Identification of a Novel Quinvirus in the Family Betaflexiviridae That Infects Winter Wheat.

Yellow mosaic disease in winter wheat is usually attributed to the infection by bymoviruses or furoviruses; however, there is still limited information on whether other viral agents are also associated with this disease. To investigate the wheat viromes associated with yellow mosaic disease, we carried out de novo RNA sequencing (RNA-seq) analyses of symptomatic and asymptomatic wheat-leaf samples obtained from a field in Hokkaido, Japan, in 2018 and 2019. The analyses revealed the infection by a novel betaflexivirus, which tentatively named wheat virus Q (WVQ), together with wheat yellow mosaic virus (WYMV, a bymovirus) and northern cereal mosaic virus (a cytorhabdovirus). Basic local alignment search tool (BLAST) analyses showed that the WVQ strains (of which there are at least three) were related to the members of the genus Foveavirus in the subfamily Quinvirinae (family Betaflexiviridae). In the phylogenetic tree, they form a clade distant from that of the foveaviruses, suggesting that WVQ is a member of a novel genus in the Quinvirinae. Laboratory tests confirmed that WVQ, like WYMV, is potentially transmitted through the soil to wheat plants. WVQ was also found to infect rye plants grown in the same field. Moreover, WVQ-derived small interfering RNAs accumulated in the infected wheat plants, indicating that WVQ infection induces antiviral RNA silencing responses. Given its common coexistence with WYMV, the impact of WVQ infection on yellow mosaic disease in the field warrants detailed investigation.

Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana.

Eukaryotes employ RNA silencing as an innate defense system against invading viruses. Dicer proteins play the most crucial role in initiating this antiviral pathway as they recognize and process incoming viral nucleic acids into small interfering RNAs. Generally, 2 successive infection stages constitute viral infection in plants. First, the virus multiplies in initially infected cells or organs after viral transmission and then the virus subsequently spreads systemically through the vasculature to distal plant tissues or organs. Thus, antiviral silencing in plants must cope with both local and systemic invasion of viruses. In a recent study using 2 sets of different experiments, we clearly demonstrated the differential requirement for Dicer-like 4 (DCL4) and DCL2 proteins in the inhibition of intracellular and systemic infection by potato virus X in Arabidopsis thaliana. Taken together with the results of other studies, here we further discuss the functional specificity of DCL proteins in the antiviral silencing pathway.

Differential contributions of plant Dicer-like proteins to antiviral defences against potato virus X in leaves and roots.

Members 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.

Complete genome sequence of Habenaria mosaic virus, a new potyvirus infecting a terrestrial orchid (Habenaria radiata) in Japan.

The complete genomic sequence of Habenaria mosaic virus (HaMV), which infects terrestrial orchids (Habenaria radiata), has been determined. The genome is composed of 9,499 nucleotides excluding the 3'-terminal poly(A) tail, encoding a large polyprotein of 3,054 amino acids with the genomic features typical of a potyvirus. Putative proteolytic cleavage sites were identified by sequence comparison to those of known potyviruses. The HaMV polyprotein showed 58 % amino acid sequence identity to that encoded by the most closely related potyvirus, tobacco vein banding mosaic virus. Phylogenetic analysis of the polyprotein amino acid sequence and its coding sequences confirmed that HaMV formed a cluster with the chilli veinal mottle virus group, most of which infect solanaceous plants. These results suggest that HaMV is a distinct member of the genus Potyvirus.

Characterization of burdock mottle virus, a novel member of the genus Benyvirus, and the identification of benyvirus-related sequences in the plant and insect genomes.

