Identification and expression of functionally conserved circadian clock genes in lichen-forming fungi

doi:10.21203/rs.3.rs-1499077/v1

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Identification and expression of functionally conserved circadian clock genes in lichen-forming fungi

https://doi.org/10.21203/rs.3.rs-1499077/v1

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Lichen-forming fungi establish stable symbioses with green algae or cyanobacteria. Many species have broad distributions, both in geographic and ecological space, making them ideal subjects to study organism-environment interactions. However, little is known about the specific mechanisms that contribute to environmental adaptation in lichen-forming fungi. The circadian clock provides a well-described mechanism that contributes to regional adaptation across a variety of species, including fungi. Here, we identify the putative circadian clock components in phylogenetically divergent lichen-forming fungi. The core circadian genes (frq, wc-1, wc-2, frh) are present across the Fungi, including 31 lichen-forming species, and their evolutionary trajectories mirror overall fungal evolution. Comparative analyses of the clock genes indicate conserved domain architecture among lichen- and non-lichen-forming taxa. We used RT-qPCR to examine the core circadian loop of two unrelated lichen-forming fungi, Umbilicaria pustulata (Lecanoromycetes) and Dermatocarpon miniatum (Eurotiomycetes), to determine that the putative frq gene is activated in a light-dependent manner similar to the model fungus Neurospora crassa. Together, these results demonstrate that lichen-forming fungi retain functional light-responsive mechanisms, including a functioning circadian clock. Our findings provide a stepping stone into investigating the circadian clock in the lichen symbiosis, e.g. its role in adaptation and in synchronizing the symbiotic interaction.

circadian clock

evolution

functional genes

genome mining

phylogenetic analysis

phylogeny

symbiosis

The circadian clock is a well-described central molecular timekeeping mechanism that regulates biochemical processes within most organisms helping them to perceive and respond to abiotic and biotic environmental cues. Three properties of circadian clock systems indicate their indispensable role in modulating organism-environment interactions. Firstly, their ubiquity: circadian systems are present in nearly all organisms across the various kingdoms of life, and appear to have arisen at least three times during the evolution of life^1–3. Circadian clock homologs present in most plants, including algae, are highly conserved⁴, while the circadian clock components present in fungi and animals also show striking sequence similarity, for example BMAL1/CLOCK in animals and WC-1 in fungi¹. In prokaryotes, cyanobacteria circadian systems have been best studied, but homologs of the cyanobacteria circadian clock genes kaiA, kaiB and kaiC are also found in other prokaryotic lineages⁵.

The adaptive value of the circadian system becomes obvious in populations that have lost circadian rhythms over the course of their evolutionary history, or “switch off” their circadian clocks due to seasonal cues; in both cases, the circadian system becomes less rhythmic under conditions where rhythmicity does not provide adaptive information to the biological system. Independently-evolved populations of the cavefish Astyanax mexicanus show widespread disruption of circadian clock gene rhythmicity, as well as a reduction in rhythmic transcription compared to surface-dwelling populations⁶. Activating overwintering mechanisms in trees involves an interplay between photoreceptors and the circadian clock, which is then disrupted for the duration of the winter⁷. Although the circadian rhythms of Svalbard reindeer are attenuated during winter months ⁸, similarly to that of other Arctic mammals such as the muskox Ovibos moschatus ⁹ and the red fox Vulpes vulpes¹⁰.

The circadian clock provides fitness and performance advantages in a variety of model systems, further supporting its role in environmental adaptation. In plants, the circadian clock aligns chemical defenses to herbivore feeding patterns, regulates drought responses, and anticipates pathogen attack (see Xu et al. 2022¹¹ for a recent review of the role of the circadian clock in plant biotic and abiotic stress responses). In fungi, the asexual reproductive patterns of Neurospora crassa have long been observed to be regulated by the circadian clock^12,13, while strains of Neurospora discreta with habitat-specific circadian rhythms maintain higher fitness under their respective habitats¹⁴.

