Takehiro Ohki

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

Amino acid substitution in the coat protein of Melon necrotic spot virus causes loss of binding to the surface of Olpidium bornovanus zoospores

Melon necrotic spot virus (MNSV) is transmitted by the fungus Olpidiumbornovanus. In this study, we used immunofluorescence microscopy to detect MNSV particles over the entire surface of the O. bornovanus zoospore; MNSV particles were not detected on the related fungus O. virulentus, which cannot transmit MNSV. The amino acid substitution Ile → Phe at position 300 in the MNSV coat protein resulted in loss of both specific binding and fungal transmission, while virion assembly and biological aspects were unaffected. Taken together, these results suggest that the MNSV coat protein acts as a ligand to the O. bornovanus zoospore as part of a fungal-vector transmission system.

High temperatures activate local viral multiplication and cell-to-cell movement of Melon necrotic spot virus but restrict expression of systemic symptoms.

The infection of melon plants by Melon necrotic spot virus (MNSV) and the development of necrotic disease symptoms are a seasonal occurrence in Japan, which take place between winter and early summer, but not during mid-summer. In this paper we investigate the effect of three different temperatures (15, 20, and 25 degrees C) on the local and systemic expression of MNSV in melon plants. Previously, the incidence of plants expressing systemic symptoms caused by MNSV and other viruses was found to be greater at temperatures less than 20 degrees C. In this study, our temperature-shift experiments support previous studies that found the expression of systemic symptoms increases as temperature falls from 25 to 20 degrees C and decreases as temperature rises from 20 to 25 degrees C. However, MNSV replication in melon cells and local viral movement within leaves following the inoculation of melon protoplasts or cotyledons were more frequent at 25 degrees C than at 15 or 20 degrees C.

The protruding domain of the coat protein of Melon necrotic spot virus is involved in compatibility with and transmission by the fungal vector Olpidium bornovanus

The Chi and W strains of Melon necrotic spot virus (MNSV) are efficiently transmitted by isolates Y1 and NW1, respectively, of the fungal vector Olpidium bornovanus. Analysis of chimeric viruses constructed by switching the coat protein (CP) gene between the two strains unveiled the involvement of the CP in the attachment of MNSV to zoospores of a compatible isolate of O. bornovanus and in the fungal transmission of the virus. Furthermore, analysis of the chimeric virus based on the Chi strain with the protruding domain of the CP from strain W suggested the involvement of the domain in compatibility with zoospore. Comparison of the three-dimensional structures between the CP of the two MNSV strains showed that many of the differences in these amino acid residues are present on the surface of the virus particles, suggesting that these affects the recognition of fungal vectors by the virus.

A New Strain of Melon necrotic spot virus that Is Unable to Systemically Infect Cucumis melo

We report a new strain of Melon necrotic spot virus (MNSV) that is unable to systemically infect Cucumis melo. A spherical virus (W-isolate), about 30 nm in diameter like a carmovirus, was isolated from watermelons with necrotic symptoms. The W-isolate had little serological similarity to MNSV, and it did not cause any symptoms in six melon cultivars susceptible to MNSV; however, the host range of the W-isolate was limited exclusively to cucurbitaceous plants, and transmission by O. bornovanus was confirmed. Its genomic structure was identical to that of MNSV, and its p89 protein and coat protein (CP) showed 81.6 to 83.2% and 74.1 to 75.1% identity to those of MNSV, respectively. Analysis of protoplast showed that the W-isolate replicated in melons at the single-cell level. Furthermore, chimeric clones carrying the CP of MNSV induced necrotic spots in melons. These results suggested that the absence of symptoms in melons was due to a lack of ability of the W-isolate to move from cell to cell. In view of these findings, we propose that the new isolate should be classified as a novel MNSV watermelon strain.

Characterization of Grapevine Algerian latent virus isolated from nipplefruit (Solanum mammosum) in Japan.

Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato bushy stunt virus nipplefruit strain (TBSV-Nf), and an unknown spherical virus were isolated from nipplefruit (Solanum mammosum) cultivated in Chiba Prefecture, Japan. The spherical virus was identified as Grapevine Algerian latent virus nipplefruit strain (GALV-Nf) from the genus Tombusvirus, based on its physical properties, serological relationships, and analysis of genomic RNA. The genomic RNA of GALV-Nf is 4731 nucleotides long and encodes five open reading frames as well as those of other tombusviruses. Nipplefruit infected with GALV-Nf had severe stunting, leaf deformation, and clear mosaic symptoms. This is the first report of an isolation of GALV in Japan.

