Study of growth-improving and sporophore-inducing endobacteria isolated from Pleurotus pulmonarius.

Several Pleurotus species (oyster mushrooms) are commercially cultivated in India owing to the favorable tropical agro-climatic conditions. However, there are only a few studies on the microbiome of mushrooms, especially oyster mushrooms. The aim of this study was to assess the effect of endobacteria on mycelial growth, spawning, sporophore development, and proximate composition of P. pulmonarius. We isolated several bacterial strains from the sporophores of P. pulmonarius and assessed the in vitro production of indole acetic acid, ammonia, and siderophores. The selected bacteria were individually supplemented with spawn, substrate, or both for sporophore production. Three of 130 isolates were selected as mycelial growth-promoting bacteria in both solid and submerged fermentation. These bacterial isolates were identified through Gram staining, biochemical characterization, and 16S rRNA sequencing. Isolate PP showed 99.24% similarity with Priestia paraflexa, whereas isolates PJ1 and PJ2 showed 99.78% and 99.65% similarities, respectively, with Rossellomorea marisflavi. The bacterial supplementation with spawn, substrate, or both, increased the biological efficiency (BE) and nutrient content of the mushrooms. The bacterial supplementation with substrate augmented BE by 64.84%, 13.73%, and 27.13% using PJ2, PP, and PJ1, respectively; under similar conditions of spawn supplementation, BE was increased by 15.24%, 47.30%, 48.10%, respectively. Overall, the supplementation of endobacteria to improve oyster mushroom cultivation may open a new avenue for sustainable agricultural practices in the mushroom industry.

The endohyphal microbiome: current progress and challenges for scaling down integrative multi-omic microbiome research.

As microbiome research has progressed, it has become clear that most, if not all, eukaryotic organisms are hosts to microbiomes composed of prokaryotes, other eukaryotes, and viruses. Fungi have only recently been considered holobionts with their own microbiomes, as filamentous fungi have been found to harbor bacteria (including cyanobacteria), mycoviruses, other fungi, and whole algal cells within their hyphae. Constituents of this complex endohyphal microbiome have been interrogated using multi-omic approaches. However, a lack of tools, techniques, and standardization for integrative multi-omics for small-scale microbiomes (e.g., intracellular microbiomes) has limited progress towards investigating and understanding the total diversity of the endohyphal microbiome and its functional impacts on fungal hosts. Understanding microbiome impacts on fungal hosts will advance explorations of how "microbiomes within microbiomes" affect broader microbial community dynamics and ecological functions. Progress to date as well as ongoing challenges of performing integrative multi-omics on the endohyphal microbiome is discussed herein. Addressing the challenges associated with the sample extraction, sample preparation, multi-omic data generation, and multi-omic data analysis and integration will help advance current knowledge of the endohyphal microbiome and provide a road map for shrinking microbiome investigations to smaller scales. Video Abstract.

Tripartite interactions comprising yeast-endobacteria systems in the gut of vector mosquitoes.

It is shown that bacteria use yeast as a niche for survival in stressful conditions, therefore yeasts may act as temporary or permanent bacterial reservoirs. Endobacteria colonise the fungal vacuole of various osmotolerant yeasts which survive and multiply in sugar-rich sources such as plant nectars. Nectar-associated yeasts are present even in the digestive system of insects and often establish mutualistic symbioses with both hosts. Research on insect microbial symbioses is increasing but bacterial-fungal interactions are yet unexplored. Here, we have focused on the endobacteria of Wickerhamomyces anomalus (formerly Pichia anomala and Candida pelliculosa), an osmotolerant yeast associated with sugar sources and the insect gut. Symbiotic strains of W. anomalus influence larval development and contribute digestive processes in adults, in addition to exerting wide antimicrobial properties for host defence in diverse insects including mosquitoes. Antiplasmodial effects of W. anomalus have been shown in the gut of the female malaria vector mosquito Anopheles stephensi. This discovery highlights the potential of utilizing yeast as a promising tool for symbiotic control of mosquito-borne diseases. In the present study, we have carried out a large Next Generation Sequencing (NGS) metagenomics analysis including W. anomalus strains associated with vector mosquitoes Anopheles, Aedes and Culex, which has highlighted wide and heterogeneous EB communities in yeast. Furthermore, we have disclosed a Matryoshka-like association in the gut of A stephensi that comprises different EB in the strain of W. anomalus WaF17.12. Our investigations started with the localization of fast-moving bacteria-like bodies within the yeast vacuole of WaF17.12. Additional microscopy analyses have validated the presence of alive intravacuolar bacteria and 16S rDNA libraries from WaF17.12 have identified a few bacterial targets. Some of these EB have been isolated and tested for lytic properties and capability to re-infect the yeast cell. Moreover, a selective competence to enter yeast cell has been shown comparing different bacteria. We suggested possible tripartite interactions among EB, W. anomalus and the host, opening new knowledge on the vector biology.

