White root rot, caused by the ascomycete Rosellinia necatrix , is a devastating disease worldwide, particularly in fruit trees in Japan. Here we report on the biological and molecular properties of a novel bipartite double-stranded RNA (dsRNA) virus encompassing dsRNA-1 (8,931 bp) and dsRNA-2 (7,180 bp), which was isolated from a field strain of R. necatrix , W779. Besides the strictly conserved 5′ (24 nt) and 3′ (8 nt) terminal sequences, both segments show high levels of sequence similarity in the long 5′ untranslated region of approximately 1.6 kbp. dsRNA-1 and -2 each possess two open reading frames (ORFs) named ORF1 to -4. Although the protein encoded by 3′-proximal ORF2 on dsRNA-1 shows sequence identities of 22 to 32% with RNA-dependent RNA polymerases from members of the families Totiviridae and Chrysoviridae , the remaining three virus-encoded proteins lack sequence similarities with any reported mycovirus proteins. Phylogenetic analysis showed that the W779 virus belongs to a separate clade distinct from those of other known mycoviruses. Purified virions ∼50 nm in diameter consisted of dsRNA-1 and -2 and a single major capsid protein of 135 kDa, which was shown by peptide mass fingerprinting to be encoded by dsRNA-1 ORF1. We developed a transfection protocol using purified virions to show that the virus was responsible for reduction of virulence and mycelial growth in several host strains. These combined results indicate that the W779 virus is a novel bipartite dsRNA virus with potential for biological control (virocontrol), named Rosellinia necatrix megabirnavirus 1 (RnMBV1), that possibly belongs to a new virus family.

High-throughput virome analyses with various fungi, from cultured or uncultured sources, have led to the discovery of diverse viruses with unique genome structures and even neo-lifestyles. Examples in the former category include splipalmiviruses and ambiviruses. Splipalmiviruses, related to yeast narnaviruses, have multiple positive-sense (+) single-stranded (ss) RNA genomic segments that separately encode the RNA-dependent RNA polymerase motifs, the hallmark of RNA viruses (members of the kingdom Orthornavirae). Ambiviruses appear to have an undivided ssRNA genome of 3∼5 kb with two large open reading frames (ORFs) separated by intergenic regions. Another narna-like virus group has two fully overlapping ORFs on both strands of a genomic segment that span more than 90% of the genome size. New virus lifestyles exhibited by mycoviruses include the yado-kari/yado-nushi nature characterized by the partnership between the (+)ssRNA yadokarivirus and an unrelated dsRNA virus (donor of the capsid for the former) and the hadaka nature of capsidless 10–11 segmented (+)ssRNA accessible by RNase in infected mycelial homogenates. Furthermore, dsRNA polymycoviruses with phylogenetic affinity to (+)ssRNA animal caliciviruses have been shown to be infectious as dsRNA–protein complexes or deproteinized naked dsRNA. Many previous phylogenetic gaps have been filled by recently discovered fungal and other viruses, which haveprovided interesting evolutionary insights. Phylogenetic analyses and the discovery of natural and experimental cross-kingdom infections suggest that horizontal virus transfer may have occurred and continue to occur between fungi and other kingdoms.

Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant- and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single- and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species. Eukaryotic genomes contain sequences that have originated from DNA viruses and reverse-transcribing viruses, i.e., retroviruses, pararetroviruses (DNA viruses), and transposons. However, the sequences of non-retroviral RNA viruses, which are unable to convert their genomes to DNA, were until recently considered not to be integrated into eukaryotic nuclear genomes. We present evidence for multiple independent events of horizontal gene transfer from a wide range of RNA viruses, including plus-sense, minus-sense, and double-stranded RNA viruses, into the genomes of distantly related plant lineages. Some non-retroviral integrated RNA viral sequences are conserved across genera within a plant family, whereas others are retained only in a limited number of species in a genus. Integration profiles of non-retroviral integrated RNA viral sequences demonstrate the potential of these sequences to serve as powerful molecular tools for deciphering phylogenetic relationships among related plants. Moreover, this study highlights plants co-opting non-retroviral RNA virus sequences, and provides insights into plant genome evolution and interplay between non-reverse-transcribing RNA viruses and their hosts.