The Mitochondrial Genome of the Glomeromycete Rhizophagus sp. DAOM 213198 Reveals an Unusual Organization Consisting of Two Circular Chromosomes

Maryam Nadimi 0 Franck O.P. Stefani 0 Mohamed Hijri 0 0 De partement de Sciences Biologiques, Universite de Montr e al, Institut de Recherche en Biologie Ve ge tale (IRBV) , Quebec , Canada Mitochondrial (mt) genomes are intensively studied in Ascomycota and Basidiomycota, but they are poorly documented in basal fungal lineages. In this study, we sequenced the complete mtDNA of Rhizophagus sp. DAOM 213198, a close relative to Rhizophagus irregularis, a widespread, ecologically and economical relevant species belonging to Glomeromycota. Unlike all other known taxonomically close relatives harboring a full-length circular chromosome, mtDNA of Rhizophagus sp. reveals an unusual organization with two circular chromosomes of 61,964 and 29,078 bp. The large chromosome contained nine protein-coding genes (atp9, nad5, cob, nad4, nad1, nad4L, cox1, cox2, and atp8), small subunit rRNA gene (rns), and harbored 20 tRNA-coding genes and 10 orfs, while the small chromosome contained five protein-coding genes (atp6, nad2, nad3, nad6, and cox3), large subunit rRNA gene (rnl) in addition to 5 tRNA-coding genes, and 8 plasmid-related DNA polymerases (dpo). Although structural variation of plant mt genomes is well documented, this study is the first report of the presence of two circular mt genomes in arbuscular mycorrhizal fungi. Interestingly, the presence of dpo at the breakage point in intergenes cox1-cox2 and rnl-atp6 for large and small mtDNAs, respectively, could be responsible for the conversion of Rhizophagus sp. mtDNA into two chromosomes. Using quantitative real-time polymerase chain reaction, we found that both mtDNAs have an equal abundance. This study reports a novel mtDNA organization in Glomeromycota and highlights the importance of studying early divergent fungal lineages to describe novel evolutionary pathways in the fungal kingdom. Introduction Mitochondria are membrane-bound organelles that are involved in several cell processes such as adenosine triphosphate (ATP) production via oxidative phosphorylation, respiration, RNA maturation, and protein synthesis. Mitochondria are also involved in cell division, growth, and death. They harbor their own genetic material that has evolved from an ancestral prokaryote genome. The endosymbiotic theory (Margulis 1971) suggests that the origin of nuclear genome of eukaryotic cells evolved in parallel to the origin of mitochondrial (mt) genome (Gray et al. 1999; Lang et al. 1999). Structure, size, and even function of mt genomes are variable among eukaryotes (Fukuhara et al. 1993; Drissi et al. 1994; Nosek and Tomaska 2003; Alverson et al. 2011; Nadimi et al. 2012; Beaudet, Nadimi, et al. 2013). Previous studies have shown that mitochondria exhibit a large diversity of genome architectures. For example, linear, circular, and fragmented mtDNAs have been described in Cucumis, Phythium, Ichthyosporean protists, Globodera pallida, Pediculus humanus capitis, Candida labiduridarum, Candida frijolesensis, and Brachionus plicatilis (Martin 1995; Armstrong et al. 2000; Burger et al. 2003; Suga et al. 2008; Alverson et al. 2011; Valach et al. 2011; Shao et al. 2012). Genome reshuffling and evolution of mtDNA structures have been observed in phylogenetically distant eukaryotic lineages as well as in closely related species (Fukuhara et al. 1993; Wilson and Williamson 1997). Conversion of circular genomes to monomeric linear genomes has been shown to occur by an insertion of linear plasmids with inverted terminal repeats (Schnare et al. 1986; Heinonen et al. 1987), resulting in the extension of mt genome size. Another feature of mtDNA is the size variability among eukaryotic lineages, spanning from ~6 kb in Plasmodium falciparum (Apicomplexa) to 11.3 Mb in the angiosperm genus Silene (Conway et al. 2000; Sloan et al. 2012). Mitochondria without any genes and organisms lacking mitochondria have been reported (reviewed in Keeling and Slamovits 2004). The gene content of mtDNA also varies broadly from 5 genes in Plasmodium (Conway et al. 2000) to 100 genes in jakobid flagellates (Burger et al. 2013), while 4050 genes are commonly observed in mtDNA of eukaryotes. Gene content, size of introns, intergenic regions, and mobile elements such as open reading frames (orfs), plasmid-related DNA polymerase sequences (dpo), and short inverted repeats (SIRs) are the major causes of polymorphism in mt genomes of eukaryotes. Arbuscular mycorrhizal fungi (AMF) are members of the phylum Glomeromycota (Sch ussler et al. 2001) and they represent an early-diverging fungal lineage dating back to the Early Devonian (Remy et al. 1994; Redecker et al. 2000). AMF are plant root-inhabiting fungi, where they form mutualistic symbiotic associations with ~80% of vascular plants (Smith and Read 2008). They promote plant growth by enhancing mineral uptake, in particular phosphorus, and protect plants against pathogens by controlling the growth of some soil fungal pathogens or by inducing plant defense responses (Ismail et al. 2011, 2013; Ismail and Hijri 2012). Recently, nuclear and mitochondrial genomics of AMF have been intensively studied (Tisserant et al. 2012, 2013; Halary et al. 2013). The first published mt genome of AMF was Rhizophagus irregularis isolate 494, (previously named Glomus intraradices and then Glomus irregulare) followed by the publication of the mt genomes of 11 taxa belonging to the genera Rhizophagus, Glomus, and Gigaspora (Lee and Young 2009; Formey et al. 2012; Nadimi et al. 2012; Pelin et al. 2012; Beaudet, Nadimi, et al. 2013; Beaudet, Terrat, et al. 2013; de la Providencia et al. 2013). AMF identification using the traditional ribosomal DNA markers of the nuclear genomes is uncertain due to high levels of intraspecific variations (Stockinger et al. 2009; Kruger et al. 2012; Schoch and Seifert 2012). Therefore, the publication of mt genomes provides useful data to identify AMF strains. For instance, sequences from intergenic and intronic regions are very divergent, which allows discrimination of closely related isolates (Formey et al. 2012; Beaudet, Terrat, et al. 2013; de la Providencia et al. 2013). Mitochondrial genome sequencing provides insights into the mtDNA evolution within Glomeromycota. Indeed, mtDNA structure in Glomeromycota has been shown to undergo different evolutionary mechanisms such as fragmented genes (Nadimi et al. 2012), lateral gene transfer, insertion/excision of mobile elements (Beaudet, Nadimi, et al. 2013; Beaudet, Terrat, et al. 2013), and transmission of SIRs (Formey et al. 2012; Beaudet, Terrat, et al. 2013). AMF mt genomes have been invaded by different types of selfish mobile genetic elements (MGEs) or mobilomes, such as homing endonuclease, plasmid-related DNA polymerase (dpo), and SIRs (Formey et al. 2012; Beaudet, Terrat, et al. 2013). However, their movement and recombination mechanisms are not clearly understood. MGEs are typically known as DNA fragments encoding enzymes and other proteins that mediate (...truncated)