| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2002: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
The 50KDa protein (50KP) encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV), a movement protein, was expressed in transgenic Nicotiana occidentalis plants. The 50KP-expressing plants (50KP-plants) complemented the syntemic spread of the 50KP-defective mutants of an infectious cDNA clone of ACLSV (pCLSF). Severity of symptoms was greatly enhanced and the accumulation of virus in upper leaves was increased in 50KP-plants inoculated with ACLSV compared with nontransgenic control plants (NT-plants). However, most 50KP-plants inoculated with Grapevine berry inner necrosis virus (GINV), another species of the genus Trichovirus, neither developed obvious symptoms nor supported virus accumulation in inoculated or upper leaves. In contrast, systemic symptoms developed and virus accumulated equally in NT- and CP-plants inoculated with GINV. After inoculation with Apple stem grooving virus (ASGV) or Apple stem pitting virus, there was no difference in symptom development and virus accumul
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ation among 50KP-, CP-, and NT-plants. ACLSV-50KP and GINV-MP (39KP) fused to Green, Yellow or Cyan fluorescent proteins (GFP, YFP or CFP) were expressed transiently in leaf cells of 50KP- and NT- plants. The intracellular and intercellular trafficking and tubule-inducing activity of 39KP were specifically interfered in leaf epidermis and protoplasts from 50KP- Plant leaves, in contrast that those of 50KP and ASGV-36KP were never interfered in cells from 50KP- plants. When 39KP-YFP was co-expressed with 50KP-CFP in leaf epidermis of NT-plants, both MPs were confined to single cells, indicating that 50KP-CFP interferes with the cell-to-cell trafficking of 39KP-YFP. Mutational analyses using 50KP deletion mutants showed that the cell-to-cell trafficking of 39KP was interfered with the deleted proteins retained the activities described above, but not with the dyslunctional 50KP. Transgenic plants expressing the 50KP-deleted proteins retained the activities showed specific resistance against GINV, in contrast with transgenic plants expressing dysfunctional 50KP did not show any resistance to the virus. From these results, it is concluded that the specific resistance of 50KP-plants against GINV is caused by the interference of the intracellular and intercellular trafficking of 39KP with 50KP. Less
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