Depside and Depsidone Synthesis in Lichenized Fungi Comes into Focus through a Genome-Wide Comparison of the Olivetoric Acid and Physodic Acid Chemotypes of Pseudevernia furfuracea

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.

Depside and depsidone synthesis in lichenized fungi comes into focus through a genome-wide comparison of the olivetoric and physodic acid chemotype of Pseudevernia furfuracea

ABSTRACTPrimary biosynthetic enzymes involved in the synthesis of lichen polyphenolic compounds depsides and depsidones are Non-Reducing Polyketide Synthases (NR-PKSs), and cytochrome P450s (CytP450). However, for most depsides and depsidones the corresponding PKSs are unknown. Additionally, in non-lichenized fungi specific fatty acyl 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 the synthesis of olivetoric and physodic acid, 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 olivetoric and physodic acid biosynthesis. This is the first study to identify the gene cluster and the FAS likely responsible for physodic and olivetoric 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.

Buellia epigaea (Pers.) Tuck , a new record of lichenized fungus species for Antarctica

Buellia epigaea, a terricolous lichenized fungal species known from numerous localities in Northern Hemisphere, but only from Australia in Southern Hemisphere, is reported from Antarctica for the first time. Here we provide morphological, anatomical, and molecular characteristics (nrITS) of this species. Besides, the differences of B. epigaea with morphologically, ecologically or phylogenetically related species are discussed.

Two new synonyms of Pertusaria coccodes (Pertusariales, Ascomycota)

Pertusaria atropallida and P. uralensis are found to represent synonyms of the lichenized fungus P. coccodes. Lectotypes are selected for P. atropallida and P. uralensis.

Testing carbon and nitrogen sources for the in vitro growth of the model lichenized fungus Endocarpon pusillum Hedw.

To improve the efficiency of isolating and culturing lichen mycobionts, we performed a growth assay on an Australian strain of the soil-crust lichenized fungus Endocarpon pusillum Hedw. This assay determined the preferred nitrogen and carbon sources of the fungus by limiting the available nitrogen or carbon sources to single compounds found in soils, plants and lichen thalli. We found that the non-proteinaceous amino acid, GABA, produced the most growth of all nutrients when provided as the sole nitrogen source but was a poor carbon source. Fructose, glucose, cellobiose and sorbitol produced the most growth of all the carbon sources tested. Ammonium, nitrate and polyamines were poor nutrient sources. These findings correspond with reports of primary metabolite pools in other lichen species and may guide future studies involving growth of recalcitrant lichen mycobionts.

Remarkable cases of parallel evolution of the placodioid thallus growth form in the Lecanographaceae (Arthoniales) with the description of a new species of Alyxoria from Mexico

The new species Alyxoria sierramadrensis is described from Mexico where it inhabits limestone rocks. The lichen developing from this fungus is characterized by a placodioid to subfoliose thallus with a white pruinose surface; rounded to shortly elongated ascomata with a black epruinose margin and a widely exposed, white pruinose hymenial disc; hyaline, 3-septate ascospores, 17–25 × 7–9 μm; the presence of anthraquinones rendering the medulla orange. Phylogenetic analyses of nuLSU, mtSSU and RPB2 sequences place this species in the genus Alyxoria (Lecanographaceae). This generic affiliation is surprising because all known Alyxoria species have a crustose thallus. Lecanographaceae mainly includes species without a thallus (lichenicolous taxa) or with a thin crustose thallus, the only exception being Simonyella variegata with a fruticose thallus. The new species belongs to the Alyxoria ochrocheila subgroup, which includes lichens also frequently known to have anthraquinones, white pruinose hymenial discs and 3-septate ascospores. Phylogenetic analyses further determined the systematic position of the monotypic genus Phoebus. This genus, considered as an Arthoniales of uncertain family affiliation, is shown to belong to the Lecanographaceae. With its placodioid thallus, it is another example of a lichenized fungus with a deviating morphology in thallus structure for the family, increasing the number of remarkable cases of parallel evolution of lichen growth forms within the Arthoniales. Phoebus hydrophobius is newly recorded for Mexico.

Coregulation of dimorphism and symbiosis by cyclic AMP signaling in the lichenized fungusUmbilicaria muhlenbergii

Umbilicaria muhlenbergiiis the only known dimorphic lichenized fungus that grows in the hyphal form in lichen thalli but as yeast cells in axenic cultures. However, the regulation of yeast-to-hypha transition and its relationship to the establishment of symbiosis are not clear. In this study, we show that nutrient limitation and hyperosmotic stress trigger the dimorphic change inU. muhlenbergii. Contact with algal cells of its photobiontTrebouxia jamesiiinduced pseudohyphal growth. Treatments with the cAMP diphosphoesterase inhibitor IBMX (3-isobutyl-1-methylxanthine) induced pseudohyphal/hyphal growth and resulted in the differentiation of heavily melanized, lichen cortex-like structures in culture, indicating the role of cAMP signaling in regulating dimorphism. To confirm this observation, we identified and characterized two Gα subunitsUmGPA2andUmGPA3. Whereas deletion ofUmGPA2had only a minor effect on pseudohyphal growth, the ΔUmgpa3mutant was defective in yeast-to-pseudohypha transition induced by hyperosmotic stress orT. jamesiicells. IBMX treatment suppressed the defect of ΔUmgpa3in pseudohyphal growth. Transformants expressing theUmGPA3G45VorUmGPA3Q208Ldominant active allele were enhanced in the yeast-to-pseudohypha transition and developed pseudohyphae under conditions noninducible to the wild type. Interestingly,T. jamesiicells in close contact with pseudohyphae ofUmGPA3G45VandUmGPA3Q208Ltransformants often collapsed and died after coincubation for over 72 h, indicating that improperly regulated pseudohyphal growth due to dominant active mutations may disrupt the initial establishment of symbiotic interaction between the photobiont and mycobiont. Taken together, these results show that the cAMP-PKA pathway plays a critical role in regulating dimorphism and symbiosis inU. muhlenbergii.