Molecular characterization of the complete genome of a novel ormycovirus infecting the ectomycorrhizal fungus Hortiboletus rubellus.

Advancements in high-throughput sequencing and the development of new bioinformatics tools for large-scale data analysis play a crucial role in uncovering virus diversity and enhancing our understanding of virus evolution. The discovery of the ormycovirus clades, a group of RNA viruses that are phylogenetically distinct from all known Riboviria members and are found in fungi, highlights the value of these tools for the discovery of novel viruses. The aim of this study was to examine viral populations in fungal hosts to gain insights into the diversity, evolution, and classification of these viruses. Here, we report the molecular characterization of a newly discovered ormycovirus, which we have named "Hortiboletus rubellus ormycovirus 1" (HrOMV1), that was found in the ectomycorrhizal fungus Hortiboletus rubellus. The bipartite genome of HrOMV1, whose nucleotide sequence was determined by HTS and RLM-RACE, consists of two RNA segments (RNA1 and RNA2) that exhibit similarity to those of previously studied ormycoviruses in their organization and the proteins they encode. The presence of upstream, in-frame AUG triplets in the 5' termini of both RNA segments suggests that HrOMV1, like certain other ormycoviruses, employs a non-canonical translation initiation strategy. Phylogenetic analysis showed that HrOMV1 is positioned within the gammaormycovirus clade. Its putative RNA-dependent RNA polymerase (RdRp) exhibits sequence similarity to those of other gammaormycovirus members, the most similarity to that of Termitomyces ormycovirus 1, with 33.05% sequence identity. This protein was found to contain conserved motifs that are crucial for RNA replication, including the distinctive GDQ catalytic triad observed in gammaormycovirus RdRps. The results of this study underscore the significance of investigating the ecological role of mycoviruses in mycorrhizal fungi. This is the first report of an ormycovirus infecting a member of the ectomycorrhizal genus Hortiboletus.

Molecular characterization of a new endornavirus inhabiting the ectomycorrhizal fungus Hygrophorus penarioides.

Viruses hosted by uncultivated fungi have been poorly studied. We carried out studies to characterize a large dsRNA segment (~20 kbp) detected in the basidiomycetous, ectomycorrhizal fungus Hygrophorus penarioides. The dsRNA was gel-purified and its randomly amplified cDNA fragments were used for high throughput sequencing (HTS). Reads were de novo assembled and BLASTx analysis revealed sequence similarity to viruses of the family Endornaviridae. The 5' and 3' terminal sequences of the dsRNA segment were determined by performing RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE). The full-length cDNA sequence of the putative endornavirus comprises 16,785 nt and contains a single, long open reading frame which encodes for a polyprotein of 5522 aa with conserved domains for cysteine-rich region, helicase, glycosyltransferase, and RNA-dependent RNA polymerase. The virus was named Hygrophorus penarioides endornavirus 1 (HpEnV1). A BLASTp search performed using the polyprotein sequence revealed that the most closely related, fully sequenced endornavirus to HpEnV1 is Ceratobasidium endornavirus B.

Novel and diverse mycoviruses co-inhabiting the hypogeous ectomycorrhizal fungus Picoa juniperi.

Viruses hosted by ectomycorrhizal fungi remain poorly studied. In this study, we detected eight new fungal viruses co-infecting a single isolate of the hypogeous ectomycorrhizal fungus Picoa juniperi using high-throughput sequencing. Phylogenetic analysis of one identified virus abbreviated as PjMTV1 revealed its closest relatives as members of the newly proposed family "Megatotiviridae". Phylogenetic analyses of two identified viruses abbreviated as PjV1 and PjV2 showed that these viruses are associated with members of the proposed family "Fusagraviridae". Phylogenetic analysis of the identified one another virus abbreviated as PjYV1 demonstrated that this virus is related to the members of the proposed family Yadokariviridae. The remaining four identified virus-like contigs were determined as segments of the bipartite dsRNA mycoviruses from the family Partitiviridae. The mycoviruses reported in this study are the first viruses described in Picoa juniperi, and PjMTV1 characterized herein is the secondly reported member of the newly proposed family "Megatotiviridae".

