Beet necrotic yellow vein virus (BNYVV) is an economically important pathogen of sugar beet and has been found worldwide, probably as the result of recent worldwide spread. The BNYVV genome consists of four or five RNA components. Here, we report analysis of sequence variation in the RNA3-p25, RNA4-p31, RNA2-CP, and RNA5-p26 genes of 73 worldwide isolates. The RNA3-p25 gene encodes virulence and avirulence factors. These four sets of gene sequences each fell into two to four groups, of which the three groups of p25 formed eight subgroups with different geographical distributions. Each of these subgroup isolates (strains) could have arisen from four original BNYVV population and their mixed infections. The genetic diversity for BNYVV was relatively small. Selection pressure varied greatly depending on the BNYVV gene and geographical location. Isolates of the Italy strain, in which p25 was subject to the strongest positive selection, were able to overcome the Rz1-host resistance gene to differing degrees, whereas other geographically limited strains could not. Resistance-breaking variants were generated by p25 amino acid changes at positions 67 and 68. Our studies suggest that BNYVV originally evolved in East Asia and has recently become a pathogen of cultivated sugar beet followed by the emergence of new resistance-breaking variants.

Replication of RNA viruses in insect cells triggers an antiviral defense that is mediated by RNA interference (RNAi) which generates viral-derived small interfering RNAs (siRNAs). However, it is not known whether an antiviral RNAi response is also induced in insects by reoviruses, whose double-stranded RNA genome replication is thought to occur within core particles. Deep sequencing of small RNAs showed that when the small brown planthopper (Laodelphax striatellus) was infected by Rice black-streaked dwarf virus (RBSDV) (Reoviridae; Fijivirus), more viral-derived siRNAs accumulated than when the vector insect was infected by Rice stripe virus (RSV), a negative single-stranded RNA virus. RBSDV siRNAs were predominantly 21 and 22 nucleotides long and there were almost equal numbers of positive and negative sense. RBSDV siRNAs were frequently generated from hotspots in the 5′- and 3′-terminal regions of viral genome segments but these hotspots were not associated with any predicted RNA secondary structures. Under laboratory condition, L. striatellus can be infected simultaneously with RBSDV and RSV. Double infection enhanced the accumulation of particular genome segments but not viral coat protein of RBSDV and correlated with an increase in the abundance of siRNAs derived from RBSDV. The results of this study suggest that reovirus replication in its insect vector potentially induces an RNAi-mediated antiviral response.

In plants, RNA silencing is part of a defense mechanism against virus infection but there is little information as to whether RNA silencing-mediated resistance functions similarly in roots and leaves. We have obtained transgenic Nicotiana benthamiana plants encoding the coat protein readthrough domain open reading frame (54 kDa) of Beet necrotic yellow vein virus (BNYVV), which either showed a highly resistant or a recovery phenotype following foliar rub-inoculation with BNYVV. These phenotypes were associated with an RNA silencing mechanism. Roots of the resistant plants that were immune to foliar rub-inoculation with BNYVV could be infected by viruliferous zoospores of the vector fungus Polymyxa betae, although virus multiplication was greatly limited. In addition, virus titer was reduced in symptomless leaves of the plants showing the recovery phenotype, but it was high in roots of the same plants. Compared with leaves of silenced plants, higher levels of transgene mRNAs and lower levels of transgene-derived small interfering RNAs (siRNAs) accumulated in roots. Similarly, in nontransgenic plants inoculated with BNYVV, accumulation level of viral RNA-derived siRNAs in roots was lower than in leaves. These results indicate that the RNA silencing-mediated resistance to BNYVV is less effective in roots than in leaves.

The transmission of viral infections between plant and fungal hosts has been suspected to occur, based on phylogenetic and other findings, but has not been directly observed in nature. Here, we report the discovery of a natural infection of the phytopathogenic fungus by a plant virus, cucumber mosaic virus (CMV). The CMV-infected strain was obtained from a potato plant growing in Inner Mongolia Province of China, and CMV infection was stable when this fungal strain was cultured in the laboratory. CMV was horizontally transmitted through hyphal anastomosis but not vertically through basidiospores. By inoculation via protoplast transfection with virions, a reference isolate of CMV replicated in and another phytopathogenic fungus, suggesting that some fungi can serve as alternative hosts to CMV. Importantly, in fungal inoculation experiments under laboratory conditions, could acquire CMV from an infected plant, as well as transmit the virus to an uninfected plant. This study presents evidence of the transfer of a virus between plant and fungus, and it further expands our understanding of plant-fungus interactions and the spread of plant viruses.

RNA3 and RNA4 of beet necrotic yellow vein virus (BNYVV) are not essential for virus multiplication, but are associated with vector-mediated infection and disease development in sugar beet roots. Here, a unique role for RNA4 in virus transmission, virulence and RNA silencing suppression was demonstrated. Mutagenic analysis revealed that the RNA4-encoded p31 open reading frame (ORF) was involved in efficient vector transmission and slight enhancement of symptom expression in some Beta species. No effects of RNA4 on virus accumulation in infected tissue were observed. Furthermore, the p31 ORF was involved in the induction of severe symptoms by BNYVV in Nicotiana benthamiana plants without affecting viral RNA accumulation. In contrast, RNA3-encoded p25, previously identified as a major contributor to symptom induction in sugar beet, had no such effect on N. benthamiana. In two different silencing suppression assays, neither p31 nor p25 was able to suppress RNA silencing in leaves, but the presence of p31 enhanced a silencing suppressor activity in roots without alteration in viral RNA accumulation. Thus, BNYVV p31 plays a multifunctional role in efficient vector transmission, enhanced symptom expression and root-specific silencing suppression.