Investigating Endobacteria that Thrive Within Mucoromycota.

Metagenomics approaches have revealed the importance of Mucoromycota in the evolution and functioning of plant microbiomes. Comprised of three subphyla (Glomeromycotina, Mortierellomycotina, and Mucoromycotina), this early diverging lineage of fungi encompasses species of mycorrhizal fungi, root endophytes, plant pathogens, and many decomposers of plant debris. Interestingly, several taxa of Mucoromycota share a common feature, that is, the presence of endobacteria within their mycelia and spores. The study of these endosymbiotic bacteria is still a challenging task. However, given recent improvements in the sensitivity of culture-free approaches, a deeper understanding of such microbial interactions is now possible and fuels an emerging research field. In this chapter, we report how Mucoromycota, in particular Mortierellomycotina, and their endobacteria can be investigated using a combination of diverse cellular biology, microscopy, and molecular techniques.

Microbe Profile: Gigaspora margarita, a multifaceted arbuscular mycorrhizal fungus.

Gigaspora margarita is a cosmopolitan arbuscular mycorrhizal fungus, which - as an obligate symbiont- requires being associated to a host plant to accomplish its life cycle. It is characterized by huge white spores, the development of extraradical auxiliary cells, and the lack of intraradical vesicles. Its genome is dominated by transposable elements and is one of the largest fungal genomes so far sequenced. G. margarita has the peculiar feature to host taxonomically different endobacteria in its cytoplasm. The development of a cured line has allowed us to demonstrate how the endobacteria have a positive impact on the fungal physiology and -with a cascade effect- on the mycorrhizal plant.

Advances and Challenges in Fluorescence in situ Hybridization for Visualizing Fungal Endobacteria.

Several bacteria have long been known to interact intimately with fungi, but molecular approaches have only recently uncovered how cosmopolitan these interactions are in nature. Currently, bacterial-fungal interactions (BFI) are inferred based on patterns of co-occurrence in amplicon sequencing investigations. However, determining the nature of these interactions, whether the bacteria are internally or externally associated, remains a grand challenge in BFI research. Fluorescence in situ hybridization (FISH) is a robust method that targets unique sequences of interest which can be employed for visualizing intra-hyphal targets, such as mitochondrial organelles or, as in this study, bacteria. We evaluate the challenges and employable strategies to resolve intra-hyphal BFI to address pertinent criteria in BFI research, such as culturing media, spatial distribution of bacteria, and abundance of bacterial 16S rRNA copies for fluorescent labeling. While these experimental factors influence labeling and detection of endobacteria, we demonstrate how to overcome these challenges thorough permeabilization, appropriate media choice, and targeted amplification using hybridization chain reaction FISH. Such microscopy imaging approaches can now be utilized by the broader research community to complement sequence-based investigations and provide more conclusive evidence on the nature of specific bacterial-fungal relationships.

Detection and Identification of Novel Intracellular Bacteria Hosted in Strains CBS 648.67 and CFCC 80795 of Biocontrol Fungi Metarhizium.

