The Périgord black truffle (Tuber melanosporum Vittad.) and the Piedmont white truffle dominate today’s truffle market^1,2. The hypogeous fruiting body of T. melanosporum is a gastronomic delicacy produced by an ectomycorrhizal symbiont³ endemic to calcareous soils in southern Europe. The worldwide demand for this truffle has fuelled intense efforts at cultivation. Identification of processes that condition and trigger fruit body and symbiosis formation, ultimately leading to efficient crop production, will be facilitated by a thorough analysis of truffle genomic traits. In the ectomycorrhizal Laccaria bicolor, the expansion of gene families may have acted as a ‘symbiosis toolbox’⁴. This feature may however reflect evolution of this particular taxon and not a general trait shared by all ectomycorrhizal species⁵. To get a better understanding of the biology and evolution of the ectomycorrhizal symbiosis, we report here the sequence of the haploid genome of T. melanosporum, which at ∼ 125 megabases is the largest and most complex fungal genome sequenced so far. This expansion results from a proliferation of transposable elements accounting for ∼ 58% of the genome. In contrast, this genome only contains ∼ 7,500 protein-coding genes with very rare multigene families. It lacks large sets of carbohydrate cleaving enzymes, but a few of them involved in degradation of plant cell walls are induced in symbiotic tissues. The latter feature and the upregulation of genes encoding for lipases and multicopper oxidases suggest that T. melanosporum degrades its host cell walls during colonization. Symbiosis induces an increased expression of carbohydrate and amino acid transporters in both L. bicolor and T. melanosporum, but the comparison of genomic traits in the two ectomycorrhizal fungi showed that genetic predispositions for symbiosis—‘the symbiosis toolbox’—evolved along different ways in ascomycetes and basidiomycetes.

Arbuscular mycorrhizal fungi (AMF) occur in the rhizosphere and in plant tissues as obligate symbionts, having key roles in plant evolution and nutrition. AMF possess endobacteria, and genome sequencing of the endobacterium Candidatus Glomeribacter gigasporarum revealed a reduced genome and a dependence on the fungal host. To understand the effect of bacteria on fungal fitness, we used next-generation sequencing to analyse the transcriptional profile of Gigaspora margarita in the presence and in the absence of its endobacterium. Genomic data on AMF are limited; therefore, we first generated a gene catalogue for G. margarita. Transcriptome analysis revealed that the endobacterium has a stronger effect on the pre-symbiotic phase of the fungus. Coupling transcriptomics with cell biology and physiological approaches, we demonstrate that the bacterium increases the fungal sporulation success, raises the fungal bioenergetic capacity, increasing ATP production, and eliciting mechanisms to detoxify reactive oxygen species. By using TAT peptide to translocate the bioluminescent calcium reporter aequorin, we demonstrated that the line with endobacteria had a lower basal intracellular calcium concentration than the cured line. Lastly, the bacteria seem to enhance the fungal responsiveness to strigolactones, the plant molecules that AMF perceive as branching factors. Although the endobacterium exacts a nutritional cost on the AMF, endobacterial symbiosis improves the fungal ecological fitness by priming mitochondrial metabolic pathways and giving the AMF more tools to face environmental stresses. Thus, we hypothesise that, as described for the human microbiota, endobacteria may increase AMF innate immunity.

As obligate symbionts of most land plants, arbuscular mycorrhizal fungi (AMF) have a crucial role in ecosystems, but to date, in the absence of genomic data, their adaptive biology remains elusive. In addition, endobacteria are found in their cytoplasm, the role of which is unknown. In order to investigate the function of the Gram-negative Candidatus Glomeribacter gigasporarum, an endobacterium of the AMF Gigaspora margarita, we sequenced its genome, leading to an ∼ 1.72-Mb assembly. Phylogenetic analyses placed Ca. G. gigasporarum in the Burkholderiaceae whereas metabolic network analyses clustered it with insect endobacteria. This positioning of Ca. G. gigasporarum among different bacterial classes reveals that it has undergone convergent evolution to adapt itself to intracellular lifestyle. The genome annotation of this mycorrhizal-fungal endobacterium has revealed an unexpected genetic mosaic where typical determinants of symbiotic, pathogenic and free-living bacteria are integrated in a reduced genome. Ca. G. gigasporarum is an aerobic microbe that depends on its host for carbon, phosphorus and nitrogen supply; it also expresses type II and type III secretion systems and synthesizes vitamin B12, antibiotics- and toxin-resistance molecules, which may contribute to the fungal host's ecological fitness. Ca. G. gigasporarum has an extreme dependence on its host for nutrients and energy, whereas the fungal host is itself an obligate biotroph that relies on a photosynthetic plant. Our work represents the first step towards unraveling a complex network of interphylum interactions, which is expected to have a previously unrecognized ecological impact.