Insects are the host and vector of diverse viruses including those that infect vertebrates, plants, and fungi. Recent wide-scale transcriptomic analyses have uncovered the existence of a number of novel insect viruses belonging to an alphavirus-like superfamily (virgavirus/negevirus-related lineage). In this study, through an in silico search using publicly available insect transcriptomic data, we found numerous virus-like sequences related to insect virga/nege-like viruses. Phylogenetic analysis showed that these novel viruses and related virus-like sequences fill the major phylogenetic gaps between insect and plant virga/negevirus lineages. Interestingly, one of the phylogenetic clades represents a unique insect-infecting virus group. Its members encode putative coat proteins which contained a conserved domain similar to that usually found in the coat protein of plant viruses in the family Virgaviridae. Furthermore, we discovered endogenous viral elements (EVEs) related to virga/nege-like viruses in the insect genomes, which enhances our understanding on their evolution. Database searches using the sequence of one member from this group revealed the presence of EVEs in a wide range of insect species, suggesting that there has been prevalent infection by this virus group since ancient times. Besides, we present detailed EVE integration profiles of this virus group in some species of the Bombus genus of bee families. A large variation in EVE patterns among Bombus species suggested that while some integration events occurred after the species divergence, others occurred before it. Our analyses support the view that insect and plant virga/nege-related viruses might share common virus origin(s).

Aphids (order Hemiptera) are important insect pests of crops and are also vectors of many plant viruses. However, little is known about aphid-infecting viruses, particularly their diversity and relationship to plant viruses. To investigate the aphid viromes, we performed deep sequencing analyses of the aphid transcriptomes from infested barley plants in a field in Japan. We discovered virus-like sequences related to nege/ kita-, flavi-, tombus-, phenui-, mononega-, narna-, chryso-, partiti-, and luteoviruses. Using RT-PCR and sequence analyses, we determined almost complete sequences of seven nege/kitavirus-like virus genomes; one of which was a variant of the Wuhan house centipede virus (WHCV-1). The other six seem to belong to four novel viruses distantly related to Wuhan insect virus 9 (WhIV-9) or Hubei nege-like virus 4 (HVLV-4). We designated the four viruses as barley aphid RNA virus 1 to 4 (BARV-1 to-4). Moreover, some nege/kitavirus-like sequences were found by searches on the transcriptome shotgun assembly (TSA) libraries of arthropods and plants. Phylogenetic analyses showed that BARV-1 forms a clade with WHCV-1 and HVLV-4, whereas BARV-2 to-4 clustered with WhIV-9 and an aphid virus, Aphis glycines virus 3. Both virus groups (tentatively designated as Centivirus and Aphiglyvirus, respectively), together with arthropod virus-like TSAs, fill the phylogenetic gaps between the negeviruses and kitaviruses lineages. We also characterized the flavi/jingmen-like and tombus-like virus sequences as well as other RNA viruses, including six putative novel viruses, designated as barley aphid RNA viruses 5 to 10. Interestingly, we also discovered that some aphid-associated viruses, including nege/kita-like viruses, were present in different aphid species, raising a speculation that these viruses might be distributed across different aphid species with plants being the reservoirs. This study provides novel information on the diversity and spread of nege/kitavirus-related viruses and other RNA viruses that are associated with aphids.

SUMMARYMembers of the plant Dicer-like (DCL) protein family are the critical components of the RNA-silencing pathway that mediates innate antiviral defence. The distinct antiviral role of each individual DCL protein has been established with mostly based on observations of aerial parts of plants. Thus, although the roots are closely associated with the life cycle of many plant viruses, little is known about the antiviral activities of DCL proteins in roots. We observed that antiviral silencing strongly inhibits potato virus X (PVX) replication in roots of some susceptible Solanaceae species. Silencing of the DCL4 homolog in Nicotiana benthamiana partially elevated PVX replication levels in roots. In Arabidopsis thaliana, which was originally considered a non-host plant of PVX, high levels of PVX accumulation in inoculated leaves were achieved by inactivation of DCL4, while in the upper leaves and roots, it required the additional inactivation of DCL2. In transgenic A. thaliana carrying the PVX amplicon with a green fluorescent protein (GFP) gene insertion in the chromosome (AMP243 line), absence of DCL4 enabled high levels of PVX-GFP accumulation in various aerial organs but not in the roots, suggesting that DCL4 is critical for intracellular antiviral silencing in shoots but not in roots, where it can be functionally compensated by other DCL proteins. Together, the high level of functional redundancies among DCL proteins may contribute to the potent antiviral activities against PVX replication in roots.