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Epulopiscium sp. type B (Lachnospiraceae) is an exceptionally large, highly polyploid, intestinal symbiont of the coral reef dwelling surgeonfish Naso tonganus. These obligate anaerobes do not form mature endospores and reproduce solely through the production of multiple intracellular offspring. This likely makes them dependent on immediate transfer to a receptive host for dispersal. During reproduction, only a small proportion of Epulopiscium mother-cell DNA is inherited. To explore the impact of this unusual viviparous lifestyle on symbiont population dynamics, we investigated Epulopiscium sp. type B and their fish hosts collected over the course of two decades, at island and reef habitats near Lizard Island, Australia. Using multi-locus sequence analysis, we found that recombination plays an important role in maintaining diversity of these symbionts and yet populations exhibit linkage disequilibrium (LD). Symbiont populations showed spatial but not temporal partitioning. Surgeonfish are long-lived and capable of traveling long distances, yet the population structures of Epulopiscium suggest that adult fish tend to not roam beyond a limited locale. Codiversification analyses and traits of this partnership suggest that while symbionts are obligately dependent on their host, the host has a facultative association with Epulopiscium. We suggest that congression of unlinked markers contributes to LD estimates in this and other recombinant populations of bacteria. The findings here inform our understanding of evolutionary processes within intestinal Lachnospiraceae populations.

Arbuscular mycorrhizal (AM) fungi form an intimate symbiosis with roots of more than 80% of land plants without eliciting a significant defense response, and how they do so is yet to be determined. Typically, plants mount a defense response upon sensing chitin in fungal walls, and to counteract this response, plant-pathogenic fungi secrete small effector proteins with chitin-binding LysM domains. In the AM fungus, Rhizophagus irregularis, a small, putatively-secreted LysM protein, which we refer to as RiSLM, is among the most highly expressed effector-like proteins during symbiosis. Here, we show that RiSLM expression is reduced during non-functional symbiosis with Medicago mutants, mtpt4-2 and vapyrin. We demonstrate that RiSLM can bind to both chitin and chitosan, and we model the protein-ligand interaction to identify possible binding sites. Finally, we have identified RiSLM homologs in five published R. irregularis isolate genomes and demonstrate that the gene is subject to a high rate of evolution and is experiencing positive selection, while still conserving putative function. Our results present important clues for elucidating a role for a LysM effector, RiSLM, in AM symbiosis.

Animals and plants interact with microbes by engaging specific surveillance systems, regulatory networks, and response modules that allow for accommodation of mutualists and defense against antagonists. Antimicrobial defense responses are mediated in both animals and plants by innate immunity systems that owe their functional similarities to convergent evolution. Like animals and plants, fungi interact with bacteria. However, the principles governing these relations are only now being discovered. In a study system of host and nonhost fungi interacting with a bacterium isolated from the host, we found that bacteria used a common gene repertoire to engage both partners. In contrast, fungal responses to bacteria differed dramatically between the host and nonhost. These findings suggest that as in animals and plants, the genetic makeup of the fungus determines whether bacterial partners are perceived as mutualists or antagonists and what specific regulatory networks and response modules are initiated during each encounter., Fungal-bacterial symbioses range from antagonisms to mutualisms and remain one of the least understood interdomain interactions despite their ubiquity as well as ecological and medical importance. To build a predictive conceptual framework for understanding interactions between fungi and bacteria in different types of symbioses, we surveyed fungal and bacterial transcriptional responses in the mutualism between Rhizopus microsporus (Rm) (ATCC 52813, host) and its Mycetohabitans (formerly Burkholderia) endobacteria versus the antagonism between a nonhost Rm (ATCC 11559) and Mycetohabitans isolated from the host, at two time points, before and after partner physical contact. We found that bacteria and fungi sensed each other before contact and altered gene expression patterns accordingly. Mycetohabitans did not discriminate between the host and nonhost and engaged a common set of genes encoding known as well as novel symbiosis factors. In contrast, responses of the host versus nonhost to endobacteria were dramatically different, converging on the altered expression of genes involved in cell wall biosynthesis and reactive oxygen species (ROS) metabolism. On the basis of the observed patterns, we formulated a set of hypotheses describing fungal-bacterial interactions and tested some of them. By conducting ROS measurements, we confirmed that nonhost fungi increased production of ROS in response to endobacteria, whereas host fungi quenched their ROS output, suggesting that ROS metabolism contributes to the nonhost resistance to bacterial infection and the host ability to form a mutualism. Overall, our study offers a testable framework of predictions describing interactions of early divergent Mucoromycotina fungi with bacteria.

