Immuno-localization Of Encoded Proteins Of Geminivirus And Associated Satellite DNA

Tobacco curly shoot virus (TbCSV) and Tomato yellow curl leaf virus-China (TYLCCNV) caused serious diseases on tobacco and tomato in Yunnan province of China. In this paper the AC4 and Rep proteins encoded by TbCSV and PCI protein of satellite DNA associated with TYLCCNV were investigated by molecular cytological methods.The AC2 and AC4 genes of TbCSV Y35 isolate were expressed with pET-30a expression vector in Escherichia coll. The expressed fusion proteins (6xHis-AC2 and 6xHis-AC4) were purified with Ni2+-NTA resin and the polyclonal antibody of Y35AC4 was then prepared. Then immuno-gold labeling with the Y35AC4 antibody indicated that TbCSV AC4 protein accumulated in the chloroplast and mitochondria of infected tobacco cells. At the same time, immuno-gold labeling with the produced Rep polyconal antibody of TbCSV Y35 isolate indicated that Rep protein localized in the nuclear of the infected tobacco cells.The polyclonal antibody of C1 protein of satellite DNA of TYLCCNV Y10 isolate had been prepared by our laboratory. Immuno-gold labeling with this antibody found that TYLCCNV CI protein didn't accumulated in tobacco cells when infected by Y10 DNA-A alone, but accumulated mainly in the nuclear of tobacco cells when infected by Y10 DNA-A and DNA together. Occasionally, the gold particles were found in the chloroplast of the infected tobacco cells. The localization of C1 protein in transgenic tobacco cells was same as that in the infected'tobacco cells. In interveinal tissue outgrowths of transgenic Nicoliana tabacum, we found that CI protein also mainly localized in the nuclear and no obvious difference was found among different cells. Minor accumulation was also found in the chloroplast.