Daniele Armaleo

Induction of a complete secondary-product pathway in a cultured lichen fungus

CULBERSON, C. F., AND ARMALEO, D. 1992. Induction of a complete secondary-product pathway in a cultured lichen fungus. Experimental Mycology 16, 52-63. Experimental studies on the secondary metabolism characteristic of lichens have been impeded by the slow growth of the fungi and by the inconsistent results of many attempts to induce the pathways in the fungi isolated from their photosynthetic partners. In the present study, a lichen-specific secondary pathway was consistently induced in a lichen fungus (Cladonia grayi) grown in the absence of the alga. The depside (4-0-demethylsphaerophorin) and two depsidones (grayanic and 4-0-demethylgrayanic acids) found in the natural lichen began to accumulate a few days after the transfer of lightly fragmented mycelia from liquid to solid medium. Induction was enhanced on drier substrates and was correlated with the proliferation of aerial hyphae, where the major product (grayanic acid) accumulated in extracellular patches visible by fluorescence microscopy. The time course was analyzed by quantitative high-performance liquid chromatography of extracts from small cultures grown on nylon filters. Induction was rapid in view of the slow growth of the fungus, and secondary productivity was comparable to that of some nonlichen fungi. These results cotm polyketides; lichen fungi; Cladonia grayi; grayanic acid; depsides; depsidones; aerial hyphae; photobiont; hyphal differentiation.

Lichen chimeras: DNA analysis suggests that one fungus forms two morphotypes

In most lichens, the symbiosis between one fungus (mycobiont) and one photosynthetic partner (photobiont) results in a uniform thallus whose morphology is distinctive for each combination of symbionts. In some lichens, two morphologically different thalli, one containing a green alga, the other a cyanobacterium, are joined together in a chimera called photosymbiodeme. The question whether the same or two different mycobionts are involved in the formation of the different chimera components (morphotypes) is relevant to lichen morphogenesis, physiology, and taxonomy, but has not been answered conclusively to date. We have developed nucleic acid extraction procedures suitable for lichens. Using Southern hybridization and the polymerase chain reaction we demonstrate the genetic near-identity of the mycobionts forming paired morphotypes in two different photosymbiodemes.

Insights from the first putative biosynthetic gene cluster for a lichen depside and depsidone

The genes for polyketide synthases (PKSs), enzymes that assemble the carbon backbones of many secondary metabolites, often cluster with other secondary pathway genes. We describe here the first lichen PKS cluster likely to be implicated in the biosynthesis of a depside and a depsidone, compounds in a class almost exclusively produced by lichen fungi (mycobionts). With degenerate PCR with primers biased toward presumed PKS genes for depsides and depsidones we identified among the many PKS genes in Cladonia grayi four (CgrPKS13-16) potentially responsible for grayanic acid (GRA), the orcinol depsidone characteristic of this lichen. To single out a likely GRA PKS we compared mRNA and GRA induction in mycobiont cultures using the four candidate PKS genes plus three controls; only CgrPKS16 expression closely matched GRA induction. CgrPKS16 protein domains were compatible with orcinol depside biosynthesis. Phylogenetically CgrPKS16 fell in a new subclade of fungal PKSs uniquely producing orcinol compounds. In the C. grayi genome CgrPKS16 clustered with a CytP450 and an o-methyltransferase gene, appropriately matching the three compounds in the GRA pathway. Induction, domain organization, phylogeny and cluster pathway correspondence independently indicated that the CgrPKS16 cluster is most likely responsible for GRA biosynthesis. Specifically we propose that (i) a single PKS synthesizes two aromatic rings and links them into a depside, (ii) the depside to depsidone transition requires only a cytochrome P450 and (iii) lichen compounds evolved early in the radiation of filamentous fungi.

A Rapid and Inexpensive Method for the Purification of DNA from Lichens and their Symbionts

A simple DNA extraction method is described, applicable to many different kinds of lichens. The method involves the use of the detergents DTAB and CTAB and yields DNA that can be directly amplified with the polymerase chain reaction or digested with restriction enzymes.

Fungal and algal gene expression in early developmental stages of lichen-symbiosis

How plants and microbes recognize each other and interact to form long-lasting relationships remains one of the central questions in cellular communication. The symbiosis between the filamentous fungus Cladonia grayi and the single-celled green alga Asterochloris sp. was used to determine fungal and algal genes upregulated in vitro in early lichen development. cDNA libraries of upregulated genes were created with suppression subtractive hybridization in the first two stages of lichen development. Quantitative PCR subsequently was used to verify the expression level of 41 and 33 candidate fungal and algal genes respectively. Induced fungal genes showed significant matches to genes putatively encoding proteins involved in self and non-self recognition, lipid metabolism, and negative regulation of glucose repressible genes, as well as to a putative d-arabitol reductase and two dioxygenases. Upregulated algal genes included a chitinase-like protein, an amino acid metabolism protein, a dynein-related protein and a protein arginine methyltransferase. These results also provided the first evidence that extracellular communication without cellular contact can occur between lichen symbionts. Many genes showing slight variation in expression appear to direct the development of the lichen symbiosis. The results of this study highlight future avenues of investigation into the molecular biology of lichen symbiosis.

