Osamu Netsu

Molecular detection of nine rice viruses by a reverse-transcription loop-mediated isothermal amplification assay

A reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay was established for the detection of nine viruses from infected rice plants, including rice black-streaked dwarf virus (RBSDV), rice dwarf virus (RDV), rice gall dwarf virus (RGDV), rice ragged stunt virus (RRSV), rice transitory yellowing virus (RTYV), rice stripe virus (RSV), rice grassy stunt virus (RGSV), rice tungro spherical virus (RTSV), and rice tungro bacilliform virus (RTBV). Virus-specific primer sets were designed from the genome sequences of these viruses. By the combination of RNA rapid extraction and RT-LAMP, these nine viruses could be detected within 2h from infected rice plants. The sensitivities of the assays were either higher than (for RSV, RTBV, and RTYV) or similar (for RDV) to those of one-step RT-PCR. Furthermore, RTBV and RTSV were detected not only in infected rice plants but also in viruliferous insect vectors. The RT-LAMP assays may facilitate studies on rice disease epidemiology, outbreak surveillance, and molecular pathology.

In Planta Recognition of a Double-Stranded RNA Synthesis Protein Complex by a Potexviral RNA Silencing Suppressor

This work reports the detailed molecular function of TGBp1, a suppressor of RNA silencing encoded by a potexvirus. TGBp1 interacts with SGS3 and RDR6 and aggregates SGS3/RDR6 bodies in the cytoplasm, thereby inhibiting dsRNA synthesis. Thus, this work sheds new light on the dsRNA synthesis–mediated secondary siRNA pathway as another general target of viral suppressors of RNA silencing. RNA silencing plays an important antiviral role in plants and invertebrates. To counteract antiviral RNA silencing, most plant viruses have evolved viral suppressors of RNA silencing (VSRs). TRIPLE GENE BLOCK PROTEIN1 (TGBp1) of potexviruses is a well-characterized VSR, but the detailed mechanism by which it suppresses RNA silencing remains unclear. We demonstrate that transgenic expression of TGBp1 of plantago asiatica mosaic virus (PlAMV) induced developmental abnormalities in Arabidopsis thaliana similar to those observed in mutants of SUPPRESSOR OF GENE SILENCING3 (SGS3) and RNA-DEPENDENT RNA POLYMERASE6 (RDR6) required for the trans-acting small interfering RNA synthesis pathway. PlAMV-TGBp1 inhibits SGS3/RDR6-dependent double-stranded RNA synthesis in the trans-acting small interfering RNA pathway. TGBp1 interacts with SGS3 and RDR6 and coaggregates with SGS3/RDR6 bodies, which are normally dispersed in the cytoplasm. In addition, TGBp1 forms homooligomers, whose formation coincides with TGBp1 aggregation with SGS3/RDR6 bodies. These results reveal the detailed molecular function of TGBp1 as a VSR and shed new light on the SGS3/RDR6-dependent double-stranded RNA synthesis pathway as another general target of VSRs.

Rice Dwarf Viruses with Dysfunctional Genomes Generated in Plants Are Filtered Out in Vector Insects: Implications for the Origin of the Virus

ABSTRACT Rice dwarf virus (RDV), with 12 double-stranded RNA (dsRNA) genome segments (S1 to S12), replicates in and is transmitted by vector insects. The RDV-plant host-vector insect system allows us to examine the evolution, adaptation, and population genetics of a plant virus. We compared the effects of long-term maintenance of RDV on population structures in its two hosts. The maintenance of RDV in rice plants for several years resulted in gradual accumulation of nonsense mutations in S2 and S10, absence of expression of the encoded proteins, and complete loss of transmissibility. RDV maintained in cultured insect cells for 6 years retained an intact protein-encoding genome. Thus, the structural P2 protein encoded by S2 and the nonstructural Pns10 protein encoded by S10 of RDV are subject to different selective pressures in the two hosts, and mutations accumulating in the host plant are detrimental in vector insects. However, one round of propagation in insect cells or individuals purged the populations of RDV that had accumulated deleterious mutations in host plants, with exclusive survival of fully competent RDV. Our results suggest that during the course of evolution, an ancestral form of RDV, of insect virus origin, might have acquired the ability to replicate in a host plant, given its reproducible mutations in the host plant that abolish vector transmissibility and viability in nature.

Transgenic strategies to confer resistance against viruses in rice plants

Rice (Oryza sativa L.) is cultivated in more than 100 countries and supports nearly half of the world’s population. Developing efficient methods to control rice viruses is thus an urgent necessity because viruses cause serious losses in rice yield. Most rice viruses are transmitted by insect vectors, notably planthoppers and leafhoppers. Viruliferous insect vectors can disperse their viruses over relatively long distances, and eradication of the viruses is very difficult once they become widespread. Exploitation of natural genetic sources of resistance is one of the most effective approaches to protect crops from virus infection; however, only a few naturally occurring rice genes confer resistance against rice viruses. Many investigators are using genetic engineering of rice plants as a potential strategy to control viral diseases. Using viral genes to confer pathogen-derived resistance against crops is a well-established procedure, and the expression of various viral gene products has proved to be effective in preventing or reducing infection by various plant viruses since the 1990s. RNA interference (RNAi), also known as RNA silencing, is one of the most efficient methods to confer resistance against plant viruses on their respective crops. In this article, we review the recent progress, mainly conducted by our research group, in transgenic strategies to confer resistance against tenuiviruses and reoviruses in rice plants. Our findings also illustrate that not all RNAi constructs against viral RNAs are equally effective in preventing virus infection and that it is important to identify the viral “Achilles’ heel” gene to target for RNAi attack when engineering plants.

Fig mosaic emaravirus p4 protein is involved in cell-to-cell movement.