Arbuscular Mycorrhizal Symbiosis Primes Tolerance to Cucumber Mosaic Virus in Tomato.

Tomato plants can establish symbiotic interactions with arbuscular mycorrhizal fungi (AMF) able to promote plant nutrition and prime systemic plant defenses against pathogens attack; the mechanism involved is known as mycorrhiza-induced resistance (MIR). However, studies on the effect of AMF on viral infection, still limited and not conclusive, indicate that AMF colonization may have a detrimental effect on plant defenses against viruses, so that the term "mycorrhiza-induced susceptibility" (MIS) has been proposed for these cases. To expand the case studies to a not yet tested viral family, that is, Bromoviridae, we investigated the effect of the colonization by the AMF Funneliformis mosseae on cucumber mosaic virus (CMV) infection in tomato by phenotypic, physiological, biochemical, and transcriptional analyses. Our results showed that the establishment of a functional AM symbiosis is able to limit symptoms development. Physiological and transcriptomic data highlighted that AMF mitigates the drastic downregulation of photosynthesis-related genes and the reduction of photosynthetic CO2 assimilation rate caused by CMV infection. In parallel, an increase of salicylic acid level and a modulation of reactive oxygen species (ROS)-related genes, toward a limitation of ROS accumulation, was specifically observed in CMV-infected mycorrhizal plants. Overall, our data indicate that the AM symbiosis influences the development of CMV infection in tomato plants and exerts a priming effect able to enhance tolerance to viral infection.

Novel and divergent bipartite mycoviruses associated with the ectomycorrhizal fungus Sarcosphaera coronaria.

Members of the family Partitiviridae are reported from a variety of fungal and plant taxa. After dsRNA-preparation, deep sequencing, and bioinformatics, we here reveal the existence of various divergent partitiviruses co-infecting the ectomycorrhizal fungus Sarcosphaera coronaria, symbiotically associated with the pine species Pinus brutia in Turkey. A total of 75 complete or nearly complete sequences related to the members of Alphapartitivirus and Betapartitivirus, were detected from the ascocarp sample of the fungal isolate. Two of the identified partitivirus genome segments encoding for partitiviral capsid protein represent evolutionarily distinct members of Alphapartitivirus, indicating that they may have diverged in the presence of long spatial isolation. In an attempt to match the two genome segments of the identified partitiviruses and distinguish individual species co-inhabiting a single host, nine possible genome segment pairs were identified.

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.

Metatranscriptomic Analysis and In Silico Approach Identified Mycoviruses in the Arbuscular Mycorrhizal Fungus Rhizophagus spp.

Arbuscular mycorrhizal fungi (AMF), including Rhizophagus spp., can play important roles in nutrient cycling of the rhizosphere. However, the effect of virus infection on AMF's role in nutrient cycling cannot be determined without first knowing the diversity of the mycoviruses in AMF. Therefore, in this study, we sequenced the R. irregularis isolate-09 due to its previously demonstrated high efficiency in increasing the N/P uptake of the plant. We identified one novel mitovirus contig of 3685 bp, further confirmed by reverse transcription-PCR. Also, publicly available Rhizophagus spp. RNA-Seq data were analyzed to recover five partial virus sequences from family Narnaviridae, among which four were from R. diaphanum MUCL-43196 and one was from R. irregularis strain-C2 that was similar to members of the Mitovirus genus. These contigs coded genomes larger than the regular mitoviruses infecting pathogenic fungi and can be translated by either a mitochondrial translation code or a cytoplasmic translation code, which was also reported in previously found mitoviruses infecting mycorrhizae. The five newly identified virus sequences are comprised of functionally conserved RdRp motifs and formed two separate subclades with mitoviruses infecting Gigasporamargarita and Rhizophagusclarus, further supporting virus-host co-evolution theory. This study expands our understanding of virus diversity. Even though AMF is notably hard to investigate due to its biotrophic nature, this study demonstrates the utility of whole root metatranscriptome.

