A microtubule-associated protein, NbMAP90, is involved in viral movement in Nicotiana benthamiana plants
| Autor: | Jao-Shien Chen, 陳昭賢 |
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| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 During reproduction, plant viruses need many host factors to assist their transcription, translation, replication, encapsidation and movement etc. In order to investigate the interaction between hosts and viruses, we used Bamboo mosaic virus (BaMV) as the material to screen the genes of Nicotiana benthamiana meaningful for viruses. BaMV belongs to the genus Potexvirus and is a single-stranded, positive-sense RNA virus. We isolated the differentially expressed genes of N. benthamiana after virus infection by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. Among the up-regulated candidates a gene fragment and its full length cDNA were cloned. The sequence was predicted to encode a 90-kDa microtubule-associated protein product and designated NbMAP90. Immunolocalization assay by confocal microscopy also showed colocalization of NbMAP90 and microtubules. On the other hand, NbMAP90 was predicted to contain a TBC (Tre-2, Bub2p, Cdc16) functional domain, suggesting its possible role in the regulation of intracellular membrane trafficking. For further understanding the roles of NbMAP90 in BaMV replication, we reduced the NbMAP90 mRNA by gene silencing strategy, infected the NbMAP90-knockdown protoplasts and leaves with virus, and assessed viral accumulation by measuring the amount of viral coat protein. There was no significant difference in BaMV coat protein accumulation between the NbMAP90-knockdown protoplasts and the control cells, indicating the viral replication was not interfered by reduction of NbMAP90. However, at 7 days post-inoculation, BaMV coat protein accumulation in the NbMAP90-knockdown leaves was 50% of that in control leaves; however, coat protein in knockdown leaves was 2% of that in control leaves in systemic leaves. Additionally, once the NbMAP90 was knocked down, the viral spreading efficiency was reduced than in the control leaves. We also infected the NbMAP90-knockdown leaves with Potato virus X (PVX) and Cucumber mosaic virus (CMV) and the results also showed coat protein reduction, indicating that effects of this gene on the population accumulation of plant viruses exhibits no species-specificity. By means of these results we suggest that NbMAP90 could be involved in BaMV cell-to-cell movement. The possible mechanism of this protein involved in BaMV life cycle in plants is discussed. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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