|Hsu, Hei Ti|
|Lin, Chen Hsuan|
Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/5/2005
Publication Date: 12/15/2005
Citation: Chen, T.C., Hsu, H.T., Jain, R.K., Huang, C.W., Lin, C.H., Liu, F.L., and Yeh, S.D. 2005. Purification and serological analyses of tospoviral nucleocapsid proteins expressed by Zucchini yellow mosaic virus vector in squash. Journal of Virological Methods. 129:113-124. Interpretive Summary: Tospovirus nucleocapsid protein (NP) genes were cloned into a virus vector and the NPs were expressed in plants, purified from infected tissues and used for serological analysis. Expression of a foreign gene in plants has numerous advantages. It can be applied in the study of biological function of the protein encoded by the gene. When expressed in larger quantity, the expressed protein can be used for production of vaccine or commercial uses. In current studies, a virus vector derived from Zucchini yellow mosaic virus was utilized for production of NPs from various tospoviruses including Tomato spotted wilt, Impatiens necrotic spot, Watermelon silver mottle, Peanut bud necrosis, and Watermelon bud necrosis viruses. The NPs were successfully expressed in Nicotiana benthamiana and purified. The method is useful in plant virus investigation. When used, serological analysis can be applied to viruses that are quarantine restricted and are not present in the investigating countries. This research is related to National Plant Health Program 303. The information reported is important to US agriculture, quarantine officials and plant pathologists.
Technical Abstract: A plant viral vector engineered from an in vivo infectious clone of Zucchini yellow mosaic virus (ZYMV) was used to express the nucleocapsid proteins (NPs) of tospoviruses in planta. The open reading frames (ORFs) of NPs of different serogroups of tospoviruses, including Tomato spotted wilt virus, Impatiens necrotic spot virus, Watermelon silver mottle virus, Peanut bud necrosis virus, and Watermelon bud necrosis virus (WBNV), were in frame inserted in between the P1 and HC-Pro genes of the ZYMV vector. Six histidine residues and an NIa protease cleavage site were added at the C-terminal region of the inserts to facilitate purification and process of free form of the expressed NPs, respectively. Approximately 1.2-2.5 mg/NPs 100 g tissues were purified from leaf extracts of zucchini squash. The expressed WBNV NP was used as an immunogen for the production of highly specific polyclonal antisera and monoclonal antibodies. The procedure provides a convenient and fast way for production of large quantities of pure NPs of tospoviruses in planta. The system also has a potential for production of any proteins of interest in cucurbits.