Projets

Importance de l’interface membranaire plante-bactérie lors de la symbiose avec rhizobium chez les légumineuses

Résumé du projet

Pour assurer leur apport en azote, la plupart des légumineuses sont capables de développer une endosymbiose avec des bactéries fixatrices d’azote appelées rhizobiums. Comprendre les mécanismes de cette symbiose rhizobium-légumineuse représente un enjeu majeur en agriculture pour optimiser la fixation d’azote des légumineuses cultivées et envisager le transfert de cette propriété aux céréales. L’étude intensive de deux légumineuses modèles a permis de montrer que lors de l’établissement de l’interaction symbiotique, les rhizobiums sont reconnus de façon spécifique par la plante hôte. Celle-ci active un programme d’infection et d’organogenèse nodulaire aboutissant à l’internalisation des bactéries au sein des cellules d’un organe racinaire spécialisé, le nodule. Tout au long de ce processus symbiotique, la plante-hôte contrôle de façon étroite l’infection par les bactéries et leur accommodation intracellulaire, par la mise en place d’une interface membranaire spécialisée. Malgré son importance, notre compréhension de la dynamique et des acteurs protéiques de cette interface reste limitée. Récemment, une approche de génétique directe menée chez la légumineuse Aeschynomene evenia a conduit à l’isolement de mutants d’infection altérés dans le gène AeORM1 prédit coder une protéine orosomucoïde (ORM). Les protéines ORM jouent un rôle majeur dans la biologie des organismes, notamment en régulant négativement la synthèse des sphingolipides de membrane, mais leur implication dans la symbiose n’a jamais été investiguée. Le projet vise à explorer le rôle d’AeORM1 dans la symbiose rhizobienne en combinant des analyses de mutant, des approches moléculaires, de microscopie et de biochimie. Il devrait ainsi permettre de mieux comprendre le rôle direct ou indirect des protéines ORM sur l’interface symbiotique avec rhizobium.   

Financement

Le projet « Symbiotic Interface » (2022-2024) est financé par l’INRAE dans le cadre de l’AAP SPE

Participants

  • Jean-François ARRIGHI (Researcher)
  • Frédéric GRESSENT (Researcher)
  • Djamel GULLY (Engineer)
  • Nico NOUWEN (Researcher)
  • Marjorie PERVENT (Technician)

A new symbiosis pathway for rhizobial infection and nodulation in legumes (SymWay)

Résumé du projet

Legumes are of prime agronomic and ecological importance thanks to their ability to develop a nitrogen-fixing symbiosis with rhizobia that are hosted in a specialized root organ, the nodule. Research performed on two model legumes has identified many genes important for this symbiosis and led to a general scheme where rhizobia produce Nod factor signal molecules that are perceived by plant LysM-RLK receptors, which, in turn, activate a Nod signalling pathway controlling an intracellular infection process through infection thread formation, concomitantly with nodule organogenesis. However, this symbiotic process is not universal. Some legumes (25% of genera) such as lupin, peanut and Aeschynomene spp., use another symbiotic mechanism not involving infection threads. It is considered simpler but remains understudied. Among Aeschynomene spp., several are remarkable because they are efficiently nodulated by photosynthetic Bradyrhizobium strains not producing Nod factors. Therefore, alternative symbiotic pathways exist to trigger nodulation without Nod factor recognition and infection thread formation.

To decipher the mechanisms of this so-called Nod-independent symbiosis, we established Aeschynomene evenia as a working model during the ANR project “AeschyNod”, and recently generated a high-quality genome for this species. By combining a forward genetic screen for nodulation mutants in A. evenia with a genomic survey of genes known to have symbiotic roles in model legumes, we demonstrated the lack of involvement in A. evenia of key genes required for rhizobial perception and intracellular infection in model legumes, while downstream components of Nod signaling are conserved. The major breakthrough of this work was the identification of two novel actors essential for establishment of this Nod-independent symbiosis, AeRLCK (Receptor-Like Cytoplasmic Kinase) and AeCRK (Cysteine-rich Receptor-like Kinase). RLCKs and CRKs are central players in plant signalling pathways, through interaction with ligand-binding receptor kinases (RLKs). We hypothesize that AeRLCK and AeCRK play key roles by interacting with receptors and other actors to mediate perception of photosynthetic Bradyrhizobium, signal transduction and infection, but their mode of action remains to be discovered.

