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• 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.

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.

Glomeromycota have been considered the most ancient group of fungi capable of positively interacting with plants for many years. Recently, other basal fungi, the Endogone Mucoromycotina fungi, have been identified as novel plant symbionts, challenging the paradigm of Glomeromycota as the unique ancestral symbionts of land plants. Glomeromycota are known to host endobacteria and recent evidences show that also some Mucoromycotina contain endobacteria. In order to examine similarities between basal groups of plant‐associated fungi, we tested whether Endogone contained endobacteria. Twenty‐nine Endogone were investigated in order to identify Mollicutes‐related endobacteria (Mre). Fruiting bodies were processed for transmission electron microscopy and molecularly investigated using fungal and Mre‐specific primers. We demonstrate that Mre are present inside 13 out of 29 Endogone: endobacteria are directly embedded in the fungal cytoplasm and their 16S rDNA sequences cluster together with the ones retrieved from Glomeromycota, forming, however, a separate new clade. Our findings provide new insights on the evolutionary relations between Glomeromycota, Mucoromycotina and endobacteria, raising new questions on the role of these still enigmatic microbes in the ecology, evolution and diversification of their fungal hosts during the history of plant–fungal symbiosis.

Strigolactones are versatile plant molecules used not only as hormones but also as a communication system to regulate arbuscular mycorrhizal symbiosis through the activation of multiple responses. Abstract Strigolactones (SLs) first evolved as regulators of simple developmental processes in very ancient plant lineages, and then assumed new roles to sustain the increasing biological complexity of land plants. Their versatility is also shown by the fact that during evolution they have been exploited, once released in the rhizosphere, as a communication system towards plant-interacting organisms even belonging to different kingdoms. Here, we reviewed the impact of SLs on soil microbes, paying particular attention to arbuscular mycorrhizal fungi (AMF). SLs induce several responses in AMF, including spore germination, hyphal branching, mitochondrial metabolism, transcriptional reprogramming, and production of chitin oligosaccharides which, in turn, stimulate early symbiotic responses in the host plant. In the specific case study of the AMF Gigaspora margarita, SLs are also perceived, directly or indirectly, by the well-characterized population of endobacteria, with an increase of bacterial divisions and the activation of specific transcriptional responses. The dynamics of SLs during AM root colonization were also surveyed. Although not essential for the establishment of this mutualistic association, SLs act as positive regulators as they are relevant to achieve the full extent of colonization. This possibly occurs through a complex crosstalk with other hormones such as auxin, abscisic acid, and gibberellins.

For more than 450 million years, arbuscular mycorrhizal fungi (AMF) have formed intimate, mutualistic symbioses with the vast majority of land plants and are major drivers in almost all terrestrial ecosystems. The obligate plant-symbiotic AMF host additional symbionts, so-called Mollicutes -related endobacteria (MRE). To uncover putative functional roles of these widespread but yet enigmatic MRE, we sequenced the genome of Dh MRE living in the AMF Dentiscutata heterogama . Multilocus phylogenetic analyses showed that MRE form a previously unidentified lineage sister to the hominis group of Mycoplasma species. Dh MRE possesses a strongly reduced metabolic capacity with 55% of the proteins having unknown function, which reflects unique adaptations to an intracellular lifestyle. We found evidence for transkingdom gene transfer between MRE and their AMF host. At least 27 annotated Dh MRE proteins show similarities to nuclear-encoded proteins of the AMF Rhizophagus irregularis , which itself lacks MRE. Nuclear-encoded homologs could moreover be identified for another AMF, Gigaspora margarita , and surprisingly, also the non-AMF Mortierella verticillata . Our data indicate a possible origin of the MRE-fungus association in ancestors of the Glomeromycota and Mucoromycotina . The Dh MRE genome encodes an arsenal of putative regulatory proteins with eukaryotic-like domains, some of them encoded in putative genomic islands. MRE are highly interesting candidates to study the evolution and interactions between an ancient, obligate endosymbiotic prokaryote with its obligate plant-symbiotic fungal host. Our data moreover may be used for further targeted searches for ancient effector-like proteins that may be key components in the regulation of the arbuscular mycorrhiza symbiosis. Significance Obligate plant-symbiotic, arbuscular mycorrhizal fungi (AMF) are major drivers of terrestrial ecosystems and host enigmatic Mollicutes -related endobacteria (MRE) in their cytoplasm. The genome analysis of a MRE living in the AMF Dentiscutata heterogama revealed it to represent a previously unidentified bacterial lineage of Mycoplasma -related species. Dh MRE shows strongly reduced metabolic capacity and underwent trans-kingdom gene transfer: its genome codes for an arsenal of eukaryotic-like putative effector proteins, with nuclear encoded homologues in AMF and Mortierella . The MRE-fungus (-plant) association probably evolved in ancestors of Glomeromycota and Mucoromycotina . This calls for a targeted search for ancient effector proteins that play crucial roles in the MRE interaction with fungal hosts, and putatively also with plants.