"Endosymbiosis" is a cohesive form of a symbiotic association. Endobacteria exist in many fungi and play important roles in fungal host biology. Metarhizium spp. are important entomopathogenic fungi for insect pest control. In the present study, we performed comprehensive ana-lyses of strains of Metarhizium bibionidarum and M. anisopliae using PCR, phylogenetics, and fluorescent electron microscopy to identify endobacteria within hyphae and conidia. The results of the phylogenetic ana-lysis based on 16S rRNA gene sequences indicated that these endobacteria were the most closely related to Pelomonas puraquae and affiliated with Betaproteobacteria. Ultrastructural observations indicated that endobacteria were coccoid and less than 500| |nm in diameter. The basic characteristics of endobacteria in M. bibionidarum and M. anisopliae were elucidated, and biological questions were raised regarding their biological functions in the Metarhizium hosts.

Third Generation Genome Sequencing Reveals That Endobacteria in Nematophagous Fungi Esteya vermicola Contain Multiple Genes Encoding for Nematicidal Proteins.

Esteya vermicola is the first recorded endoparasitic nematophagous fungus with high infectivity capacity, attacking the pinewood nematode Bursaphelenchus xylophilus which causes pine wilt disease. Endosymbionts are found in the cytoplasm of E. vermicola from various geographical areas. We sequenced the genome of endobacteria residing in E. vermicola to discover possible biological functions of these widespread endobacteria. Multilocus phylogenetic analyses showed that the endobacteria form a previously unidentified lineage sister to Phyllobacterium myrsinacearum species. The number of genes in the endobacterium was 4542, with 87.8% of the proteins having a known function. It contained a high proportion of repetitive sequences, as well as more Acyl-CoA synthetase genes and genes encoding the electron transport chain, compared with compared with plant-associated P. zundukense Tri 48 and P. myrsinacearum DSM 5893. Thus, this symbiotic bacterium is likely to be more efficient in regulating gene expression and energy release. Furthermore, the endobacteria in nematophagous fungi Esteya vermicola contained multiple nematicidal subtilase/subtilisin encoding genes, so it is likely that endobacteria cooperate with the host to kill nematodes.

Effect of Endosymbiotic Bacteria on Fungal Resistance Toward Heavy Metals.

Most studies on metal removal or tolerance by fungi or bacteria focus on single isolates, without taking into consideration that some fungi in nature may be colonized by endobacteria. To address this knowledge gap, we investigated the tolerance and removal of diverse metals with two fungal species: Linnemannia elongata containing Burkholderia-related endobacteria and Benniella erionia containing Mollicute-related endobacteria. Isogenic lines of both species were generated with antibiotic treatments to remove their respective endobacteria. Experiments involved comparing the isogenic lines and wild type fungi in relation to the minimum inhibitory concentration for the metals, the fungal ability to remove these different metals via atomic adsorption spectroscopy, and the interaction of the metals with specific functional groups of the fungi and fungi-bacteria to determine the role of the bacteria via attenuated total reflection fourier transformed infrared (ATR-FTIR). Finally, we determined the influence of different metal concentrations, associated with moderate and high fungal growth inhibition, on the presence of the endobacteria inside the fungal mycelium via quantitative real-time PCR. Results showed that the presence of the endosymbiont increased B. erionia resistance to Mn2+ and increased the removal of Fe2+ compared to isogenic lines. The absence of the endosymbiont in L. elongata increased the fungal resistance toward Fe2+ and improved the removal of Fe2+. Furthermore, when the bacterial endosymbiont was present in L. elongata, a decrease in the fungal resistance to Ca2+, Fe2+, and Cr6+was noticeable. In the ATR-FTIR analysis, we determined that C-H and C = O were the major functional groups affected by the presence of Cu2+, Mn2+, and Fe2+ for L. elongata and in the presence of Cu2+ and Ca2+ for B. eronia. It is noteworthy that the highest concentration of Pb2+ led to the loss of endobacteria in both L. elongata and B. eronia, while the other metals generally increased the concentration of endosymbionts inside the fungal mycelium. From these results, we concluded that bacterial endosymbionts of fungi can play a fundamental role in fungal resistance to metals. This study provides the first step toward a greater understanding of symbiotic interactions between bacteria and fungi in relation to metal tolerance and remediation.

