Elaboration d'une stratégie analytique pour l'identification de métabolites clés impliqués dans la symbiose mycorhizienne entre Rhizophagus irregularis-Medicago truncatula

La symbiose mycorhizienne à arbuscule (MA) est une interaction bénéfique entre les racines de la plupart des plantes et certains champignons du sol appartenant aux Gloméromycètes. La plante bénéficie d'une meilleure nutrition hydrique et minérale. En échange elle fournit au champignon MA, symbiote obligatoire, sa source de carbone qui lui permet d'accomplir son cycle de vie. Si quelques signaux symbiotiques impliqués dans les phases précoces de la symbiose MA ont été caractérisés ces dernières années, nos connaissances sont très limitées concernant les molécules régulatrices de la symbiose échangées entre les partenaires in planta. Dans ce contexte, nous avons élaboré une stratégie analytique dans le but d'identifier des métabolites clés impliqués dans la symbiose mycorhizienne. Nous avons associé une analyse différentielle du métabolome de racines mycorhizées et non mycorhizées à une analyse de l'expression de gènes spécifique de la symbiose mycorhizienne. L'analyse métabolique couplant la chromatographie liquide ultra performante (UPLC) et la spectrométrie de masse haute résolution (Q-TOF) avec des analyses statistiques non supervisées de type OPLS-DA, a permis de mettre en évidence 71 métabolites au moins 10 fois plus présents dans les racines mycorhizées. Parmi ces métabolites une cinquantaine n'avait jamais été identifiée comme caractéristiques de la mycorhization, dont la propionyl-carnitine. Quant à l'analyse transcriptomique, elle avait pour but de sélectionner parmi ces métabolites criblés ceux pouvant avoir un rôle de signaux régulateurs de la symbiose, reflété par leur activité sur le transcriptome du champignon MA. Nous avons réalisé une analyse de l'expression de gènes spécifiques de la symbiose sur des spores de Rhizophagus irregularis stimulées par des extraits racinaires fractionnés par HPLC. Par la complémentarité des deux analyses (transcriptomique et métabolique), les métabolites davantage présents dans les racines mycorhizées et détectés dans les fractions HPLC qui modulent l'expression de gènes fongiques sont identifiés comme des candidats potentiels.The arbuscular mycorrhizal (MA) symbiosis is a mutualist interaction between soil fungi (Glomeromycota) and roots of most plant species. During symbiosis AM fungi provide plants with minerals and water, and obtain in return photoassimilates to carry out its life cycle. If some symbiotic signals involved in the early stages of the AM symbiosis have been characterized in recent years, our knowledge is very limited on regulatory molecules exchanged between the symbiotic partners in planta. In this context, we have developed an analytical strategy in order to identify key metabolites involved in mycorrhizal symbiosis. We associate a differential analysis of the metabolome of mycorrhizal roots and not mycorrhizal an analysis of the expression of genes specific mycorrhizal symbiosis. The analysis of metabolic coupling the high performance liquid chromatography (UPLC) and high resolution mass spectrometry (Q-TOF) analysis with statistical unsupervised type OPLS-DA, has highlighted 71 metabolites at least 10 times more prevalent in mycorrhizal roots. Among these metabolites fifty had never been identified as characteristics mycorrhization, including propionyl-carnitine. As for the transcriptomic analysis, it was to select among these those screened metabolites may have a role in regulating the symbiosis signals reflected by their activity on the transcriptome MA of the fungus. We conducted an analysis of the expression of specific genes on the symbiosis of spores of Rhizophagus irregularis stimulated by root extracts fractionated by HPLC. The complementarity of the two analysis (transcriptomics and metabolic), more metabolites present in mycorrhizal roots and detected in HPLC fractions that modulate the expression of fungal genes were identified as potential candidates

Combining Metabolomics and Gene Expression Analysis Reveals that Propionyl- and Butyryl-Carnitines Are Involved in Late Stages of Arbuscular Mycorrhizal Symbiosis

International audienceThe arbuscular mycorrhizal (AM) symbiosis is a widespread mutualistic association between soil fungi (Glomeromycota) and the roots of most plant species. AM fungi are obligate biotrophs whose development is partially under the control of their plant host. We explored the possibility to combine metabolomic and transcriptomic approaches to find putative mycorrhiza-associated metabolites regulating AM fungal development. Methanol extracts of Medicago truncatula roots colonized or not with the AM fungus Rhizophagus irregularis were analyzed and compared by ultra-high-performance liquid chromatography (UHPLC), high-resolution mass spectrometry (Q-TOF), and multivariate statistical discrimination. We detected 71 mycorrhiza-associated analytes exclusively present or at least 10-fold more abundant in mycorrhizal roots. To identify among these analytes those that could regulate AM fungal development, we fractionated by preparative and semi-preparative HPLC the mycorrhizal and non-mycorrhizal root extracts and established how the 71 analytes were distributed among the fractions. Then we tested the activity of the fractions on germinating spores of R. irregularis by quantifying the expression of 96 genes known for their diverse in planta expression patterns. These investigations reveal that propionyl- and butyryl-carnitines accumulated in mycorrhizal roots. The results suggest that these two molecules regulate fungal gene expression in planta and represent interesting candidates for further biological characterization

Metabolite profiling of pea roots in response to phosphate availability

The arbuscular mycorrhizal (AM) symbiosis is a mutualistic association between soil fungi (Glomeromycota) and roots of most plant species. A recent study showed that high phosphate fertilization could inhibit mycorrhizal colonization at a very early stage, before hyphopodium formation. The authors proposed that inhibiting and/or stimulatory compounds might be present in roots grown under high phosphate or low phosphate, respectively. To further address this question, we performed metabolite profiling analyses of extracts of pea roots grown under low and high phosphate concentrations. Ultra high performance liquid chromatography (UHPLC) was coupled with high resolution (HR) mass spectrometry (Q-TOF) and multivariate statistical analysis. This allowed the detection of 34 ions discriminating the two conditions. A majority (28 ions) were more abundant in roots grown under low phosphate concentration, and among them four were specific of this condition. The results suggest that the regulation of AM symbiosis by phosphate may involve the synthesis or accumulation of stimulatory compounds in roots grown under low phosphate