The Effect of Phosphorylation of Triple Gene Block Protein 1 (TGBp1) on Viral Replication and Cell-to-Cell Movement of Bamboo Mosaic Virus in Nicotiana benthamiana

• Main Authors: Lan-Hui Wang, 王蘭蕙
• Other Authors: Dr. Chi-Ping Cheng
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/90265907350259181577

碩士 === 慈濟大學 === 生命科學系碩士班 === 102 === Bamboo mosaic virus (BaMV) belongs to the genus Potexvirus which contains five open reading frames (ORFs). Three of the five ORFs are triple gene block protein 1, 2, 3 (TGBp1, 2, 3) which involved in the viral movement between plant cells. To investigate the effect of phosphorylation of BaMV TGBp1 on cell-to-cell movement in Nicotiana benthamiana, we first used NetPhos 2.0 to predict the potential phosphorylation positions and BaMV infectious clone which expresses GFP was used to create the TGBp1 mutants. Threonine-58 and Threonine-111 were changed into Aspartate (T58D and T111D, respectively) or Alanine (T58A and T111A), and Serine-15, 18, and 247 were changed into Aspartate (S15D, S18D and S247D). In Planta experiments, we transfected the plasmid which contains wild type or mutant TGBp1 sequences into Nicotiana benthamiana leaves. By measuring fluorescent area, mutants S15D and S18D as well as T58D and T111D with mimicking constant phosphorylation or T111A which cannot be phosphorylated have reduced GFP foci area and intensity. I further studied whether these TGBp1 mutants influence BaMV replication by protoplast assays. By measuring the coat protein accumulation, S15D was significantly reduced comparing to the wild type, whereas which of T111D was not. These results indicate that mimicking constant phosphorylation on Serine-15 affects replication of BaMV and constant phosphorylation on Threonine-111 dramatically reduces the cell-to-cell movement of BaMV since its replication efficiency in the protoplasts was not affected. In summary, this study resolves the phosphorylation status on several amino acids of BaMV TGBp1 which play important roles for BaMV replication and cell-to-cell movement in N. benthamiana.