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.

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.

La biologie du champignon mycorhizien a arbuscules Rhizophagus irregularis DAOM197198 a la lumiere de la genomique et de la transcriptomique. Les Glomeromycetes sont des champignons symbiotiques mutualistes associes aux racines des plantes. La majorite des especes vegetales sont capables de s'associer a ces champignons. Cette association favorise le recrutement de sels mineraux du sol par les plantes, en echange le champignon retire du carbone de son hote. La mise en place de cette symbiose consiste en une succession d'etapes de developpement fongique sous controle de molecules signaletiques. Mes travaux de recherche ont porte sur la description des programmes genetiques qui sous-tendent le developpement du champignon Rhizophagus irregularis isolat DAOM197198 lors de l'etablissement de la symbiose. Le champignon a ete cultive dans plusieurs conditions marquant les points clef de son developpement, et les ARN ont ete sequences. Les donnees RNAseq obtenues ont ete utilisees pour definir les modeles de gene de l'assemblage du genome. R. Irregularis possede un genome haploide et homocaryotique (Tisserant*, Malbreil* et al. , 2013). Les strigolactones (molecules signales de la plante) regulent de l'ordre de 300 genes durant la phase pre-symbiotique, et ce, de maniere sequentielle. A travers l'etude du transcriptome de R. Irregularis en association avec trois especes vegetales phylogenetiquement eloignees, j'ai montre qu'il existe un set de 262 genes fortement induits quel que soit l'hote. Ces resultats ont permis d'affiner la recherche de candidats pertinents impliques dans la symbiose et le developpement dans les tissus vegetaux. Enfin le couplage d'approches transcriptomiques et metabolomiques ont permis l'identification des propionyl- et butyryl-carnitine, qui sont potentiellement impliquees dans la symbiose.

Arbuscular mycorrhizal (AM) symbiosis is the most widespread mutualistic association. It concerns 80% of land plants and involves fungi belonging to the phylum Glomeromycota. Benefits to the host plants due to this symbiosis range from nutrient supply to protection against pathogens. AM fungi are important components of the soil microbiome and are of great interest for managing sustainable agriculture, provided that their life cycle is better understood. Recently, major advances in the genomics of the model AM fungus Rhizophagus irregularis DAOM197198 have been published, offering new tools to investigate the biology of this symbiosis. In this chapter, we provide an overview of the efforts that were necessary to reach these results, from the discovery of these fungi and the description of their mutualistic incidence to their in vitro cultivation and on to genomics. The genome of DAOM197198 is estimated at ca. 150 Mb. It is haploid and less polymorphic than expected. Although it is an obligate biotrophic fungus, very little gene loss was observed. We put the Rhizophagus gene repertoire in perspective with previous investigations performed on the physiology of AM fungi: germination, early signalling with host plants, plant invasion, metabolism (phosphorous, carbon and nitrogen) and sexuality. Clearly, the publication of the genome of R. irregularis DAOM197198 is a turning point in the study of AM fungi, and large areas of their biology that still remain to be elucidated will now become accessible for investigation.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

The 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 arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.