Arbuscular mycorrhizal fungi (AMF) belong to Glomeromycotina, and are mutualistic symbionts of many land plants. Associated bacteria accompany AMF during their lifecycle to establish a robust tripartite association consisting of fungi, plants and bacteria. Physical association among this trinity provides possibilities for the exchange of genetic materials. However, very few horizontal gene transfer (HGT) from bacteria or plants to AMF has been reported yet. In this study, we complement existing algorithms by developing a new pipeline, Blast2hgt, to efficiently screen for putative horizontally derived genes from a whole genome. Genome analyses of the glomeromycete identified 19 fungal genes that had been transferred between fungi and bacteria/plants, of which seven were obtained from bacteria. Another 18 genes were found to be recently acquired from either plants or bacteria. In the genome, gene duplication has contributed to the expansion of three foreign genes. Importantly, more than half of the foreign genes were expressed in various transcriptomic experiments, suggesting that these genes are functional in . Functional annotation and available evidence showed that these acquired genes may participate in diverse but fundamental biological processes such as regulation of gene expression, mitosis and signal transduction. Our study suggests that horizontal gene influx through endosymbiosis is a source of new functions for , and HGT might have played a role in the evolution and symbiotic adaptation of this arbuscular mycorrhizal fungus.

Endogonales (Mucoromycotina), composed of Endogonaceae and Densosporaceae, is the single known non-Dikarya order with ectomycorrhizal members. Furthermore, they 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.

Nitrogen (N) availability and soil microbiota exert significant impacts on plant metabolic systems and yield. Different studies have indicated that yield and nitrogen use efficiency (NUE) of pearl millet (Pennisetum glaucum L.R.Br.) can be improved by the inoculation of N-fixing bacteria. However, the interactive effects of different N sources and bacteria inoculation on growth, productivity, and NUE of pearl millet have been less explored. Therefore, individual and interactive effects of different N sources (organic and inorganic) and bacteria inoculation on growth, productivity, and NUE of pearl millet were investigated in this study. Two different N sources, i.e., organic (farmyard manure) and inorganic (urea) alone or in 50% + 50% combinations (urea + FYM), were used to supply the recommended amount of N. Similarly, seeds were inoculated with two different N-fixing bacteria, i.e., endobacteria (Enterobacter sp. MN17) and rhizobacteria (Stenotrophomonas maltophilia FA-9). Urea + farmyard manure (FYM) and seed inoculation rhizobacteria improved soil attributes, yield-related traits, grain quality, NUE, and net economic returns. Soil porosity was significantly improved by seed inoculation with both bacteria and FYM application. Similarly, seed inoculation with rhizobacteria increased soil organic carbon by 45.45% and 34.88% during the 1st and 2nd year of the study, respectively. Urea + FYM application combined with rhizobacteria seed inoculation improved the number of grains per ear (23.49 and 23.63%), 1000-grain weight (5.76% and 7.85%), grain yield (23.19% and 25.0%), and NUE (33.83 and 48.15%). Similarly, grain quality was significantly improved by seed inoculation with both bacteria. Likewise, urea + FYM combined with rhizobacteria improved nitrogen use efficiency (NUE) by 33.83% and 48.15% in 2020 and 2021, respectively, compared to no N application and no seed inoculation. The highest economic returns (1506.4 and 1506.9 USD) were noted for urea + FYM application combined with rhizobacteria seed inoculation. Therefore, urea + FYM application combined with rhizobacteria (S. maltophilia FA-9) seed inoculation seemed a viable approach to improve grain yield, grain quality, NUE, and net economic returns of pearl millet.

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. 1R11iZ3xLrWDQ_XwZ4bfc5 Video Abstract

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.