Thèse le Profil Protéique et Arn du Bactéroïde Fixateur d'Azote chez les Plantes de Medicago H/F - Doctorat.Gouv.Fr

Établissement : Université Paris-Saclay GS Biosphera - Biologie, Société, Ecologie & Environnement, Ressources, Agriculture & Alimentation École doctorale : Sciences du Végétal : du gène à l'écosystème Laboratoire de recherche : I2BC - Institut de Biologie Intégrative de la Cellule Direction de la thèse : Emanuele BIONDI ORCID 0000000195338191 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-06T23:59:59 Au cours de la symbiose entre la bactérie Sinorhizobium meliloti et la légumineuse Medicago sativa, les cellules bactériennes se différencient en bactéroïdes polyploïdes allongés au niveau intracellulaire à l'intérieur des nodules racinaires. Ces cellules reprogramment entièrement leur composition protéique et leur métabolisme afin de fixer l'azote atmosphérique. Dans le cadre de ce projet de doctorat, le candidat explorera le protéome des bactéroïdes et leur relation avec les peptides NCR capables de pénétrer à l'intérieur de la cellule bactérienne. Parallèlement, le transcriptome des bactéroïdes et la localisation de plusieurs gènes hautement exprimés seront étudiés. L'objectif est d'obtenir la carte la plus détaillée possible des fonctions exprimées dans les bactéroïdes et, si possible, de mettre en évidence leur lien avec les peptides NCR. In the rhizobium-legume symbiosis, the symbiotic bacteria are housed inside root nodules where they reduce nitrogen to ammonia for transfer to the plant in exchange of an exclusive niche and abundant nutrient sources. The efficiency of the legume symbiosis, which can entirely satisfy the plant's nitrogen needs, relies largely on the close and extensive contact between the bacterial symbiont and the host. This intimacy between the symbionts is attained in specific cells of the nodule, called symbiotic cells. Mature nodules contain thousands of these plant symbiotic cells, which are each infected with thousands of intracellular rhizobia, the bacteroids, confined in organelle-like vesicle, called symbiosome. The endosymbiotic bacteroids are adapted to intracellular life and are metabolically differentiated bacteria, expressing to extreme high levels the nitrogenase enzyme that catalyzes nitrogen reduction and other metabolic functions supporting nitrogen fixation (1).
In legumes belonging to the genus Medicago (including the forage crop M. sativa - i.e. alfalfa - and the model species M. truncatula), bacteroid differentiation of the symbiont Sinorhizobium meliloti involves also additional and irreversible, hence terminal, cellular modifications (2). They include cell enlargement (bacteroids are 5- to 10-fold longer and up to 2-fold broader than their free-living counterparts), genome amplification (polyploidy reaching over 30 In the rhizobium-legume symbiosis. These dramatic changes in the bacterial physiology are controlled by the activity of hundreds of Nodule Cystene-Rich (NCR) peptides which may have several different intracellular activities including perturbation of translation, protein stability, metabolism and cell cycle (3, 4, 5).
Indeed, the mechanism of terminal bacteroid differentiation to improved symbiosis remains largely to be discovered.
The objectives of this thesis project are:
- Characterize the proteome of free living/bacteroids/NCR-treated cells.
- Analyze the expression of key genes of symbiosis using RNA fish.
- Characterized the NCR targets in the bacterial cell
This project is organized in three tasks. Briefly the PhD candidate will first characterize the proteome of Sinorhizobium meliloti in collaboration with the Mass Spectrometry facility of I2BC, focusing on bacteria and (in bacteroids) plant proteins that may be introduced in the bacterial cell. Particular emphasis will be given to small proteins (NCR peptides from the plant) in bacteroids. The second task will focus on the analysis of several genes that are highly expressed in bacteroids (same are already known, for example nitrogenase subunits, and other will be discovered by the first task). The third task will focus on targets in the bacterial cell of NCRs identify in the bacteroid proteome in the first task and also in collaboration with Eva Kondorosi who has unpublished results of the interactome of tens of NCR peptides.
Here below a more detailed project plan:

1) Analysis of bacteroid/NCR-treated proteomes. Bacteroids will be extracted as previously described and proteins will be extracted and analyzed in different ways in order to characterized the whole proteome of soluble proteins and also the proteome enriched in small proteins, as previously described. Previous results have already characterized enzymes that are enriched in bacteroids (such as Nitrogenase complex and other enzymes related to known symbiotic processes). In parallel, free-living cells will be treated with known NCRs (published and unpublished in collaboration with Eva Kondorosi) in order to characterized the proteome variation upon treatments with sub-lethal doses of synthetic peptides. Further analysis will be performed crossing data from bacteroids and NCR-treated cells in order to find common bacteroid specific proteins. In the second part of the thesis, the construction of specific mutants coding for those proteins could be envisioned.
2) Analysis of highly expressed genes in bacteroids. Several preliminary data suggest that the bacteroid cell organizes its space by creating less dense regions of the cytoplasm for genes highly expressed in order to perform an exceptional rate of expression (the case for nif genes coding the enzyme Nitrogenase). First a deep RNAseq analysis will be performed at the sequencing facility of I2BC in order to full elucidate the gens that are expressed in bacteroids and possibly non-coding RNAs that may enter inside the bacterial cell. We will also monitor several candidates such as nif genes, as positive controls, and genes found with task 1 using RNA FISH as previously described (Mohapatra et al., 2020) using epifluorescence and Latex-SIM high resolution microscopy (I2BC imaging platform). The goal is to demonstrate that highly expressed genes are expressed in zones that are less dense. This analysis will be performed in wild type cells and mutants of DNA organizing proteins (Smc, MucR, HU etc) that are available in the host lab.
3) Analysis of the interaction between NCRs and bacteroid proteins. The hypothesis is that several of the most expressed and typical proteins in bacteroids may be targets of NCR peptides. This part of analysis will take advantage on preliminary results in the lab of Eva Kondorosi that has been characterizing the interactome of several, not-characterized so far, peptides. NCR found in task 1 inside bacteroids and abundant proteins will be crossed with interactomics results in order to find interesting pairs (NCR-protein) that will be analyzed affinity purification using tagged NCRs as targets for trapping target proteins in plants. Mutants of those NCR-targeted proteins will be constructed in order to explore their role during symbiosis, as previously described (6).

- Positionnement du projet dans le contexte national ; international, originalité, partenariats et collaborations, risques potentiels / Position of the project in the national ; international context, partnerships and collaborations, potential risks (1/2 page max.)

This thesis is a fundamental research project, which is driven by the curiosity to understand how the rhizobium-legume symbiosis functions in general and specifically here, to understand how the bacterial differentiation process is regulated by the plants. The project has nevertheless implications for important societal challenges in agronomy and health. Symbiotic nitrogen fixation by legumes has a tremendous ecological impact because it is the major natural nitrogen input in the biosphere. Moreover, symbiotic nitrogen fixation in crop plants represents a potential solution for the reduction of the use of synthetic nitrogen fertilizers in agriculture. The symbiotic interaction enables legume plants to grow in the absence of nitrogen fertilizers, as the symbiotic bacteria provide them with ammonium reduced from atmospheric nitrogen. The approach here proposed in order to understand the behavior of the bacterial symbiont genome and its modification is novel and innovative as these biological aspects has never been investigated before in detail and in a systematic way. Exploring the role of the genome modifications involved in bacteroid differentiation could lead to the improvement of nitrogen-fixing capacities of the microsymbiont. Moreover, bacteroid differentiation is not occurring in all legumes and especially in major crops (e.g. bean, soybean). Thus, the project may also provide the basic knowledge in order to develop new strategies to engineer bacteroid differentiation in such hosts.

Le candidat idéal aura de préférence une expérience dans le domaine de la microbiologie moléculaire. Des connaissances en biologie moléculaire sont également recommandées. Un atout supplémentaire serait une expérience dans l'étude de la différenciation symbiotique. Enfin, un intérêt pour la biologie végétale et une expérience de travail avec les plantes seront très appréciés.