Transcriptional Profiles of a Foliar Fungal Endophyte (Pestalotiopsis, Ascomycota) and Its Bacterial Symbiont (Luteibacter, Gammaproteobacteria) Reveal Sulfur Exchange and Growth Regulation during Early Phases of Symbiotic Interaction.

Symbiosis with bacteria is widespread among eukaryotes, including fungi. Bacteria that live within fungal mycelia (endohyphal bacteria) occur in many plant-associated fungi, including diverse Mucoromycota and Dikarya. Pestalotiopsis sp. strain 9143 is a filamentous ascomycete isolated originally as a foliar endophyte of Platycladus orientalis (Cupressaceae). It is infected naturally with the endohyphal bacterium Luteibacter sp. strain 9143, which influences auxin and enzyme production by its fungal host. Previous studies have used transcriptomics to examine similar symbioses between endohyphal bacteria and root-associated fungi such as arbuscular mycorrhizal fungi and plant pathogens. However, currently there are no gene expression studies of endohyphal bacteria of Ascomycota, the most species-rich fungal phylum. To begin to understand such symbioses, we developed methods for assessing gene expression by Pestalotiopsis sp. and Luteibacter sp. when grown in coculture and when each was grown axenically. Our assays showed that the density of Luteibacter sp. in coculture was greater than in axenic culture, but the opposite was true for Pestalotiopsis sp. Dual-transcriptome sequencing (RNA-seq) data demonstrate that growing in coculture modulates developmental and metabolic processes in both the fungus and bacterium, potentially through changes in the balance of organic sulfur via methionine acquisition. Our analyses also suggest an unexpected, potential role of the bacterial type VI secretion system in symbiosis establishment, expanding current understanding of the scope and dynamics of fungal-bacterial symbioses. IMPORTANCE Interactions between microbes and their hosts have important outcomes for host and environmental health. Foliar fungal endophytes that infect healthy plants can harbor facultative endosymbionts called endohyphal bacteria, which can influence the outcome of plant-fungus interactions. These bacterial-fungal interactions can be influential but are poorly understood, particularly from a transcriptome perspective. Here, we report on a comparative, dual-RNA-seq study examining the gene expression patterns of a foliar fungal endophyte and a facultative endohyphal bacterium when cultured together versus separately. Our findings support a role for the fungus in providing organic sulfur to the bacterium, potentially through methionine acquisition, and the potential involvement of a bacterial type VI secretion system in symbiosis establishment. This work adds to the growing body of literature characterizing endohyphal bacterial-fungal interactions, with a focus on a model facultative bacterial-fungal symbiosis in two species-rich lineages, the Ascomycota and Proteobacteria.

Symbiotic and toxinogenic Rhizopus spp. isolated from soils of different papaya producing regions in Mexico.

Mucoralean fungi from the genus Rhizopus are common inhabitants of terrestrial ecosystems, being some pathogens of animals and plants. In this study, we analyzed the symbiotic and toxinogenic potential of Rhizopus species derived from agricultural soils dedicated to the production of papaya (Carica papaya L.) in Mexico. Four representative strains of soil-derived Rhizopus spp. were analyzed employing molecular, microscopic, and metabolic methods. The ITS phylogenies identified the fungi as Rhizopus microsporus HP499, Rhizopus delemar HP475 and HP479, and Rhizopus homothallicus HP487. We discovered that R. microsporus HP499 and R. delemar HP475 harbor similar endofungal bacterial symbionts that belong to the genus Mycetohabitans (Burkholderia sensu lato) and that none of the four fungi were associated with Narnavirus RmNV-20S and RmNV-23S. Intriguingly, the interaction between R. delemar - Mycetohabitans showed different phenotypes from known R. microsporus - Mycetohabitans symbioses. Elimination of bacteria in R. delemar HP475 did not cause a detrimental effect on fungal growth or asexual reproduction. Moreover, metabolic and molecular analyses confirmed that, unlike symbiotic R. microsporus HP499, R. delemar HP475 does not produce rhizoxin, one of the best-characterized toxins produced by Mycetohabitans spp. The rhizoxin (rhi) biosynthetic gene cluster seems absent in this symbiotic bacterium. Our study highlights that the symbioses between Rhizopus and Mycetohabitans are more diverse than anticipated. Our findings contribute to expanding our understanding of the role bacterial symbionts have in the pathogenicity, biology and evolution of Mucorales.

