Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes

Abstract

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

Fig. 1.—

Comparison of Glomus irregulare DAOM-234179, Glomus sp. DAOM-240422, and G. cerebriforme mitochondrial genomes. The circular-mapping genomes were opened upstream of rnl to allow for easier comparisons. Genes on the outer and inner circumference are transcribed in a clockwise and counterclockwise direction, respectively. Gene and corresponding product names are atp6, 8, 9, ATP synthase subunit 6; cob, apocytochrome b; cox1–3, cytochrome c oxidase subunits; nad1–4, 4L, 5–6, NADH dehydrogenase subunits; rnl, rns, large and small subunit rRNAs; A-W, tRNAs, the letter corresponding to the amino acid specified by the particular tRNA followed by their anticodon. ORFs smaller than 100 amino acids are not shown.

Fig. 2.—

(A) Linear genome representation to compare the mitochondrial synteny between Glomus irregulare DAOM-234179, Glomus sp. DAOM-240422, and G. cerebriforme. The linear-mapping genomes were opened upstream of rnl to allow for easier comparisons. Corresponding gene clusters between G. irregulare DAOM-234179 and Glomus sp. DAOM-240422 are underlined in red, green, and blue. The newly formed intergenic regions of Glomus sp. DAOM-240422 are boxed in gray and tagged with a number. (B) Nucleotide identity comparison with BLASTn between the reshuffled Glomus sp. DAOM-240422 intergenic regions (atp9-cox1 [box 1], nad4L-rnl [box 2], and cox3-rns [box 3]) with their putative homologous sequence in G. irregulare DAOM-234179. Homologous regions are indicated by the projections with their corresponding percent identity.

Fig. 3.—

Similarity network of dpo sequences inserted in AMF mitochondrial genomes. (A) Each node represents a dpo insert colored by mitochondrial genome. The node size is proportional to the sequence length. Two nodes are connected by an edge when they share significant similarity (reciprocal best BLAST hit with a minimum of 1E−10 score, and 30% minimum identity covering at least 30% of the smallest sequence). The layout was produced by Cytoscape, using an edge-weighted force-directed model, which brings closer sequences sharing more similarity. Twelve homology groups were formed (labeled G1–G12). There are 97 nodes on that network with 336 edges. (B) Same network but nodes are colored by their mitochondrial intergenic region localization. The legend shows a linear size relationship between the smallest dpo sequence (Glomus irregulare DAOM-240415_cox3-rnl_4, 129 bp) and the largest one (G. cerebriforme atp6-atp9, 3444 bp).

Fig. 4.—

(A) Sub-cluster of the global network of shared amino acid similarities between the Glomeromycota DPO proteins and all homologous sequences found on GenBank. Building this data set was performed using translated Glomeromycota sequences as queries for a BLASTp search on the GenBank nr database (minimal e-value threshold of 1E−40, 20% minimal similarity covering at least 20% of the smallest sequence). The network layouts were further produced by Cytoscape software, using an edge-weighted force-directed model, meaning that genes sharing more protein similarity appear closer in the display. There are 135 nodes in that network with 2,520 edges. (B) DPO protein maximum likelihood tree obtained with the rtREV + CAT phylogenetic model. The bacterial/viral cluster was used to root the tree. Number on branches indicates bootstrap support values (<60% cut-off) and Bayesian inference values, respectively. The Bayesian analyses gave a similar topology (data not shown). The tree includes AMF (black), other fungi (orange), plants (green), virus (red), and bacteria (blue).