Tomofumi Mochizuki

Shoot meristem tissue of tobacco inoculated with Cucumber mosaic virus is infected with the virus and subsequently recovers from infection by RNA silencing

Distribution of Cucumber mosaic virus (CMV) in shoot meristem tissue of CMV-inoculated tobacco was successively analyzed with immunohistochemical microscopy and in situ hybridization. CMV signals were detected in the tissue at 7 days postinoculation (dpi), but then they decreased and disappeared after 14 dpi. Detailed observation confirmed CMV invasion of shoot apical meristem at 6–8 dpi. Short interfering RNA corresponding to CMV RNAs was first detected at 7 dpi and was detected up to 24 dpi. These results suggest that the shoot meristem tissue is infected with CMV but subsequently recovers from the infection by RNA silencing.

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.

Single amino acid substitutions at residue 129 in the coat protein of cucumber mosaic virus affect symptom expression and thylakoid structure

The symptomatic effect of the amino acid type at residue 129 in the coat protein of cucumber mosaic virus was investigated in tobacco using coat protein mutants of the pepo strain in which proline 129 was substituted with 19 other amino acids. These mutants caused six types of symptoms: white mosaic, pale green mosaic, veinal chlorosis, veinal necrosis, systemic necrosis, and necrotic local lesions. Transmission electron microscopy revealed that the chloroplasts of plants showing the three former types of symptoms contained few thylakoid membranes. Cytopathic effects characteristic of cells from plants showing the three latter symptom types were not observed.

The 2b protein of cucumber mosaic virus is essential for viral infection of the shoot apical meristem and for efficient invasion of leaf primordia in infected tobacco plants

It has been reported previously that a 2b protein-defective mutant of the cucumber mosaic virus (CMV) Pepo strain (Delta 2b) induces only mild symptoms in systemically infected tobacco plants. To clarify further the role of the 2b protein as an RNA silencing suppressor in mosaic symptom expression during CMV infection, this study monitored the sequential distribution of Delta 2b in the shoot meristem and leaf primordia (LP) of inoculated tobacco. Time-course histochemical observations revealed that Delta 2b was distributed in the shoot meristem at 7 days post-inoculation (p.i.), but could not invade shoot apical meristem (SAM) and quickly disappeared from the shoot meristem, whereas wild-type (Pepo) transiently appeared in SAM from 4 to 10 days p.i. In LP, Delta 2b signals were detected only at 14 and 21 days p.i., whereas dense Pepo signals were observed in LP from 4 to 18 days p.i. Northern blot analysis showed that small interfering RNA (siRNA) derived from Delta 2b RNA accumulated earlier in the shoot meristem and LP than that of Pepo. However, a similar amount of siRNA was detected in both Pepo- and Delta 2b-infected plants at late time points. Tissue printing analysis of the inoculated leaves indicated that the areas infected by Pepo increased faster than those infected by Delta 2b, whereas accumulation of Delta 2b in protoplasts was similar to that of Pepo. These findings suggest that the 2b protein of the CMV Pepo strain determines virulence by facilitating the distribution of CMV in the shoot meristem and LP via prevention of RNA silencing and/or acceleration of cell-to-cell movement.

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.

Antiviral RNA Silencing Is Restricted to the Marginal Region of the Dark Green Tissue in the Mosaic Leaves of Tomato Mosaic Virus-Infected Tobacco Plants

ABSTRACT Mosaic is a common disease symptom caused by virus infection in plants. Mosaic leaves of Tomato mosaic virus (ToMV)-infected tobacco plants consist of yellow-green and dark green tissues that contain large and small numbers of virions, respectively. Although the involvement of RNA silencing in mosaic development has been suggested, its role in the process that results in an uneven distribution of the virus is unknown. Here, we investigated whether and where ToMV-directed RNA silencing was established in tobacco mosaic leaves. When transgenic tobaccos defective in RNA silencing were infected with ToMV, little or no dark green tissue appeared, implying the involvement of RNA silencing in mosaic development. ToMV-related small interfering RNAs were rarely detected in the dark green areas of the first mosaic leaves, and their interior portions were susceptible to infection. Thus, ToMV-directed RNA silencing was not effective there. By visualizing the cells where ToMV-directed RNA silencing was active, it was found that the effective silencing occurs only in the marginal regions of the dark green tissue (∼0.5 mm in width) and along the major veins. Further, the cells in the margins were resistant against recombinant potato virus X carrying a ToMV-derived sequence. These findings demonstrate that RNA silencing against ToMV is established in the cells located at the margins of the dark green areas, restricting the expansion of yellow-green areas, and consequently defines the mosaic pattern. The mechanism of mosaic symptom development is discussed in relation to the systemic spread of the virus and RNA silencing.

Coat protein mutations in an attenuated Cucumber mosaic virus encoding mutant 2b protein that lacks RNA silencing suppressor activity induces chlorosis with photosynthesis gene repression and chloroplast abnormalities in infected tobacco plants.

In tobacco plants, the Cucumber mosaic virus (CMV) pepo strain induces mosaic symptoms, including pale green chlorosis and malformed tissues. Here, we characterized the involvement of 2b protein and coat protein (CP) in the development of mosaic symptoms. A 2b mutant (R46C) that lacks viral suppressor of RNA silencing (VSR) activity showed an asymptomatic phenotype with low levels of virus accumulation. Tomato spotted wilt virus NSs protein did not complement the virulence of the R46C, although it did restore high-level virus accumulation. However, R46C mutants expressing mutated CP in which the amino acid P129 was mutated to A, E, C, Q, or S induced chlorosis that was associated with reduced expression of chloroplast and photosynthesis related genes (CPRGs) and abnormal chloroplasts with fewer thylakoid membranes. These results suggest that the CP of the CMV pepo strain acquires virulence by amino acid mutations, which causes CPRG repression and chloroplast abnormalities.

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.

Amino acid 129 in the coat protein of Cucumber mosaic virus primarily determines invasion of the shoot apical meristem of tobacco plants

We previously reported that a strain of Cucumber mosaic virus (Pepo CMV) invaded the shoot apical meristem (SAM, tunica corpus) of tobacco plants at 6–8 days postinoculation (dpi), contrary to earlier observations. To identify a viral factor determining the ability to invade the SAM, we inoculated plants with two other CMV strains, MY17 and Y, and tested the three strains in this study. Immunohistochemical microscopy revealed that MY17 CMV invaded the SAM at 7 dpi, the same as Pepo CMV, but Y CMV did not, even at 21 dpi. Using RNA pseudorecombinants between Pepo and Y CMV, we found that Pepo RNA 2 affected the rate of SAM invasion, and Pepo RNA 3 was required for successful SAM invasion. Inoculation with RNA 1 and RNA 2 from Y CMV and RNA 3 containing the chimeric coat protein (CP) gene between Pepo and Y CMV or a Y RNA 3 point mutant containing a Ser-to-Pro substitution at position 129 in CP (Y129P) revealed that amino acid 129 of CP is the determinant for successful SAM invasion. The rate of SAM invasion of the pseudorecombinants and Y129P was consistent with the efficiency of cell-to-cell movement in the inoculated leaves, implying that SAM invasion by CMV strains may be due to efficient cell-to-cell movement.