Characterization of AeRLCK and AeCRK is crucial to uncover the molecular basis of the Nod independent symbiosis. Our main goals in the SymWay project are i) to characterize the biological functions of AeRLCK and AeCRK, ii) to identify AeRLCK and AeCRK interacting proteins and their roles in nodulation, and iii) to understand how some legume species have evolved a Nod-independent symbiotic process through the recruitment of these actors. For these purposes, our consortium involves three partners, including two leaders in the study of the rhizobium-legume symbiosis; an expert on the Nod-independent process in Aeschynomene and a specialist in the characterization of symbiotic receptors. The third partner has strong expertise in bioinformatics. Together, we will make use of a series of genomic, genetic, transcriptomic, molecular biology and protein biochemistry approaches and exploit comparative symbiotic systems.

Deciphering the molecular mechanisms of the Nod-independent symbiosis, through characterization of AeRLCK and AeCRK, should lead to a new paradigm for how the rhizobium-legume symbiosis can be established and, thus, significantly broaden our views on the evolution and diversity of the mechanisms underpinning this highly beneficial plant-microbe interaction. In turn, acquired knowledge in Aeschynomene legumes could be transferred to crop legumes using a similar infection process (no infection threads) via breeding programs. This project is also expected to provide important novel leads to design adapted strategies aimed at transferring nitrogen-fixing symbiosis to cereals.

Financement

The SymWay project is supported by an ANR grant (2022-2026).

Partenaires

  • PHIM (MSLT group)
  • LIPME
  • GenoToul

Participants

  • Jean-François ARRIGHI (Researcher)         
  • Clémence CHAINTREUIL (Engineer)
  • Eric GIRAUD (Researcher)
  • Frédéric GRESSENT (Researcher)
  • Djamel GULLY (Engineer)               
  • Nico NOUWEN (Researcher)          
  • Marjorie PERVENT (Technician)
  • Natasha HORTA (Ph.D student)

Receptor proteins and redox control at the interface of symbiosis & immunity (Duality)

Résumé du projet

Plants benefit from two major symbiotic interactions with soil microbes, the Arbuscular Mycorrhizal (AM) and the Rhizobium-Legume (RL) symbioses, to improve their mineral nutrition. They generally establish such associations following recognition of symbiotic chitin-derived molecules called Myc-LCOs and Nod factors (NFs) when they are produced by AM fungi and rhizobia, respectively. These symbiotic signal molecules are perceived by members of the plant LysM-domain Receptor-Like Kinase (LysM-RLK) family which also controls the perception of pathogenic chitinic molecules. In the model legume Medicago truncatula, we have shown that four LysM-RLK receptors are particularly important for symbiosis and/or immunity; MtNFP, MtLYK3, MtLYR3 and MtLYK9. We have also evidenced the presence of these four LysM-RLKs in Aeschynomene evenia, which is an exception in legumes in not requiring NFs for the RL symbiosis. How specific signal recognition through these LysM-RLKs leads to the distinction of symbionts from pathogens and what might be their functions in a NF-independent context is not well understood.

Upon perception of symbiotic signals or pathogenic elicitors an antagonistic regulation of Reactive Oxygen Species (ROS) and nitrogen species homeostasis, including hydrogen peroxide and nitric oxide (NO), constitutes one of the earliest known plant responses. In M. truncatula, symbiotic signals induce a rapid change in ROS and can block a pathogen-induced ROS burst, both effects being dependent on MtNFP. This supports close connections between MtNFP and ROS regulation for symbiosis and immunity. We have also shown that ROS and NO can have positive roles in both the AM and the RL symbioses, and that in A. evenia, ROS production and cell collapse are early steps of NF-independent symbiosis. We hypothesise that (i) the regulation of the cellular redox state via LysM receptors is a critical component of the mechanisms that enables M. truncatula to distinguish between symbiotic and pathogenic signals, (ii) LysM-RLK and ROS/NO are involved differently in the establishment of the RL symbiosis in A. evenia. Therefore, the spatio-temporal patterns and the genetic control of the cellular redox state during symbiotic and immune-related signalling need to be characterized.

The main goal of the DUALITY project is to study the dual roles of LysM-RLK receptors and redox state in symbiosis and immunity. Our objectives are to determine (1) the plant cellular redox state in different biotic conditions, as well as in both NF-dependent and NF-independent nodulation; (2) the roles of key LysM-RLKs in redox state regulation; (3) the variations to LysM-RLKs and their roles in influencing redox control to enable NF-independent nodulation; and (4) how MtNFP controls interactions with contrasting outcomes. To tackle to such issues, DUALITY ideally brings together 4 partners who are leader in the fields of LysM receptors and symbiotic signalling in M. truncatula (P1), redox signalling (P2), plant immunity (P3) and Nod factor-independent symbiosis in A. evenia (P4). The project will exploit remarkable tools such as biosensors for in vivo spatio-temporal analysis of redox signaling; recently obtained genetic and genomic resources for A. evenia; new M. truncatula plant mutants (in receptors and redox signaling), and new MtNFP interacting proteins.