The central circadian oscillator of N. crassa is composed of the negative element frequency (frq) and its interactions with the White Collar Complex (WCC), composed of proteins encoded by the white collar-1 (wc-1) and white collar-2 (wc-2) genes. WCC activates transcription of frq, which ultimately represses its own expression by affecting the phosphorilation of WC-1 and WC-2^15,16. Another essential component of the circadian regulator is Frequency Interacting RNA Helicase (FRH), which plays a role in regulating FRQ expression by variably protecting FRQ against ubiquitin-mediated degradation and suppressing frq expression via interaction with the WCC¹⁷. This central clock oscillator in turn regulates a variety of downstream transcriptional and post-translational modifications¹⁸. The N. crassa circadian clock also exhibits a temperature compensation mechanism, consistent with other circadian clock systems in plants and animals^19,20.

The interacting circadian clocks in symbiotic systems are a growing topic of interest^21,22. Driven by a recognition of the key role of the gut microbiota on human health, host circadian clock inputs have been found to influence diurnal oscillations in gut microbiota in mice and humans²³. In plants, the microbiome influences host circadian clock function in the rhizosphere²⁴. Tightly-regulated circadian systems have been observed corals and algae^25,26, as well as between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacteria Vibrio fischeri²⁷. The arbuscular mycorrhizal fungus Rhizoglomus irregulare also contains a functioning circadian clock system²⁸, which has been hypothesized to play a role in the AMF-plant symbiosis.

The lichen symbiosis is composed of a fungal partner (mycobiont) and a photosynthesizing partner, either a green alga and/or a cyanobacterium (photobiont), plus a more or less specific suite of associated prokaryotic and eukaryotic microorganisms (e.g. ^29,30). The lichen lifestyle – a fungal nutritional mode that relies on photosynthetic products of internally accommodated algal symbionts – occurs in unrelated lineages across the fungal tree of life, but is most prevalent in the Leotiomyceta (e.g. Lecanoromycetes, Eurotiomycetes, Dothideomycetes) within the Ascomycota³¹. Lichens are ubiquitous in the landscape, thriving in a diverse range of habitats across nearly all ecosystems³². This ubiquity is likely related to lichens’ capability to withstand extreme abiotic stresses, such as complete desiccation^33,34. This ability to withstand stress may be due to variable stress response pathways relative to other sessile organisms; the lichen Endocarpon pustillum maintains active transcription of metabolism-associated genes during osmotic stress that are largely suppressed in plants and fungi³⁵.

Although lichens have been acknowledged as a symbiosis of interest to explore symbiotic circadian clock systems^22,36, little work has been done to elucidate the circadian clock mechanism in lichens aside from the identification of the putative frq ortholog in the lichen-forming fungus Umbilicaria pustulata³⁷. An important prerequisite step to further studies of the lichen circadian clock is to determine how conserved the core circadian clock and photosensory machinery is across phylogenetically unrelated mycobionts, as well as to determine whether this core machinery is functional.

Here, we investigate the presence of putative circadian clock components across the Fungi, focusing on major lineages in the Ascomycota that include lichens. We performed this investigation using a two-pronged approach: a phylogenetic analysis of putative homologs of the core circadian clock genes frq, wc-1, wc-2, and frh, and functional validation of one core mechanism in the fungal circadian clock. To investigate the presence of circadian clock homologs in lichen-forming fungi, we queried genomes of lichen-forming and non-lichen-forming fungi available in GenBank and JGI. To investigate whether frq homologs in lichen-forming fungal lineages maintain their primary circadian function as targets of the WCC, we then investigated the transcript abundance of frq putative homologs in response to light in Dermatocarpon miniatum (Ascomycota, Eurotiomycetes) and U. pustulata (Ascomycota, Lecanoromycetes), two species belonging to different Ascomycete classes.

We find that homologs of the core fungal circadian clock genes, frq, wc-1, wc-2, and frh are present in lichen-forming Lecanoromycetes, Eurotiomycetes and Dothideomycetes, and that these orthologs contain strongly conserved protein-coding sequences in the functional domains of these genes. We demonstrate the light-dependent expression of the core clock gene frq in two highly diverged lichen-forming lineages, U. pustulata and D. miniatum. Taken together, these results demonstrate that lichen mycobionts retain functional light-responsive mechanisms, including a functioning circadian clock, similar to those of non-lichen-forming filamentous fungi.

