Structurally distinct mitoviruses: are they an ancestral lineage of the Mitoviridae exclusive to arbuscular mycorrhizal fungi (Glomeromycotina)?

Mitoviruses in the family Mitoviridae are the mitochondria-replicating "naked RNA viruses" with genomes encoding only the replicase RNA-dependent RNA polymerase (RdRp) and prevalent across fungi, plants, and invertebrates. Arbuscular mycorrhizal fungi in the subphylum Glomeromycotina are obligate plant symbionts that deliver water and nutrients to the host. We discovered distinct mitoviruses in glomeromycotinian fungi, namely "large duamitovirus," encoding unusually large RdRp with a unique N-terminal motif that is endogenized in some host genomes. More than 400 viral sequences similar to the large duamitoviruses are present in metatranscriptome databases. They are globally distributed in soil ecosystems, consistent with the cosmopolitan distribution of glomeromycotinian fungi, and formed the most basal clade of the Mitoviridae in phylogenetic analysis. Given that glomeromycotinian fungi are the only confirmed hosts of these viruses, we propose the hypothesis that large duamitoviruses are the most ancestral lineage of the Mitoviridae that have been maintained exclusively in glomeromycotinian fungi.

Establishing a quality management framework for commercial inoculants containing arbuscular mycorrhizal fungi.

Microbial inoculants containing arbuscular mycorrhizal (AM) fungi are potential tools in increasing the sustainability of our food production systems. Given the demand for sustainable agriculture, the production of such inoculants has potential economic value and has resulted in a variety of commercial inoculants currently being advertised. However, their use is limited by inconsistent product efficacy and lack of consumer confidence. Here, we propose a framework that can be used to assess the quality and reliability of AM inoculants. First, we set out a range of basic quality criteria which are required to achieve reliable inoculants. This is followed by a standardized bioassay which can be used to test inoculum viability and efficacy under controlled conditions. Implementation of these measurements would contribute to the adoption of AM inoculants by producers with the potential to increase sustainability in food production systems.

Plant Foraging Strategies Driven by Distinct Genetic Modules: Cross-Ecosystem Transcriptomics Approach.

Plants have evolved diverse strategies for foraging, e.g., mycorrhizae, modification of root system architecture, and secretion of phosphatase. Despite extensive molecular/physiological studies on individual strategies under laboratory/greenhouse conditions, there is little information about how plants orchestrate these strategies in the field. We hypothesized that individual strategies are independently driven by corresponding genetic modules in response to deficiency/unbalance in nutrients. Roots colonized by mycorrhizal fungi, leaves, and root-zone soils were collected from 251 maize plants grown across the United States Corn Belt and Japan, which provided a large gradient of soil characteristics/agricultural practice and thus gene expression for foraging. RNA was extracted from the roots, sequenced, and subjected to gene coexpression network analysis. Nineteen genetic modules were defined and functionally characterized, from which three genetic modules, mycorrhiza formation, phosphate starvation response (PSR), and root development, were selected as those directly involved in foraging. The mycorrhizal module consists of genes responsible for mycorrhiza formation and was upregulated by both phosphorus and nitrogen deficiencies. The PSR module that consists of genes encoding phosphate transporter, secreted acid phosphatase, and enzymes involved in internal-phosphate recycling was regulated independent of the mycorrhizal module and strongly upregulated by phosphorus deficiency relative to nitrogen. The root development module that consists of regulatory genes for root development and cellulose biogenesis was upregulated by phosphorus and nitrogen enrichment. The expression of this module was negatively correlated with that of the mycorrhizal module, suggesting that root development is intrinsically an opposite strategy of mycorrhizae. Our approach provides new insights into understanding plant foraging strategies in complex environments at the molecular level.

Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus.

