Zujian Wu

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.

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.

Biogenesis, Function, and Applications of Virus-Derived Small RNAs in Plants

RNA silencing, an evolutionarily conserved and sequence-specific gene-inactivation system, has a pivotal role in antiviral defense in most eukaryotic organisms. In plants, a class of exogenous small RNAs (sRNAs) originating from the infecting virus called virus-derived small interfering RNAs (vsiRNAs) are predominantly responsible for RNA silencing-mediated antiviral immunity. Nowadays, the process of vsiRNA formation and the role of vsiRNAs in plant viral defense have been revealed through deep sequencing of sRNAs and diverse genetic analysis. The biogenesis of vsiRNAs is analogous to that of endogenous sRNAs, which require diverse essential components including dicer-like (DCL), argonaute (AGO), and RNA-dependent RNA polymerase (RDR) proteins. vsiRNAs trigger antiviral defense through post-transcriptional gene silencing (PTGS) or transcriptional gene silencing (TGS) of viral RNA, and they hijack the host RNA silencing system to target complementary host transcripts. Additionally, several applications that take advantage of the current knowledge of vsiRNAs research are being used, such as breeding antiviral plants through genetic engineering technology, reconstructing of viral genomes, and surveying viral ecology and populations. Here, we will provide an overview of vsiRNA pathways, with a primary focus on the advances in vsiRNA biogenesis and function, and discuss their potential applications as well as the future challenges in vsiRNAs research.

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.

Pc4, a putative movement protein of Rice stripe virus, interacts with a type I DnaJ protein and a small Hsp of rice

Rice stripe virus (RSV) infects rice and causes great yield reduction in some Asian countries. In this study, rice cDNA library was screened by a Gal4-based yeast two-hybrid system using pc4, a putative movement protein of RSV, as the bait. A number of positive colonies were identified and sequence analysis revealed that they might correspond to ten independent proteins. Two of them were selected and further characterized. The two proteins were a J protein and a small Hsp, respectively. Interactions between Pc4 and the two proteins were confirmed using coimmunoprecipitation. Implications of the findings that pc4 interacted with two chaperone proteins were discussed.

Rice ragged stunt virus segment S6-encoded nonstructural protein Pns6 complements cell-to-cell movement of Tobacco mosaic virus-based chimeric virus

The protein(s) that support intercellular movement of Rice ragged stunt virus (RRSV) have not yet been identified. In this study, the role of three nonstructural proteins Pns6, Pns7 and Pns10 in cell-to-cell movement were determined with a movement-deficient Tobacco mosaic virus (TMV) vector. The results showed that only the Pns6 could complement the cell-to-cell movement of the movement-deficient TMV in Nicotiana tabacum Xanthi nc and N. benthamiana plants, and both N- and C-terminal 50 amino acids of Pns6 were essential for the cell-to-cell movement. Transient expression in epidermal cells from N. benthamiana showed that the Pns6-eGFP fusion protein was present predominantly along the cell wall as well as a few punctate sites perhaps indicating plasmodesmata. Taken together with previous finding that the Pns6 has nucleic acid-binding activity (Shao et al., 2004), the possible role of Pns6 in cell-to-cell movement of RRSV were discussed.

GsLRPK, a novel cold-activated leucine-rich repeat receptor-like protein kinase from Glycine soja, is a positive regulator to cold stress tolerance

Plant LRR-RLKs serve as protein interaction platforms, and as regulatory modules of protein activation. Here, we report the isolation of a novel plant-specific LRR-RLK from Glycine soja (termed GsLRPK) by differential screening. GsLRPK expression was cold-inducible and shows Ser/Thr protein kinase activity. Subcellular localization studies using GFP fusion protein indicated that GsLRPK is localized in the plasma membrane. Real-time PCR analysis indicated that temperature, salt, drought, and ABA treatment can alter GsLRPK gene transcription in G. soja. However, just protein induced by cold stress not by salinity and ABA treatment in tobacco was found to possess kinase activity. Furthermore, we found that overexpression of GsLRPK in yeast and Arabidopsis can enhance resistance to cold stress and increase the expression of a number of cold responsive gene markers.