Association of Bamboo mosaic virus satellite RNA encoded P20 with mitochondria

• Main Authors: Guan-Zhi Wang, 王冠智
• Other Authors: Yau-Heiu Hsu
• Format: Others
• Language: zh-TW
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/72801568774076713448

碩士 === 國立中興大學 === 生物科技學研究所 === 96 === Bamboo mosaic virus (BaMV), is a single-stranded positive-sense RNA virus with flexuous rod-shaped morphology. The satellite RNA (full-length 836 nt) associated with BaMV, designated as satBaMV RNA, encodes a 20 kDa nonstructural protein (P20). Using several programs to predict the subcellular locatizaion of P20, we found transmembrane domain and mitochondrial targeting signal (MTS), typical viral proteins that can localize on mitochondria. We used discontinuous percoll gradient to isolate mitochondria of Nicotiana benthamiana. The mitochondrial fraction was confirmed by a mitochondrial specific dye Mitotracker Orange CMTMRos and antibody of cytochrome c. Using western blot analysis P20 was detected in mitochondrial enriched fraction from satBaMV transgenic line 2-6 that was inoculated with BaMV. Protoplasts were prepared from inoculated leaves of N. benthamiana co-infected with BaMV-S viral RNA and in vitro transcripts of pBSGFP, pBSGFPP20 and pBSP20GFP, respectively. By Confocal microscopy, protoplasts from pBSGFPP20 infected leave, similar to pBSGFP displayed the green fluorescence which scattered in cytoplasm. The protoplasts contained P20GFP had obvious GFP signal that co-localized and surrounded with mitochondria of diameter at least 2μm at low expression, but the fluorescence were too strong to recognize if P20GFP had transported into mitochondria. When P20GFP gradual accumulated, the phenomenon which green fluorescence co-localize with mitochondria was lost and the 2μm diameter mitochondria were disappeared. The Protease K protection assay showed the mitochondrial membrane could be preserved completely 7 days after isolation. However P20 within mitochondrial fraction from N. benthamiana line 2-6 challenged with BaMV-S could not be protected by mitochondrial membrane after Protease K treatment. No appearance of products once MTS predicted by MitoProt II were cleaved as most of mitochondrial import mechanism. Our data support a hypothesis that P20 of satBaMV interacted with mitochondria that may be just at early phase of viral life cycle.