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial interaction between fungi and land plants and promotes global phosphate cycling in terrestrial ecosystems. AM fungi are recognised as obligate symbionts that require root colonisation to complete a life cycle involving the production of propagules, asexual spores. Recently, it has been shown that Rhizophagus irregularis can produce infection-competent secondary spores asymbiotically by adding a fatty acid, palmitoleic acid. Furthermore, asymbiotic growth can be supported using myristate as a carbon and energy source for their asymbiotic growth to increase fungal biomass. However, the spore production and the ability of these spores to colonise host roots were still limited compared to the co-culture of the fungus with plant roots. Here we show that a combination of two plant hormones, strigolactone and jasmonate, induces the production of a large number of infection-competent spores in asymbiotic cultures of Rhizophagus clarus HR1 in the presence of myristate and organic nitrogen. Inoculation of asymbiotically-generated spores promoted the growth of host plants, as observed for spores produced by symbiotic culture system. Our findings provide a foundation for the elucidation of hormonal control of the fungal life cycle and the development of inoculum production schemes.

Application of Virus-Induced Gene Silencing to Arbuscular Mycorrhizal Fungi.

Downregulation of AM fungal genes using a plant viral vector is feasible. A partial sequence of a target fungal gene is cloned into the multicloning site of CMV2-A1 vector developed from RNA2 of Cucumber mosaic virus Y strain, and the RNA2, together with RNA1 and RNA3 of the virus, are in vitro-transcribed. Inoculation of Nicotiana benthamiana with these viral RNAs results in reconstitution of the virus in the plant, which triggers silencing of the fungal gene. Here, we describe not only the methods but also several tips for successful application of virus-induced gene silencing to AM fungi.

Secretion of acid phosphatase from extraradical hyphae of the arbuscular mycorrhizal fungus Rhizophagus clarus is regulated in response to phosphate availability.

Arbuscular mycorrhizal (AM) fungi increase phosphate (P) uptake by plants. Organic phosphate comprises 30-80% of total P in most agricultural soils. Some plants can utilize organic phosphate by secreting acid phosphatase (ACP) from their roots, especially under low P conditions. Although secretion of ACP from extraradical hyphae of AM fungi has been reported, the specific factors that affect the secretion of ACP are unknown. The objective of the present study was to investigate whether secretion of ACP from extraradical hyphae is induced by low P conditions. First, specimens of Allium fistulosum were either inoculated with the AM fungus Rhizophagus clarus strain CK001 or remained uninoculated and were grown in soil with 0.5 g P2O5 kg-1 soil or without P fertilization using two-compartment pots. Soil solution was collected using mullite ceramic tubes 45 days after sowing. The soil solution was analyzed for ACP activity by using p-nitrophenylphosphate. Second, Ri T-DNA transformed roots (i.e., hairy roots) of Linum usitatissimum inoculated with R. clarus were grown on solid minimal media with two P levels applied (3 and 30 µM P) using two-compartment Petri dishes under in vitro conditions. Hyphal exudates, extraradical hyphae, and hairy roots were collected and analyzed for ACP activity. ACP activity in the soil solution of the hyphal compartment in the A. fistulosum inoculation treatment was higher without P fertilization than with P fertilization. AM colonization also was higher without P fertilization than with P fertilization. In the in vitro two-compartment culture, ACP activity of hyphal exudates and extraradical hyphae were higher under the 3-µM treatment than under the 30-µM treatment. These findings suggest that the secretion of ACP from the extraradical hyphae of R. clarus into the hyphosphere is promoted under low P conditions.

Nestedness in Arbuscular Mycorrhizal Fungal Communities in a Volcanic Ecosystem: Selection of Disturbance-tolerant Fungi along an Elevation Gradient.

Arbuscular mycorrhizal (AM) fungi play a significant role in the establishment and resilience of vegetation in harsh environments, such as volcanic slopes, in which soil is frequently disturbed by ash falling and erosion. We characterized AM fungal communities associated with a pioneer grass in a volcanic slope based on the disturbance tolerance of the fungi, addressing the hypothesis that soil disturbance is a major ecological filter for AM fungi in volcanic ecosystems and, thus, fungi that are more tolerant to soil disturbance are selected at higher elevations (i.e. nearer to the crater). Paired soil-core samples were collected from the rhizosphere of Miscanthus sinensis between the vegetation limit and forest limit on a volcanic slope and used in a trap culture with M. sinensis seedlings, in which one of the paired samples was sieved to destroy hyphal networks (disturbance treatment), while the other was not (intact treatment). Seedlings were grown in a greenhouse for two months, and the roots were subjected to molecular analysis of fungal communities. AM fungal diversity decreased with increasing elevations, in which nested structure was observed. Community dissimilarity between the disturbed and intact communities decreased with increasing elevations, suggesting that communities at higher elevations were more robust against soil disturbance. These results suggest that AM fungi that are more tolerant to soil disturbance are more widely distributed across the ecosystem, that is, they are generalists. The wide distribution of disturbance-tolerant fungi may have significant implications for the rapid resilience of vegetation after disturbance in the ecosystem.

