Scrambled and not-so-tiny genomes of fungal endosymbionts

Symbiosis, defined as prolonged and close association of two or more different organisms, is a major driving force for expansion of biological diversity from genes to ecosystems. In the most intimate form, the symbiont lives inside the cells of its host, is heritable, and the two parties could no longer survive independently. Such heritable endosymbiosis blurs the line of what constitutes an individual organism and is considered the origin of organelles in present-day eukaryotic cells (1). Over the past decade, our understanding of the mutualistic endosymbionts of insects has been greatly expanded through examination of their genomes (2–4). It seemed that once a stable obligate symbiosis is developed, the endosymbiont genomes would undergo extreme levels of reduction and become highly static. These observations parallel those found in animal mitochondria (5) and angiosperm chloroplasts (6), which begs the question of how universal these evolutionary trends are among heritable endosymbionts. In PNAS two companion articles (7, 8) provide a first glimpse of the endosymbiont genomes from a group of ancient and diverse fungi.

The fungal hosts discussed in these papers, arbuscular mycorrhizal fungi (AMF) belonging to the phylum Glomeromycota (9) (Fig. 1), are themselves obligate symbionts of land plants. In their mutualistic relationships these root-associated fungi provide the plants with mineral nutrients and water and in return receive carbohydrates derived from photosynthesis. These fungal–plant associations could be traced back to >400 Ma and are thought to be pivotal in the origin of the land plants and the functioning of terrestrial ecosystems (10). Most AMF harbor heritable endosymbionts named Mollicutes-related endobacteria (MRE) (11), which have unknown biological roles. To infer the functions of MRE, the authors of these two studies (7, 8) performed genome sequencing of MRE from four diverse AMF species, including Dentiscutata heterogama (Dh), Racocetra verrucosa (Rv), Claroideoglomus etunicatum (Ce), and Rhizophagus clarus (Rc) (Fig. 1 and Table 1).

Fig. 1.

Consensus phylogeny of AMF in the phylum Glomeromycota. Adapted from ref. 9; genera with insufficient support are omitted and the four orders are highlighted by colored background. The four endosymbiont genomes characterized in the companion articles (7, 8) are labeled in bold by the species name abbreviations of their hosts.

Table 1.

Key statistics of MRE genomes in comparison with representative Mycoplasma species and insect symbionts

Genome	No. of chromosomal contigs or scaffolds	Combined size, kbp	%GC	No. of CDS	No. of CDS/kbp	Accession no.
Dh MRE	2	709	33.9	668	0.94	PRJEB8356
Rv MRE	99	1,228	33.6	1,842	1.50	JQIB01000000
Ce MRE	67	663	34.3	1,078	1.63	JPXH01000000
Rc MRE	34	740	32.0	1,032	1.39	JPXG01000000
Mycoplasma hominis	1	665	27.1	541	0.81	NC_013511
Mycoplasma pneumoniae	1	811	40.0	684	0.84	NC_017504
Mycoplasma genitalium	1	580	31.7	509	0.88	NC_000908
Mycoplasma mycoides	1	1,212	24.0	1,017	0.84	NC_005364
“Candidatus Nasuia deltocephalinicola”	1	112	17.1	137	1.22	CP006059
“Candidatus Sulcia muelleri”	1	190	24.1	187	0.98	CP010105
“Candidatus Moranella endobia”	1	538	43.5	406	0.75	CP002243
Buchnera aphidicola	1	641	26.3	564	0.88	BA000003

MRE are related to Mycoplasma in the class Mollicutes (11), which is a group of wall-less bacteria that are mostly animal pathogens. These four MRE genomes have a size range of ∼663–1,228 kilobase pairs (kbp) and a GC content of ∼32–34%, which are comparable to those found among horizontally transmitted Mycoplasma (Table 1). The extreme genome reduction (i.e., down to the size range of ∼112–641 kbp), such as that observed in the heritable endosymbionts of insects (2–4), has not occurred in these MRE despite their long evolutionary history of being heritable endosymbionts (>400 Ma) in comparison with those insect endosymbionts (<300 Ma) (12).

