Scopolia mild mottle virus: a new tobamovirus isolated from a Scopolia japonica plant in Japan.

A tobamovirus was isolated from leaves of a Scopolia japonica plant showing mild yellowing. Back-inoculation of healthy Scopolia japonica with the isolated virus induced mild mottle on upper leaves. Phylogenetic analysis based on coat protein and replicase protein sequences revealed that the newly isolated tobamovirus was most closely related to yellow tailflower mild mottle virus (YTMMV). The newly isolated tobamovirus shared the highest nucleotide sequence identity (71%) with YTMMV, which is lower than the cutoff (90%) set for species demarcation in the genus Tobamovirus. Thus, our result suggested that scopolia mild mottle virus (SMMoV) is a new tobamovirus that infects Scopolia japonica plants in Japan.

Effect of decomposing oil palm trunk fibers on plant growth and soil microbial community composition.

Oil palm trunks (OPT) are logged for replantation and the fiber residues are disposed of into the palm plantation area. The fiber residues are expected to increase soil fertility through recycling of carbon and minerals via fiber decomposition. This study investigated the effects of OPT fiber disposal and other lignocellulosic biomass on plant growth and microbial diversity in the soil environment. Four treatment plots were tested: (A) soil+OPT fiber (1:20), (B) soil+sugarcane bagasse (1:20), (C) soil+cellulose powder (1:20), and (D) unamended soil as a negative control. Low plant height, decreased chlorophyll content, and low biomass was observed in corn grown on soil mixed with OPT fiber, cellulose, and sugarcane bagasse, when compared with those of the control. The plants grown with OPT fiber were deficient in total nitrogen and magnesium when compared with those without fiber amendment, which suggested that nitrogen and minerals in soil might be taken up by changing microflora because of the OPT fibers presence. To confirm differences in the soil microflora, metagenomics analysis was performed on untreated soil and soil from each lignocellulose treatment. The microflora of soils mixed with OPT fiber, cellulose and sugarcane bagasse revealed substantial increases in bacteria such as families Cytophagaceae and Oscillospiraceae, and two major fungal genera, Trichoderma and Trichocladium, that are involved in lignocellulose degradation. OPT fiber resulted in a drastic increase in the ratios and amounts of Trichocladium in the soil when compared with those of cellulose and sugarcane bagasse. These results indicate that unregulated disposal of OPT fiber into plantation areas could result in nutrient loss from soil by increasing the abundance of microorganisms involved in lignocellulose decomposition.

Draft genome sequence data of Clostridium thermocellum PAL5 possessing high cellulose-degradation ability.

Clostridium thermocellum is a potent cellulolytic bacterium. C. thermocellum strain PAL5, was derived from strain S14 that was isolated from bagasse paper sludge, possesses higher cellulose-degradation ability than representative strains ATCC27405 and DSM1313. In this work, we determined the draft genome sequence of C. thermocellum PAL5. Genomic DNA was used for whole-genome sequencing using the Illumina HiSeq 2500. We obtained 215 contigs of >200 bp (N50, 78,366 bp; mean length, 17,378 bp). The assembled data were subjected to the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Annotation Pipeline, and 3198 protein-coding sequences, 53 tRNA genes, and 4 rRNA genes were identified. The data are accessible at NCBI (the accession number SBHL00000000). Our data resource will facilitate further studies of efficient cellulose-degradation using C. thermocellum.

Genome segments encoding capsid protein-like variants of Pyrus pyrifolia cryptic virus.

According to previous studies, three double-stranded (ds) RNA molecules (dsRNA1, 2, and 3) detected in Japanese pear are transmitted to the next generation with high frequency through both ovules and pollen. Nucleotide sequence analysis of dsRNA1-encoding RNA-dependent RNA polymerase (RdRp) has suggested that these dsRNAs are related to a cryptovirus named Pyrus pyrifolia cryptic virus (PpCV). In this study, purified dsRNA prepared from a PpCV-infected Japanese pear cultivar was subjected to next-generation deep sequencing. This sequencing generated two de novo assembled contigs corresponding to dsRNA2 and 3, with BLAST analysis of the predicted amino acid sequences indicating homology to capsid proteins (CPs) of the cryptoviruses persimmon cryptic virus and Sinapis alba cryptic virus 1, respectively. Relationships between the two contigs and dsRNA2 and 3 were confirmed by northern blot hybridization with probes generated using primers designed from the assembled contigs. Rapid amplification of cDNA ends analyses of 5'- and 3'-terminal sequences of dsRNA2 and 3 revealed that these two dsRNAs consist of 1523 and 1481bp, respectively. The 5'-terminal sequences (AGAAUUUC) of dsRNA1, 2 and 3 were found to be conserved. Phylogenetic analysis of deduced amino acid sequences of the two CP-like variants indicated that PpCV belongs to Deltapartitivirus (Partitiviridae). Our results imply that PpCV is tri-segmented.

Nucleotide sequences of Japanese isolates of citrus vein enation virus.

The genomic sequences of five Japanese isolates of citrus vein enation virus (CVEV) isolates that induce vein enation were determined and compared with that of the Spanish isolate VE-1. The nucleotide sequences of all Japanese isolates were 5,983 nt in length. The genomic RNA of Japanese isolates had five potential open reading frames (ORF 0, ORF 1, ORF 2, ORF 3, and ORF 5) in the positive-sense strand. The nucleotide sequence identity among the Japanese isolates and Spanish isolate VE-1 ranged from 98.0% to 99.8%. Comparison of the partial amino acid sequences of ten Japanese isolates and three Spanish isolates suggested that four amino acid residues, at positions of 83, 104, and 113 in ORF 2 and position 41 in ORF 5, might be unique to some Japanese isolates.