Allocation of Carbon from an Arbuscular Mycorrhizal Fungus, Gigaspora margarita, to Its Gram-Negative and Positive Endobacteria Revealed by High-Resolution Secondary Ion Mass Spectrometry.

Arbuscular mycorrhizal fungi are obligate symbionts of land plants; furthermore, some of the species harbor endobacteria. Although the molecular approach increased our knowledge of the diversity and origin of the endosymbiosis and its metabolic possibilities, experiments to address the functions of the fungal host have been limited. In this study, a C flow of the fungus to the bacteria was investigated. Onion seedlings colonized with Gigaspora margarita, possessing Candidatus Glomeribacter gigasporarum (CaGg, Gram-negative, resides in vacuole) and Candidatus Moeniiplasma glomeromycotorum (CaMg, Gram-positive, resides in the cytoplasm,) were labelled with 13CO2. The 13C localization within the mycorrhiza was analyzed using high-resolution secondary ion mass spectrometry (SIMS). Correlative TEM-SIMS analysis of the fungal cells revealed that the 13C/12C ratio of CaGg was the lowest among CaMg and mitochondria and was the highest in the cytoplasm. By contrast, the plant cells, mitochondria, plastids, and fungal cytoplasm, which are contributors to the host, showed significantly higher 13C enrichment than the host cytoplasm. The C allocation patterns implied that CaMg has a greater impact than CaGg on G. margarita, but both seemed to be less burdensome to the host fungus in terms of C cost.

Symbiotic responses of Lotus japonicus to two isogenic lines of a mycorrhizal fungus differing in the presence/absence of an endobacterium.

As other arbuscular mycorrhizal fungi, Gigaspora margarita contains unculturable endobacteria in its cytoplasm. A cured fungal line has been obtained and showed it was capable of establishing a successful mycorrhizal colonization. However, previous OMICs and physiological analyses have demonstrated that the cured fungus is impaired in some functions during the pre-symbiotic phase, leading to a lower respiration activity, lower ATP, and antioxidant production. Here, by combining deep dual-mRNA sequencing and proteomics applied to Lotus japonicus roots colonized by the fungal line with bacteria (B+) and by the cured line (B-), we tested the hypothesis that L. japonicus (i) activates its symbiotic pathways irrespective of the presence or absence of the endobacterium, but (ii) perceives the two fungal lines as different physiological entities. Morphological observations confirmed the absence of clear endobacteria-dependent changes in the mycorrhizal phenotype of L. japonicus, while transcript and proteomic datasets revealed activation of the most important symbiotic pathways. They included the iconic nutrient transport and some less-investigated pathways, such as phenylpropanoid biosynthesis. However, significant differences between the mycorrhizal B+/B- plants emerged in the respiratory pathways and lipid biosynthesis. In both cases, the roots colonized by the cured line revealed a reduced capacity to activate genes involved in antioxidant metabolism, as well as the early biosynthetic steps of the symbiotic lipids, which are directed towards the fungus. Similar to its pre-symbiotic phase, the intraradical fungus revealed transcripts related to mitochondrial activity, which were downregulated in the cured line, as well as perturbation in lipid biosynthesis.

Wide distribution of the Ustilago maydis-bacterium endosymbiosis in naturally infected maize plants.