Metagenomic Analyses of the Viruses Detected in Mycorrhizal Fungi and Their Host Orchid.

In nature, mycorrhizal association with soilborne fungi is indispensable for orchid families. Fungal structures from compatible endo-mycorrhizal fungi in orchid cells are digested in cells to be supplied to orchids as nutrition. Because orchid seeds lack the reserves for germination, they keep receiving nutrition through mycorrhizal formation from seed germination until shoots develop (leaves) and become photoautotrophic. Seeds of all orchid species surely geminate with the help of their own fungal partners, and this specific partnership has been acquired for a long evolutional history between orchids and fungi.We have studied the interactions between orchids and mycorrhizal fungi and recently conducted transcriptome analyses (RNAseq) by a next-generation sequencing (NGS) approach. It is possible that orchid RNA isolated form naturally grown plants is contaminated with RNAs derived from mycorrhizal fungi in the orchid cells. To avoid such contamination, we here prepared aseptically germinated orchid plants (i.e., fungus-free plants) together with a pure-cultured fungal isolate and field-growing orchid samples. In the cDNA library prepared from orchid and fungal tissues, we found that partitivirus-like sequences were common in an orchid and its mycorrhizal fungus. These partitivirus-like sequences were closely related by a phylogenetic analysis, suggesting that transmission of an ancestor virus between the two organisms occurred through the specific relation of the orchid and its associated fungus.

The virome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals the first report of DNA fragments corresponding to replicating non-retroviral RNA viruses in fungi.

Arbuscular Mycorrhizal Fungi (AMF) are key components of the plant microbiota. AMF genetic complexity is increased by the presence of endobacteria, which live inside many species. A further component of such complexity is the virome associated to AMF, whose knowledge is still very limited. Here, by exploiting transcriptomic data we describe the virome of Gigaspora margarita. A BLAST search for viral RNA-dependent RNA polymerases sequences allowed the identification of four mitoviruses, one Ourmia-like narnavirus, one Giardia-like virus, and two sequences related to Fusarium graminearum mycoviruses. Northern blot and RT-PCR confirmed the authenticity of all the sequences with the exception of the F. graminearum-related ones. All the mitoviruses are replicative and functional since both positive strand and negative strand RNA are present. The abundance of the viral RNA molecules is not regulated by the presence or absence of Candidatus Glomeribacter gigasporarum, the endobacterium hosted by G. margarita, with the exception of the Ourmia-like sequence which is absent in bacteria-cured spores. In addition, we report, for the first time, DNA fragments corresponding to mitovirus sequences associated to the presence of viral RNA. These sequences are not integrated in the mitochondrial DNA and preliminary evidence seems to exclude integration in the nuclear genome.

Novel and divergent viruses associated with Australian orchid-fungus symbioses.

Terrestrial orchids represent a symbiotic union between plants and mycorrhizal fungi. This study describes the occurrence and nature of viruses associated with one population of wild Pterostylis sanguinea orchids, including their fungal symbionts, over two consecutive years. A generic sequencing approach, which combined dsRNA-enrichment from plant and mycelial tissues, random amplification and high throughput shotgun sequencing was used to identify novel viruses. The majority of the virus-like sequences represent partial genomes, and their identification is based solely on de novo assembly of sequencing data. In orchid leaf tissues we found three isolates of a novel totivirus and an unclassified virus; both resemble fungus-infecting viruses. Two isolates of Ceratobasidium sp that were isolated from orchid underground stems contained at least 20 viruses, 16 of which were previously described as alphapartitiviruses and betapartitiviruses. A novel hypovirus and a mitovirus were genetically distant from existing members of the genera and did not readily fit into recognised subgroups.

Occurrence of similar mycoviruses in pathogenic, saprotrophic and mycorrhizal fungi inhabiting the same forest stand.

