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What is in Umbilicaria pustulata? A metagenomic approach to reconstruct the holo-genome of a lichen
(2020)
Lichens are valuable models in symbiosis research and promising sources of biosynthetic genes for biotechnological applications. Most lichenized fungi grow slowly, resist aposymbiotic cultivation, and are poor candidates for experimentation. Obtaining contiguous, high-quality genomes for such symbiotic communities is technically challenging. Here, we present the first assembly of a lichen holo-genome from metagenomic whole-genome shotgun data comprising both PacBio long reads and Illumina short reads. The nuclear genomes of the two primary components of the lichen symbiosis—the fungus Umbilicaria pustulata (33 Mb) and the green alga Trebouxia sp. (53 Mb)—were assembled at contiguities comparable to single-species assemblies. The analysis of the read coverage pattern revealed a relative abundance of fungal to algal nuclei of ∼20:1. Gap-free, circular sequences for all organellar genomes were obtained. The bacterial community is dominated by Acidobacteriaceae and encompasses strains closely related to bacteria isolated from other lichens. Gene set analyses showed no evidence of horizontal gene transfer from algae or bacteria into the fungal genome. Our data suggest a lineage-specific loss of a putative gibberellin-20-oxidase in the fungus, a gene fusion in the fungal mitochondrion, and a relocation of an algal chloroplast gene to the algal nucleus. Major technical obstacles during reconstruction of the holo-genome were coverage differences among individual genomes surpassing three orders of magnitude. Moreover, we show that GC-rich inverted repeats paired with nonrandom sequencing error in PacBio data can result in missing gene predictions. This likely poses a general problem for genome assemblies based on long reads.
Lichen-forming fungi are symbiotic organisms that synthesize unique natural products with potential for new drug leads. Here, we explored the pharmacological activity of six lichen extracts (Evernia prunastri, Pseudevernia furfuracea, Umbilicaria pustulata, Umbilicaria crustulosa, Flavoparmelia caperata, Platismatia glauca) in the context of cancer and inflammation using a comprehensive set of 11 functional and biochemical in vitro screening assays. We assayed intracellular Ca2+ levels and cell migration. For cancer, we measured tumor cell proliferation, cell cycle distribution and apoptosis, as well as the angiogenesis-associated proliferation of endothelial cells (ECs). Targeting inflammation, we assayed leukocyte adhesion onto ECs, EC adhesion molecule expression, as well as nitric oxide production and prostaglandin (PG)E2 synthesis in leukocytes. Remarkably, none of the lichen extracts showed any detrimental influence on the viability of ECs. We showed for the first time that extracts of F. caperata induce Ca2+ signaling. Furthermore, extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca reduced cell migration. Interestingly, F. caperata extracts strongly decreased tumor cell survival. The proliferation of ECs was significantly reduced by E. prunastri, P. furfuracea, and F. caperata extracts. The extracts did not inhibit the activity of inflammatory processes in ECs. However, the pro-inflammatory activation of leukocytes was inhibited by extracts from E. prunastri, P. furfuracea, F. caperata, and P. glauca. After revealing the potential biological activities of lichen extracts by an array of screening tests, a correlation analysis was performed to evaluate particular roles of abundant lichen secondary metabolites, such as atranorin, physodic acid, and protocetraric acid as well as usnic acid in various combinations. Overall, some of the lichen extracts tested in this study exhibit significant pharmacological activity in the context of inflammation and/or cancer, indicating that the group lichen-forming fungi includes promising members for further testing.