Transcriptomics and metabarcoding approaches for the study of lichen symbionts

This research has focused on two main aspects of lichen symbioses: the characterization of stress responses in lichen photobionts and the fungal diversity present inside the lichen thallus. The study of the molecular mechanisms that underlie the stress tolerance considered a member of the most common genus of lichenized green algae, Trebouxia gelatinosa. Lichens can cope with extreme environmental conditions and this capacity is linked to the desiccation tolerance, in particular of their associated photobionts. In nature, lichen thalli continuously undergo cycles of dehydration and rehydration and in desiccated states they are extremely resistant. A transcriptome analysis on T. gelatinosa was performed under control conditions, in desiccated state and after rehydration. Desiccation and rehydration affected mainly the gene expression of components of the photosynthetic apparatus, the ROS-scavenging system, heat shock proteins (HSPs) and desiccation related proteins (DRPs). The obtained data were used to investigate in more detail the relationship between the expression of genes of interest and physiological parameters, important to understand the global response of T. gelatinosa under environmental stresses. Oxidative stress and desiccation were considered. The antioxidant activity of the photobiont was studied by quantifying the gene expression of ROS scavenging and stress-related enzymes on samples treated with hydrogen peroxide (H2O2). The data produced have been evaluated in relation to the chlorophyll a fluorescence (ChlaF) activity. H2O2 treatments produced dose and time dependent oxidative effects. The gene expression in dependence to the cell water content during desiccation was also evaluated. From the hydrated to the desiccated state, both relative water content (RWC) and water potential (Ψ) were monitored. The turgor loss point was found as a key moment that triggered changes in gene transcription. Lichens are regarded as ecological microniches as their thalli harbor complex fungal communities (mycobiota) consisting of species with divergent trophic and ecological strategies. Complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based and high throughput sequencing (HTS) have been applied. This research assessed the taxonomic composition (species diversity) of a well-characterized, alpine rock lichen community which included both thalli symptomatically infected by lichenicolous fungi and asymptomatic thalli. Taxonomic composition was assessed by HTS of the ITS2 barcode. Chaetothyriales was the major component of the observed lichen mycobiomes; Basidiomycota were recorded in the least amount. Sequences representative of morphologically characterized lichenicolous fungi were predicted and were used to assess whether there was an asymptomatic presence of these taxa in other thalli.