Significance Endosymbiotic bacteria are found in diverse fungi, but little is known about how they communicate with their hosts. Some plant pathogenic bacteria use type III-translocated TAL effectors to control host transcription, and TAL-like proteins are encoded in genomes of the fungal endosymbiotic bacterium Mycetohabitans rhizoxinica. In this paper, we present evidence that these proteins are, like TAL effectors, type III-secreted, nuclear-localizing effectors that perturb host transcription and show that one enhances tolerance of the fungal host to cell membrane stress. Our characterization of an effector in a bacterial–fungal symbiosis opens a new door to molecular understanding of these interkingdom partnerships. Our findings also provide insight into the functional diversity and evolution of the TAL effector protein family., Symbioses of bacteria with fungi have only recently been described and are poorly understood. In the symbiosis of Mycetohabitans (formerly Burkholderia) rhizoxinica with the fungus Rhizopus microsporus, bacterial type III (T3) secretion is known to be essential. Proteins resembling T3-secreted transcription activator-like (TAL) effectors of plant pathogenic bacteria are encoded in the three sequenced Mycetohabitans spp. genomes. TAL effectors nuclear-localize in plants, where they bind and activate genes important in disease. The Burkholderia TAL-like (Btl) proteins bind DNA but lack the N- and C-terminal regions, in which TAL effectors harbor their T3 and nuclear localization signals, and activation domain. We characterized a Btl protein, Btl19-13, and found that, despite the structural differences, it can be T3-secreted and can nuclear-localize. A btl19-13 gene knockout did not prevent the bacterium from infecting the fungus, but the fungus became less tolerant to cell membrane stress. Btl19-13 did not alter transcription in a plant-based reporter assay, but 15 R. microsporus genes were differentially expressed in comparisons both of the fungus infected with the wild-type bacterium vs. the mutant and with the mutant vs. a complemented strain. Southern blotting revealed btl genes in 14 diverse Mycetohabitans isolates. However, banding patterns and available sequences suggest variation, and the btl19-13 phenotype could not be rescued by a btl gene from a different strain. Our findings support the conclusion that Btl proteins are effectors that act on host DNA and play important but varied or possibly host genotype-specific roles in the M. rhizoxinica–R. microsporus symbiosis.

The recent accumulation of newly discovered fungal-bacterial mutualisms challenges the paradigm that fungi and bacteria are natural antagonists. To understand the mechanisms that govern the establishment and maintenance over evolutionary time of mutualisms between fungi and bacteria, we studied a symbiosis of the fungus Rhizopus microsporus (Mucoromycotina) and its Burkholderia endobacteria. We found that nonhost R. microsporus, as well as other mucoralean fungi, interact antagonistically with endobacteria derived from the host and are not invaded by them. Comparison of gene expression profiles of host and nonhost fungi during interaction with endobacteria revealed dramatic changes in expression of lipid metabolic genes in the host. Analysis of the host lipidome confirmed that symbiosis establishment was accompanied by specific changes in the fungal lipid profile. Diacylglycerol kinase (DGK) activity was important for these lipid metabolic changes, as its inhibition altered the fungal lipid profile and caused a shift in the host-bacterial interaction into an antagonism. We conclude that adjustments in host lipid metabolism during symbiosis establishment, mediated by DGKs, are required for the mutualistic outcome of the Rhizopus-Burkholderia symbiosis. In addition, the neutral and phospholipid profiles of R. microsporus provide important insights into lipid metabolism in an understudied group of oleaginous Mucoromycotina. Lastly, our study revealed that the DGKs involved in the symbiosis form a previously uncharacterized clade of DGK domain proteins.

Arbuscular mycorrhizal fungi (AMF, Glomeromycotina), in addition to forming symbioses with the majority of land plants, harbor vertically transmitted endosymbiotic bacteria 'Candidatus Glomeribacter gigasporarum' (CaGg) and 'Candidatus Moeniiplasma glomeromycotorum' (CaMg). CaGg is a nonessential mutualist of AMF, whereas the lifestyle of CaMg is unknown. To start unraveling the interactions between AMF and their endosymbionts in nature, we examined diversity and distribution of AMF-associated endobacteria in North Atlantic dunes at Cape Cod. Of nearly 500 foredune AMF isolates successfully genotyped during a systematic study, 94% were classified as Gigasporaceae. Two percent of all AMF spores harbored CaGg, and 88% contained CaMg. CaGg was found only in the Gigasporaceae, whereas CaMg was present in Gigasporaceae, Acaulosporaceae, and Diversisporaceae. Incidence of CaGg across AMF was not affected by any of the environmental parameters measured, whereas distribution of CaMg in one of the fungal hosts was impacted by plant density. CaMg populations associated with AMF individuals displayed high levels of genetic diversity but no evidence of gene flow, suggesting that host physical proximity is not sufficient to facilitate horizontal transmission of CaMg. Finally, in addition to a novel lineage of CaGg, we discovered that AMF likely harbor Burkholderia-related bacteria with close phylogenetic affinity to free-living Burkholderia and endobacteria of other Mucoromycota fungi.

