Taiyun Wei

Silencing by RNAi of the gene for Pns12, a viroplasm matrix protein of Rice dwarf virus, results in strong resistance of transgenic rice plants to the virus

The non-structural protein Pns12 of Rice dwarf virus is one of the early proteins expressed in cultured insect cells, and it is one of 12 proteins that initiate the formation of the viroplasm, the putative site of viral replication. Pns4 is also a non-structural protein, visible as minitubules after nucleation of the viroplasm. We introduced Pns12- and Pns4-specific RNA interference (RNAi) constructs into rice plants. The resultant transgenic plants accumulated short interfering RNAs specific to the constructs. The progeny of rice plants with Pns12-specific RNAi constructs, after self-fertilization, were strongly resistant to viral infection. By contrast, resistance was less apparent in the case of rice plants with Pns4-specific RNAi constructs, and delayed symptoms appeared in some plants of each line. Our results suggest that interference with the expression of a protein that is critical for viral replication, such as the viroplasm matrix protein Pns12, might be a practical and effective way to control viral infection in crop plants.

The Spread of Rice Dwarf Virus among Cells of Its Insect Vector Exploits Virus-Induced Tubular Structures

ABSTRACT Various cytopathological structures, known as inclusion bodies, are formed upon infection of cultured leafhopper cells by Rice dwarf virus, a member of the family Reoviridae. These structures include tubules of approximately 85 nm in diameter which are composed of the nonstructural viral protein Pns10 and contain viral particles. Such tubular structures were produced in heterologous non-host insect cells that expressed Pns10 of the virus. These tubules, when associated with actin-based filopodia, were able to protrude from the surface of cells and to penetrate neighboring cells. A binding assay in vitro revealed the specific binding of Pns10 to actin. Infection of clusters of cells was readily apparent 5 days after inoculation at a low multiplicity of infection with the virus, even in the presence of neutralizing antibodies. However, treatment of host cells with drugs that inhibited the elongation of actin filaments abolished the extension of Pns10 tubules from the surface of cells, with a significant simultaneous decrease in the extent of infection of neighboring cells. These results together revealed a previously undescribed aspect of the intercellular spread of Rice dwarf virus, wherein the virus exploits tubules composed of a nonstructural viral protein and actin-based filopodia to move into neighboring cells.

Genetic diversity and population structure of rice stripe virus in China

Rice stripe virus (RSV) is one of the most economically important pathogens of rice and is repeatedly epidemic in China, Japan and Korea. The most recent outbreak of RSV in eastern China in 2000 caused significant losses and raised serious concerns. In this paper, we provide a genotyping profile of RSV field isolates and describe the population structure of RSV in China, based on the nucleotide sequences of isolates collected from different geographical regions during 1997-2004. RSV isolates could be divided into two or three subtypes, depending on which gene was analysed. The genetic distances between subtypes range from 0.050 to 0.067. The population from eastern China is composed only of subtype I/IB isolates. In contrast, the population from Yunnan province (southwest China) is composed mainly of subtype II isolates, but also contains a small proportion of subtype I/IB isolates and subtype IA isolates. However, subpopulations collected from different districts in eastern China or Yunnan province are not genetically differentiated and show frequent gene flow. RSV genes were found to be under strong negative selection. Our data suggest that the most recent outbreak of RSV in eastern China was not due to the invasion of new RSV subtype(s). The evolutionary processes contributing to the observed genetic diversity and population structure are discussed.

Development of an Insect Vector Cell Culture and RNA Interference System To Investigate the Functional Role of Fijivirus Replication Protein

ABSTRACT An in vitro culture system of primary cells from white-backed planthopper, an insect vector of Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus, was established to study replication of the virus. Viroplasms, putative sites of viral replication, contained the nonstructural viral protein P9-1, viral RNA, outer-capsid proteins, and viral particles in virus-infected cultured insect vector cells, as revealed by transmission electron and confocal microscopy. Formation of viroplasm-like structures in non-host insect cells upon expression of P9-1 suggested that the matrix of viroplasms observed in virus-infected cells was composed basically of P9-1. In cultured insect vector cells, knockdown of P9-1 expression due to RNA interference (RNAi) induced by synthesized double-stranded RNA (dsRNA) from the P9-1 gene strongly inhibited viroplasm formation and viral infection. RNAi induced by ingestion of dsRNA strongly abolished viroplasm formation, preventing efficient viral spread in the body of intact vector insects. All these results demonstrated that P9-1 was essential for viroplasm formation and viral replication. This system, combining insect vector cell culture and RNA interference, can further advance our understanding of the biological activities of fijivirus replication proteins.

