The lichen symbiosis re-viewed through the genomes of Cladonia grayi and its algal partner Asterochloris glomerata

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Algal virus; Coculture; Fungi; Gene expression; Gene family evolution; Horizontal gene transfer; Plant-fungal interactions; Symbiont autonomy; Symbiosis genes.

Fig. 1

The lichen Cladonia grayi. The most conspicuous parts of the Cladonia thallus are the goblet-shaped podetia that support the sexual and vegetative reproductive structures: the goblets’ upper margins are covered with brown fungal apothecia, sites of meiotic spore production and ejection into the air; the podetial surfaces are covered with green vegetative propagules called soredia, which are tiny alga-fungus packets detached by rain and wind and able to grow and differentiate into full thalli. Soredia are continuously produced and extruded onto the podetial surface from the underlying fungal tissue, which has algae embedded in it. The ground is covered with the less conspicuous, leaf-shaped parts of Cladonia called squamules (yellow arrowhead), which are tiny but fully differentiated lichen thalli with typical medullar, algal, and cortical layers. The grass-like bodies are bryophyte initials. The focus-stacked photograph was taken in D.A.’s lab by Thomas Barlow, who holds the copyright and consents to its use in this study

Fig. 2

Phylogenies, genome sizes and sequence distribution. Left side: Fungal (top) and algal (bottom) PhyML trees (LG + G + F + I) for C. grayi and A. glomerata involving, respectively, a random sample of 6000 and 4000 ungapped sites extracted from a concatenated alignment of 2137 and 683 orthologous protein families containing 794,828 and 159,356 ungapped sites. Bootstrap support values label internodes. Scales indicate nucleotide substitutions per site. Right side: Bars are proportional to genome size, and different shadings indicate the proportions of recent and older sequence replicas or of unique sequences. Duplicated sequences in genomes were revealed by BLAST alignment of the genomic sequence against itself at the nucleotide (BLASTN) or amino acid (TBLASTX) levels. The duplicated regions include regular genes as well as repeated elements (not yet fully characterized), but microsatellites and low complexity sequences were filtered out. Sequences that matched in both BLASTN and TBLASTX searches were only counted in the BLASTN category. Only alignments with e-values <1e^− 15 in both the BLASTN and TBLASTX analyses were considered

Fig. 3

A viral insertion in the Asterochloris genome. a GC content and gene distribution. The diagram represents a 1 Mb genomic region produced by joining scaffolds 120 and 80 at their inverted repeat-containing edges (purple triangles). The % GC content is proportional to the height and color intensity of the orange-yellow band. Genes are indicated by rectangles whose color represents the category of their best match in Genbank (BLASTP e-value <1e^− 5). The blue or red segments perpendicular to the Kb line are repeated sequences or gaps, respectively. The low GC region in yellow represents the remnant of a viral insertion (Additional file 4). b Origins of best matches. Most genes in the low GC region of A. glomerata are viral or prokaryotic in origin, in contrast to those in the genome as a whole

Fig. 4

Cladonia MAT loci and their evolution. a Configurations of the MAT loci in three Cladonia species. The top diagrammed alignment is based on the alignment between the annotated C. grayi MAT1–1 region (scaffold_00075:76000–90,000 at [67]) and a provisional sequence of the C. grayi MAT1–2 region (accession MH795990). The C. grayi MAT1–1 and MAT1–2 regions are drawn above the basepair indicator line. Under the line are the MAT1–1 regions derived from the genomes of two other Cladonia species (Additional file 5). In C. grayi, the conserved flanking regions are gray, while the unrelated central regions are stippled differently for each mating type. Dark or gray arrows represent genes and gene-segments. CLAGR_008123-RA is considered a putative MAT1–1-7 ortholog because of its location and its BLAST hits to MAT1–1-7 orthologs from Trichophyton and other fungi. b Evolutionary model. Horizontal colored arrows represent MAT idiomorphs. The central line represents the MAT locus configuration of a possible homothallic Cladonia ancestor, and the vertical arrows represent the putative transitions towards the present heterothallic MAT1–1 (orange) or MAT1–2 (blue) configurations (Additional file 5). The graded shading in the deletion triangle leading to MAT1–2 symbolizes the deletion’s undefined left boundary beyond MAT1–1-7