We have previously described that laboratory strains of Ustilago maydis, a fungal pathogen of maize and its ancestor teosinte, harbor an intracellular bacterium that enables the fungus to fix nitrogen. However, it is not clear whether other strains isolated from nature also harbor endosymbiotic bacteria, and whether these fix nitrogen for its host. In the present study, we isolated U. maydis strains from naturally infected maize. All the isolated strains harbored intracellular bacteria as determined by PCR amplification of the 16S rRNA gene, and some of them showed capacity to fix nitrogen. That these are truly bacterial endosymbionts were shown by the fact that, after thorough treatments with CuSO4 followed by serial incubations with antibiotics, the aforementioned bacterial gene was still amplified in treated fungi. In all, these data support the notion that U. maydis-bacterium endosymbiosis is a general phenomenon in this species.

The Mosaic Architecture of NRPS-PKS in the Arbuscular Mycorrhizal Fungus Gigaspora margarita Shows a Domain With Bacterial Signature.

As obligate biotrophic symbionts, arbuscular mycorrhizal fungi (AMF) live in association with most land plants. Among them, Gigaspora margarita has been deeply investigated because of its peculiar features, i.e., the presence of an intracellular microbiota with endobacteria and viruses. The genome sequencing of this fungus revealed the presence of some hybrid non-ribosomal peptide synthases-polyketide synthases (NRPS-PKS) that have been rarely identified in AMF. The aim of this study is to describe the architecture of these NRPS-PKS sequences and to understand whether they are present in other fungal taxa related to G. margarita. A phylogenetic analysis shows that the ketoacyl synthase (KS) domain of one G. margarita NRPS-PKS clusters with prokaryotic sequences. Since horizontal gene transfer (HGT) has often been advocated as a relevant evolutionary mechanism for the spread of secondary metabolite genes, we hypothesized that a similar event could have interested the KS domain of the PKS module. The bacterial endosymbiont of G. margarita, Candidatus Glomeribacter gigasporarum (CaGg), was the first candidate as a donor, since it possesses a large biosynthetic cluster involving an NRPS-PKS. However, bioinformatics analyses do not confirm the hypothesis of a direct HGT from the endobacterium to the fungal host: indeed, endobacterial and fungal sequences show a different evolution and potentially different donors. Lastly, by amplifying a NRPS-PKS conserved fragment and mining the sequenced AMF genomes, we demonstrate that, irrespective of the presence of CaGg, G. margarita, and some other related Gigasporaceae possess such a sequence.

Chemical Mediators at the Bacterial-Fungal Interface.

Interactions among microbes are key drivers of evolutionary progress and constantly shape ecological niches. Microorganisms rely on chemical communication to interact with each other and surrounding organisms. They synthesize natural products as signaling molecules, antibiotics, or modulators of cellular processes that may be applied in agriculture and medicine. Whereas major insight has been gained into the principles of intraspecies interaction, much less is known about the molecular basis of interspecies interplay. In this review, we summarize recent progress in the understanding of chemically mediated bacterial-fungal interrelations. We discuss pairwise interactions among defined species and systems involving additional organisms as well as complex interactions among microbial communities encountered in the soil or defined as microbiota of higher organisms. Finally, we give examples of how the growing understanding of microbial interactions has contributed to drug discovery and hypothesize what may be future directions in studying and engineering microbiota for agricultural or medicinal purposes.

Gigaspora margarita and Its Endobacterium Modulate Symbiotic Marker Genes in Tomato Roots under Combined Water and Nutrient Stress.

As members of the plant microbiota, arbuscular mycorrhizal fungi (AMF) may be effective in enhancing plant resilience to drought, one of the major limiting factors threatening crop productivity. AMF host their own microbiota and previous data demonstrated that endobacteria thriving in Gigaspora margarita modulate fungal antioxidant responses. Here, we used the G. margarita-Candidatus Glomeribacter gigasporarum system to test whether the tripartite interaction between tomato, G. margarita and its endobacteria may improve plant resilience to combined water/nutrient stress. Tomato plants were inoculated with spores containing endobacteria (B+) or not (B-), and exposed to combined water/nutrient stress. Plants traits, AM colonization and expression of AM marker genes were measured. Results showed that mycorrhizal frequency was low and no growth effect was observed. Under control conditions, B+ inoculated plants were more responsive to the symbiosis, as they showed an up-regulation of three AM marker genes involved in phosphate and lipids metabolism compared with B- inoculated or not-inoculated plants. When combined stress was imposed, the difference between fungal strains was still evident for one marker gene. These results indicate that the fungal endobacteria finely modulate plant metabolism, even in the absence of growth response.

