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Stage:174
From Master 2 Recherche Biosciences Végétales
Laboratoire d'accueil : Laboratoire des Interactions Plantes Micro-organismes (LIPM), INRA, 24 Chemin de Borde Rouge – Auzeville, CS 52627, 31326 Castanet Tolosan cedex
Equipe d'accueil : Clare Gough et Julie Cullimore : Signaux symbiotiques et leur perception transduction.
Encadrant(e)(s) : Julie Cullimore, Julie.Cullimore@toulouse.inra.fr
Contexte scientifique et objectifs
The production of lipochitooligosaccharidic Nod factors (LCOs) by Rhizobia bacteria is essential for establishing the beneficial nitrogen-fixing symbiosis with legume plants. These factors are also used to improve the production of both legume and non-legume plants in agriculture, produced by the company Novozymes. Knowledge of how these factors are perceived by the plant is necessary in order to further improve the use of these molecules and of symbioses in sustainable agriculture.
Nod factor perception is very specific between Rhizobia species and their plant hosts. In the model legume Medicago truncatula, the production of O-sulphated Nod factors by its rhizobial symbiont Sinorhizobium meliloti, is very important for eliciting responses in the root epidermal and cortical cells. The low concentrations (pM-nM) and structural specificity for inducing these Nod factor responses suggest these signaling molecules are perceived by high affinity receptors. These responses (and indeed elicitation of nitrogen fixing nodules by S. meliloti) are dependent on the lysin motif receptor like kinase (LysM-RLK) called NFP (NOD FACTOR PERCEPTION). NFP is also involved in the perception of Myc factors produced by mycorrhizal fungi and in resistance to pathogens. It thus may act as an adaptor protein for different protein partners leading to differential signal transduction in different plant-microbe interactions (see Gough and Jacquet 2013; Cullimore and Gough 2013).
LysM domain proteins are known to be involved in the perception of chitin and other N-acetyl glucosamine containing molecules and in some cases direct binding has been observed. For example in rice, chitin binds to the LysM protein CEBiP which then dimerizes with a LysM-RLK (CERK1) to transduce the signal via a cytoplasmic kinase (see Gust et al. 2012). In the case of NFP, our work has shown that it does not directly bind Nod factors to high affinity (unpublished work), suggesting that Nod factor signaling requires additional proteins for perception, in addition to transduction. Our group is using genetic (see project by F. Debellé), molecular and proteomic approaches (this project) to identify such proteins.
Programme de recherches :
Purification of protein complexes is now widely used in plants to identify receptor protein interactors. To undertake such an approach we have produced stable transgenic lines of NFP tagged with the FLAG epitope and expressed either from a root epidermal (extensin) promoter (line ProEXT-NFP-FLAG) or from its own promoter (line ProNFP-NFP-FLAG). By solubilizing protein complexes from these roots and immunopurification with an anti-FLAG antibody, a proteomic approach is being used to identify the co-immunopurified proteins. Certain protein candidates have already been identified. Depending on the state of the project, the proteomic approach will be continued to establish whether the candidate proteins interact with NFP either before or after Nod factor addition and/or after Rhizobium inoculation. The main part of the work will be to use a combination of molecular genetic, biochemical and physiological tests to functionally characterize certain candidates. This will involve analyzing their i) protein domain interaction with NFP through co-expression in vitro or in heterologous systems – Nicotiana benthamiana leaves or yeast, ii) biological role using reverse genetics (RNAi) using the rapid M. truncatula transformation system with Agrobacterium rhizogenes, followed by phenotyping of the roots for Nod factor responses and nodulation.
The project is envisaged to be continued as a PhD thesis project to identify how a NFP protein complex specifically recognizes sulphated Nod factors and how it connects to the downstream symbiotic signaling pathways. This work should help us understand how Rhizobium specifically interacts with its legume host to establish the agronomically important legume-Rhizobia symbiosis, knowledge which could also help the goal to establish a nitrogen-fixing Rhizobia symbiosis with cereals.
Techniques et matériels
Molecular biology : PCR, cloning, sequencing. Transformation and protein expression in
Nicotiana benthamiana and roots of M. truncatula, using Agrobacterium.
Biochemistry : Protein purification, co-immuoprecipitation, gel electrophoresis, Western blotting, interactions recepto-ligand.
Références
Gough and Jacquet (2013) TIPS 18, 566-574
Cullimore J, Gough C (2013) Lipochitooligosaccharide perception and the basis of partner recognition in root endosymbioses. In Molecular Microbiology of the Rhizosphere Vol. I, Ed. Frans J. de Bruijn, Wiley-Blackwell. Pp 483-494.
Fliegmann et al (2013) ACS Chem Biol 8, 1900-1906
Gust et al (2012) TIPS, 17, 495-502