Fig. 5

Flagellar proteins. a Number of candidate flagella proteins in chlorophytes. Reference C. reinhardtii proteins of the CiliaCut protein set (blue bars) and flagella proteome (green bars) were searched for putative orthologs in sequenced motile and non-motile chlorophytes using the reciprocal best BLASTP hit criterion. b The 314 candidate A. glomerata flagella proteins identified from multiple sources of evidence (see Methods). c Distribution of flagella proteins across Chlorophytes. The left cladogram shows the likely evolutionary relationships of sequenced Chlorophytes. The ƒ mark indicates organisms known to build motile flagella. Crei: Chlamydomonas reinhardtii; Volvox: Volvox carteri; C169: Coccomyxa subellipsoidea C-169; NC64A: Chlorella variabilis NC64A; Otau: Ostreococcus tauri; Oluc: Ostreococcus lucimarinus; M. CCMP: Micromonas pusilla CCMP1545; M. RCC: Micromonas sp. RC299. Presence (dot) or absence (circle) of putative orthologs identified by reciprocal best BLASTP hit of C. reinhardtii outer dynein proteins, inner dynein proteins, radial spoke proteins, central pair proteins and intraflagellar transport proteins

Fig. 6

Cultures of C. grayi and A. glomerata reconstituted on filters in Petri dishes

Fig. 7

Differential fungal (C. grayi) and algal (A. glomerata) gene expression in coculture vs. monoculture. RPKM expression ratios are sorted from high to low. Genes considered unaffected in coculture are labeled gray (Co/Mo ~ 1). Those labeled black above or below the gray range are considered induced or repressed, respectively. Induction and repression thresholds correspond respectively to 2 and 0.5 for the fungus and 1.3 and 0.77 for the alga. Notice the smaller range of differential expression induction displayed by the alga under our experimental conditions (Additional file 6)

Fig. 8

Classes of genes differentially induced during early fungus-alga interactions in coculture. The pie charts divide the induced genes for each symbiont into three broad classes (numbers of genes in parentheses). The “better defined” genes are subdivided in groups roughly comparable between the symbionts (gray and white boxes). The area of each box is proportional to the percent of genes it contains relative to all better defined genes (265 for the fungus and 243 for the alga). The number behind each group’s name indicates its enrichment factor relative to the whole genome (see Methods). The hatched areas represent groups with less than 10 genes each. The p values for the enrichment of the indicated groups within the induced genes are all < 0.05, and most are << 10^− 3

Fig. 9

Secreted proteins among the proteins induced in coculture. The small black symbols coalescing into a curve represent the Co/Mo ratios of the genes induced in coculture; the circles represent the corresponding protein sizes, gray for non-secreted, black for secreted proteins. The average sizes (# of amino acids) of a) all genome proteins, b) all induced proteins, c) all induced and secreted proteins are a) 477, b) 381, c) 341 for the fungus and a) 447, b) 420, c) 436 for the alga. Proteins were considered secreted only if they scored as such in all three programs SignalP [109], TargetP [110], and TMHMM [111]. In TMHMM, a transmembrane domain prediction program, a protein was considered compatible with secretion only if it had either no predicted TM domains or only one at the N terminus. The data used in this figure are in Additional file 6

