La biologie du champignon mycorhizien à arbuscules Rhizophagus irregularis DAOM 197198 à la lumière de la génomique et de la transcriptomique

La biologie du champignon mycorhizien à arbuscules Rhizophagus irregularis DAOM197198 à la lumière de la génomique et de la transcriptomique. Les Glomeromycètes sont des champignons symbiotiques mutualistes associés aux racines des plantes. La majorité des espèces végétales sont capables de s'associer à ces champignons. Cette association favorise le recrutement de sels minéraux du sol par les plantes, en échange le champignon retire du carbone de son hôte. La mise en place de cette symbiose consiste en une succession d'étapes de développement fongique sous contrôle de molécules signalétiques. Mes travaux de recherche ont porté sur la description des programmes génétiques qui sous-tendent le développement du champignon Rhizophagus irregularis isolat DAOM197198 lors de l'établissement de la symbiose. Le champignon a été cultivé dans plusieurs conditions marquant les points clef de son développement, et les ARN ont été séquencés. Les données RNAseq obtenues ont été utilisées pour définir les modèles de gène de l'assemblage du génome. R. irregularis possède un génome haploïde et homocaryotique (Tisserant*, Malbreil* et al., 2013). Les strigolactones (molécules signales de la plante) régulent de l'ordre de 300 gènes durant la phase pré-symbiotique, et ce, de manière séquentielle. A travers l'étude du transcriptome de R. irregularis en association avec trois espèces végétales phylogénétiquement éloignées, j'ai montré qu'il existe un set de 262 gènes fortement induits quel que soit l'hôte. Ces résultats ont permis d'affiner la recherche de candidats pertinents impliqués dans la symbiose et le développement dans les tissus végétaux. Enfin le couplage d'approches transcriptomiques et métabolomiques ont permis l'identification des propionyl- et butyryl-carnitine, qui sont potentiellement impliquées dans la symbiose.The biology of the arbuscular mycorrhizal fungus Rhizophagus irregularis DAOM197198 enlighten by genomics and transcriptomics. Glomeromycota are mutualistic fungi associated with plant roots. The vast majority of plant species are able to form a symbiosis with these organisms. This association improves plant nutrition via a better water and mineral recruitment from the soil. In return, the fungus receives carbon compounds. Symbiosis establishment is achieved by a step by step development, led by signal exchanges. My work was focused on describing genetic programs supporting the fungal development of Rhizophagus irregularis DAOM197198, during symbiosis establishment. The fungus was grown in several conditions representing key point of its development, and RNA were sequenced by illumina. RNAseq data obtained were then used to define the gene models on the genome assembly. R. irregularis has a haploid and homocaryotic genome (Tisserant*, Malbreil* et al., 2013). Strigolactones (plant signal molecules) affect the expression of around 300 genes during the pre-symbiotic development, in a sequential manner. By studying the R. irregularis transcriptomes in association with 3 phylogenetically distant plants, we report that a set of 262 genes are highly induced whatever the host is. These results allowed refining the number of candidate genes possibly playing an important role in the symbiotic development. Finally, by coupling metabolomic and transcriptomic approaches, two molecules (propionyl- and butyryl-carnitine) could be identified and might play a role in late steps of symbiosis establishment

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

The Comparison of Expressed Candidate Secreted Proteins from Two Arbuscular Mycorrhizal Fungi Unravels Common and Specific Molecular Tools to Invade Different Host Plants

Arbuscular mycorrhizal fungi (AMF), belonging to the fungal phylum Glomeromycota, form mutualistic symbioses with roots of almost 80% of land plants. The release of genomic data from the ubiquitous AMF Rhizophagus irregularis revealed that this species possesses a large set of putative secreted proteins (RiSPs) that could be of major importance for establishing the symbiosis. In the present study, we aimed to identify SPs involved in the establishment of AM symbiosis based on comparative gene expression analyses. We first curated the secretome of the R. irregularis DAOM 197198 strain based on two available genomic assemblies. Then we analyzed the expression patterns of the putative RiSPs obtained from the fungus in symbiotic association with three phylogenetically distant host plants—a monocot, a dicot and a liverwort—in comparison with non-symbiotic stages. We found that 33 out of 84 RiSPs induced in planta were commonly up-regulated in these three hosts. Most of these common RiSPs are small proteins of unknown function that may represent putative host non-specific effector proteins. We further investigated the expressed secretome of Gigaspora rosea, an AM fungal species phylogenetically distant from R. irregularis. G. rosea also presents original symbiotic features, a narrower host spectrum and a restrictive geographic distribution compared to R. irregularis. Interestingly, when analyzing up-regulated G. rosea SPs (GrSPs) in different hosts, a higher ratio of host-specific GrSPs was found compared to RiSPs. Such difference of expression patterns may mirror the restrained host spectrum of G. rosea compared to R. irregularis. Finally, we identified a set of conserved SPs, commonly up-regulated by both fungi in all hosts tested, that could correspond to common keys of AMF to colonize host plants. Our data thus highlight the specificities of two distant AM fungi and help in understanding their conserved and specific strategies to invade different hosts

Algal ancestor of land plants was preadapted for symbiosis

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis