Studies on the symptom determinants of geminivirus

• Main Authors: kao-Wei Dai, 戴國偉
• Other Authors: 胡仲祺
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/90962515967545770890

碩士 === 國立中興大學 === 生物科技學研究所 === 96 === Geminiviruses are characterized by circular single-stranded DNA genomes with one or two components which are encapsidated in geminate semi-icosahedral particles and replicate via a double-stranded DNA intermediate. Previous studies have revealed the ability of geminiviruses to alter the cell-cycle processes and inflict various type of symptoms on different host plants, indicating that geminiviruses possess the potential to interact with host factor and regulate the symptom expression in a species-specific manner. However, the responsive viral protein and the regulatory mechanism remained to be elucidated. It has been shown by our group that Nicotiana benthamiana plants infected with Ageratum yellow vein virus Nan-Tou Strain (AYVV-NT) display severe upward leaf curling symptoms. In contrast, the leaves of those infected by Tomato leaf curl virus Tai-Nan strain (TLCV-TN) or Squash leaf curl virus Yun-Lin strain (SqLCV-YL) curl downward. Therefore, the aim of this study is to identify and characterize sequences or genes in geminivirus genome invplved in symptom determination. Using the infectious clone of AYVV-NT and TLCV-TN as the experiment materials, recombinant viruses were constructed to locate the viral sequence or genes that influence the disease phenotype. In the first approach, chimeric viruses were constructed by exchanging fragments of the viral genomes generated by restriction enzymes. The resulted chimeric monomers were used as the templates to construct infectious clones using the simplified method developed previously in our laboratory. Results of the inoculation assays on N. benthamiana revealed that symptoms were determined by viral genome spanning nt 1968 ~ 138, which include the intergenic region (IR) 、AC4 gene and 5’-portion of Rep gene. By loss-of-function mutations, it was found that AC4 is likely involved in the leaf curl symptoms, although further fine mapping is required to identify the exact symptom determinant. The second approach is the co-inoculation of AYVV-NT and TLCV-TN in N. benthamiana plants. No recombinant virus was isolated from the co-inoculated plants. The result revealed that the direction of leaf curling was controlled by the difference in population sizes of the two viruses, similar to the control of phenotypes in classic quantitative genetics. The third approach is to generate random recombinants by DNA shuffling, by which no infectious recombinant virus was obtained to date. Collectively, the results revealed the primary viral determinants of the direction of leaf curling symptom. It is expected that through the use of the infectious recombinant geminiviruses, the underlying mechanism for the control symptoms will be elucidated.