Secretome Analysis from the Ectomycorrhizal Ascomycete Cenococcum geophilum

Maíra de Freitas Pereira, Claire Veneault-Fourrey, Patrice Vion, Fréderic Guinet, Emmanuelle Morin, Kerrie W. Barry, Anna Lipzen, Vasanth Singan, Stephanie Pfister, Hyunsoo Na, Megan Kennedy, Simon Egli, Igor Grigoriev, Francis Martin, Annegret Kohler, Martina Peter

Cenococcum geophilum is an ectomycorrhizal fungus with global distribution in numerous habitats and associates with a large range of host species including gymnosperm and angiosperm trees. Moreover,C. geophilumis the unique ectomycorrhizal species within the clade Dothideomycetes, the largest class of Ascomycetes containing predominantly saprotrophic and many devastating phytopathogenic fungi. Recent studies highlight that mycorrhizal fungi, as pathogenic ones, use effectors in form of Small Secreted Proteins (SSPs) as molecular keys to promote symbiosis. In order to better understand the biotic interaction ofC. geophilumwith its host plants, the goal of this work was to characterize mycorrhiza-induced small-secreted proteins (MiSSPs) that potentially play a role in the ectomycorrhiza formation and functioning of this ecologically very important species. We combined different approaches such as gene expression profiling, genome localization and conservation of MiSSP genes in differentC. geophilumstrains and closely related species as well as protein subcellular localization studies of potential targets of MiSSPs in interacting plants using in tobacco leaf cells. Gene expression analyses ofC. geophiluminteracting withPinus sylvestris(pine) andPopulus tremula×Populus alba(poplar) showed that similar sets of genes coding for secreted proteins were up-regulated and only few were specific to each host. Whereas pine induced more carbohydrate active enzymes (CAZymes), the interaction with poplar induced the expression of specific SSPs. We identified a set of 22 MiSSPs, which are located in both, gene-rich, repeat-poor or gene-sparse, repeat-rich regions of theC. geophilumgenome, a genome showing a bipartite architecture as seen for some pathogens but not yet for an ectomycorrhizal fungus. Genome re-sequencing data of 15C. geophilumstrains and two close relativesGlonium stellatumandLepidopterella palustriswere used to study sequence conservation of MiSSP-encoding genes. The 22 MiSSPs showed a high presence-absence polymorphism among the studiedC. geophilumstrains suggesting an evolution through gene gain/gene loss. Finally, we showed that six CgMiSSPs target four distinct sub-cellular compartments such as endoplasmic reticulum, plasma membrane, cytosol and tonoplast. Overall, this work presents a comprehensive analysis of secreted proteins and MiSSPs in different genetic level ofC. geophilumopening a valuable resource to future functional analysis.