Fig. 10

A predicted ribitol transporter in C. grayi. a Differential transcription in coculture vs. monoculture of five putative sugar transporters in C. grayi. They were the top five BLAST hits obtained by querying the genome with the sequences of two functionally validated fungal D-sorbitol/D-mannitol/ribitol transporters [117]. Only CLAGR_004844-RA is induced in coculture (Co/Mo on Y axis; 1 means no induction.). b CLAGR_004844-RA amino acid sequence. The 12 transmembrane domains [118] are indicated in bold purple. Consensus amino acids for sugar transporters [119, 120] are highlighted in cyan. c Protein phylogeny (PhyML, 100 bootstraps) of the five C. grayi transporters. The green branches correspond to nodes with bootstrap support ≥70%. The C. grayi proteins are labeled brown. The other transporters are identified by GI number and by fungal species. Taxa are also labeled as ascomycetes (Asco) or basidiomycetes (Basidio), the latter used as outgroup. The CLAGR_004844-RA clade is highlighted yellow. Bar indicates amino acid substitutions/site

Fig. 11

Polyketide Synthase (PKS) and Non-Ribosomal Peptide Synthetase (NRPS) genes in C. grayi. The three protein categories in a, b, c are named on the right. CLAGR_009784 is a PKS-NRPS hybrid. The only PKS whose likely downstream product (grayanic acid) is known is CLAGR_002732 [34]. The length of the horizontal lines is proportional to gene length (vertical gray lines delimit 3-Kb segments). Graphic symbols for protein domains are indicated at top. Genes with a suz_1 prefix were reannotated manually. See Additional file 7 for further details

Fig. 12

Protein family tree of archaeal ATPases. The phylogram was constucted using FastTree [144] on a MAFFT [145] amino acid alignment of 91 putative archaeal ATPases from prokaryotes and eukaryotes. Branches with bootstrap values ≥0.77 are thickened. Bar indicates amino acid substitutions/site. To the ATPases present in the published Galdieria phylogeny [146], we added all the proteins above Methanocaldococcus_j__MJ0632 in this figure. The eukaryotic taxa shown represent most of those currently known to harbor putative archaea-derived ATPases. ATPases from Galdieria (Gs) are marked red, from green algae and plants green, from fungi brown. All branches in black are prokaryotic, except for the amoeba Dictyostelium at the base. Branch labels include the taxon name or symbol and a protein identifier. The Asterochloris (Aster) proteins are indicated by their gene names in the JGI database [68]. The phylogeny suggests several independent HGT events, but it cannot exclude a very ancient HGT from Archaea to a common eukaryotic ancestor followed by losses in most eukaryotes. Asterochloris, Galdieria, and Selaginella have the largest families of archaeal ATPases (with 26, 12, and 7 members, respectively). See also Additional file 7

Fig. 13

A unique set of Gα subunits is present in Cladonia. The protein phylogeny (PhyML, 100 bootstraps) of the eight C. grayi Gα subunits clusters into three major MAG A, MAG B, and MAG C clades (highlighted). The unique MAG C paralogs are shown on the bottom. The green branches correspond to nodes with bootstrap support ≥67%. The C. grayi proteins are labeled brown. Bar indicates amino acid substitutions/site. See also Additional file 8

Fig. 14

Evolution of transcription factor/regulator families in fungi (left) and algae (right). All the species used and numerical data are listed in Additional file 9. The C. grayi and A. glomerata abbreviations are bolded. Area of symbols is proportional to the change observed. Green circles: number of families gained, red circles: number of families lost. Green triangles: number of expanded families, red triangles: number of contracted families

Fig. 15

Nitrate assimilation gene clustering in Chlorophytes. In the upper part of the figure, the algal phylogeny (left) and the corresponding taxa (right) bracket the gene clusters and unclustered paralogs in each taxon. Gene and cluster lengths are to scale; color codes and acronyms are listed below the 5 kb bar. Phylogeny and clusters were obtained as described in Methods. The lower part of the figure displays as vertical bars the expression levels of the nitrate assimilation genes in the alga grown alone or with the fungus. The full names of the taxa listed from top to bottom are: Micromonas pusilla CCMP1545; Micromonas RCC299; Ostreococcus tauri; Ostreococcus lucimarinus; Ostreococcus sp. RCC809; Chlamydomonas reinhardtii; Volvox carteri; Chlorella variabilis NC64A; Coccomyxa subellipsoidea C-169; Asterochloris glomerata. All the corresponding genome data are at [164]