Mitochondrial genome of Phlebia radiata is the second largest (156 kbp) among fungi and features signs of genome flexibility and recent recombination events

Abstract

Mitochondria are eukaryotic organelles supporting individual life-style via generation of proton motive force and cellular energy, and indispensable metabolic pathways. As part of genome sequencing of the white rot Basidiomycota species Phlebia radiata, we first assembled its mitochondrial genome (mtDNA). So far, the 156 348 bp mtDNA is the second largest described for fungi, and of considerable size among eukaryotes. The P. radiata mtDNA assembled as single circular dsDNA molecule containing genes for the large and small ribosomal RNAs, 28 transfer RNAs, and over 100 open reading frames encoding the 14 fungal conserved protein subunits of the mitochondrial complexes I, III, IV, and V. Two genes (atp6 and tRNA-IleGAU) were duplicated within 6.1 kbp inverted region, which is a unique feature of the genome. The large mtDNA size, however, is explained by the dominance of intronic and intergenic regions (sum 80% of mtDNA sequence). The intergenic DNA stretches harness short (≤ 200 nt) repetitive, dispersed and overlapping sequence elements in abundance. Long self-splicing introns of types I and II interrupt eleven of the conserved genes (cox1,2,3; cob; nad1,2,4,4L,5; rnl; rns). The introns embrace a total of 57 homing endonucleases with LAGLIDADGD and GYI-YIG core motifs, which makes P. radiata mtDNA to one of the largest known reservoirs of intron-homing endonucleases. The inverted duplication, intergenic stretches, and intronic features are indications of dynamics and genetic flexibility of the mtDNA, not fully recognized to this extent in fungal mitochondrial genomes previously, thus giving new insights for the evolution of organelle genomes in eukaryotes.

Figure 1. Gene map of Phlebia radiata mtDNA.

Colour of the scale (kb) bar indicates orientation of transcription: clockwise (CW, white), counter-clockwise (CCW, grey). Bars mark protein-coding (blue) and RNA (red) genes, and alternative C-termini in atp6 and cox2 are depicted (orange). Intron type is indicated in colour: group I (light grey), group II (yellow), and uncertain (white). Within introns, the hypothetical and identified ORFs are indicated: over >200 amino-acid long ORFs (turquoise), and homing endonuclease domains GIY-YIG (black) and LAGLIDADG (dark grey). The transparent ribbon illustrates location of the 6,076 bp inversion-duplication. Asterisk indicates putative tRNA-Ile^CAU. The inner circle (scale at 12 o’clock in linear units) plots nucleotide bias (G/C skew) up to hexamers along each mtDNA position; G/C (red), A/T (blue), total strand bias (black), placing oriC around 11∶30 o’clock as the largest bias of G over C.

Figure 2. Contribution of the various features of P. radiata mtDNA to genome size.

Conserved coding sequence refers to the conserved fungal mitochondrial proteome ORFs, rRNAs and tRNAs. Significant ORFs refer to additional identified and hypothetical protein coding sequences with E-value<0.001 obtained by BLASTp queries. Freestanding refers to intronless. Intronic significant ORFs include homing endonucleases, also exon-to-exon-fused ORFs, excluding intergenic ORF HEGs (see text).

Figure 3. Schematic view of the C-termini regions of P. radiata mtDNA atp6 and cox2 genes.

Green lines denote atp6 coding sequence region and the last exon of cox2. Spheres/ovals represent LAGLIDADG 1 (green) and GIY-YIG (grey) homing endonuclease domains. A) Region of 201 bp (orange) with high 1 vs. 1 sequence similarity, corresponding to the 3′-end of the atp6 gene. Codon Adaptation Index (CAI) values are shown for the atp6 N-terminal region (blue) and for the regions of high sequence similarity. Reference codon usage is from: 1) atp6 N-terminus, 2) atp6 N-terminus and atp8-9, 3) atp6 N-terminus, atp8-9, cox1-3, nad1-5 and nad4L. B) Separated by three LAGLIDADG 1 domains and a GIY-YIG domain, two regions of high 1 vs. 1 sequence similarity (orange) exist for the last 66 bp of the cox2 gene. Image was generated with ExPASy PROSITE MyDomains Image Creator (http://prosite.expasy.org/mydomains/) and edited in Inkscape version 0.48.2 (http://inkscape.org/).

Figure 4. Codon usage of the P. radiata mitochondrial genome protein-coding gene open reading frames in comparison to selected Basidiomycota and Ascomycota species.

Neighbor-joining tree with topology derived from codon on codon root mean squared difference distance matrix (Table S3, Table S4). Agaricomycotina, blue; Ustilaginomycotina, red; Pucciniomycotina, black; Ascomycota species Gibberella zeae = Fusarium graminearum, green, as outgroup. Scale bar indicates nucleotide changes per site.

Figure 5. Phylogeny of fungal mitochondrial proteomes.

The statistically most likely tree was derived by Bayesian inference from a multi-gene superalignment of mtDNA-encoded proteins (2 019 aa positions, 44 taxa). Posterior consensus support values are depicted for branching, and nodes receiving ≤0.8 support were collapsed into polytomies. The tree was rooted from mid-point. Colours refer to phyla or sub-phyla: turquoise, Chytridiomycota; yellow, Monoblepharidomycota; orange brown, Glomeromycota; dark blue, Ascomycota; pink, Ustilaginomycotina (Basidiomycota); red, Pucciniomycotina (Basidiomycota); bright green, Agaricomycotina (Basidiomycota); Blastocladiomycota node as outgroup. Scale bar indicates amino-acid substitutions per site. For species and mtDNA accession, see Table 5.

Figure 6. Dispersed and inverted repeat sequences in P. radiata mtDNA.

Colors: black, conserved protein-coding, rRNA and tRNA genes; grey, introns; white, intergenic regions. The large 6.2 kb duplication-inversion is excluded from the figures. A) Red ribbons connect regions of significant (E-value <1×10⁻⁷) nucleotide sequence similarity. B) Coloured ribbons connect similar sequence regions. Only clusters with at least 6 similar repeat members are shown. The sequences were clustered as a function of similarity in CD-HIT Suite (h-cd-hit-est run with consecutive 0.75, 0.80, and 0.90 cut-off values) from a sequence set that returned <0.001 1 vs. 1 blastn values.