Thèse Découverte de Nouveaux Peptides Actifs Impliqués dans le Développement des Racines et des Nodules chez les Légumineuses. 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 : IPS2 - Institut de Sciences des Plantes de Paris-Saclay Direction de la thèse : Marie-Laure MARTIN ORCID 0000000340009600 Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-06T23:59:59 Les légumineuses, telles que Medicago truncatula, établissent des relations symbiotiques avec des rhizobiums fixateurs d'azote, un processus nécessitant une signalisation complexe entre la plante et la bactérie. Les peptides jouent un rôle crucial dans cette interaction, et grâce à de nouvelles données single cell RNAseq (scRNAseq) récemment publiée, l'équipe d'accueil a identifié de nouveaux peptides qui restent entièrement non caractérisés.
Ce projet de doctorat vise à identifier et à caractériser fonctionnellement de ces nouveaux peptides et leur implication dans le développement des racines et des nodules. Une liste de 200 peptides candidats a déjà été sélectionnée par l'équipe d'accueil.
La première tâche de ce projet de doctorat consiste en un criblage à haut débit dans des protoplastes de M. truncatula utilisant une approche CRISPR activatrice (CRISPR-Act), une technique de régulation génétique qui utilise le système CRISPR-Cas9 pour augmenter l'expression de gènes endogènes spécifiques. À cette fin, une bibliothèque d'ARN guides (gRNAs) ciblant les promoteurs des peptides candidats sera transfectée dans des protoplastes exprimant le CRISPR-Act, conduisant à leur surexpression. L'effet des changements induits par la surexpression des peptides sur le transcriptome des cellules sera analysé par scRNAseq.
Dans une seconde partie, les 10 peptides les plus prometteurs seront testés fonctionnellement in vivo chez M. truncatula afin d'évaluer leurs rôles dans le développement racinaire et la nodulation.
Ce projet interdisciplinaire intègre des techniques de pointe et des collaborations avec des groupes de recherche de premier plan à l'IPS2 et à Toulouse. Il permettra au doctorant ou à la doctorante d'acquérir des compétences précieuses en bioinformatique, CRISPR-Act, transformation de protoplastes et transcriptomique unicellulaire.
Cette stratégie permettra d'identifier de nouveaux peptides régulateurs de la symbiose mais aussi d'établir le CRISPR-Act couplé au scRNAseq comme un outil puissant pour la génomique fonctionnelle à haut débit chez les plantes. Plants from the legume family, such as Medicago truncatula, have the capacity to establish a symbiosis with nitrogen-fixing soil bacteria collectively called rhizobia. These bacteria, hosted in root-derived organs called nodules, fix atmospheric nitrogen for the benefit of the plant within nodules, while being fed by plant photosynthetically-derived polysaccharides. Medicago truncatula (Medicago) has emerged as a model for legume research; its well-annotated genome and the various published protocols make it ideal to study nodulation.
Studies in Medicago have revealed that the nodulation process involves numerous peptides. These peptides can act locally, promoting or tempering nodulation, such as the CLAVATA3/EMBRYO SURROUNDING REGION (CLE) or NODULE-SPECIFIC CYSTEINE RICH (NCR) family peptides. Others act at a long distance, such as the root-produced C-TERMINALLY ENCODED PEPTIDE (CEP)-family peptides, that trigger a systemic signaling cascade after being transported to the shoot, and regulating optimal nodule number.Because of their small size and hormone-like effect on plant growth, exogenous application of synthetic peptides can mimic the effects of plant-produced peptides, making them excellent candidates for sustainably improving crop performance without genetic modification. Although the functions of several peptides have been described, new genomic and transcriptomic data, especially single cell RNAseq approaches in several legume families have revealed for the first time a large number of peptide expressed cell specifically and which have never been characterized[1]. The host team have selected the most promising candidate peptides based on (1) their sequence conservation across legume species, (2) the peptides presenting known conserved functional motifs, and (3) the peptides expressed mostly in specific cell types such as pericycle, endodermis, cortex and epidermis, which are primarily involved in the plant rhizobium interaction and lateral root formation. In this PhD project, we plan to develop a strategy to rapidly and effectively screen the numerous candidate peptides and identify the ones involved in M. truncatula root and nodule development.
This approach relies on the use a CRISPR-Activator construct combined with a library of guide RNAs (gRNAs) targeting the promoters of all candidate peptides, leading to their overexpression. Then, scRNAseq is used to identify active peptides affecting cell transcriptomes (See graphical summary below). Peptides triggering the most interesting transcriptional response will be further studied in whole plants for their role in root development and symbiotic nodulation. Task1- High-throughput CRISPR-Act screen associated to scRNAseq
To be able to efficiently and rapidly identify the most active peptides among the 200 selected candidates, the PhD candidate will develop a novel screening method using a CRISPR-based activator system to overexpress peptide candidate genes and characterize their effects by scRNAseq.
The CRISPR-Act system uses a mutated Cas9 protein that recognizes DNA without cutting it and that is fused to transcriptional activator domains. gRNAs can then be used to recruit the CRISPR-Act complex to specific genes promoters, leading to their overexpression[2-3]. The PhD candidate will use this system to induce expression of candidate peptides by transfecting Medicago root cells with a pooled library of CRISPR-Act vectors, followed by selection of transfected cells and scRNAseq.
(1) Design of gRNAs targeting the region directly upstream of the transcription start site of candidate peptide genes will be done using a specialized bioinformatic pipeline and they will select the two best gRNAs for each. Insertion of the gRNA into the CRISPR-Act plasmid requires a simple step, and can be done rapidly.
(2) The cell walls of Medicago roots will be digested using commonly used protocols to obtain plant cells called protoplasts. These cells will then be transfected with the pooled CRISPR-Act plasmids to express the CRISPR-Act system and the mStayGold gene, an optimized version of GFP, as a transfection marker. mStayGold expression will be used to select transfected cells using Fluorescence-Assisted Cell Sorting (FACS) for scRNAseq using the BD Rhapsody platform, a technique already established in the lab.
(3) Analysis of scRNAseq data will provide information for each cell on: (a) which gRNA is expressed; (b) whether this results in candidate gene overexpression; (c) how this affects the transcriptome; and (c) which cell type is most strongly affected. We will include negative control gRNAs not targeting any genes, and positive control gRNAs targeting well-characterized peptides such as NCRs. We will build a Gene Regulatory Network (GRN) using MINI-EX, that will reveal what other genes are most likely involved in the signal transduction pathways induced by the candidates. They will select the 10 candidates with the strongest effect on the transcriptome, specifically on genes involved in root development, symbiosis, and pathogen response and/or expressed in the cell types producing nodules (cortex, epidermis, endodermis) for further investigation.
In this task, the PhD candidate will be trained on plasmid cloning and protoplasts transfection. In addition, they will also be trained on scRNAseq data analysis on R and python, skills that are currently extremely sought for their next scientific journey.

