1 2 Arbuscular mycorrhizal (AM) fungi that belong to the phylum Glomeromycota associate 3 with most land plants and supply mineral nutrients to the host plants. One of the four viral 4 segments found by deep-sequencing of dsRNA in the AM fungus Rhizophagus clarus strain 5 RF1 showed similarity to mitoviruses and is characterized in this report. The genome 6 segment is 2,895 nucleotides in length, and the largest ORF was predicted by applying either 7 the mold mitochondrial or the universal genetic code. The ORF encodes a polypeptide of 820 8 amino acids with a molecular mass of 91.2 kDa and conserves the domain of the mitovirus 9RdRp superfamily. Accordingly, the dsRNA was designated as R. clarus mitovirus 1 strain 10 RF1 (RcMV1-RF1). Mitoviruses are localized exclusively in mitochondria and thus 11 generally employ the mold mitochondrial genetic code. The distinct codon usage of 12 RcMV1-RF1, however, suggests that the virus is potentially able to replicate not only in 13 mitochondria but also in the cytoplasm. RcMV1-RF1 RdRp showed the highest similarity to 14 the putative RdRp of a mitovirus-like ssRNA found in another AM fungus, followed by RdRp 15 of a mitovirus in an ascomycotan ectomycorrhizal fungus. The three mitoviruses found in the 16 three mycorrhizal fungi formed a deeply branching clade that is distinct from the two major 17 clades in the genus Mitovirus. 18 Kitahara et al. 3

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.

No abstract