Identification and phylogenetic analysis of putative circadian clock genes

The four core fungal clock genes frq, wc-1, wc-2, frh of the model fungus Neurospora crassa were used as queries to search for homologs in the genomes of the lichen-forming and non-lichen-forming fungi via reciprocal BLAST in GenBank, through the Genome Portal of the Joint Genome Institute³⁸ and from Calchera et al. 2019³⁹; see Table S1.

Putative clock genes recovered from a broad range of fungal taxa across the Ascomycota, Basidiomycota, and Mucoromycota were used for the phylogenetic analysis. We made sure to include lichenized (and if possible non-lichenized) representatives of most Leotiomycete lineages that include lichens (Lecanoromyctes, Eurotiomycetes, Dothideomycetes). Amino acid sequence alignments were performed using MAFFT v7.450⁴⁰. After alignment, sequences were trimmed using TrimAl v1.2⁴¹, removing all columns with gaps in more than 20% of sequences in the alignment. Phylogenetic trees were inferred by maximum likelihood using RAxML version 8⁴² using the GAMMA BLOSUM62 model and 1,000 bootstrap replicates. The presence of functional domains between lichen-forming and non-lichen-forming lineages was performed using the protein domain and visualization tool DomainViz⁴³.

Study site and sample collection

Lichen thalli of Dermatocarpon miniatum and Umbilicaria pustulata were harvested from co-localized (ca. 600 m apart) sites in the vicinity of Eppstein, in the Taunus region of Germany (50°08′N 08°24′E). Single thallus fragments were taken from multiple populations (4 populations of U. pustulata and 5 populations of D. miniatum) to minimize disturbance. U. pustulata populations were located on horizontal or gently sloping, sun-exposed rock faces. D. miniatum populations were located on vertical, well-shaded rock faces. Samples were collected and briefly cleaned of debris before being transferred to labeled paper bags before immediate transfer to a growth chamber.

Growth conditions and light exposure treatment

After sample collection, lichen thalli were radially divided into two pieces and placed in individually wrapped 5 cm-diameter Petri dishes with sterile blotting paper and ca. 200 ul of sterile deionized water. Petri dishes were subsequently incubated for 72h in a plant growth chamber (CLF Plant Climatics GmbH, Wertingen, DE) at 16^o C under a light:dark 12:12 h light regime with 30 µmol m^− 2 s^− 1 of light being provided during the light phase. After 72h, all Petri dishes were wrapped in aluminum foil and placed in an additional paper box to induce a 24h dark incubation period. Shortly before 8:00 (dark:dark 24 = circadian time 0, subjective dawn), control thalli were harvested into liquid nitrogen. Aluminum foil was then removed and light-treated Petri dishes were exposed for 20 minutes. Petri dishes were kept equidistant from light sources in the growth chamber during light exposure before being harvested for RNA extraction.

Transcript abundance

After tissue harvesting from lichen thalli (150 mg), RNA was extracted with TRI Reagent (Zymo Research Europe GmbH, Freiburg, DE) according to the manufacturer’s instructions. Total RNA was quantified using a NanoPhotometer P 300 (Implen GmbH, Munich, DE) and cDNA was synthesized from 500 ng of totalRNA using RevertAid H-Minus first-strand cDNA synthesis kit (MBI Fermentas) according to the supplied protocol. Reverse transcription quantitative PCR (RT-qPCR) was per-formed in an CFX Opus 96 cycler (Bio-Rad Laboratories GmbH, Feldkirchen, DE) using the Luna Universal qPCR Master Mix kit (New England Biolabs GmbH, Frankfurt am Main, DE). Umbilicaria pustulata and Dermatocarpon miniatum actin-like 6A was used as reference genes.

Statistical analyses

All data were analyzed using R v.3.4.2⁴⁴ and RStudio v.1.0.153⁴⁵. Pairwise post-hoc comparisons were made using the R package EMMEANS⁴⁶ using Šidàk-adjusted contrasts after significant results were observed in a two-way ANOVA.

Homologs of core circadian oscillator and key input/output genes are present and highly conserved across lichen-forming fungal lineages

In order to investigate how widespread circadian clock-associated genes are across fungal lineages, we recovered putative homologs from the core circadian clock loop (frq, wc-1, wc-2, frh) and peripheral genes across a wide set of lichen-forming and non-lichen-forming fungi (see Table S1). Putative homologs were included in the phylogenetic analysis after reciprocal BLAST using Neurospora crassa protein sequences.