The mycoplasma-related endobacteria (MRE), representing a recently discovered lineage of Mollicutes, are widely distributed across arbuscular mycorrhizal fungi (AMF, Glomeromycota). AMF colonize roots of most terrestrial plants and improve plant mineral nutrient uptake in return for plant-assimilated carbon. The role of MRE in the biology of their fungal hosts is unknown. To start characterizing this association, we assessed partitioning of MRE genetic diversity within AMF individuals and across the AMF phylogeographic range. We further used molecular evolution patterns to make inferences about MRE codivergence with AMF, their lifestyle and antiquity of the Glomeromycota-MRE association. While we did not detect differentiation between MRE derived from different continents, high levels of diversity were apparent in MRE populations within AMF host individuals. MRE exhibited significant codiversification with AMF over ecological time and the absence of codivergence over evolutionary time. Moreover, genetic recombination was evident in MRE. These patterns indicate that, while MRE transmission is predominantly vertical, their complex intrahost populations are likely generated by horizontal transmission and recombination. Based on predictions of evolutionary theory, we interpreted these observations as a suggestion that MRE may be antagonists of AMF. Finally, we detected a marginally significant signature of codivergence of MRE with Glomeromycota and the Endogone lineage of Mucoromycotina, implying that the symbiosis between MRE and fungi may predate the divergence between these two groups of fungi.

Arbuscular mycorrhizal fungi (AMF, subphylum Glomeromycotina) are symbionts of most terrestrial plants. They commonly harbour endobacteria of a largely unknown biology, referred to as MRE ( M ollicutes /mycoplasma-related endobacteria). Here, we propose to accommodate MRE in the novel genus ‘Candidatus Moeniiplasma.’ Phylogeny reconstructions based on the 16S rRNA gene sequences cluster ‘Ca. Moeniiplasma ’ with representatives of the class Mollicutes , whereas phylogenies derived from amino acid sequences of 19 genes indicate that it is a discrete lineage sharing ancestry with the members of the family Mycoplasmataceae . Cells of ‘Ca. Moeniiplasma ’ reside directly in the host cytoplasm and have not yet been cultivated. They are coccoid, ~500 nm in diameter, with an electron-dense layer outside the plasma membrane. However, the draft genomes of ‘Ca. Moeniiplasma ’ suggest that this structure is not a Gram-positive cell wall. The evolution of ‘Ca. Moeniiplasma ’ appears to be driven by an ultrarapid rate of mutation accumulation related to the loss of DNA repair mechanisms. Moreover, molecular evolution patterns suggest that, in addition to vertical transmission, ‘Ca. Moeniiplasma ’ is able to transmit horizontally among distinct Glomeromycotina host lineages and exchange genes. On the basis of these unique lifestyle features, the new species ‘Candidatus Moeniiplasma glomeromycotorum’ is proposed.

Heritable endobacteria, which are transmitted from one host generation to the next, are subjected to evolutionary forces that are different from those experienced by free-living bacteria. In particular, they suffer consequences of Muller’s ratchet, a mechanism that leads to extinction of small asexual populations due to fixation of slightly deleterious mutations combined with the random loss of the most-fit genotypes, which cannot be recreated without recombination. Mycoplasma-related endobacteria (MRE) are heritable symbionts of fungi from two ancient lineages, Glomeromycota (arbuscular mycorrhizal fungi) and Mucoromycotina . Previous studies revealed that MRE maintain unusually diverse populations inside their hosts and may have been associated with fungi already in the early Paleozoic. Here we show that MRE are vulnerable to genomic degeneration and propose that they defy Muller’s ratchet thanks to retention of recombination and genome plasticity. We suggest that other endobacteria may be capable of raising similar defenses against Muller’s ratchet.