Light might regulate divergently depside and depsidone accumulation in the lichen Parmotrema hypotropum by affecting thallus temperature and water potential.

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.

Sizing the fungal and algal genomes of the lichen Cladonia grayi through quantitative PCR

Using a method based on quantitative PCR, we determined that the nuclear genome sizes for the mycobiont and photobiont of the lichen Cladonia grayi are 28.6 Mb and 106.7 Mb, respectively. This is the first genome size determination for lichens, and suggests that between 20,000 and 25,000 genes function in C. grayi. The mycobiont genome size is near the middle of the range observed within the Pezizomycota, the subphylum containing all known ascomycete lichen fungi. The genome size of the photobiont (the green alga Asterochloris sp.) is near the lower end of its class, the Trebouxiophyceae. Genomes in this size range can be sequenced at relatively low cost with current pyrosequencing-based methods. The genome sizing method requires very small amounts of precisely quantified DNA and should be applicable to any lichen whose symbionts can be reliably isolated from one another. Since the symbionts used in this project were isolated from soredia, the lichen’s vegetative propagules, we also describe a method for the establishment of axenic symbiont cultures from large numbers of soredia.

Mitotic intragenic recombination as a consequence of heteroduplex formation in Aspergillus nidulans

SummaryA diploid strain of Aspergillus nidulans with two heteroallelic mutations in the pabaA cistron (right arm of the first chromosome) has been studied. Part of the paba-independent colonies which have been examined was heterogeneous, i.e. they showed conidia of different colour and genotype. The genetic analysis of the various type of these heterogeneous colonies leads to the conclusion that, in Aspergillus nidulans, mitotic intragenic recombination is, in most cases, consequence of a single-strand break and exchange followed by the formation of a very long hybrid-DNA region (in our case a maximum of 22 meiotic units); the selected characteristics arise mainly by gene-conversion.Furthermore, data show a high negative interference between the selected crossing-over and a second crossing-over on the left arm and probably also on different chromosomes. The latter exchange occurs, as the former, between subchromatidic units.

Effect of ?-isopropylmalate on the synthesis of RNA and protein in Neurospora

SummaryThe leu-3/α-IPM (α-isopropylmalate) regulatory system, previously shown to control several genes of leucine, isoleucine, valine, and histidine biosynthesis, appears likely to be involved also in the regulation of overall RNA and protein synthesis in Neurospora. Upon addition of α-IPM the synthesis of all major species of stable RNA was found to be transiently inhibited by approximately 50%. A similar reduction was observed in overall protein synthesis. The inhibition was dependent in both cases on a functional leu-3 gene product, in conformance with previously established patterns of α-IPM dependent gene regulation. The overt resemblance of the phenomenon described here to the ‘stringent response’ of bacteria is noted but neither the mechanism of inhibition nor the precise role of α-IPM in the process has been established.

A method for simultaneous targeted mutagenesis of all nuclear rDNA repeats in Saccharomyces cerevisiae using CRISPR-Cas9

Saccharomyces cerevisiae has been the prime model to study the assembly and functionality of eukaryotic ribosomes. Within that vast landscape, the specific problem of mutagenizing all 150 nuclear rRNA genes was bypassed using strains whose chromosomal copies had been deleted and substituted by plasmid-borne rDNA. Work with these strains has produced important insights, but nucleolar structure is altered and such yeast-specific approaches are elaborate and not transferable to most other eukaryotes. We describe here a simple CRISPR-Cas9 based method to place targeted mutations in all 150 chromosomal rDNA repeats in yeast. The procedure per se is not expected to alter the nucleolus and is potentially applicable also to other eukaryotes. Yeast was transformed with a plasmid bearing the genes for Cas9 and for the guide RNA, engineered to target a site in the SSU region. Our mutagenesis plan included insertion of a spliceosomal intron in the normally intronless yeast nuclear rDNA. Despite the potential lethality of cutting all 150 rDNA repeats at the same time, yeast survived the Cas9 attack through inactivation of the cut sites either by point mutations or by inserting the intron, which was spliced out correctly from the rRNA transcript. In each mutant strain the same mutation was present in all rDNA repeats and was stably inherited even after removal of the Cas9 plasmid.