Submitted to: American Phytopathological Society
Publication Type: Abstract Only
Publication Acceptance Date: 6/29/1998
Publication Date: N/A
Technical Abstract: Viroids are the smallest known phytopathogens and infect many economically important crop plants. Potato spindle tuber viroid (PSTVd) is a covalently closed circular RNA molecule of 359 nucleotides that replicates autonomously, presumably within the nucleus of host cells. To approach the identification of specific nuclear targeting sequence elements residing in the viroid molecule, we have developed an in vivo reporter system using green fluorescent protein (GFP) as the reporter molecule. The coding region of GFP was interrupted by insertion of an intron derived from IV2 of the potato ST-LS-1 gene. A complete PSTVd sequence was embedded within the intron, and this construct was delivered delivered into Nicotiana benthamiana via a potato virus X (PVX)-based vector. The intron-containing GFP reporter gene expressed as a subgenomic RNA in the cytoplasm will not produce a functional GFP unless the subgenomic RNA is targeted to the nucleus where the intron can be removed and the spliced RNA returned to the cytoplasm and translated. The appearance of green fluorescence in leaf tissues inoculated with constructs containing a full-length PSTVd molecule embedded in the intron indicates that nuclear targeting and RNA splicing events have occurred.