This work is anticipated to provide breakthroughs to explain the mechanisms by which some members of an important family of plant receptors largely involved in plant-microbe interactions, transmit microbial signaling via changes in redox balance that control defence to pathogens and  symbiosis establishment. In turn, such knowledge may provide leads into how to simultaneously maintain efficient symbiosis and increase disease resistance in order to promote a smart agriculture.

Financement

The DUALITY project is supported by an ANR grant (2021-2025).

Partenaires

  • LIPME
  • ISA
  • LRSV
  • PHIM (MSLT group)

Participants

  • Jean-François ARRIGHI (Researcher)         
  • Eric GIRAUD (Researcher)
  • Frédéric GRESSENT (Researcher)
  • Djamel GULLY (Engineer)               
  • Nico NOUWEN (Researcher)          
  • Marjorie PERVENT (Technician)
  • Maëlle RIOS (Engineer)

ET-Nod : Effectors Triggering Nodulation In legumes

Résumé du projet

Legumes play a major agronomical and ecological role due to their ability to fix atmospheric nitrogen during symbiosis with rhizobia. The main legume crops are tropical species (soybean, peanut, mungbean, …) that represent more than 85% of the grain legume production. These species are all nodulated by Bradyrhizobium strains which contain nodulation genes (nod genes) necessary for the synthesis of key symbiotic signals, named Nod factors (NFs), but also T3SS genes that encode for the Type 3 secretion system. This secretory machinery, initially identified in animal and plant bacterial pathogens, permits the delivery of effector proteins inside the host cells where they interfere with various host processes including suppression of immune responses and favour the infection. For a long time, it was assumed that nodulation absolutely required NFs to trigger nodule organogenesis and infection. The T3SS machinery on the other hand was viewed as an accessory equipment, which modulates the efficiency and the host spectrum of the bacteria. However, it has been shown that some legume species of the Aeschynomene genus but also the cultivar Glycine max cv. Enrie are nodulated by Bradyrhizobium strains in the absence of NF synthesis. In this case, the establishment of the interaction requires that the bacteria has a functional T3SS indicating that specific type 3 effectors can directly activate the nodulation signalling pathway in legumes, bypassing the perception of Nod Factors.

Previously, we have demonstrated that in the Bradyrhizobium strain ORS3257 this T3SS-dependent symbiosis relies on a cocktail of at least five effectors playing synergistic and complementary roles in nodule organogenesis, infection and repression of plant immune responses (Teulet et al. 2019). Among them, we identified the nuclear-targeted ErnA effector, which is highly conserved among bradyrhizobia, as a key actor for nodule organogenesis. Furthermore, preliminary data indicate that other Bradyrhizobium strains can use other Type 3 effectors, distinct of ErnA, to trigger nodulation in legumes.

The recent discovery that a single effector protein is sufficient to induce nodule organogenesis without the need of NFs is a paradigm shift in the field and indicates that legume nodulation programs are not exclusively controlled by NFs.

Our main goals in the current ET-Nod project are to decipher the molecular mechanisms by which ErnA activates nodulation in Aeschynomene, to identify new effectors (named ET-Nods) behaving like ErnA in the triggering of nodulation and to characterize the importance of this effector family in the symbiotic efficiency of agronomically important legumes.

For this purposes, our consortium, involving specialists in plant symbiosis and pathogenesis will i) combine biochemical, genetic and omic approaches to characterize the molecular target(s) and interactome of ErnA, ii) develop at the level of the Bradyrhizobium genus a comparative genomic analysis coupled to a mutagenesis approach to identify new ET-Nod effectors and iii) investigate, using bacterial and plant genetics, the role played by ErnA and ET-Nod effectors in various Bradyrhizobium strains during symbioses with legume crops (soybean, peanut, cowpea …).

The knowledge acquired during this project could be exploited in agronomy to improve yield of several legume crops and to design new strategies aimed at transferring nodulation to transfer nitrogen-fixing symbiosis to cereals.

Financement

The ET-Nod project is supported by an ANR grant (2021-2025).

Partenaires

  • PHIM (MSLT group)
  • I2BC
  • LIPME

Participants

  • Eric GIRAUD (Researcher)
  • Jean-François ARRIGHI (Researcher)         
  • Clémence CHAINTREUIL (Engineer)
  • Djamel GULLY (Engineer)               
  • Nico NOUWEN (Researcher)          
  • Alicia CAMUEL (Ph.D student)