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.

Strong resistance against Rice grassy stunt virus is induced in transgenic rice plants expressing double-stranded RNA of the viral genes for nucleocapsid or movement proteins as targets for RNA interference.

Rice grassy stunt virus (RGSV), a member of the genus Tenuivirus, causes significant economic losses in rice production in South, Southeast, and East Asian countries. Growing resistant varieties is the most efficient method to control RGSV; however, suitable resistance genes have not yet been found in natural rice resources. One of the most promising methods to confer resistance against RGSV is the use of RNA interference (RNAi). It is important to target viral genes that play important roles in viral infection and proliferation at an early stage of viral replication. Our recent findings obtained from an RNAi experiment with Rice stripe virus (RSV), a tenuivirus, revealed that the genes for nucleocapsid and movement proteins were appropriate targets for RNAi to confer resistance against RSV. In this study, we transformed rice plants by introducing an RNAi construct of the RGSV genes for the nucelocapsid protein pC5 or movement protein pC6. All progenies from self-fertilized transgenic plants had strong resistance against RGSV infection and did not allow the proliferation of RGSV. Thus, our strategy to target genes for nucleocapsid and movement proteins for conferring viral resistance might be applicable to the plant viruses in the genus Tenuivirus.

Hairpin RNA derived from the gene for Pns9, a viroplasm matrix protein of Rice gall dwarf virus, confers strong resistance to virus infection in transgenic rice plants.

The nonstructural Pns9 protein of Rice gall dwarf virus (RGDV) accumulates in viroplasm inclusions, which are structures that appear to play an important role in viral morphogenesis and are commonly found in host cells infected by viruses in the family Reoviridae. An RNA interference construct was designed to target the gene for Pns9 of RGDV, namely Trigger_G9. The resultant transgenic plants accumulated short interfering RNAs specific for the construct. All progenies from self-fertilized transgenic plants had strong and heritable resistance to RGDV infection and did not allow the propagation of RGDV. By contrast, our transgenic plants remained susceptible to Rice dwarf virus, another phytoreovirus. There were no significant changes in the morphology of our transgenic plants compared with non-inoculated wild-type rice plants, suggesting that genes critical for the growth of rice plants were unaffected. Our results demonstrate that the resistance to RGDV of our transgenic rice plants is not due to resistance to the vector insects but to specific inhibition of RGDV replication and that the designed trigger sequence is functioning normally. Thus, our strategy to target a gene for viroplasm matrix protein should be applicable to plant viruses that belong to the family Reoviridae.

Immunity to Rice black streaked dwarf virus, a plant reovirus, can be achieved in rice plants by RNA silencing against the gene for the viroplasm component protein.

The nonstructural protein P9-1 of Rice black streaked dwarf virus has been confirmed to accumulate in viroplasms, the putative sites of viral replication, in infected plants and insects. We transformed rice plants by introducing an RNA interference construct against the P9-1-encoding gene. The resultant transgenic plants accumulated short interfering RNAs specific to the construct. All progenies produced by self-fertilization of these transgenic plants with induced RNA interference against the gene for P9-1 were resistant to infection by the virus. Our results demonstrated that interfering with the expression of a viroplasm component protein of plant reoviruses, which plays an important role in viral proliferation, might be a practical and effective way to control plant reovirus infection in crop plants.

Targeting specific genes for RNA interference is crucial to the development of strong resistance to rice stripe virus.

Rice 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.

Cross-protection against bean yellow mosaic virus (BYMV) and clover yellow vein virus by Attenuated BYMV isolate M11.

Attenuated isolate M11 of Bean yellow mosaic virus (BYMV), obtained after exposing BYMV-infected plants to low temperature, and its efficacy in cross-protecting against infection by BYMV isolates from gladiolus, broad bean (Vicia faba) and white clover (Trifolium repens) was assessed with western blotting and reverse transcription-polymerase chain reaction. The level of cross-protection varied depending on the challenge virus isolates. Cross-protection was complete against BYMV isolates from gladiolus, but incomplete against BYMV isolates from other hosts. M11 also partially cross-protected against an isolate of Clover yellow vein virus. A comparison of the nucleotide sequence of M11 and those of BYMV isolates from gladiolus and from other hosts showed higher homology among gladiolus isolates than the homology between gladiolus isolates and nongladiolus isolates. In the phylogenetic trees, constructed using the nucleotide sequences of an overall polyprotein of the genomes, five gladiolus isolates clustered together, completely separated from the three BYMV isolates from other hosts. A comparison of the amino acid sequences between M11 and its parental isolate IbG, and analysis of recombinant infectious clones between M11 and IbG revealed that an amino acid at position 314 was involved in the attenuation of BYMV.

The structure of melon necrotic spot virus determined at 2.8 A resolution.

The structure of melon necrotic spot virus (MNSV) was determined at 2.8 A resolution. Although MNSV is classified into the genus Carmovirus of the family Tombusviridae, the three-dimensional structure of MNSV showed a higher degree of similarity to tomato bushy stunt virus (TBSV), which belongs to the genus Tombusvirus, than to carnation mottle virus (CMtV), turnip crinkle virus (TCV) or cowpea mottle virus (CPMtV) from the genus Carmovirus. Thus, the classification of the family Tombusviridae at the genus level conflicts with the patterns of similarity among coat-protein structures. MNSV is one of the viruses belonging to the genera Tombusvirus or Carmovirus that are naturally transmitted in the soil by zoospores of fungal vectors. The X-ray structure of MNSV provides us with a representative structure of viruses transmitted by fungi.