The complete nucleotide sequence of the burdock mottle virus (BdMoV) isolated from an edible burdock plant (Arctium lappa) in Japan has been determined. BdMoV has a bipartite genome, whose organization is similar to RNA1 and RNA2 of benyviruses, beet necrotic yellow vein virus (BNYVV), beet soil-borne mosaic virus (BSBMV), and rice stripe necrosis virus (RSNV). BdMoV RNA1 (7038 nt) contains a single open reading frame (ORF) encoding a 249-kDa polypeptide that consists of methyl-transferase, helicase, papain-like protease, AlkB-like, and RNA-dependent RNA polymerase domains. The AlkB-like domain sequence is not present in the proteins encoded by other known benyviruses, but is found in replication-associated proteins of viruses mainly belonging to the families Alfaflexiviridae and Betaflexiviridae. BdMoV RNA2 (4315 nt) contains six ORFs that are similar to those of benyviruses: these are coat protein (CP), CP readthrough, triple gene block movement and cysteine-rich proteins. Phylogenetic analyses showed that BdMoV is more closely related to BNYVV and BSBMV than to RSNV. Database searches showed that benyvirus replicase-related sequences are present in the chromosomes of a chickpea plant (Cicer arietinum) and a blood-sucking insect (Rhodnius prolixus). Some other benyvirus-related sequences are found in the transcriptome shotgun libraries of a few species of plants and a bark beetle. Our results show that BdMoV is a distinct species of the genus Benyvirus and that ancestral and extant benyviruses may have infected or currently infect a wide range of hosts, including plants and insects.

Orchid fleck virus structural proteins N and P form intranuclear viroplasm-like structures in the absence of viral infection.

Orchid fleck virus (OFV) has a unique two-segmented negative-sense RNA genome that resembles that of plant nucleorhabdoviruses. In infected plant cells, OFV and nucleorhabdoviruses induce an intranuclear electron-lucent viroplasm that is believed to be the site for virus replication. In this study, we investigated the molecular mechanism by which OFV viroplasms are produced in vivo. Among OFV-encoded proteins, the nucleocapsid protein (N) and the putative phosphoprotein (P) were present in nuclear fractions of OFV-infected Nicotiana benthamiana plants. Transient coexpression of N and P, in the absence of virus infection, was shown to be sufficient for formation of an intranuclear viroplasm-like structure in plant cells. When expressed independently as a fluorescent protein fusion product in uninfected plant cells, N protein accumulated throughout the cell, while P protein accumulated in the nucleus. However, the N protein, when coexpressed with P, was recruited to a subnuclear region to induce a large viroplasm-like focus. Deletion and substitution mutagenesis demonstrated that the P protein contains a nuclear localization signal (NLS). Artificial nuclear targeting of the N-protein mutant was insufficient for formation of viroplasm-like structures in the absence of P. A bimolecular fluorescence complementation assay confirmed interactions between the N and P proteins within subnuclear viroplasm-like foci and interactions of two of the N. benthamiana importin-α homologues with the P protein but not with the N protein. Taken together, our results suggest that viroplasm formation by OFV requires nuclear accumulation of both the N and P proteins, which is mediated by P-NLS, unlike nucleorhabdovirus viroplasm utilizing the NLS on protein N.

The cysteine-rich proteins of beet necrotic yellow vein virus and tobacco rattle virus contribute to efficient suppression of silencing in roots.

Many plant viruses encode proteins that suppress RNA silencing, but little is known about the activity of silencing suppressors in roots. This study examined differences in the silencing suppression activity of different viruses in leaves and roots of Nicotiana benthamiana plants. Infection by tobacco mosaic virus, potato virus Y and cucumber mosaic virus but not potato virus X (PVX) resulted in strong silencing suppression activity of a transgene in both leaves and roots, whereas infection by beet necrotic yellow vein virus (BNYVV) and tobacco rattle virus (TRV) showed transgene silencing suppression in roots but not in leaves. For most viruses tested, viral negative-strand RNA accumulated at a very low level in roots, compared with considerable levels of positive-strand genomic RNA. Co-inoculation of leaves with PVX and either BNYVV or TRV produced an increase in PVX negative-strand RNA and subgenomic RNA (sgRNA) accumulation in roots. The cysteine-rich proteins (CRPs) BNYVV p14 and TRV 16K showed weak silencing suppression activity in leaves. However, when either of these CRPs was expressed from a PVX vector, there was an enhancement of PVX negative-strand RNA and sgRNA accumulation in roots compared with PVX alone. Such enhancement of PVX sgRNAs was also observed by expression of CRPs of other viruses and the well-known suppressors HC-Pro and p19 but not of the potato mop-top virus p8 CRP. These results indicate that BNYVV- and TRV-encoded CRPs suppress RNA silencing more efficiently in roots than in leaves.

