Author
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Zhao, Yan |
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Owens, Robert |
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Hammond, Rosemarie |
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Submitted to: BARC Poster Day
Publication Type: Abstract Only Publication Acceptance Date: 3/10/1998 Publication Date: N/A Citation: N/A Interpretive Summary: Technical Abstract: Viroids are the smallest known phytopathogens and infect many economically important crop plants. The viroid RNA genome replicates autonomously without a helper virus--presumably within the nucleus of host cells. Although there is ample information regarding the structure of potato spindle tuber viroid (PSTVd), the precise nature of the nuclear targeting signals residing in the viroid RNA molecule has yet to be determined. To approach the identification of specific sequence elements that are required for PSTVd movement into the host cell nucleus, we have developed an in vivo reporter system employing a modified version of the jellyfish green fluorescent protein (GFP). The encoding region of GFP was artificially interrupted by inserting an intron derived from IV2 of the maize ST-LS1 gene. A series of complete or partial PSTVd sequences was in turn embedded in the intron, and this series of intron-containing GFP reporter genes was then delivered into Nicotiana benthamiana via a tobacco mosaic virus (TMV) -based vector. Once inside the host plant cells, the intron-containing GFP reporter genes are expressed as a TMV subgenomic RNA in the cytoplasm. A functional GFP will not be produced, however, unless (i) the subgenomic RNA is targeted to the nucleus where the intron can be precisely removed, and (ii) the spliced mRNA is returned to the cytoplasm and translated. The appearance of green fluorescence in leaf tissues examined under the microscope indicates that nuclear targeting and RNA splicing events have occurred. Our preliminary results show that the construct with a full-length PSTV genomic strand embedded in the intron was able to produce functional GFP. |
