Rice dwarf virus (RDV) replicates in and is transmitted by a leafhopper vector in a persistent-propagative manner. Previous cytopathologic and genetic data revealed that tubular structures, constructed by the nonstructural viral protein Pns10, contain viral particles and are directly involved in the intercellular spread of RDV among cultured leafhopper cells. Here, we demonstrated that RDV exploited these virus-containing tubules to move along actin-based microvilli of the epithelial cells and muscle fibers of visceral muscle tissues in the alimentary canal, facilitating the spread of virus in the body of its insect vector leafhoppers. In cultured leafhopper cells, the knockdown of Pns10 expression due to RNA interference (RNAi) induced by synthesized dsRNA from Pns10 gene strongly inhibited tubule formation and prevented the spread of virus among insect vector cells. RNAi induced after ingestion of dsRNA from Pns10 gene strongly inhibited formation of tubules, preventing intercellular spread and transmission of the virus by the leafhopper. All these results, for the first time, show that a persistent-propagative virus exploits virus-containing tubules composed of a nonstructural viral protein to traffic along actin-based cellular protrusions, facilitating the intercellular spread of the virus in the vector insect. The RNAi strategy and the insect vector cell culture provide useful tools to investigate the molecular mechanisms enabling efficient transmission of persistent-propagative plant viruses by vector insects.

The P9-1 protein of Rice black streaked dwarf virus accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in viruses in the family Reoviridae. Crystallographic analysis of P9-1 revealed structural features that allow the protein to form dimers via hydrophobic interactions. Each dimer has carboxy-terminal regions, resembling arms, that extend to neighboring dimers, thereby uniting sets of four dimers via lateral hydrophobic interactions, to yield cylindrical octamers. The importance of these regions for the formation of viroplasm-like inclusions was confirmed by the absence of such inclusions when P9-1 was expressed without its carboxy-terminal arm. The octamers are vertically elongated cylinders resembling the structures formed by NSP2 of rotavirus, even though there are no significant similarities between the respective primary and secondary structures of the two proteins. Our results suggest that an octameric structure with an internal pore might be important for the functioning of the respective proteins in the events that occur in the viroplasm, which might include viral morphogenesis.

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Vector insect cells infected with Rice gall dwarf virus, a member of the family Reoviridae, contained the virus-associated microtubules adjacent to the viroplasms, as revealed by transmission electron, electron tomographic, and confocal microscopy. The viroplasms, putative sites of viral replication, contained the nonstructural viral proteins Pns7 and Pns12, as well as core protein P5, of the virus. Microtubule-depolymerizing drugs suppressed the association of viral particles with microtubules and prevented the release of viruses from cells without significantly affecting viral multiplication. Thus, microtubules appear to mediate viral transport within and release of viruses from infected vector cells. Rice gall dwarf virus (RGDV), Rice dwarf virus (RDV), andWound tumor virus, members of the genus Phytoreovirus in the family Reoviridae, multiply both in plants and in invertebrate insect vectors. Each virus exists as icosahedral particles of approximately 65 to 70 nm in diameter, with two concentric layers (shells) of proteins that enclose a core (1, 13). The viral genome of RGDV consists of 12 segmented double-stranded RNAs that encode six structural (P1, P2, P3, P5, P6, and P8) and six nonstructural (Pns4, Pns7, Pns9, Pns10, Pns11, and Pns12) proteins (reference 21 and references therein). The core capsid is composed of P3, the major protein, which encloses P1, P5, and P6 (12). The outer layer consists of two proteins, namely, P2 and P8 (10, 12).Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of viruses in the family Reoviridae. After infecting insect vector cell monolayers (VCMs) in culture with RDV, Wei et al. (19) examined the generation of RDV particles in and at the periphery of such viroplasms. VCMs are also useful for studies of RGDV, allowing detailed analysis of the synchronous replication and multiplication of this virus (14). In order to identify the viroplasms in RGDV-infected VCMs, we examined the subcellular localization of Pns7, Pns12, P5, and RGDV particles by confocal immunofluorescence microscopy. Pns7 and Pns12 of RGDV correspond to Pns6 and Pns11, respectively, which are components of the viroplasm of RDV (12,19). RGDV P5 is a counterpart of RDV P5, a core protein that locates inside the viroplasm in RDV-infected cells. We inoculated VCMs with RGDV, purified by the method reported in reference 15, at a multiplicity of infection (MOI) of 1; fixed them 48 h postinfection (p.i.); probed the cells with Pns7-, Pns12-, P5-, and viral-antigen-specific antibodies (11, 12) that had been conjugated to fluorescein isothiocyanate (FITC) (Sigma, St. Louis, MO) or rhodamine (Sigma); and examined them by confocal microscopy, as described previously (19). In RGDV-infected cells, Pns7, Pns12, and P5 were detected as punctate inclusions (Fig. 1). Immunostained viral antigens formed ringlike structures around the punctate inclusions. When the images were merged, Pns7, Pns12, and P5 were colocalized in the punctate inclusions, indicating that th...

SummaryRice stripe virus (RSV) has a serious negative effect on rice production in temperate regions of East Asia. Focusing on the putative importance of the selection of target sequences for RNA interference (RNAi), we analysed the effects of potential target sequences in each of the coding genes in the RSV genome, using transgenic rice plants that expressed a set of inverted-repeat (IR) constructs. The reactions of inoculated transgenic T 1 plants to RSV were divided subjectively into three classes, namely highly resistant, moderately resistant and lacking enhanced resistance to RSV, even though plants that harboured any constructs accumulated transgene-specific siRNAs prior to inoculation with RSV. Transgenic plants that harboured IR constructs specific for the gene for pC3, which encodes nucleocapsid protein, and for pC4, which encodes a viral movement protein, were immune to infection by RSV and were more resistant to infection than the natural resistant cultivars that have been used to control the disease in the field. By contrast, the IR construct specific for the gene for pC2, which encodes a glycoprotein of unknown function, and for p4, which encodes a major non-structural protein of unknown function, did not result in resistance. Our results indicate that not all RNAi constructs against viral RNAs are equally effective in preventing RSV infection and that it is important to identify the viral 'Achilles heel' for RNAi attack in the engineering of plants.