Fungal viruses (mycoviruses) are considered to be highly host specific, but phylogenetic analysis supports the occasional occurrence of horizontal transmission between species. We used an extensive sampling strategy to investigate whether similar viruses occur in more than one fungal species of the same forest habitat. Mycelial samples were collected from in-growth mesh bags (N = 259), fruiting bodies (N = 173) and cultured isolates (N = 68) at a forest site where the spatial distribution of viral infections in clonal individuals of the wood decay fungus Heterobasidion parviporum was mapped in detail earlier. The investigation revealed previously known Heterobasidion viruses in ∼2% of the single or pooled mycorrhizal samples from mesh bags, ∼3% of the fruiting body samples and none of the fungal cultures analyzed. Novel virus strains distinct from known Heterobasidion viruses were detected in cultures of ectomycorrhizal fungi (Lactarius tabidus, L. rufus) and saprotrophic fungi (Megacollybia platyphylla, Mucoraceae spp.). Overall, our results support the view that mycoviruses do not readily cross species borders. Regarding potential virocontrol applications, the introduction of Heterobasidion viruses into natural habitats is not expected to cause a major infection pressure towards the indigenous fungal community. However, the ecological consequences of the putative interspecies virus transmission events detected require further investigation.

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.

A unique mitovirus from Glomeromycota, the phylum of arbuscular mycorrhizal fungi.

Arbuscular mycorrhizal (AM) fungi that belong to the phylum Glomeromycota associate with most land plants and supply mineral nutrients to the host plants. One of the four viral segments found by deep-sequencing of dsRNA in the AM fungus Rhizophagus clarus strain RF1 showed similarity to mitoviruses and is characterized in this report. The genome segment is 2,895 nucleotides in length, and the largest ORF was predicted by applying either the mold mitochondrial or the universal genetic code. The ORF encodes a polypeptide of 820 amino acids with a molecular mass of 91.2 kDa and conserves the domain of the mitovirus RdRp superfamily. Accordingly, the dsRNA was designated as R. clarus mitovirus 1 strain RF1 (RcMV1-RF1). Mitoviruses are localized exclusively in mitochondria and thus generally employ the mold mitochondrial genetic code. The distinct codon usage of RcMV1-RF1, however, suggests that the virus is potentially able to replicate not only in mitochondria but also in the cytoplasm. RcMV1-RF1 RdRp showed the highest similarity to the putative RdRp of a mitovirus-like ssRNA found in another AM fungus, followed by RdRp of a mitovirus in an ascomycotan ectomycorrhizal fungus. The three mitoviruses found in the three mycorrhizal fungi formed a deeply branching clade that is distinct from the two major clades in the genus Mitovirus.

A novel virus-like double-stranded RNA in an obligate biotroph arbuscular mycorrhizal fungus: a hidden player in mycorrhizal symbiosis.

Arbuscular mycorrhizal (AM) fungi form mutualistic associations with most land plants and enhance phosphorus uptake of the host plants. Fungal viruses (mycoviruses) that possess a double-stranded RNA (dsRNA) genome often affect plant-fungal interactions via altering phenotypic expression of their host fungi. The present study demonstrates, for the first time, the presence of dsRNAs, which are highly likely to be mycoviruses, in AM fungi. dsRNA was extracted from mycelia of Glomus sp. strain RF1, purified, and subjected to electrophoresis. The fungus was found to harbor various dsRNA segments that differed in size. Among them, a 4.5-kbp segment was termed Glomus sp. strain RF1 virus-like medium dsRNA (GRF1V-M) and characterized in detail. The GRF1V-M genome segment was 4,557 nucleotides in length and encoded RNA-dependent RNA polymerase and a structural protein. GRF1V-M was phylogenetically distinct and could not be assigned to known genera of mycovirus. The GRF1V-M-free culture line of Glomus sp. strain RF1, which was raised by single-spore isolation, produced twofold greater number of spores and promoted plant growth more efficiently than the GRF1V-M-positive lines. These observations suggest that mycoviruses in AM fungi, at least some of them, have evolved under unique selection pressures and are a biologically active component in the symbiosis.