The enigmatic genome of Chara australis virus.

Most of the genomic sequence of Chara australis virus (CAV), previously called Chara corallina virus, has been determined. It is a ssRNA molecule of 9065 nt with at least four ORFs. At its 5' end is an ORF encoding a protein of 227 kDa, distantly homologous to the multifunctional replicases of benyviruses and rubiviruses. Next is an ORF encoding a protein of 44 kDa, homologous to the helicases of pestiviruses. The third ORF encodes an unmatched protein of 38 kDa that is probably a movement protein. The fourth and 3'-terminal ORF encodes a protein of 17.7 kDa homologous to the coat proteins of tobamoviruses. The short methyltransferase region of the CAV replicase matches only the C-terminal motif of benyvirus methyltransferases. This and other clues indicate that approximately 11% and 2% of the 5' and 3' termini of the complete CAV genome, respectively, are missing from the sequence. The aligned amino acid sequences of the CAV proteins and their nearest homologues contain many gaps but relationships inferred from them were little affected by removal of these gaps. Sequence comparisons show that three of the CAV genes may have diverged from the most closely related genes of other viruses 250-450 million years ago, and the sister relationship between the genes of CAV and those of benyviruses and tobamoviruses, mirroring the ancient sister relationship between charophytes (i.e. the algal host of CAV) and embryophytes (i.e. the plant hosts of tobamoviruses and benyviruses), is congruent with this possibility.

The evolutionary history of Beet necrotic yellow vein virus deduced from genetic variation, geographical origin and spread, and the breaking of host resistance.

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.

Identification and characterization of structural proteins of orchid fleck virus.

Orchid fleck virus (OFV) has a bipartite negative-sense RNA genome with sequence similarities to plant rhabdoviruses. The non-enveloped bullet-shaped particles of OFV are similar to those of the internal ribonucleoprotein (RNP)-M protein structure of rhabdoviruses, but they are about half the size of typical plant rhabdoviruses. Purified preparations contained intact bullet-shaped and filamentous particles. The filamentous particles showed a tightly coiled coil structure or a coiled structure with a helical twist, which resembles the RNP complex of rhabdoviruses. OFV bullet-shaped particles were structurally stable in solutions containing 2% Triton X-100 and 0.8 M NaCl. Western blot analyses revealed that the bullet-shaped particles contained N, P and M proteins, while filamentous particles contained mainly N and P proteins. In addition, a small amount of the L protein was detected in both types of particles. Thus, the structural proteins of OFV have properties similar to those of rhabdoviruses, except that the particles are non-enveloped and are relatively resistant to detergent-treatment under high-salt conditions.

Identification of amino acids of the beet necrotic yellow vein virus p25 protein required for induction of the resistance response in leaves of Beta vulgaris plants.

The RNA3-encoded p25 protein of beet necrotic yellow vein virus (BNYVV) is responsible for the production of rhizomania symptoms of sugar beet roots (Beta vulgaris subsp. vulgaris). Here, it was found that the presence of the p25 protein is also associated with the resistance response in rub-inoculated leaves of sugar beet and wild beet (Beta vulgaris subsp. maritima) plants. The resistance phenotype displayed a range of symptoms from no visible lesions to necrotic or greyish lesions at the inoculation site, and only very low levels of virus and viral RNA accumulated. The susceptible phenotype showed large, bright yellow lesions and developed high levels of virus accumulation. In roots after Polymyxa betae vector inoculation, however, no drastic differences in virus and viral RNA accumulation levels were found between plants with susceptible and resistant phenotypes, except at an early stage of infection. There was a genotype-specific interaction between BNYVV strains and two selected wild beet lines (MR1 and MR2) and sugar beet cultivars. Sequence analysis of natural BNYVV isolates and site-directed mutagenesis of the p25 protein revealed that 3 aa residues at positions 68, 70 and 179 are important in determining the resistance phenotype, and that host-genotype specificity is controlled by single amino acid changes at position 68. The mechanism of the occurrence of resistance-breaking BNYVV strains is discussed.

