi pour façonner l'architecture pariétale favorisant l'infection par les rhizobiums. Ce projet devrait fournir des connaissances nouvelles et pertinentes sur la manière dont la paroi cellulaire, principale barrière de la plante face aux éléments extérieurs, peut être remodelée pour favoriser l'internalisation de partenaires bactériens compatibles. Legumes engage in symbiotic relationship with soil proteobacteria, collectively known as rhizobia, to gain the nitrogen they need for growth. Symbiotic engagement begins in the rhizosphere, when rhizobia secreted lipo-chitooligosaccharides are perceived by host membrane receptors at the root hair(1). This triggers a cascade of calcium-regulated signaling events, activating key symbiotic transcription factors that orchestrate downstream developmental programs to facilitate rhizobia infection and nodule organogenesis(2).
The transport of rhizobia from the root surface to the developing nodule is a crucial stage in this process, which in most legume species occurs via the creation of a specialized host derived compartment called the infection thread(3, 4). The infection thread is a unique transcellular, polarized, tip-growing structure. However, its sub-cellular structure is inside-out, with the host created apoplastic space and cell wall located inside, surrounded by the plasma membrane. Studying this cell specific process in root hairs has been challenging, until adapted tissue and cell specific transcriptomics and live cell imaging methods were developed in model legumes, such as Medicago truncatula (M. truncatula) (5). Notably, these studies revealed that the host dynamically modifies and organizes the infection thread cell wall to support polarized growth, via the target secretion of specialized cell wall proteins exclusively in the growing infection thread tip (3, 4, 6-9). These cell wall regulators likely act in concert to generate the suitable mechanical properties that sustain infection thread tip growth. However, the precise details of their mechanistic functions and the overall cell wall landscape that enables unique inverted tip growth remains unclear. This proposal aims to reveal how plant cell wall proteins shape the physicochemical properties of the cell wall to facilitate inverted tip growth, using the M. truncatula-Sinorhizobium meliloti legume-rhizobia model system.
Cell wall components at the infection thread cell wall space are only beginning to be identified. These include polysaccharide degrading enzymes (e.g. infection thread specific pectate lyase NPL1) and the cell wall bound PRP ENOD11, which belongs to the family of hydroxyproline-rich glycoproteins (HRGPs)(3, 6). Although ENOD11 was identified over two decades ago as a well-known early symbiotic marker gene strongly induced by compatible rhizobia, its precise molecular function remains enigmatic(3, 8). Studies in Arabidopsis inferred that HRGP proteins may influence the mechanical properties of the cell wall by forming interconnected supramolecular structures from interactions with surrounding polysaccharides(10). Thus, we hypothesize that ENOD11 creates structural networks by interacting with surrounding polysaccharides, shaping specific cell wall architectures along the growing region of the infection thread, which in turn influence cell wall porosity and the targeted localization of cell wall enzymes. Like ENOD11, the PRP subfamily shows diverse expression patterns and biological roles across plant species (11). However, their precise molecular function in shaping the cell wall remains unclear, as each protein's role depends on specific motifs and O-glycosylation patterns. Preliminary analyses of ENOD11 RNAi roots and a TILLING mutant show reduced root colonization and underdeveloped nodules with compatible rhizobia, suggesting infection defects. These findings indicate a role for ENOD11 in facilitating infection thread expansion, though its exact function remains unknown.
This project will investigate the role proline-rich protein (PRP) ENOD11, an early symbiotic marker gene, in influencing the infection thread cell wall by using the well-established Medicago-rhizobia symbiotic system. By combining functional genomics, biochemistry, and live-cell imaging, this project aims to address three objectives: (1) What is the functional role of ENOD11 during infection thread development? (2) Which polysaccharide polymers interact with ENOD11, and how do they assemble in the cell wall? (3) Do alterations in matrix porosity influence the targeted localization of cell wall enzymes? This project combines live-cell imaging, functional genomics, and biochemical approaches to investigate the role of ENOD11 in symbiotic interaction. Using the well-established Medicago- Sinorhizobium meliloti (S. meliloti) model, the student will analyze ENOD11 function using available mutant lines together with S. meliloti strains expressing fluorescent or -galactosidase reporters. These will enable characterizing symbiotic defects in early infection thread development and eventual nodule formation by confocal microscopy and X-gal staining histochemical quantification methods, respectively. (Obj1). The student will also be trained to perform protein purification using affinity chromatography columns (His-tag or Strep-tag) with an FPLC system. The protein will then be further purified using size-exclusion chromatography and subjected to mass spectrometry analysis to identify potential O-glycosylation modifications. The student will also engage in exploratory experiments to identify interacting polysaccharides using the purified protein (Obj2). Finally, FRAP (Fluorescence Recovery After Photobleaching) will be used to characterize the porosity of the infection thread matrix and to determine how ENOD11 contributes to this property by comparing wildtype and enod11 mutant backgrounds (Obj3).

Master 2 ou équivalent en biologie moléculaire/cellulaire, de préférence en biochimie des protéines