Bacterial endosymbionts are critical to the existence of many eukaryotes. Among them, vertically transmitted endobacteria are uniquely typified by reduced genomes and molecular evolution rate acceleration relative to free-living taxa. These patterns are attributable to genetic drift-dominated degenerative processes associated with reproductive dependence on the host. The degenerative evolution scenario is well supported in endobacteria with strict vertical transmission, such as essential mutualists of insects. In contrast, heritable endosymbionts that are nonessential to their hosts and engage occasionally in horizontal transmission are expected to display deviations from the degenerative evolution model. To explore evolution patterns in such nonessential endobacteria, we focused on Candidatus Glomeribacter gigasporarum ancient heritable mutualists of fungi. Using a collection of genomes, we estimated in Glomeribacter mutation rate at 2.03 × 10(-9) substitutions per site per year and effective population size at 1.44 × 10(8) Both fall within the range of values observed in free-living bacteria. To assess the ability of Glomeribacter to purge slightly deleterious mutations, we examined genome-wide dN/dS values and distribution patterns. We found that these dN/dS profiles cluster Glomeribacter with free-living bacteria as well as with other nonessential endosymbionts, while distinguishing it from essential heritable mutualists of insects. Finally, our evolutionary simulations revealed that the molecular evolution rate acceleration in Glomeribacter is caused by limited recombination in a largely clonal population rather than by increased fixation of slightly deleterious mutations. Based on these patterns, we propose that genome evolution in Glomeribacter is nondegenerative and exemplifies a departure from the model of degenerative evolution in heritable endosymbionts.

Many eukaryotes interact with heritable endobacteria to satisfy diverse metabolic needs. Some of these interactions are facultative symbioses, in which one partner is not essential to the other. Facultative symbioses are expected to be transitional stages along an evolutionary trajectory toward obligate relationships. We tested this evolutionary theory prediction in Ca. Glomeribacter gigasporarum, nonessential endosymbionts of arbuscular mycorrhizal fungi (Glomeromycota). We found that heritable facultative mutualisms can be both ancient and evolutionarily stable. We detected significant patterns of codivergence between the partners that we would only expect in obligate associations. Using codiverging partner pairs and the fungal fossil record, we established that the Glomeromycota‐Glomeribacter symbiosis is at least 400 million years old. Despite clear signs of codivergence, we determined that the Glomeribacter endobacteria engage in recombination and host switching, which display patterns indicating that the association is not evolving toward reciprocal dependence. We postulate that low frequency of recombination in heritable endosymbionts together with host switching stabilize facultative mutualisms over extended evolutionary times.

The rate of global deposition of Cd, Pb, and Zn has decreased over the past few decades, but heavy metals already in the soil may be mobilized by local and global changes in soil conditions and exert toxic effects on soil microorganisms. We examined in vitro effects of Cd, Pb, and Zn on critical life stages in metal-sensitive ecotypes of arbuscular mycorrhizal (AM) fungi, including spore germination, presymbiotic hyphal extension, presymbiotic sporulation, symbiotic extraradical mycelium expansion, and symbiotic sporulation. Despite long-term culturing under the same low-metal conditions, two species, Glomus etunicatum and Glomus intraradices , had different levels of sensitivity to metal stress. G. etunicatum was more sensitive to all three metals than was G. intraradices . A unique response of increased presymbiotic hyphal extension occurred in G. intraradices exposed to Cd and Pb. Presymbiotic hyphae of G. intraradices formed presymbiotic spores, whose initiation was more affected by heavy metals than was presymbiotic hyphal extension. In G. intraradices grown in compartmentalized habitats with only a portion of the extraradical mycelium exposed to metal stress, inhibitory effects of elevated metal concentrations on symbiotic mycelial expansion and symbiotic sporulation were limited to the metal-enriched compartment. Symbiotic sporulation was more sensitive to metal exposure than symbiotic mycelium expansion. Patterns exhibited by G. intraradices spore germination, presymbiotic hyphal extension, symbiotic extraradical mycelium expansion, and sporulation under elevated metal concentrations suggest that AM fungi may be able to survive in heavy metal-contaminated environments by using a metal avoidance strategy.