Putative homologs for lichen-forming taxa from a variety of clades were recovered, ranging from the class Lecanoromycetes to the classes Dothideomycetes and Eurotiomycetes. In order to explore potential variation between lichen-forming and non-lichen-forming fungal lineages, non-lichen-forming lineages from these classes were included when available from GenBank. Given that circadian clock homologs have been identified in lineages such as Rhizophagus irregularis in the Mucoromycota²⁸ and have been predicted by functional annotation in the Basidiomycota, we further included lineages in these clades in the phylogenetic analysis. Circadian homologs for wc-1 and wc-2, as well as for frq and other circadian clock genes, are present across most lichen-forming and select non-lichen-forming fungal clades ; nonetheless, some putative homologs are missing in some lineages, including notably frq in the Trichocomaceae (e.g. Aspergillus spp. and Penicillium spp.) within the Eurotiomycetes and in several Umbilicaria spp. in the lichen-forming Lecanoromycetes (Fig. 1).

Domain architecture of putative core circadian clock homologs is broadly conserved across lichen-forming and non-lichen-forming taxa

The phylogeny inferred for circadian clock homologs of WC-1 demonstrates a broad consensus with overall fungal evolution, including the phylogenetic placement of the lichenized taxa (Fig. 2a). In order to investigate whether functional domains in the core fungal circadian clock proteins were conserved, we compared the presence of functional domains between lichen-forming and non-lichen-forming lineages in the Ascomycota as well as the Basidiomycota/Mucoromycota in the Pfam (Fig. 2b-d) and PROSITE (Fig. 2e-g) protein domain databases using the protein domain and visualization tool DomainViz⁴³.

The evolution of wc-1 (Fig. 2), wc-2, frq and frh (Fig. S1-3) largely mirrors known class-level relationships of the fungal tree of life. Clock genes of lichen-forming taxa fall within the respective fungal classes (Lecanoromycetes, Eurotiomycetes, Dothideomycetes) with high support, rather than forming a monophyletic clade. This suggests that there are no shared signatures of lichenization in the analyzed clock genes.

The results demonstrate that two key regions of the Neurospora WC-1 protein are broadly conserved between lichen-forming Ascomycota, non-lichen-forming Ascomycota, and Basidiomycota/Mucoromycota lineages. The first of these is the PAS region, originally identified in the Drosophila melanogaster period clock (PER), Ah receptor nuclear translocator (ARNT) and single-minded (SIM) proteins, and which is involved in protein dimerization, light perception, light regulation and circadian rhythm regulation. At the C-terminal end, there is broad conservation across all three investigated lineages in the GATA-type zinc finger motif (Fig. 1b-g), involved in protein localization to a set of consensus sequences in the regulatory regions of many light- and clock-regulated genes⁴⁷.

Function of the core circadian clock gene frequency is conserved in two highly diverged lichen-forming fungal lineages

To investigate whether frq homologs in lichen-forming fungal lineages maintain their primary circadian function as targets of the WCC, we investigated the transcript abundance of frq putative homologs in response to light in Dermatocarpon miniatum (Ascomycota, Eurotiomycetes) and Umbilicaria pustulata (Ascomycota, Lecanoromycetes), two species belonging to different Ascomycete classes (Fig. 1, asterisks). Thalli were harvested from a colocalized site in the Taunus mountains of Germany (Fig. 2a) and were acclimatized for 72h under standardized conditions of 12:12 light:dark at 16° C, following which half of the thalli were exposed to 20 minutes of light (30 µE) before harvesting (Fig. 2b). All primers (Table S2) were previously tested for each species and no fluorescence was detected in negative controls (dH₂0 and RT- controls).

After significant gene:treatment interactions were detected in ANOVA for both species, post-hoc tests revealed significantly increased frq transcript abundance in both D. miniatum (p = 0.0012, Šidàk-adjusted contrasts) and U. pustulata (p = 0.0101, Šidàk-adjusted contrasts) after exposure to light, while transcript abundance of wc-1 was not significantly affected by the light treatment in either species (Fig. 2c). Statistical results for each species are summarized in Table S3.

