Analysis of the tripartite interaction between legumes, symbiotic nitrogen-fixing bacteria and aphids.

President of the jury :                       

• Pr. Cécile SABOURAULT - Pr., CNRS, Université of Côte d'Azur Nice

Rapporteurs :                     

• Pr. Hubert CHARLES - Pr., INSA, Lyon.
• Dr. Pierre-Emmanuel COURTY - Dr., INRAE, Dijon.         

Examiners

• Pr. Véronique GRUBER - Pr., IPS2, Université Paris-Saclay Paris.

Thesis Director :

• Pr. Marylène POIRIÉ - Pr., INRAE, Université Côte d'Azur Nice
• Pr. Pierre FRENDO - Pr., INRAE, Université Côte d'Azur Nice

Abstract :

Symbiotic bacteria are key drivers in shaping their host plants, aiding their adaptation to environmental challenges. Previous analysis has shown that symbiosis between Medicago truncatula and Sinorhizobium meliloti primes plant defence against the pea aphid Acyrthosiphon pisum. Within this framework, my research investigates how Medicago-aphid interaction is influenced and modulated by the plant symbiotic bacteria partner.

First, I have investigated how nitrogen-fixing symbiosis (NFS) in the legume M. truncatula A17 modulates leaf metabolism during aphid infestation. Metabolomic analysis by LC-MS and GC-MS and defence gene expression analysis by RT-qPCR were performed in leaves of both NFS and nitrate-fed (non-inoculated; NI) plants with and without aphid infestation (Amp). Our results showed that accumulation of primary and specialized metabolites was modulated by both NFS and aphid infestation. Amongst the 194 identified metabolites, 62 defence-related compounds such as salicylate, pipecolate, gentisic acid and several soluble sugars were differentially regulated by aphid infestation in both NFS and NI conditions. Interestingly, 19 metabolites, including triterpenoid saponins, accumulated specifically in NFS_Amp conditions. Gene expression analysis showed that aphid-infested plants exhibited significantly higher expression of Pathogenesis Related Protein 1, a marker for the salicylic acid pathway, under both NFS and NI conditions. Proteinase Inhibitor, a marker of the jasmonic acid pathway, was also induced by aphid infestation, but with significantly higher expression in NFS conditions compared to NI conditions. We also observed in aphid-infested plants significantly higher expression of Chalcone isomerase, flavonol synthase, hydroxyisoflavone-O-methyl transferase and Pterocarpan synthase, while D-pinitol dehydrogenase was only significantly induced in NI infested leaves. Thus, in addition to being a plant nitrogen provider, NFS stimulates accumulation of specialized Medicago defences metabolites upon aphid attack.

Second, since aphids are phloem-feeding herbivores and the phloem is a vital organ for solute transport and defence within the plant, I have also analysed the M. truncatula plant phloem metabolome and proteome on NFS and NI plants, with and without aphid infestation. Proteins were analysed from “in stacking gel” by LC-MSMS and untargeted metabolomic was done both by hydrophobic C18 and hydrophilic HILIC LC-MS. 61 defence-related metabolites like salicylic acid, pipecolic acid, DIBOA, and 12 defence-related proteins like PR-10, Hevein, ABA-responsive protein, were significantly modulated by aphid infestation in both NFS and NI conditions. Using MixOmics analysis to search for correlation between metabolites and proteins, I found that developmental proteins such as amine oxidase and the aspartic proteinase nepenthesin-like were negatively regulated when defence metabolites like salicylic acid and p-hydroxybenzoic acid were produced during infestation, whereas defence related proteins like plastocyanin-like domain protein were positively regulated. This translates in the reduced production of carboxylic acids and an overall increase in phenolics and isoflavones during infestation. Although we found no plant metabolites or proteins significantly modulated by NFS during aphid infestation, we found the accumulation of 37 aphid cellular and membrane proteins in the plant phloem while feeding, of which 20 were significant to NFS conditions during infestation including many cellular and membrane linked proteins.

These findings lay the groundwork for incorporating symbiotic interactions into future research and exploring their practical applications as a potential enhancer of plant defences against herbivores. This approach could offer a novel form of biological control, contributing to integrated pest management strategies.

Key words : Sinorhizobium meliloti, Signalisation redox, Régulation transcriptionnelle, Protéomique, S-glutathionylation.

In person or via Zoom : https://univ-cotedazur.zoom.us/j/82444390837?pwd=WbfxdOabAejL89XqMrn1nwJ44Pengv.1

Meeting ID : 824 4439 0837

Secret code : 874462