Stimulation of asymbiotic sporulation in arbuscular mycorrhizal fungi by fatty acids.

Arbuscular mycorrhizal (AM) fungi are obligate symbionts that depend on living host plants to complete their life cycle1,2. This feature, which leads to their unculturability in the absence of plants, strongly hinders basic research and agricultural application of AM fungi. However, at least one AM fungus can grow and develop fertile spores independently of a host plant in co-culture with the bacterium Paenibacillus validus3. The bacteria-derived substances are thought to act as stimulants or nutrients for fungal sporulation, but these molecules have not been identified. Here, we show that (S)-12-methyltetradecanoic acid4,5, a methyl branched-chain fatty acid isolated from bacterial cultures, stimulates the branching of hyphae germinated from mother spores and the formation of secondary spores in axenic culture of the AM fungus Rhizophagus irregularis. Extensive testing of fatty acids revealed that palmitoleic acid induces more secondary spores than the bacterial fatty acid in R. irregularis. These induced spores have the ability to infect host plant roots and to generate daughter spores. Our work shows that, in addition to a major source of organic carbon6-9, fatty acids act as stimulants to induce infection-competent secondary spores in the asymbiotic stage and could provide the key to developing the axenic production of AM inoculum.

Globular structures in roots accumulate phosphorus to extremely high concentrations following phosphorus addition.

Crops with improved uptake of fertilizer phosphorus (P) would reduce P losses and confer environmental benefits. We examined how P-sufficient 6-week-old soil-grown Trifolium subterraneum plants, and 2-week-old seedlings in solution culture, accumulated P in roots after inorganic P (Pi) addition. In contrast to our expectation that vacuoles would accumulate excess P, after 7 days, X-ray microanalysis showed that vacuolar [P] remained low (<12 mmol kg-1 ). However, in the plants after P addition, some cortex cells contained globular structures extraordinarily rich in P (often >3,000 mmol kg-1 ), potassium, magnesium, and sodium. Similar structures were evident in seedlings, both before and after P addition, with their [P] increasing threefold after P addition. Nuclear magnetic resonance (NMR) spectroscopy showed seedling roots accumulated Pi following P addition, and transmission electron microscopy (TEM) revealed large plastids. For seedlings, we demonstrated that roots differentially expressed genes after P addition using RNAseq mapped to the T. subterraneum reference genome assembly and transcriptome profiles. Among the most up-regulated genes after 4 hr was TSub_g9430.t1, which is similar to plastid envelope Pi transporters (PHT4;1, PHT4;4): expression of vacuolar Pi-transporter homologs did not change. We suggest that subcellular P accumulation in globular structures, which may include plastids, aids cytosolic Pi homeostasis under high-P availability.

Impact of Introduction of Arbuscular Mycorrhizal Fungi on the Root Microbial Community in Agricultural Fields.

Arbuscular mycorrhizal (AM) fungi are important members of the root microbiome and may be used as biofertilizers for sustainable agriculture. To elucidate the impact of AM fungal inoculation on indigenous root microbial communities, we used high-throughput sequencing and an analytical pipeline providing fixed operational taxonomic units (OTUs) as an output to investigate the bacterial and fungal communities of roots treated with a commercial AM fungal inoculum in six agricultural fields. AM fungal inoculation significantly influenced the root microbial community structure in all fields. Inoculation changed the abundance of indigenous AM fungi and other fungal members in a field-dependent manner. Inoculation consistently enriched several bacterial OTUs by changing the abundance of indigenous bacteria and introducing new bacteria. Some inoculum-associated bacteria closely interacted with the introduced AM fungi, some of which belonged to the genera Burkholderia, Cellulomonas, Microbacterium, Sphingomonas, and Streptomyces and may be candidate mycorrhizospheric bacteria that contribute to the establishment and/or function of the introduced AM fungi. Inoculated AM fungi also co-occurred with several indigenous bacteria with putative beneficial traits, suggesting that inoculated AM fungi may recruit specific taxa to confer better plant performance. The bacterial families Methylobacteriaceae, Acetobacteraceae, Armatimonadaceae, and Alicyclobacillaceae were consistently reduced by the inoculation, possibly due to changes in the host plant status caused by the inoculum. To the best of our knowledge, this is the first large-scale study to investigate interactions between AM fungal inoculation and indigenous root microbial communities in agricultural fields.