Fig mosaic virus (FMV), a member of the newly formed genus Emaravirus, is a segmented negative-strand RNA virus. Each of the six genomic FMV segments contains a single ORF: that of RNA4 encodes the protein p4. FMV-p4 is presumed to be the movement protein (MP) of the virus; however, direct experimental evidence for this is lacking. We assessed the intercellular distribution of FMV-p4 in plant cells by confocal laser scanning microscopy and we found that FMV-p4 was localized to plasmodesmata and to the plasma membrane accompanied by tubule-like structures. A series of experiments designed to examine the movement functions revealed that FMV-p4 has the capacity to complement viral cell-to-cell movement, prompt GFP diffusion between cells, and spread by itself to neighbouring cells. Altogether, our findings demonstrated that FMV-p4 shares several properties with other viral MPs and plays an important role in cell-to-cell movement.

The nonstructural protein pC6 of rice grassy stunt virus trans-complements the cell-to-cell spread of a movement-defective tomato mosaic virus

The nonstructural protein pC6 encoded by rice grassy stunt virus is thought to correspond functionally to the nonstructural protein pC4 of rice stripe virus, which can support viral cell-to-cell movement. In a trans-complementation experiment with a movement-defective tomato mosaic virus, pC6 and pC4 facilitated intercellular transport of the virus. Transient expression of pC6, fused with green fluorescent protein, in epidermal cells was predominantly observed close to the cell wall as well as in a few punctate structures, presumably associated with plasmodesmata. These results suggest that pC6 has a role similar to that of pC4 in viral cell-to-cell movement.

Three-Dimensional Analysis of the Association of Viral Particles with Mitochondria during the Replication of Rice Gall Dwarf Virus

Examination of cultured insect vector cells that had been infected with Rice gall dwarf virus (RGDV), using transmission electron microscopy and confocal microscopy, revealed the presence of clusters of virus-coated mitochondria around viroplasms in which replication and assembly of RGDV occurred, suggesting a role for mitochondria in supplying the energy required for viral morphogenetic processes. Electron tomography revealed that RGDV particles on the surface of mitochondria are arrayed in an orderly but loose manner, unlike tightly packaged particles in vesicular compartments, suggesting the presence of counterpart molecules on the surface of mitochondria. The viral particles in close proximity to mitochondria were aligned along intermediate filaments, which might serve as scaffolds for the anchorage of these particles. RGDV has a putative mitochondrion-targeting sequence on the outer surface of the outer-capsid protein P8. The arrangement of RGDV particles around mitochondria suggests that the region of the P8 protein containing the mitochondrion-targeting sequence might attach to a molecule like a receptor on the outer mitochondrial membrane. Our analysis demonstrates the three-dimensional arrangement and molecular basis for the mitochondrial proximity of RGDV particles during viral replication.

Peanut stunt virus 2b cistron plays a role in viral local and systemic accumulation and virulence in Nicotiana benthamiana

To analyze the role of the 2b protein (2bP) of Peanut stunt virus (PSV) in the viral infection cycle, we constructed PSV mutants that express either no 2bP or N-terminal-truncated 2bP. The accumulation of wild-type and mutant viruses in tobacco protoplasts indicated that the 2b cistron is not essential for viral replication. Viral accumulation in Nicotiana benthamiana plants suggested that the 2b cistron is responsible for viral accumulation in inoculated and upper leaves and has a role in virulence. The involvement of eight N-terminal amino acids of 2bP in these functions is discussed.

The movement protein encoded by gene 3 of rice transitory yellowing virus is associated with virus particles.

Gene 3 in the genomes of several plant-infecting rhabdoviruses, including rice transitory yellowing virus (RTYV), has been postulated to encode a cell-to-cell movement protein (MP). Trans-complementation experiments using a movement-defective tomato mosaic virus and the P3 protein of RTYV, encoded by gene 3, facilitated intercellular transport of the mutant virus. In transient-expression experiments with the GFP-fused P3 protein in epidermal leaf cells of Nicotiana benthamiana, the P3 protein was associated with the nucleus and plasmodesmata. Immunogold-labelling studies of thin sections of RTYV-infected rice plants using an antiserum against Escherichia coli-expressed His(6)-tagged P3 protein indicated that the P3 protein was located in cell walls and on virus particles. In Western blots using antisera against E. coli-expressed P3 protein and purified RTYV, the P3 protein was detected in purified RTYV, whilst antiserum against purified RTYV reacted with the E. coli-expressed P3 protein. After immunogold labelling of crude sap from RTYV-infected rice leaves, the P3 protein, as well as the N protein, was detected on the ribonucleocapsid core that emerged from partially disrupted virus particles. These results provide evidence that the P3 protein of RTYV, which functions as a viral MP, is a viral structural protein and seems to be associated with the ribonucleocapsid core of virus particles.

Recent progress in research on cell-to-cell movement of rice viruses

To adapt to plants as hosts, plant viruses have evolutionally needed the capacity to modify the host plasmodesmata (PD) that connect adjacent cells. Plant viruses have acquired one or more genes that encode movement proteins (MPs), which facilitate the cell-to-cell movement of infectious virus entities through PD to adjacent cells. Because of the diversity in their genome organization and in their coding sequences, rice viruses may each have a distinct cell-to-cell movement strategy. The complexity of their unusual genome organizations and replication strategies has so far hampered reverse genetic research on their genome in efforts to investigate virally encoded proteins that are involved in viral movement. However, the MP of a particular virus can complement defects in cell-to-cell movement of other distantly related or even unrelated viruses. Trans-complementation experiments using a combination of a movement-defective virus and viral proteins of interest to identify MPs of several rice viruses have recently been successful. In this article, we reviewed recent research that has advanced our understanding of cell-to-cell movement of rice viruses.