Eva Kondorosi

Hongrie / France

Prix Balzan 2018 pour l'écologie chimique

Structure of Symbiotic Peptides and Their Regulation: A Frontier in Chemical Ecology

During the last decades the study of symbiotic nitrogen fixation has developed into a frontier research area in chemical ecology. In symbiosis, the partners not only provide missing nutrients, but also communicate with each other and affect each other’s physiology with chemical signals and effectors. A remarkable example of such chemical interactions occurs in symbiosis of legume plants with Rhizobium soil bacteria during the formation of nitrogen-fixing root nodules. The symbiosis is initiated by chemical signals, by flavonoids excreted from the root triggering in the bacterium partner the synthesis of lipochitooligosaccharide Nod factors required for nodule organogenesis and infection of nodule cells with rhizobia. Research conducted by Kondorosi’s team has demonstrated that in Medicago nodules, both the infected plant cells and the intracellular bacteria undergo a coordinated multistep differentiation process. This involves astonishingly similar molecular mechanisms in the eukaryote and prokaryote partners, such as the loss of cell division ability and genome amplification by genome duplications accompanied with extreme cell enlargement. From the plant side, the differentiation steps of the bacteria are directed by 700 to 800 plant peptides. Most of them are nodule-specific cysteine-rich NCR peptides, encoded by small genes which have evolved specifically for symbiosis with exclusive expression in the symbiotic cells, involving different sets of genes at the early, intermediary and late stages. These peptides can interact with the bacterial cell envelope and with intracellular bacterial targets and alter their composition and metabolism. At present, the function of only a few NCR peptides is known. Researchers have yet to answer questions as to why so many peptides are required, how many are essential, what activities they represent, what the structural requirements are and how their production is regulated.

Eva Kondorosi envisions a three-year Balzan research project with two components, each to be carried out by a postdoctoral research fellow. Its aims are to obtain insights on first-order questions concerning i) the identification of bacterial targets of selected NCR peptides and the structure-function relationships of NCR peptides and ii) the regulation of NCR genes. The project will be carried out in the Institute of Plant Biology at the Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary, where two or three postdoctoral fellows will work under the supervision of the Principle Investigator, Eva Kondorosi. The project will be administrated at the office of Academia Europaea.

i) The first part of the project will be carried out by two postdoctoral fellows. Salome Kyralova, a structural biologist from the Charles University in Prague, will join Kondorosi’s team as a postdoctoral fellow to carry out structure-function relationship studies on selected NCR peptides. The other postdoctoral fellow will return from maternity leave and will focus on the identification of bacterial targets of NCR peptides. The following research questions will be answered:
• What are the peptides’ bacterial targets?
• How does the formation of disulfide bridges influence the peptides’ properties?
• Which amino acids are crucial for the activity?
• What is the conformation of the peptides and how does this enable them to interact with many different bacterial targets?

The structural studies will be carried out on NCR peptides purified from the heterologous expression system as well as synthetic NCRs with and without formation of disulfide bridges and using their derivatives; shorter peptides or full length and shorter peptides with altered amino acid composition. To meet this aim, the team will integrate CD spectroscopy, N15 and C13-NMR, mass spectrometry and testing in planta the requirements for activity with replacement of the wild type peptides with mutated or hybrid peptides. Bacterial targets will be identified with pull down experiments coupled to mass spectrometry.

ii) The second part of the project will study the regulation of NCR genes; the postdoctoral position will be internationally advertised. This work will involve the delimitation of promoter regions, the identification of transcription factors, cis-elements and likely signals from the bacterium provoking differential gene expression. Methodology will include yeast one-hybrid screens, DNA pull-down experiments and gel retardation assays coupled with proteomics as well as with data mining of RNAseq results of wild-type and mutant nodules. The team predicts that altered composition of the bacterial membranes and cell envelope might serve as signals for consecutive activation of NCR genes in coordination with differentiation of the bacterium partner. This hypothesis will be tested with transcriptome analysis of nodules induced by various bacterial mutants, blocked in the synthesis of membrane and cell surface components.

At the conclusion of the project, results will be published in high-impact, peer-reviewed international scientific journals (including PNAS and Science). Moreover, they will be presented in scientific conferences (such as the International Nitrogen Fixation Conference in 2019 and the European Nitrogen Fixation Conference in 2020) and disseminated to the broader scientific community and general public at meetings like the Annual Academia Europaea Conference in 2019.