Takeshi Osaki

Amino acid 129 of cucumber mosaic virus coat protein determines local symptom expression and systemic movement in Tetragonia expansa, Momordica charantia and Physalis floridana

Local symptom expression and systemic movement of Cucumber mosaic virus (CMV) in Tetragonia expansa, Momordica charantia and Physalis floridana were mapped to the amino acid at position 129 of CMV coat protein (CP), using pseudorecombinants, chimeric RNAs, a site-directed mutant of RNA 3 and four strains of CMV : pepo-, SO-, MY17- and Y-CMV. Local and systemic symptoms caused by three strains, pepo-, SO- and MY17-CMV, and those by Y-CMV differed in the three host species. The three strains expressed local chlorotic spots at 24°C and systemic chlorotic spots and ringspots at 36°C, whereas Y-CMV developed local necrotic spots at 24°C but no systemic symptoms at 36°C in T. expansa. In M. charantia the three strains caused systemic chlorotic spots, whereas Y-CMV caused local necrotic spots. The three caused systemic mosaic and Y-CMV systemic necrosis in P. floridana. With pseudorecombinants combined with pepo- and Y-CMV RNAs, CMV RNA 3 was responsible for symptom expression and systemic infection. Inoculation with Y-CMV RNA 1, RNA 2 and chimeric RNA 3s exchanged CP gene fragments between pepo- and Y-CMV showed that NruI-XhoI fragment of CP was essential for symptom expression. Comparative analysis of the NruI-XhoI fragments revealed that only the amino acid at position 129 was common among the three strains but different from that of Y-CMV. Inoculation with a point mutant constructed by substituting one nucleotide resulting in an amino acid change from Ser to Pro at position 129 in Y-CMV CP verified the previous experiments. These results indicate that the amino acid at position 129 of CMV CP is the determinant for local symptom expression and systemic movement in the three host species. CMV CP containing Ser at position 129 may induce resistant responses in these plants.

Movement of cucumber mosaic virus is restricted at the interface between mesophyll and phloem pathway in Cucumis figarei.

Viral movement in the leaf tissues of a resistant host, Cucumis figarei, inoculated with the pepo strain of Cucumber mosaic virus (CMV) and incubated at 24°C or 36°C was investigated by fluorescence in situ hybridization (FISH), leaf-press blotting, tissue printing and immunogold-silver staining techniques. Observation by FISH revealed that at 24°C most infection sites with CMV at 0.01 mg/ml or 0.1 mg/ml were limited to a single cell during the incubation period, that the number of infection sites increased from 24hpi (hours post inoculation) to 80 hpi in the leaves inoculated with CMV at 0.5 mg/ml, and that the size as well as the number of infection sites rapidly increased with time in the leaves inoculated with CMV at 2.0 mg/ml. These results suggested that one factor for the resistance of C. figarei at 24°C might be an inhibition of viral movement in and out of the infection sites. Leaf-press blotting and tissue blotting indicated that CMV remained in the infection sites at 24°C, whereas it spread from the inoculated leaves to other parts of the plants through vascular systems at 36°C. Immunogold-silver staining demonstrated that at 24°C CMV infected bundle sheath (BS) cells in minor veins, whereas at 36°C it invaded not only BS cells, but also phloem parenchyma (PP)/ companion cell (CC) or PP/intermediary cell (IC) complexes in minor veins in the regions with chlorotic symptoms. These results indicated that at 24°C CMV had difficulty in passing through the interface between BS and PP/CC or PP/ IC complexes and that viral entry from mesophyll to the phloem pathway was inhibited in the inoculated leaves.