The genome of Rhizophagus clarus HR1 reveals a common genetic basis for auxotrophy among arbuscular mycorrhizal fungi.

BACKGROUND: Mycorrhizal symbiosis is one of the most fundamental types of mutualistic plant-microbe interaction. Among the many classes of mycorrhizae, the arbuscular mycorrhizae have the most general symbiotic style and the longest history. However, the genomes of arbuscular mycorrhizal (AM) fungi are not well characterized due to difficulties in cultivation and genetic analysis. In this study, we sequenced the genome of the AM fungus Rhizophagus clarus HR1, compared the sequence with the genome sequence of the model species R. irregularis, and checked for missing genes that encode enzymes in metabolic pathways related to their obligate biotrophy. RESULTS: In the genome of R. clarus, we confirmed the absence of cytosolic fatty acid synthase (FAS), whereas all mitochondrial FAS components were present. A KEGG pathway map identified the absence of genes encoding enzymes for several other metabolic pathways in the two AM fungi, including thiamine biosynthesis and the conversion of vitamin B6 derivatives. We also found that a large proportion of the genes encoding glucose-producing polysaccharide hydrolases, that are present even in ectomycorrhizal fungi, also appear to be absent in AM fungi. CONCLUSIONS: In this study, we found several new genes that are absent from the genomes of AM fungi in addition to the genes previously identified as missing. Missing genes for enzymes in primary metabolic pathways imply that AM fungi may have a higher dependency on host plants than other biotrophic fungi. These missing metabolic pathways provide a genetic basis to explore the physiological characteristics and auxotrophy of AM fungi.

Dissection of niche competition between introduced and indigenous arbuscular mycorrhizal fungi with respect to soybean yield responses.

Arbuscular mycorrhizal (AM) fungi associate with most land plants and deliver phosphorus to the host. Identification of biotic/abiotic factors that determine crop responses to AM fungal inoculation is an essential step for successful application of the fungi in sustainable agriculture. We conducted three field trials on soybean with a commercial inoculum and developed a new molecular tool to dissect interactions between the inoculum and indigenous fungi on the MiSeq sequencing platform. Regression analysis indicated that sequence read abundance of the inoculum fungus was the most significant factor that determined soybean yield responses to the inoculation, suggesting that dominance of the inoculum fungus is a necessary condition for positive yield responses. Agricultural practices (fallow/cropping in the previous year) greatly affected the colonization levels (i.e. read abundances) of the inoculum fungus via altering the propagule density of indigenous AM fungi. Analysis of niche competition revealed that the inoculum fungus competed mainly with the indigenous fungi that are commonly distributed in the trial sites, probably because their life-history strategy is the same as that of the inoculum fungus. In conclusion, we provide a new framework for evaluating the significance of environmental factors towards successful application of AM fungi in agriculture.

How do arbuscular mycorrhizal fungi handle phosphate? New insight into fine-tuning of phosphate metabolism.

Contents Summary 1116 I. Introduction 1116 II. Foraging for phosphate 1117 III. Fine-tuning of phosphate homeostasis 1117 IV. The frontiers: phosphate translocation and export 1119 V. Conclusions and outlook 1120 Acknowledgements 1120 References 1120 SUMMARY: Arbuscular mycorrhizal fungi form symbiotic associations with most land plants and deliver mineral nutrients, in particular phosphate, to the host. Therefore, understanding the mechanisms of phosphate acquisition and delivery in the fungi is critical for full appreciation of the mutualism in this association. Here, we provide updates on physical, chemical, and biological strategies of the fungi for phosphate acquisition, including interactions with phosphate-solubilizing bacteria, and those on the regulatory mechanisms of phosphate homeostasis based on resurveys of published genome sequences and a transcriptome with reference to the latest findings in a model fungus. For the mechanisms underlying phosphate translocation and export to the host, which are major research frontiers in this field, not only recent advances but also testable hypotheses are proposed. Lastly, we briefly discuss applicability of the latest tools to gene silencing in the fungi, which will be breakthrough techniques for comprehensive understanding of the molecular basis of fungal phosphate metabolism.