Task2- Functional validation of the candidate peptides
To validate the role of the candidate peptides, the 10 peptides displaying the strongest transcriptional responses affecting genes of interest, and the most conserved across legume species will be selected and functionally tested in vivo in Medicago. To that end we will generate chimeric M. truncatula plants using the hairy root technique. This technique allows the production of transformed roots in 3-4 weeks while the upper part of the plant stays untransformed. gRNAs will be expressed in constitutively expressing CRISPR-Act plants for root and nodulation phenotyping. Effect on rhizobium infection, nodule number, nodule development will be studied. Additional scRNAseq will be planned in vivo to integrate the observed phenotypes and validate or improve the GRN generated in Task2, leading to a global integration of the role of the candidate peptides.

In this task, the PhD candidate will acquire M. truncatula hairy root production techniques, Arabidopsis stable transformation, root and nodulation phenotyping skills, and additional cloning knowledge. They will be involved in the writing of a publication, presenting the validated active peptides alongside of the CRISPR-Act approach used for high-throughput discovery of new genes, a strategy that was never used before in plants.

Risk management:

- The efficiency of the CRISPR-Act system was already validated in the Guillotin-Lab in Arabidopsis and corn.
- However, it is possible that only few of the candidate peptides trigger strong transcriptional effects. Hence, as a safeguard, 10 peptides already known to be involved in root development and nodulation will be included in the screen as positive control. While the role of these peptides is already known, we do not know which cell type responds to them and, for most of them, we do not know the transcriptomic effect induced by their overexpression. This alternative approach, despite based on already known peptides regulating nodulation, will thus allow producing new knowledge.
- As another safeguard and if the activation system is not efficient enough, we will perform exogenous peptide treatments on 50 selected peptides on roots and perform bulk RNAseq to identify the one triggering the most interesting effects.
- Crispr Act hairy root system is already in use in the lab and show promising results in overexpressing candidate genes. Yet if for some candidate this approach is not enough, candidate will be overexpressed using more robust 35S promoters.

Le ou la candidat(e) doit être titulaire d'un diplôme de Master (Master 2) avec une formation en biologie moléculaire ou en sciences des plantes. Une bonne connaissance des techniques de biologie moléculaire est requise. Une expérience préalable en symbiose ou en biologie des peptides sera appréciée.

Niveau d'anglais requis : B2 (intermédiaire supérieur) : vous pouvez utiliser la langue efficacement et vous exprimer avec précision.