The fungal circadian system, as elucidated initially in Neurospora crassa, is composed of three primary oscillators: a FRQ/WCC-dependent oscillator (FWO) as well as FRQ-independent (WC-FLO) and oscillators that are both FRQ- and WCC-independent (FLO)⁴⁸.The main FRQ/WCC-dependent oscillator, of which frq, wc-1, wc-2 and frh are the core components, was initially thought to be limited to Ascomycota, but has since been identified in various lineages across the Basidiomycota and Mucoromycota. Although a frq homolog has been previously identified in the genome of Umbilicaria pustulata within the lichen-forming Lecanoromycetes³⁷, there have been no previous reports on the presence or absence of circadian clock genes across lichen-forming fungi more broadly. The identification of putative homologs of circadian and light-sensing genes in a wide range of lichen-forming fungal lineages, combined with the conserved light-activated function of the core circadian clock component frq in two highly diverged lichen-forming fungi, points to the conservation of the circadian clock mechanism in mycobionts. Lichen-forming fungi exist in stable symbioses with algae and cyanobacteria, both of which have relatively well-understood circadian clocks in free-living conditions^49,50. The elucidation of the well-conserved fungal circadian mechanism in mycobionts thus opens the door for the investigation of how the circadian systems of myco- and photobionts interact and coordinate.

An increasing amount of attention has gone into holobiont chronobiology and the characteristics of circadian systems in symbiotic interactions²², although the vast majority of investigations into the evolution and function of circadian clock components have been made in free-living organisms. To date, most holobiont chronobiology studies deal with microbiotic effects of prokaryotes and unicellular prokaryotes on host chronobiology^21–24. Nonetheless, important inroads into the role of rhythmicity have been made in eukaryotic-eukaryotic systems ranging from arbuscular mycorrhizal fungal interactions and their host plants²⁸ as well as Symbiodinium algae and their coral host species^25,26. The lichen symbiosis is composed of partners with highly specialized and often well-investigated roles^51,52, and outputs from one symbiont (such as photosynthates) may affect the production of compounds by the other symbiont⁵³. Each symbiont produces compounds in a seasonally-variable manner^54,55, which may be regulated at least in part by the photosensory and circadian machinery of the symbionts. Lichen symbioses are also remarkably stable and geographically widespread, and the exchange of symbionts along gradients has been reported (e.g. ^56–58, providing an excellent model in which to study the effect of each partner’s circadian system in the holobiont.

While we identified putative homologs of all components of the core circadian clock machinery in nearly all lineages, some of the core circadian clock genes were missing in some lineages. The gene frq appears to be missing from certain isolated lichen-forming fungal lineages in the Umbilicariaceae, as well as from the Trichocomaceae (Aspergillus spp. and Penicillium spp.) within the Eurotiomycetes. The main paradigm of the evolution of frq in the Fungi has changed considerably over the last decades. Originally identified in Neurospora crassa (Ascomycota, Sordariomycetes), initial investigations of its evolution restricted its presence to within the Sordariomycetes, Leotiomycetes and Dothideomycetes within the Ascomycota ⁴⁸. More recent investigations have expanded this understanding considerably, and frq is understood to have evolved at least before the divergence of Mucoromycotina and Zoopagomycota from Dikarya ³⁷.

In our present study, the putative homologs of frq could not be found in the genomes of certain lichen-forming fungal lineages (U. phaea, U. deusta, U. spodochroa, U. freyi; see Davydov et al. 2017⁵⁹ for an overview of the Umbilicariaceae). Little life history or ecological rationale can at present be given for a parsimonious loss of the frq homolog in these lineages alone; interestingly, we were able to find a copy for one of these species (U. spodochroa) as well as for other lineages that do not have their genomes sequenced (Fig. S4), using a degenerate primer design approach (Table S2). It may be that frq is present in many more species but simply remains undetected in some genomes because of issues with local genome quality, genome assembly, annotation, and gene prediction. For the other circadian clock components investigated in this study, we found that frh and wc-1 were occasionally missing in single disparate lineages: wc-2 in Alternaria alternata and wc-2 and frh in Sordaria fimicola. Of the four genes investigated in this study, only the gene wc-1 was identified in every lineage without exception.