Stringent Expression Control of Pathogenic R-body Production in Legume Symbiont Azorhizobium caulinodans.

R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than CaedibacterAzorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.IMPORTANCECaedibacter species, which are obligate endosymbiotic bacteria of paramecia, produce R bodies, and R-body-producing endosymbionts that are released from their hosts are pathogenic to symbiont-free paramecia. Besides Caedibacter species, R bodies have also been observed in a few free-living bacteria, but the significance of R-body production in these bacteria is still unknown. Recent advances in genome sequencing technologies revealed that many Gram-negative bacteria possess reb genes encoding R-body components, and interestingly, many of them are animal and plant pathogens. Azorhizobium caulinodans, a microsymbiont of the tropical legume Sesbania rostrata, also possesses reb genes. In this study, we demonstrate that A. caulinodans has ability to kill the host plant cells by producing R bodies, suggesting that pathogenicity conferred by an R body might be universal in bacteria possessing reb genes. Furthermore, we provide the first insight into the molecular mechanism underlying the expression of R-body production in response to environmental factors, such as temperature and 2-oxoglutarate.

Nestedness in Arbuscular Mycorrhizal Fungal Communities along Soil pH Gradients in Early Primary Succession: Acid-Tolerant Fungi Are pH Generalists.

Soil acidity is a major constraint on plant productivity. Arbuscular mycorrhizal (AM) fungi support plant colonization in acidic soil, but soil acidity also constrains fungal growth and diversity. Fungi in extreme environments generally evolve towards specialists, suggesting that AM fungi in acidic soil are acidic-soil specialists. In our previous surveys, however, some AM fungi detected in strongly acidic soils could also be detected in a soil with moderate pH, which raised a hypothesis that the fungi in acidic soils are pH generalists. To test the hypothesis, we conducted a pH-manipulation experiment and also analyzed AM fungal distribution along a pH gradient in the field using a synthesized dataset of the previous and recent surveys. Rhizosphere soils of the generalist plant Miscanthus sinensis were collected both from a neutral soil and an acidic soil, and M. sinensis seedlings were grown at three different pH. For the analysis of field communities, rhizosphere soils of M. sinensis were collected from six field sites across Japan, which covered a soil pH range of 3.0-7.4, and subjected to soil trap culture. AM fungal community compositions were determined based on LSU rDNA sequences. In the pH-manipulation experiment the acidification of medium had a significant impact on the compositions of the community from the neutral soil, but the neutralization of the medium had no effect on those of the community from the acidic soil. Furthermore, the communities in lower -pH soils were subsets of (nested in) those in higher-pH soils. In the field communities a significant nestedness pattern was observed along the pH gradient. These observations suggest that the fungi in strongly acidic soils are pH generalists that occur not only in acidic soil but also in wide ranges of soil pH. Nestedness in AM fungal community along pH gradients may have important implications for plant community resilience and early primary succession after disturbance in acidic soils.

Aquaporin-mediated long-distance polyphosphate translocation directed towards the host in arbuscular mycorrhizal symbiosis: application of virus-induced gene silencing.

Arbuscular mycorrhizal fungi translocate polyphosphate through hyphae over a long distance to deliver to the host. More than three decades ago, suppression of host transpiration was found to decelerate phosphate delivery of the fungal symbiont, leading us to hypothesize that transpiration provides a primary driving force for polyphosphate translocation, probably via creating hyphal water flow in which fungal aquaporin(s) may be involved. The impact of transpiration suppression on polyphosphate translocation through hyphae of Rhizophagus clarus was evaluated. An aquaporin gene expressed in intraradical mycelia was characterized and knocked down by virus-induced gene silencing to investigate the involvement of the gene in polyphosphate translocation. Rhizophagus clarus aquaporin 3 (RcAQP3) that was most highly expressed in intraradical mycelia encodes an aquaglyceroporin responsible for water transport across the plasma membrane. Knockdown of RcAQP3 as well as the suppression of host transpiration decelerated polyphosphate translocation in proportion to the levels of knockdown and suppression, respectively. These results provide the first insight into the mechanism underlying long-distance polyphosphate translocation in mycorrhizal associations at the molecular level, in which host transpiration and the fungal aquaporin play key roles. A hypothetical model of the translocation is proposed for further elucidation of the mechanism.