RNA4-encoded p31 of beet necrotic yellow vein virus is involved in efficient vector transmission, symptom severity and silencing suppression in roots.

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.

Orchid fleck virus is a rhabdovirus with an unusual bipartite genome.

Orchid fleck virus (OFV) has an unusual bipartite negative-sense RNA genome with clear sequence similarities to those of nucleorhabdoviruses. The OFV genome consists of two single-stranded RNA molecules, RNA1 and RNA2 that are 6413 and 6001 nt long, respectively, with open reading frame (ORF) information in the complementary sense. RNA1 encodes 49 (ORF1), 26 (ORF2), 38 (ORF3), 20 (ORF4) and 61 kDa (ORF5) proteins, and RNA2 encodes a single protein of 212 kDa (ORF6). ORF1, ORF5 and ORF6 proteins had significant similarities (21-38 % identity) to the nucleocapsid protein (N), glycoprotein (G) and polymerase (L) gene products, respectively, of other rhabdoviruses, especially nucleorhabdoviruses, whereas ORF2, ORF3 and ORF4 proteins had no significant similarities to other proteins in the international databases. Similarities between OFV and rhabdoviruses were also found in the sequence complementarity at both termini of each RNA segment (the common terminal sequences are 3'-UGUGUC---GACACA-5'), the conserved intergenic sequences and in being negative sense. It was proposed that a new genus Dichorhabdovirus in the family Rhabdoviridae of the order Mononegavirales should be established with OFV as its prototype member and type species.

Lower levels of transgene silencing in roots is associated with reduced DNA methylation levels at non-symmetrical sites but not at symmetrical sites.

Transgene transcripts were recently shown to accumulate at higher levels in roots, relative to leaves, of silenced-transgenic Nicotiana benthamiana plants and to be inversely related with the accumulation of small interfering RNAs (siRNAs), suggesting that RNA silencing is less active in roots than in leaves (Andika et al., 2005. Mol. Plant-Microbe Interact. 18: 194). Here we show that the lower transgene RNA silencing activity in roots was associated with lower transgene methylation levels at non-symmetrical CpNpN context but not at symmetrical CpG or CpNpG context in three sets of transformant plants with different exogenous genes. In contrast, such a difference between roots and leaves was not observed for the Tnt1 retrotransposon: no Tnt1 transcript was detected in roots or in leaves of N. benthamiana, while equal levels of Tnt1-derived siRNA accumulation and Tnt1 methylation were found. From our data and previously reported information, we suggest that roots have less of an activity that acts at the step of generation of siRNAs.

Evidence that RNA silencing-mediated resistance to beet necrotic yellow vein virus is less effective in roots than in leaves.

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.

Orchid fleck virus: Brevipalpus californicus mite transmission, biological properties and genome structure.

Orchid fleck virus (OFV) causes necrotic or chlorotic ring spots and fleck symptoms in many orchid species world-wide. The virus has non-enveloped, bacilliform particles of about 40 nm x 100-150 nm and is sap-transmissible to several plant species. OFV is transmitted by the mite Brevipalpus californicus (Banks) in a persistent manner and efficiently transmitted by both adults and nymphs, but not by larvae. Viruliferous mites retain their infectivity for 3 weeks on a virus-immune host. The genome of OFV consists of two molecules of 6431 (RNA1) and 6001 nucleotides (RNA2). The RNAs have conserved and complementary terminal sequences. RNA1 contains five open reading frames (ORF), and RNA2 encodes a single ORF. Although some of the encoded proteins of OFV have sequences similar to those of proteins of plant rhabdoviruses, OFV differs from viruses in the family Rhabdoviridae in having a bipartite genome.