The P7-1 protein of southern rice black-streaked dwarf virus, a fijivirus, induces the formation of tubular structures in insect cells

Southern rice black-streaked dwarf virus (SRBSDV), an insect- and plant-infecting reovirus of the genus Fijivirus, induced the formation of virus-containing tubules in infected plant and insect vector cells. Expression of the nonstructural protein P7-1 of SRBSDV in insect cells by a recombinant baculovirus resulted in the formation of tubules with dimensions and appearance similar to those found in SRBSDV-infected cells. These tubules protruded from the cell surface, supporting the hypothesis that the P7-1 protein contains two putative transmembrane domains that are necessary for the formation of tubules. Furthermore, the self-interaction of SRBSDV P7-1 protein indicates that this protein has the capacity to form homodimers or oligomers to assemble the proposed helical symmetry structure of tubules. Taken together, our results indicate that SRBSDV P7-1 has the intrinsic ability of self-interaction to form tubules growing from the cell surface in the absence of other viral proteins.

Entry of Rice Dwarf Virus into Cultured Cells of Its Insect Vector Involves Clathrin-Mediated Endocytosis

ABSTRACT Electron microscopy revealed that the entry of Rice dwarf virus (RDV) into insect vector cells involved endocytosis via coated pits. The treatment of cells with drugs that block receptor-mediated or clathrin-mediated endocytosis significantly reduced RDV infectivity. However, the drug that blocks caveola-mediated endocytosis had a negligible effect on such infection. Infection was also inhibited when cells had been pretreated with bafilomycin A1, which interferes with acidification of endosomes. Moreover, immunofluorescence staining indicated that the virus is internalized into early endosomes. Together, our data indicate that RDV enters insect vector cells through receptor-mediated, clathrin-dependent endocytosis and is sequestered in early endosomes.

p2 of Rice stripe virus (RSV) interacts with OsSGS3 and is a silencing suppressor

A rice cDNA library was screened by a galactosidase 4 (Gal4)-based yeast two-hybrid system with Rice stripe virus (RSV) p2 as bait. The results revealed that RSV p2 interacted with a rice protein exhibiting a high degree of identity with Arabidopsis thaliana suppressor of gene silencing 3 (AtSGS3). The interaction was confirmed by bimolecular fluorescence complementation assay. SGS3 has been shown to be involved in sense transgene-induced RNA silencing and in the biogenesis of trans-acting small interfering RNAs (ta-siRNAs), possibly functioning as a cofactor of RNA-dependent RNA polymerase 6 (RDR6). Given the intimate relationships between virus and RNA silencing, further experiments were conducted to show that p2 was a silencing suppressor. In addition, p2 enhanced the accumulation and pathogenicity of Potato virus X in Nicotiana benthamiana. Five genes that have been demonstrated to be targets of TAS3-derived ta-siRNAs were up-regulated in RSV-infected rice. The implications of these findings are discussed.

Tubular Structure Induced by a Plant Virus Facilitates Viral Spread in Its Vector Insect

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.

Association of Rice Gall Dwarf Virus with Microtubules Is Necessary for Viral Release from Cultured Insect Vector Cells

ABSTRACT 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.

The early secretory pathway and an actin-myosin VIII motility system are required for plasmodesmatal localization of the NSvc4 protein of Rice stripe virus

Plant viruses utilize movement proteins to gain access to plasmodesmata (PD) for cell-to-cell propagation. While the NSvc4 protein of Rice stripe virus (RSV) is implicated in the passage of viruses from cell to cell, its role remains to be elucidated. We examined the mechanisms by which RSV NSvc4 is targeted to PD in cell walls. NSvc4 accumulated at PD when expressed as a fusion with yellow fluorescent protein in leaf cells of Nicotiana benthamiana. NSvc4 was targeted to PD via the endoplasmic reticulum-to-Golgi secretory pathway, and the actomyosin motility system was required for the delivery of NSvc4 to PD. Moreover, it appeared that NSvc4 utilized myosin VIII-1 rather than myosin XI for trafficking to PD. Taken together, our data reveal that the targeting of NSvc4 to PD exploits the early secretory pathway and the actin-myosin VIII motility system in the leaves of a non-host plant, N. benthamiana.