Arbuscular mycorrhizal fungi (AMF) are important members of the plant microbiome. They are obligate biotrophs that colonize the roots of most land plants and enhance host nutrient acquisition. Many AMF themselves harbor endobacteria in their hyphae and spores. Two types of endobacteria are known in Glomeromycota: rod-shaped Gram-negative Candidatus Glomeribacter gigasporarum, CaGg, limited in distribution to members of the Gigasporaceae family, and coccoid Mollicutes-related endobacteria, Mre, widely distributed across different lineages of AMF. The goal of the present study is to investigate the patterns of distribution and coexistence of the two endosymbionts, CaGg and Mre, in spore samples of several strains of Gigaspora margarita. Based on previous observations, we hypothesized that some AMF could host populations of both endobacteria. To test this hypothesis, we performed an extensive investigation of both endosymbionts in G. margarita spores sampled from Cameroonian soils as well as in the Japanese G. margarita MAFF520054 isolate using different approaches (molecular phylotyping, electron microscopy, fluorescence in situ hybridization and quantitative real-time PCR). We found that a single AMF host can harbour both types of endobacteria, with Mre population being more abundant, variable and prone to recombination than the CaGg one. Both endosymbionts seem to retain their genetic and lifestyle peculiarities regardless of whether they colonize the host alone or together. These findings show for the first time that fungi support an intracellular bacterial microbiome, in which distinct types of endobacteria coexist in a single cell.

Progress in understanding the biology of arbuscular mycorrhizal fungi is hampered by the limited number of species that can be successfully propagated and studied in vitro . We report the establishment of monoxenic cultures of Glomus etunicatum in association with excised Ri T-DNA transformed carrot roots. The fungus can be propagated in vitro using monoxenically formed resting spores and/or colonized root fragments. Modified White's medium buffered with 10 mM MES (pH 6) or MOPSO (pH 6.5) was most optimal for the host root growth as well as for G. etunicatum spore germination and mycorrhiza formation. The number of resting spores formed in vitro correlated positively with the length of roots occupied by arbuscular mycorrhizal structures, including arbuscules and vesicles. Spores first appeared in dual cultures within two weeks of root inoculation. Sporulation was asynchronous and continued until root senescence. Under applied culture conditions, spores achieved mature appearance within 5–7 d after their initiation. Approximately 6% of monoxenic spores were aborted at different stages of their development. Although G. etunicatum spores formed in vitro exhibited general morphological and anatomical similarity to soil-borne inoculum, they were significantly smaller and had thicker spore walls than their soil-borne counterparts. Caution should, therefore, be exercised in utilizing the in vitro system as a model of growth and development of glomalean fungi in soil.

Many eukaryotes interact with heritable endobacteria to satisfy diverse metabolic needs. Some of these interactions are facultative symbioses, in which one partner is not essential to the other. Facultative symbioses are expected to be transitional stages along an evolutionary trajectory toward obligate relationships. We tested this evolutionary theory prediction in Ca. Glomeribacter gigasporarum, nonessential endosymbionts of arbuscular mycorrhizal fungi (Glomeromycota). We found that heritable facultative mutualisms can be both ancient and evolutionarily stable. We detected significant patterns of codivergence between the partners that we would only expect in obligate associations. Using codiverging partner pairs and the fungal fossil record, we established that the Glomeromycota-Glomeribacter symbiosis is at least 400 million years old. Despite clear signs of codivergence, we determined that the Glomeribacter endobacteria engage in recombination and host switching, which display patterns indicating that the association is not evolving toward reciprocal dependence. We postulate that low frequency of recombination in heritable endosymbionts together with host switching stabilize facultative mutualisms over extended evolutionary times.

The prediction that progressive coupling of host and symbiont metabolic and reproductive interests leads to reduced mixing of symbiont lineages has been verified extensively in maternally transmitted bacterial endosymbionts of insects. To test whether this prediction is also applicable to associations of bacteria with fungi, we explored patterns of molecular evolution in two lineages of mutualistic endosymbionts of fungi: the Burkholderia endosymbionts of Rhizopus microsporus (Mucormycotina) and Candidatus Glomeribacter gigasporarum endosymbionts of arbuscular mycorrhizal fungi (Glomeromycota). We compared these two lineages with the closely related Candidatus Tremblaya princeps endosymbionts of mealybugs (Hemiptera, Coccoidea, Pseudococcidae) and to free-living Burkholderia species. To make inferences about the life histories of the endosymbionts, we relied on the empirically validated predictions of the nearly neutral theory of molecular evolution that a reduction of the effective population size increases the rate of fixation of slightly deleterious mutations. Our analyses showed that the slightly deleterious mutation accumulation patterns in the Burkholderia endosymbionts of Rhizopus were nearly indistinguishable from those in their free-living relatives. In contrast, Ca. Glomeribacter showed unique patterns of molecular evolution that differentiated them from both the Burkholderia endosymbionts of Rhizopus and from the Ca. Tremblaya endosymbionts of insects. These findings imply that reduced mixing of symbiont lineages is not a universal feature of symbioses between fungi and endocellular bacteria.