Other than not having experienced extreme genome reduction, these MRE are different from most insect endosymbionts by possessing highly dynamic genomes. For the MRE associated with Rv and Rc AMF, a single phylotype was found based on the 16S rRNA gene sequences. However, multiple recombinant types of chromosomal contigs were found, which prevents the assembly of these contigs into a single sequence representing the chromosome of each species. This intraspecific genome heterogeneity, in which strains share identical 16S rRNA but have diverged in their gene content, have been reported in a few recent studies on insect symbionts (13–15). These findings challenge the common practice of using 16S rRNA sequences for defining operational taxonomic units and also our definition of species in bacteria. The case of Ce MRE, for which multiple 16S phylotypes were found within individual fungal spores, seems to support the interpretation of this genome heterogeneity as a form of sympatric speciation (14). Curiously, the Dh MRE (7) differs from the other three MRE (8) in lacking intraspecific genome heterogeneity (such that the genome is well-assembled) and having a much lower gene density (Table 1). The explanations for these differences remain to be investigated.

Despite these differences, both studies agreed on several key findings. First, ∼3–5% of the protein-coding sequences (CDS) in these MRE genomes seem to have been acquired from their fungal hosts. Intriguingly, most of these genes contain protein domains involved in eukaryotic cell processes, such as leucine-rich repeats (LRR; protein–protein interactions), heterokaryon incompatibility (HET; nonself recognition and programmed cell death in fungi), and sentrin/small ubiquitin-like modifier (SUMO; eukaryotic posttranslational modification). It is conceivable that these genes are likely involved in the MRE–AMF interactions and contributed to the evolutionary success of MRE. Second, the aforementioned genome heterogeneity and putative horizontal gene transfers were likely to have been promoted by the presence of mobile elements and repeated sequences. The main evidence includes finding transposase genes at rearrangement junctions and the presence of recombinase A (recA; homologous recombination). Notably, traces of plectroviral invasion were found in Rv and Ce MRE. This Mollicutes-specific phage has been linked to genome rearrangements and horizontal gene transfers in Spiroplasma (16). Taken together, these factors contributing to MRE genome plasticity may be the key to counteract the degenerative evolution observed in other heritable endosymbionts, although how MRE escape from elevated levels of genetic drift (17, 18) is unknown. Finally, the metabolic capabilities of these MRE seemed to be quite limited. Similar to their sister lineage (either the Hominis or the Pneumoniae group within Mycoplasma—the two studies disagreed on this point), MRE lost genes for cell wall biosynthesis, the tricarboxylic acid cycle, and oxidative phosphorylation. The absence of these genes may reflect the ancestral state of the MRE–Hominis–Pneumoniae clade and supports a high level of host dependency for MRE. Particularly, the DNA replication initiator (dnaA) and cell division protein (ftsZ) are absent in Dh MRE (7), and several of the DNA/RNA polymerase subunits are absent in the other three MRE (8). These findings suggest that the cell functioning and proliferation of MRE may be under the control of their AMF hosts, which is likely the outcome of long-term coevolution. However, one big caveat of these gene content analyses is that functional annotation was not available for >50% of the MRE genes, so these inferences should be made cautiously.

In conclusion, these two studies on MRE genomes provide an opportunity to compare and contrast the evolutionary patterns between ancient endosymbionts of fungi and insects. The similarities, and more interestingly the differences, expand what we know about these peculiar life forms. For future studies, heritable endosymbionts of other major eukaryotic branches would certainly be of interest. Perhaps somewhat disappointingly, the exact functions of MRE remain largely a mystery despite our having four genome sequences available. This frustration is shared by many genome analyses, particularly for those lineages that are highly divergent from model organisms. Unfortunately, no easy solution is available to address this issue. A possible alternative approach to dissect this complex biological system may involve attacking from the host’s side. Few of the AMF lineages seem to have lost their MRE symbionts (11), and among them Rhizophagus irregularis is the only AMF species whose genome sequence is available (19). Future comparisons among these MRE-free AMF with those still maintaining their symbionts, or experimental reintroduction/replacement of symbionts (20), could provide some further clues to solve this puzzle.