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Intracellular accommodation of nitrogen-fixing symbiotic bacteria – BugsInACell

Intracellular accommodation of endosymbiotic bacteria within plant cells.

Role of antimicrobial peptides in plant-bacteria symbioses, in the bacterial differentiation and in trophic exchanges.

Role of antimicrobial peptides in symbiosis.

Nitrogen fixing root symbioses of legume plants with rhizobia and of actinorhizal plants with Frankia constitute a major input of combined nitrogen in the biosphere. These endosymbioses rely on the intimated contact between thousands of intracellular bacteria and their host cells in the root nodules of the plant. The role of host-produced antimicrobial peptides in maintaining such a large bacterial population and their functioning is the objective of BugsInACell.

The in vivo role of host-peptides is studied by a combination of cell biology approaches and transcriptomics, proteomics and metabolomics as well as the determination of physiological parameters.
In vitro studies rely on the use of synthetic peptides and cell biology, physiology and standard bacteriological methods for defining their impact on bacterial functioning.
We use bacterial genetics to identify bacterial adaptations required to respond to these host-produced effector peptides.

We have shown that antimicrobial peptides are used in symbiotic interactions with bacteria in plants as diverse as legumes and actinorhizal plants. This suggests that such peptides are widespread in symbiotic interactions and are efficient tools to manage large endosymbiont populations.
These peptides may have different functions and effects on the symbiotic bacteria. In legumes, these peptides induce the bacteria in a terminally differentiated state but also induce them in a state which is symbiotically more proficient. In actinorhizal plants, these peptides may promote metabolic exchanges between the bacterial cells and their host cell such as the release by the bacteria of the nitrogen-rich amino acids glutamate or glutamine. This trophic effect of the peptides is probably also improving the efficiency of the symbiosis.
Finally, we identified several bacterial functions which are required for their response to the host produced peptides.

This project will enhance our knowledge on the requirements for the intracellular accommodation of bacteria during the legume and actinorhizal symbioses. The detailed characterization of the role of the host peptides may pave the way for novel biotechnology approaches to improve nitrogen fixation in existing symbiotic systems which are agronomically important and which are functioning sub-optimally. Our work constitutes also an essential step towards transfer of nitrogen-fixing symbiosis to crop plants lacking nitrogen-fixation capability which is nowadays an actively pursued goal by different laboratories in the world.

1. Carro L, Pujic P, Alloisio N, Fournier P, Boubakri H, Hay AE, Poly F, François P, Hocher V, Mergaert P, Balmand S, Rey M, Heddi A & Normand P. 2015. Alnus peptides modify membrane porosity and induce the release of N-rich metabolites from nitrogen fixing Frankia. ISME J. doi:10.1038/ismej.2014.257.
(this article was published in the best microbial ecology journal (IF 2013 9.24)).

2. Silipo A, Vitiello G, Gully D, Sturiale L, Chaintreuil C, Fardoux J, Gargani D, Lee HI, Kulkarni G, Busset N, Marchetti R, Palmigiano A, Moll H, Engel R, Lanzetta R, Paduano L, Parrilli M, Chang WS, Holst O, Newman DK, Garozzo D, D'Errico G, Giraud E*, Molinaro A*. (2014) Covalently linked hopanoid-lipid A improves outer-membrane resistance of a Bradyrhizobium symbiont of legumes. Nat Commun 5:5106. * Co-senior authors (IF 10.742)

3. Mardirossian, M., Grzela, R., Giglione, C., Meinnel, T., Gennaro, R., Mergaert, P., and Scocchi, M. (2014) The host antimicrobial peptide Bac71-35 binds to bacterial ribosomal proteins and inhibits protein synthesis. Chem. Biol. 21, 1639–1647. (IF 6.586)

4. Guefrachi, I., Nagymihaly, M., Pislariu, C.I., Van de Velde, W., Ratet, P., Mars, M., Udvardi, M.K., Kondorosi, E., Mergaert*, P., and Alunni, B. (2014). Extreme specificity of NCR gene expression in Medicago truncatula. BMC Genomics 15, 712. (IF 4.04)

Nitrogen fixing root symbioses of legume plants with soil bacteria collectively called rhizobia and of actinorhizal plants with Frankia have a tremendous ecological and agronomic impact. These plants constitute a major input of combined nitrogen in the biosphere, they are key pioneering plants in ecological successions and legume or actinorhizal crops require no or little nitrogen fertilizer for growth.
These endosymbioses require the development of nodules, root organs formed by the host plant to house the nitrogen-fixing bacteria. Recent progress in the genetics and molecular biology of nodule organogenesis in legumes has made the prospect of transferring the symbiosis to cereals realistic in a foreseeable future. The ability of legumes and actinorhizal plants to acquire sufficient nitrogen solely from the symbiosis relies on the intimate contact between thousands of intracellular (endosymbiotic) rhizobia or Frankia bacteria and their host cells in the root nodules. However, the understanding how a eukaryotic cell can maintain such a large bacterial population or how bacteria can chronically resist in a plant cell is lagging far behind the progress that has been made on nodule organogenesis. Thus, successful engineering of nitrogen fixing symbiosis in crops will require first a significant knowledge improvement of this central aspect of the symbiosis. The endosymbiotic rhizobia in the nodule cells, called bacteroids, are differentiated bacteria which have a specific metabolism. In many legumes their formation is accompanied with a morphological transformation which is imposed by the host plant. In Medicago and related legumes, nodule-specific antimicrobial peptides, called NCRs, have been shown to control bacteroid differentiation. However, the mechanisms that control bacteroid differentiation in legumes with other bacteroid morphotypes are unknown. Endosymbiont differentiation is also taking place in the actinorhizal symbiosis by the formation of nitrogen-fixing Frankia vesicles. Like in legumes, also this process is under the control of unknown host plant factors.
We will study a model system of Bradyrhizobium strains interacting with Aeschynomene (A. evenia and A. afraspera) and soybean legumes in which bacteroids have three different host-dependent morphotypes. We will determine for these morphotypes parameters such as nitrogen fixation efficiency, and the bacteroid metabolome, proteome and transcriptome. These data will permit to identify morphotype-specific or –enhanced pathways and indicate whether particular cell morphotypes represent an advantage to the host plant as it is suggested by a few preliminary studies.
A second objective is the identification of bacterial determinants required for bacteroid differentiation. For this, genetic and transcriptomic approaches will be used and candidate functions will be characterized in detail.
Third, we will identify the plant effectors that affect bacteroid differentiation. Bioassays based on the change of bacterial morphology or the activation of genes specifically up-regulated during bacteroid morphogenesis will be developed. This global approach will be associated with the evaluation of candidate effectors (nodule-specific antimicrobial peptides) through biochemical and cell biology assays.
Fourth, we will explore whether mechanisms for endosymbiont control operating in legumes are conserved in the evolutionary distant actinorhizal symbiosis. Nodule-specific antimicrobial peptides have been identified in the actinorhizal plants Alnus glutinosa and Casuarina glauca and the role of these peptides in vesicle formation will be studied by in vivo and in vitro approaches.
This project will enhance our knowledge on the requirements for the intracellular accommodation of bacteria during the legume and actinorhizal symbioses and therefore constitutes an essential step towards transfer of nitrogen-fixing symbiosis to crop plants lacking nitrogen-fixation capability.

Project coordination

Peter MERGAERT (Organisme de recherche)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.


LEM-CNRS Ecologie microbienne UMR CNRS

Help of the ANR 500,507 euros
Beginning and duration of the scientific project: September 2013 - 42 Months

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