Cucumber mosaic virus establishes systemic infection at increased temperature following viral entrance into the phloem pathway of Tetragonia expansa

ABSTRACT A potential regulatory site for Cucumber mosaic virus (CMV, pepo strain) movement necessary to establish systemic infection was identified through immunological and hybridization studies on Tetragonia expansa, which was systemically infected by CMV at 36 degrees C but not at 24 degrees C. In inoculated leaves, cell-to-cell movement of CMV was enhanced at 36 degrees C compared with that observed at 24 degrees C. CMV was distributed in the phloem cells of minor veins as well as epidermal and mesophyll cells at both 36 and 24 degrees C. CMV was detected in the petioles of inoculated leaves, stems, and petioles of uninoculated upper leaves at 36 degrees C, whereas CMV was detected only in the petioles of inoculated leaves and in stems at 24 degrees C. CMV moved into the phloem and was transported to the stem within 24 h postinoculation (hpi) at 36 degrees C. However, it did not accumulate in the petioles of the upper leaves until 36 hpi. In petioles of inoculated leaves at 24 degrees C, CMV was detected in the external phloem but not in the internal phloem. From these results, we conclude that systemic infection is established after viral entrance into the phloem pathway in T. expansa at 36 degrees C.

Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and affects pathogenicity in cowpea.

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.

Tobacco mosaic virus is transmissible from tomato to tomato by pollinating bumblebees.

Tobacco mosaic virus (TMV) was detected by ELISA, electron microscopy and/or bioassay from bumblebee (Bumbus terrestris), pollen clumps, nest materials and bee-visited anthers of flowers from greenhouses in which tomatoes had been pollinated by bees and were severely infected with TMV. Experimental bee-mediated transmission of TMV in greenhouse tomatoes demonstrated that the bumblebees transported TMV from plant to plant and that they spread the virus in greenhouses. This is the first report describing TMV transmission by bumblebees.

Purification and partial characterization of figaren, an RNase-like novel antiviral protein from Cucumis figarei

An antiviral protein, designated figaren, was purified from leaves of Cucumis figarei and partially characterized. Column chromatography, SDS-polyacrylamide gel electrophoresis and periodic acid-Schiff staining revealed that figaren is a glycoprotein with a molecular weight of 23 kDa. Figaren was stable at pH 2 to 12 and below 90°C. N-terminal amino acid sequencing indicated that figaren contained the conserved region for the S-allele-associated ribonucleases (RNases). In-gel RNase assay showed that figaren digested yeast RNAs. Figaren also digested double-stranded RNAs extracted from Cucumber mosaic virus (CMV)-infected tobacco tissues. Fluorescence in situ hybridization revealed that figaren and RNases (beef pancrease and RNase T1 at 1 μg/ml similarly inhibited CMV infection in cowpea leaves. Figaren and the RNases at 5-500 ng/ml had similar inhibitory effect on local lesion formation by CMV. These data suggest that figaren is a novel RNase-like antiviral protein.

Effect of Figaren, an antiviral protein from Cucumis figarei, on Cucumber mosaic virus infection

Local lesion formation on cowpea leaves was more than 50% inhibited by treatment with a 23 kDa RNase-like glycoprotein from Cucumis figarei, figaren, from 24 hr before to 1 hr after inoculation with Cucumber mosaic virus (CMV). CMV accumulation detected by ELISA in tobacco leaves treated with figaren 6 or 0 hr before inoculation with CMV was suppressed. When upper leaves of tobacco plants were treated with figaren and inoculated 10 min later with CMV, mosaic symptoms were delayed for 5–7 days on most of the tobacco plants, and some plants remained asymptomatic. From fluorescence in situ hybridization, infection sites were present in figaren-treated cowpea or melon leaves after inoculation with CMV, though the sites were reduced in number and size compared with those in water-treated control leaves. The amount of CMV RNAs and CMV antigen in melon protoplasts inoculated with CMV and subsequently incubated with figaren similarly increased with time as did that in the control. ELISA and local lesion assays indicated that CMV infection on the upper surfaces of the leaves of tobacco, melon, cowpea and C. amaranticolor whose lower surfaces had been treated with figaren 5–10 min before CMV inoculation was almost completely inhibited. Figaren did not inhibit CMV infection on the opposite untreated leaf halves of melon, cowpea and C. amaranticolor, whereas it almost completely inhibited CMV infection on the untreated halves of leaves of tobacco. CMV infection was not inhibited in the untreated upper or lower leaves of the four plants. These data suggest that figaren does not completely prevent CMV invasion but does inhibit the initial infection processes. It may also induce localized acquired resistance in host plants.