1998 Fiscal Year Final Research Report Summary
Molecular analysis of plant resistance response triggered by virus infection
| Project/Area Number |
08044220
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research . |
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| Research Field |
Molecular biology
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| Research Institution | The University of Tokyo (1997-1998)
Teikyo University (1996) |
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Principal Investigator |
WATANABE Yuichiro Univ. of Tokyo, 大学院・総合文化研究科, 助教授 (60183125)
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| Co-Investigator(Kenkyū-buntansha) |
KUMAR Dinesh USDA plant Gene Expression Center, Senior Researcher, 植物遺伝子発現研究所, 主任研究員
BEACHY Roger, N. The Scripps Research Institue, Division of Plant Biology, Professor, 植物生物学部門, 教授
OKADA Yoshimi Teikyo University, Dept. Biosciences, Professor, 理工学部, 教授 (30011703)
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| Project Period (FY) |
1996 – 1998
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| Keywords | N gene / apoptosis / necrosis / tobacco mosaic virus / movement protein / replicase |
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| Research Abstract |
The N gene-mediated hypersensitive response (HR) in tobacco provides a high degree of resistance against most tobamoviruses by halting the progress of infection at the site of inoculation. A previous report indicated a role for the 126/183-kDa replicase in induction of the HR in tobacco containing the N gene (H. S. Padgett and R. N. Beachy, Plant Cell 5 : 577-586, 1993). Chimeric virus genomes were constructed in which the gene encoding the 126/183-kDa proteins of the HR-eliciting pathogen, tobacco mosaic virus (TMV), and the resistance breaking tobamovirus, Ob, were exchanged. Inoculation of the chimeric viruses to leaves of Nicotiana tabacum cv. Xanthi NN confirmed that either the replicase protein of TMV or its mRNA was responsible for induction of HR. An expression vector based on the Ob virus was used to express fragments of various replicase genes. With this approach, it was determined that the HR is caused by a portion of the replicase protein extending from amino acid 692 to 11
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16. Consistent with this result, Ob mutants that induce the HR on NN tobacco were found to carry mutations within the same portion of the replicase gene. The N gene- mediated HR is inactive at high temperatures, yet these mutants were able to overcome the HR at significantly lower temperature than could TMV, indicating that the temperature sensitivity of the N gene response is manifested at the level of interaction between the virus and the defense response mechanism.
The P30 movement protein (MP) of tomato mosaic tobamovirus (ToMV) is synthesized in the early stages of infection and is phosphorylated in vivo. Here, we determined that serine 37 and serin 238 in the ToMV MP are sites of phosphorylation. MP mutants in which serine was replaced by alanine at positions 37 and 238 (LQ37A238A) or at position 37 only (LQ37A) were not phosphorylated, and mutant viruses did not infect tobacco or tomato plants,. By contrast, mutation of serine 238 to alanine did not affect the infectivity of the virus (LQ238A). To investigate the sub- cellular localization of mutant MPs, we constructed viruses that expressed each mutant MP fused with the green fluorescent protein (GFP) of Aequorea victoria. Wide-type and mutant LQ238A MP fusion proteins showed distinct temporally regulated patterns of MP-GFP localization in protoplasts and formation of fluorescent ring-shaped infection sites on Nicotiana benthamiana. However mutant virus LQ37A MP-GFP did not show a distinct pattern of localization or formation of fluorescent rings. Pulse-chase experiments revealed that MP produced by mutant virus LQ37A was less stable that wide-type and LQ238A MPs. MP which contained threonine at position 37 was phosphorylated, but the stability of the MP in vivo was very low. These studies suggest that the presence of serine at position 37 or phosphorylation of serine 37 is essential for intracellular localization and stability of the MP, which is necessary for the protein to function. Less
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