1995 Fiscal Year Final Research Report Summary
DEVELOPMENT OF A SYSTEM FOR INTRODUCTION AND EXPRESSION OF PLANT REOVIRUS GENES USING OR INSECT TISSUE CULTURE CELLS.
| Project/Area Number |
05454129
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Applied molecular and cellular biology
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| Research Institution | HOKKAIDO UNIVERSITY |
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Principal Investigator |
UYEDA Ichiro Hokkaido Univ., Fac.of Agr., Pro., 農学部, 教授 (10113523)
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| Co-Investigator(Kenkyū-buntansha) |
HATAYA Tatsuji Hokkaido Univ., Fac.of Agr., Assist., 農学部, 助手 (20241367)
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| Project Period (FY) |
1993 – 1995
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| Keywords | Plant reoviruses / Vector insects / Tissue culture |
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| Research Abstract |
Attempts to introduce viral genomic RNAs with or without a helper virus were unsuccessful. Therefore, isolation of the minimum infective units, that is an RNA polymerase and genomic RNA complex, was investigated.
RNA polymerase-genomic RNA complex of rice dwarf virus was isolated. After treatment of high concentration of MgCl_2, purified virions were subjected to equilibrium density gradient centrifugation in CsCl, resulting in removal of outer capsid polypeptide P8. The isolated core particle is consisted of P1, P3, and P7 polypeptides and genomic RNAs. The core particles retain an RNA polymerase activity. The core is further subjected to CsTFA equilibrium density gradient centrifugation to remove P1 and P7 from the core. The major polypeptide of the resultant empty particles was P3. P7 banded at higher density in the gradient tube, suggesting that it binds to genomic RNAs. In North-Western blotting analyzes, genomic dsRNAs specifically bound to P7, but not other structural proteins. Base on these results, it was hypothesized that P7 playd a critical role in an active RNA polymerase-genomic RNA complex.
Cell culture of planthopper was successfully established. After 61-73 transfers in the same medium as that for leafhoppers, 5 cell lines were established. Cell are about a half size of leafhoppers and grow slowly. Upon inoculation with rice black-streaked dwarf virus, infection is demonstrated by indirect immuno fluorescent staining. This is the first use of planthoppers in plant virus research.
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