Difference in Striga-susceptibility is reflected in strigolactone secretion profile, but not in compatibility and host preference in arbuscular mycorrhizal symbiosis in two maize cultivars.

Strigolactones released from plant roots trigger both seed germination of parasitic weeds such as Striga spp. and hyphal branching of the symbionts arbuscular mycorrhizal (AM) fungi. Generally, strigolactone composition in exudates is quantitatively and qualitatively different among plants, which may be involved in susceptibility and host specificity in the parasite-plant interactions. We hypothesized that difference in strigolactone composition would have a significant impact on compatibility and host specificity/preference in AM symbiosis. Strigolactones in root exudates of Striga-susceptible (Pioneer 3253) and -resistant (KST 94) maize (Zea mays) cultivars were characterized by LC-MS/MS combined with germination assay using Striga hermonthica seeds. Levels of colonization and community compositions of AM fungi in the two cultivars were investigated in field and glasshouse experiments. 5-Deoxystrigol was exuded exclusively by the susceptible cultivar, while the resistant cultivar mainly exuded sorgomol. Despite the distinctive difference in strigolactone composition, the levels of AM colonization and the community compositions were not different between the cultivars. The present study demonstrated that the difference in strigolactone composition has no appreciable impact on AM symbiosis, at least in the two maize cultivars, and further suggests that the traits involved in Striga-resistance are not necessarily accompanied by reduction in compatibility to AM fungi.

Up-regulation of genes involved in N-acetylglucosamine uptake and metabolism suggests a recycling mode of chitin in intraradical mycelium of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi colonize roots and form two kinds of mycelium, intraradical mycelium (IRM) and extraradical mycelium (ERM). Arbuscules are characteristic IRM structures that highly branch within host cells in order to mediate resource exchange between the symbionts. They are ephemeral structures and at the end of their life span, arbuscular branches collapse from the tip, fungal cytoplasm withdraws, and the whole arbuscule shrinks into fungal clumps. The exoskeleton of an arbuscule contains structured chitin, which is a polymer of N-acetylglucosamine (GlcNAc), whereas a collapsed arbuscule does not. The molecular mechanisms underlying the turnover of chitin in AM fungi remain unknown. Here, a GlcNAc transporter, RiNGT, was identified from the AM fungus Rhizophagus irregularis. Yeast mutants defective in endogenous GlcNAc uptake and expressing RiNGT took up (14)C-GlcNAc, and the optimum uptake was at acidic pH values (pH 4.0-4.5). The transcript levels of RiNGT in IRM in mycorrhizal Lotus japonicus roots were over 1000 times higher than those in ERM. GlcNAc-6-phosphate deacetylase (DAC1) and glucosamine-6-phosphate isomerase (NAG1) genes, which are related to the GlcNAc catabolism pathway, were also induced in IRM. Altogether, data suggest the existence of an enhanced recycling mode of GlcNAc in IRM of AM fungi.

Detection and characterization of mycoviruses in arbuscular mycorrhizal fungi by deep-sequencing.

Fungal viruses (mycoviruses) often have a significant impact not only on phenotypic expression of the host fungus but also on higher order biological interactions, e.g., conferring plant stress tolerance via an endophytic host fungus. Arbuscular mycorrhizal (AM) fungi in the phylum Glomeromycota associate with most land plants and supply mineral nutrients to the host plants. So far, little information about mycoviruses has been obtained in the fungi due to their obligate biotrophic nature. Here we provide a technical breakthrough, "two-step strategy" in combination with deep-sequencing, for virological study in AM fungi; dsRNA is first extracted and sequenced using material obtained from highly productive open pot culture, and then the presence of viruses is verified using pure material produced in the in vitro monoxenic culture. This approach enabled us to demonstrate the presence of several viruses for the first time from a glomeromycotan fungus.