The rapid induction of frq expression by light has long been a characteristic phenotype of the main FWO oscillator in N. crassa (since e.g. Crosthwaite et al. 1995⁶⁰). This phenotype of light-induced frq expression has been observed in other Ascomycota such as the soil-living saprophyte Pyronema confluens (Pezizomycetes)⁶¹, as well as in the Mucoromycota, such as the arbuscular mycorrhizal fungus R. irregularis²⁸. The presence of frq, wc-1, and wc-2 alone, however, is not enough to infer a functional FWO oscillator: in the plant pathogen Verticillium dahliae, the frq homolog is present, but is largely unresponsive to light⁶². It is thus noteworthy that, in the present study, we observe that lichen-forming fungi from two highly diverged lineages, the Lecanoromycetes (U. pustulata) and the Eurotiomycetes (D. miniatum) both display frq light-dependent responses similar to the canonical responses of N. crassa, pointing to a functionally-conserved core circadian clock mechanism in at least two highly-diverged groups of lichen-forming fungi.

Circadian rhythms are central to the functioning of symbiotic interactions, as evidenced by the squid-Vibrio²⁷, coral-Symbiodinium^25,26, and plant-mycorrhiza²⁸ symbioses. The lichen system is an unexplored model of symbiosis with respect to the circadian clock that has great potential in contributing to our understanding of organism-organism, and organism-environment interactions. Having identified the core components of the fungal side of the lichen symbiosis and determined their functional conservation in at least two broadly diverged lineages of lichen-forming fungi, we can now address questions like: How does the circadian system influence seasonal and diurnal dynamics in the lichen symbiosis, such as growth, nitrogen fixation, photosynthesis, or ascospore discharge? Which seasonal and diurnal rhythms are important in this mutualism in the first place? To what extent do the circadian clocks of mycobiont and photobiont interact and interdepend? Is there circadian regulation in partner recruitment, establishment, and maintenance of the symbiotic association? What is the role of the circadian clock machinery in climatic niche specialization? An in-depth knowledge of the lichen circadian clock and its outputs and contributions to the symbiosis has the potential to enhance our understanding of symbiotic systems more broadly, from geographic distributions to interactions at the cellular and the molecular levels.

Acknowledgements

We thank Miklós Bálint and Leonie Schardt for access to and assistance in using the CFX Opus 96 cycler and Anjuli Calchera for helpful discussions and assistance with genome annotation and gene discovery.

Author contributions

H.V., F.D.G. and I.S. conceived the project and planned experiments; H.V., D.M. and I.S. collected the samples; H.V. and J.O. performed experimental work; H.V., D.M., G.S. and F.D.G. processed genomic data; H.V. analyzed the data and H.V., F.D.G. and I.S. wrote the manuscript. All authors contributed to the various drafts and gave final approval for publication.

Funding

Part of this work was funded by the LOEWE-Centre for Translational Biodiversity Genomics through the Hessen State Ministry of Higher Education, Research and the Arts (HMWK).

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Data availability

NCBI/GenBank Biosample IDs for the genomes utilized in this study published in Singh et al. 2022 (lichen/fungus): Umbilicaria freyi: SAMN27294873/SAMN26992773; Umbilicaria deusta: SAMN27294874/SAMN26992774; Umbilicaria hispanica (Lasallia hispanica): SAMN27294875/SAMN26992775; Umbilicaria phaea: SAMN27294876/SAMN26992776; Umbilicaria spodochroa: SAMN27294878/SAMN26992778; Umbilicaria subpolyphylla: SAMN27294879/SAMN26992779; Umbilicaria grisea: SAMN27294880/SAMN26992780; Dermatocarpon miniatum: SAMN27294881/SAMN26992781. JGI Project IDs for the genomes utilized in this study: Sclerophora sanguinea: 1052667; Xanthoria parietina: 16820; Usnea florida: 1051215; Lobaria pulmonaria: 1006353; Cladonia grayi: Cgr/DA2myc/ss v2.0 (portal name). NCBI/GenBank Biosample IDs for all 44 remaining accession numbers/IDs of published genomes used in this study can be found in Table S1.

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No competing interests reported.

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Identification and expression of functionally conserved circadian clock genes in lichen-forming fungi

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Abstract

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Introduction

Materials And Methods

Identification and phylogenetic analysis of putative circadian clock genes

Study site and sample collection

Growth conditions and light exposure treatment

Transcript abundance

Statistical analyses

Results

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

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