Characterization of Tomato bushy stunt virus newly isolated from nipplefruit (Solanum mammosum) in Japan.

Tomato bushy stunt virus (TBSV) was isolated from nipplefruit (Solanum mammosum) in Chiba Prefecture, Japan, with mosaic symptoms, leaf deformation, and stunting. The genomic structure of the TBSV-nipplefruit strain (TBSV-Nf) is identical to that of the TBSV-cherry strain (TBSV-Ch). Transcripts from its full-length cDNA clone without a cap analog at the 5′ terminus infected Nicotiana glutinosa, Nicotiana benthamiana, S. mammosum, Lycopersicon esculentum, Chenopodium amaranticolor, and Chenopodium quinoa, which displayed symptoms identical to those of the parental virus. Although TBSV-Ch systemically infected nipplefruit, systemic symptoms caused by TBSV-Nf were more severe than those by TBSV-Ch. This virus is the first TBSV discovered in Japan and the first TBSV ever isolated from nipplefruit.

Functional degeneration of the resistance gene nsv against Melon necrotic spot virus at low temperature

The single recessive gene, nsv, which confers resistance against Melon necrotic spot virus (MNSV), has recently been used to develop virus-resistant melon cultivars in Japan. However, the Chiba isolate of MNSV, a common isolate in Japan, infected resistant cultivars when inoculated melon plants were grown at 15°C. Viral RNAs accumulated in protoplasts from resistant cultivars at both 15 and 20°C. Mechanical inoculation of the cotyledons caused MNSV to spread throughout the leaves at 15°C, but not at 20°C. These results support our novel hypothesis that a temperature-sensitive inactivation of disease resistance genes occurs at the nsv locus in melon cultivars with the resistance gene grown at temperatures below 20°C.

Comparisons of ribosomal DNA-internal transcribed spacer sequences and biological features among Olpidium bornovanus isolates from Cucurbitaceae-cultivating soil in Japan

The chytrid fungus Olpidium bornovanus is an obligate plant parasite that acts as a vector to transmit Melon necrotic spot virus in cultivated soil. Here, we conducted a molecular phylogenetic analysis of 16 isolates of O. bornovanus taken from soil in which Cucurbitaceae plants had been grown in various locations throughout Japan. The ribosomal DNA and internal transcribed spacer region sequences of the 16 O. bornovanus isolates were divided into four molecular phylogenetic groups, designated O.bor-A to O.bor-D. Biological features of O. bornovanus isolates such as host specificity varied consistently with their molecular phylogenetic groups.

Species Composition of Alate Aphids (Hemiptera: Aphididae) Harboring Potato Virus Y and the Harbored Virus Strains in Hokkaido, Northern Japan

Abstract Many studies have evaluated transmission abilities of laboratory-reared aphids for potato virus Y (PVY), but few have focused on PVY-harboring species of field-collected aphids and the strains of PVY harbored by aphids. In the present study, we collected alate aphids in yellow pan traps in potato fields with Japanese commercial cultivars in Hokkaido, northern Japan in single 24-h periods during the tuber bulking stage and examined whether individual whole aphids harbored PVY by nested RT-PCR. PVY-positive individuals were identified to species using the gene sequence for cytochrome c oxidase subunit I and, when needed, morphological data and distribution records. In addition, individual strains of PVY harbored were determined using partial sequences of coat protein. Among 1,857 aphids trapped, 195 aphids had PVY and comprised 19 species; 17 species were identified to species-group taxa. Most of the aphid species detected as PVY positive colonize weeds that are common around potato fields in Hokkaido. Five species-group taxa had not been reported previously as a vector aphid of PVY and might be new PVY-vector species. PVYNTN was most frequently detected from PVY-positive aphids as found recently in PVY-infected potatoes in commercial fields in Hokkaido. Two or three PVY strains were rarely detected from a single aphid, and no obvious difference was found in the proportion of the harbored PVY strains among positive aphid species. The first documentation of the species composition of PVY-harboring aphids and the strains of PVY harbored in East Asia should aid understanding of the epidemiology of PVY in Japan.