Lichens, encompassing 20,000 known species, are symbioses between specialized fungi (mycobionts), mostly ascomycetes, and unicellular green algae or cyanobacteria (photobionts). Here we describe the first parallel genomic analysis of the mycobiont Cladonia grayi and of its green algal photobiont Asterochloris glomerata. We focus on genes/predicted proteins of potential symbiotic significance, sought by surveying proteins differentially activated during early stages of mycobiont and photobiont interaction in coculture, expanded or contracted protein families, and proteins with differential rates of evolution. A) In coculture, the fungus upregulated small secreted proteins, membrane transport proteins, signal transduction components, extracellular hydrolases and, notably, a ribitol transporter and an ammonium transporter, and the alga activated DNA metabolism, signal transduction, and expression of flagellar components. B) Expanded fungal protein families include heterokaryon incompatibility proteins, polyketide synthases, and a unique set of G-protein α subunit paralogs. Expanded algal protein families include carbohydrate active enzymes and a specific subclass of cytoplasmic carbonic anhydrases. The alga also appears to have acquired by horizontal gene transfer from prokaryotes novel archaeal ATPases and Desiccation-Related Proteins. Expanded in both symbionts are signal transduction components, ankyrin domain proteins and transcription factors involved in chromatin remodeling and stress responses. The fungal transportome is contracted, as are algal nitrate assimilation genes. C) In the mycobiont, slow-evolving proteins were enriched for components involved in protein translation, translocation and sorting. The surveyed genes affect stress resistance, signaling, genome reprogramming, nutritional and structural interactions. The alga carries many genes likely transferred horizontally through viruses, yet we found no evidence of inter-symbiont gene transfer. The presence in the photobiont of meiosis-specific genes supports the notion that sexual reproduction occurs in Asterochloris while they are free-living, a phenomenon with implications for the adaptability of lichens and the persistent autonomy of the symbionts. The diversity of the genes affecting the symbiosis suggests that lichens evolved by accretion of many scattered regulatory and structural changes rather than through introduction of a few key innovations. This predicts that paths to lichenization were variable in different phyla, which is consistent with the emerging consensus that ascolichens could have had a few independent origins.

(2011). Insights from the first putative biosynthetic gene cluster for a lichen depside and depsidone. Mycologia: Vol. 103, No. 4, pp. 741-754.

Depsides and depsidones are the most common secondary products uniquely produced in lichens by the fungal symbiont, and they accumulate on the outer surface of its hyphae. Their biological roles are subject to debate. Quantitatively the compounds typical of a given lichen can vary dramatically from thallus to thallus. Several studies have addressed whether this variability is correlated with the light reaching different thalli, but the conclusions are contradictory. We addressed the question with the lichen Parmotrema hypotropum growing on unshaded, vertical tree trunks, a controlled natural environment where the light absorbed by each thallus over its lifetime is the only major position-dependent variable. The exact north-east-south-west orientation of each thallus was used to calculate its yearly light exposure based on astronomical and meteorological considerations. The calculated irradiation around the trunk, distributed over a continuous 40-fold intensity range, then was compared with the amount of compound per unit thallus weight, determined by quantitative thin layer chromatography. P. hypotropum accumulates the depside atranorin in the cortex and the depsidone norstictic acid in the medulla and around the algae. A direct correlation was observed between the yearly amount of light reaching the lichen and the amount of atranorin. In contrast, the amount of norstictic acid decreased with increasing light. Although we did not measure thallus temperature and water potential, a unifying interpretation of these and other published data is that depside/depsidone accumulation in lichens is mediated by localized changes in temperature and water potential produced by light absorption within each thallus. This suggests water relations-based functions for depsides and depsidones.

Primary biosynthetic enzymes involved in the synthesis of lichen polyphenolic compounds depsides and depsidones are non-reducing polyketide synthases (NR-PKSs), and cytochrome P450s. However, for most depsides and depsidones the corresponding PKSs are unknown. Additionally, in non-lichenized fungi specific fatty acid synthases (FASs) provide starters to the PKSs. Yet, the presence of such FASs in lichenized fungi remains to be investigated. Here we implement comparative genomics and metatranscriptomics to identify the most likely PKS and FASs for olivetoric acid and physodic acid biosynthesis, the primary depside and depsidone defining the two chemotypes of the lichen Pseudevernia furfuracea. We propose that the gene cluster PF33-1_006185, found in both chemotypes, is the most likely candidate for the olivetoric acid and physodic acid biosynthesis. This is the first study to identify the gene cluster and the FAS likely responsible for olivetoric acid and physodic acid biosynthesis in a lichenized fungus. Our findings suggest that gene regulation and other epigenetic factors determine whether the mycobiont produces the depside or the depsidone, providing the first direct indication that chemotype diversity in lichens can arise through regulatory and not only through genetic diversity. Combining these results and existing literature, we propose a detailed scheme for depside/depsidone synthesis.

We connect ribosome biogenesis to desiccation tolerance in lichens, widespread symbioses between specialized fungi (mycobionts) and unicellular phototrophs. We test whether the introns present in the nuclear ribosomal DNA of lichen mycobionts contribute to their anhydrobiosis. Self-splicing introns are found in the rDNA of several eukaryotic microorganisms, but most introns populating lichen rDNA are unable to self-splice, being either catalytically impaired group I introns, or spliceosomal introns ectopically present in rDNA. Although the mycobiont’s splicing machinery removes all introns from rRNA, Northern analysis indicates delayed post-transcriptional removal during rRNA processing, suggesting interference with ribosome assembly. To study the effects of lichen introns in a model system, we used CRISPR to introduce a spliceosomal rDNA intron from the lichen fungus Cladonia grayi into all nuclear rDNA copies of Saccharomyces cerevisiae, which lacks rDNA introns. Three intron-bearing yeast mutants were constructed with the intron inserted either in the 18S rRNA genes, the 25S rRNA genes, or in both. The mutants removed the introns correctly but had half the rDNA genes of the wildtype, grew 4.4–6 times slower, and were 40–1700 times more desiccation tolerant depending on intron position and number. Intracellular trehalose, a disaccharide implicated in desiccation tolerance, was detected at low concentration. Our data suggest that the interference of the splicing machinery with ribosome assembly leads to fewer ribosomes and proteins and to slow growth and increased desiccation tolerance in the yeast mutants. The relevance of these findings for slow growth and desiccation tolerance in lichens is discussed.