We identified 163 AP2/EREBP (APETALA2/ethylene-responsive element-binding protein) genes in rice. We analyzed gene structures, phylogenies, domain duplication, genome localizations and expression profiles. Conserved amino acid residues and phylogeny construction using the AP2/ERF conserved domain sequence suggest that in rice the OsAP2/EREBP gene family can be classified broadly into four subfamilies [AP2, RAV (related to ABI3/VP1), DREB (dehydration-responsive element-binding protein) and ERF (ethylene-responsive factor)]. The chromosomal localizations of the OsAP2/EREBP genes indicated 20 segmental duplication events involving 40 genes; 58 redundant OsAP2/EREBP genes were involved in tandem duplication events. There were fewer introns after segmental duplication. We investigated expression profiles of this gene family under biotic stresses [infection with rice viruses such as rice stripe virus (RSV), rice tungro spherical virus (RTSV) and rice dwarf virus (RDV, three virus strains S, O and D84)], and various abiotic stresses. Symptoms of virus infection were more severe in RSV infection than in RTSV and RDV infection. Responses to biotic stresses are novel findings and these stresses enhance the ability to identify the best candidate genes for further functional analysis. The genes of subgroup B-5 were not induced under abiotic treatments whereas they were activated by the three RDV strains. None of the genes of subgroups A-3 were differentially expressed by any of the biotic stresses. Our 44K and 22K microarray results suggest that 53 and 52 non-redundant genes in this family were up-regulated in response to biotic and abiotic stresses, respectively. We further examined the stress responsiveness of most genes by reverse transcription-PCR. The study results should be useful in selecting candidate genes from specific subgroups for functional analysis.

Cytoplasmic inclusion bodies, known as viroplasms or viral factories, are assumed to be the sites of replication of members of the family Reoviridae. Immunocytochemical and biochemical analyses were carried out to characterize the poorly understood viroplasms of the phytoreovirus Rice dwarf virus (RDV). Within 6 h of inoculation of cells, viroplasms, namely discrete cytoplasmic inclusions, were formed that contained the non-structural proteins Pns6, Pns11 and Pns12 of RDV, which appeared to be the constituents of the inclusions. Formation of similar inclusions in non-host insect cells upon expression of Pns12 in a baculovirus system and the association of molecules of Pns12 in vitro suggested that the inclusions observed in RDV-infected cells were composed basically of Pns12. Core proteins P1, P3, P5 and P7 and core virus particles were identified in the interior region of the inclusions. In contrast, accumulation of the outer capsid proteins P2, P8 and P9 and of intact virus particles was evident in the peripheral regions of the inclusions. These observations suggest that core particles were constructed inside the inclusions, whereas outer capsid proteins were assembled at the periphery of the inclusions. Viral inclusions were shown to be the sites of viral RNA synthesis by labelling infected cells with 5-bromouridine 59-triphosphate. The number of viroplasms decreased with time post-inoculation as their sizes increased, suggesting that inclusions might fuse with one another during the virus-propagation process. Our results are consistent with a model, proposed for vertebrate reoviruses, in which viroplasms play a pivotal role in virus assembly.

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Rice stripe disease, caused by rice stripe virus (RSV), is one of the major virus diseases in east Asia. Rice plants infected with RSV usually show symptoms such as chlorosis, weakness, necrosis in newly emerged leaves and stunting. To reveal rice cellular systems influenced by RSV infection, temporal changes in the transcriptome of RSV-infected plants were monitored by a customized rice oligoarray system. The transcriptome changes in RSV-infected plants indicated that protein-synthesis machineries and energy production in the mitochondrion were activated by RSV infection, whereas energy production in the chloroplast and synthesis of cell-structure components were suppressed. The transcription of genes related to host-defence systems under hormone signals and those for gene silencing were not activated at the early infection phase. Together with concurrent observation of virus concentration and symptom development, such transcriptome changes in RSV-infected plants suggest that different sets of various host genes are regulated depending on the development of disease symptoms and the accumulation of RSV. INTRODUCTIONRice stripe disease is the most severe virus disease of rice in east Asia. Typical symptoms are chlorosis and weakness on newly emerged leaves. The plant becomes considerably stunted when affected at the early growth stages (Ou, 1972). Rice stripe disease also causes necrosis of newly emerged leaves (Takahashi et al., 1991). The causal virus is Rice stripe virus (RSV), which belongs to the genus Tenuivirus (Falk & Tsai, 1998). RSV is transmitted by small brown planthopper (SBPH; Laodelphax striatellus), Terthron albovittatum, Unkanodes sapporonus and Unkanodes albifascia (Falk & Tsai, 1998;Ou, 1972). RSV has a thin, filamentous shape and no envelope. The genome consists of four single-stranded RNA segments; RNA1 is negative-sense and RNAs 2-4 are ambisense. Viral mRNAs transcribed from viral RNA or viral cRNA by RNA-dependent RNA polymerase are released to the cytoplasm. Subsequently, a 59-capped short ribonucleotide leader cleaved from the host mRNA is added to the viral mRNAs by cap-snatching. The 59-capped RSV RNA is transcribed efficiently in host cells (Falk & Tsai, 1998;Shimizu et al., 1996). Genes encoding a gene-silencing suppressor and movement proteins were also identified in the RSV genome (Lu et al., 2009;Xiong et al., 2008Xiong et al., , 2009. Although extensive functional analysis of the RSV genome has been conducted, there have been no reports on the interaction between RSV and rice plants, which may clarify the mechanisms behind the appearance of disease symptoms.Rice is a model cereal plant, for which many genomic and transcriptome resources and tools are already available (Liang et al., 2008;Ouyang et al., 2007; Rice Annotation Project, 2008; Rice Full-length cDNA Consortium, 2003). The elucidation of genome sequences and structures in diverse organisms has led to the development of various high-throughput genome and transcriptome analytical tools. Numerous transcriptome profiles in...

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.

The complete nucleotide sequence of RNA 1, the largest genomic segment of rice stripe virus (RSV), was determined using two sets of overlapping cDNA clones.

An analysis, using microarrays, of gene expression in rice plants infected with Rice dwarf virus revealed significant decreases in levels of expression of genes that are involved in the formation of cell walls, reflecting the stunted growth of diseased plants. The expression of plastid-related genes also was suppressed, as anticipated from the white chlorotic appearance of infected leaves. By contrast, the expression of defense- and stress-related genes was enhanced after viral infection. These results suggest that virus-infected rice plants attempt to survive viral infection and replication by raising the levels of expression of defense- and stress-related genes while suppressing the expression of genes required for the elongation of cells and photosynthesis.