Aposymbiosis of a Burkholderiaceae-Related Endobacterium Impacts on Sexual Reproduction of Its Fungal Host.

Bacterial endosymbionts inhabit diverse fungal lineages. Although the number of studies on bacteria is increasing, the mechanisms by which bacteria affect their fungal hosts remain unclear. We herein examined the homothallic isolate, Mortierella sugadairana YTM39, harboring a Burkholderiaceae-related endobacterium, which did not produce sexual spores. We successfully eliminated the bacterium from fungal isolates using ciprofloxacin treatment and asexual spore isolation for germinated asexual spores. Sexual spore formation by the fungus was restored by eliminating the bacterium from isolates. These results indicate that sexual reproduction by the fungus was inhibited by the bacterium. This is the first study on the sexual spore infertility of fungal hosts by endofungal bacteria.

Phylogenomics of Endogonaceae and evolution of mycorrhizas within Mucoromycota.

Endogonales (Mucoromycotina), composed of Endogonaceae and Densosporaceae, is the only known non-Dikarya order with ectomycorrhizal members. They also form mycorrhizal-like association with some nonspermatophyte plants. It has been recently proposed that Endogonales were among the earliest mycorrhizal partners with land plants. It remains unknown whether Endogonales possess genomes with mycorrhizal-lifestyle signatures and whether Endogonales originated around the same time as land plants did. We sampled sporocarp tissue from four Endogonaceae collections and performed shotgun genome sequencing. After binning the metagenome data, we assembled and annotated the Endogonaceae genomes. We performed comparative analysis on plant-cell-wall-degrading enzymes (PCWDEs) and small secreted proteins (SSPs). We inferred phylogenetic placement of Endogonaceae and estimated the ages of Endogonaceae and Endogonales with expanded taxon sampling. Endogonaceae have large genomes with high repeat content, low diversity of PCWDEs, but without elevated SSP/secretome ratios. Dating analysis estimated that Endogonaceae originated in the Permian-Triassic boundary and Endogonales originated in the mid-late Silurian. Mycoplasma-related endobacterium sequences were identified in three Endogonaceae genomes. Endogonaceae genomes possess typical signatures of mycorrhizal lifestyle. The early origin of Endogonales suggests that the mycorrhizal association between Endogonales and plants might have played an important role during the colonization of land by plants.

Genome and evolution of the arbuscular mycorrhizal fungus Diversispora epigaea (formerly Glomus versiforme) and its bacterial endosymbionts.

Arbuscular mycorrhizal (AM) fungi form endosymbioses with most plants, and they themselves are hosts for Mollicutes/Mycoplasma-related endobacteria (MRE). Despite their significance, genomic information for AM fungi and their MRE are relatively sparse, which hinders our understanding of their biology and evolution. We assembled the genomes of the AM fungus Diversispora epigaea (formerly Glomus versiforme) and its MRE and performed comparative genomics and evolutionary analyses. The D. epigaea genome showed a pattern of substantial gene duplication and differential evolution of gene families, including glycosyltransferase family 25, whose activities are exclusively lipopolysaccharide biosynthesis. Genes acquired by horizontal transfer from bacteria possibly function in defense against foreign DNA or viruses. The MRE population was diverse, with multiple genomes displaying characteristics of differential evolution and encoding many MRE-specific genes as well as genes of AM fungal origin. Gene family expansion in D. epigaea may enhance adaptation to both external and internal environments, such as expansion of kinases for signal transduction upon external stimuli and expansion of nucleoside salvage pathway genes potentially for competition with MRE, whose genomes lack purine and pyrimidine biosynthetic pathways. Collectively, this metagenome provides high-quality references and begins to reveal the diversity within AM fungi and their MRE.