Nuclear targeting by fragmentation of the potato spindle tuber viroid genome.
Transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a Potato virus X-based expression vector previously demonstrated the nuclear targeting capability of the 359 nucleotide Potato spindle tuber viroid (PSTVd) RNA genome. To further delimit the putative nuclear-targeting signal, PSTVd subgenomic fragments were embedded within the intron, and recombinant reporter RNAs were inoculated onto Nicotiana benthamiana plants. Appearance of green fluorescence in leaf tissue inoculated with PSTVd-fragment-containing constructs indicated shuttling of the RNA into the nucleus by fragments as short as 80 nucleotides in length. Plant-to-plant variation in the timing of intron removal and subsequent GFP fluorescence was observed; however, earliest and most abundant GFP expression was obtained with constructs containing the conserved hairpin I palindrome structure and embedded upper central conserved region. Our results suggest that this conserved sequence and/or the stem-loop structure it forms is sufficient for import of PSTVd into the nucleus.
Abraitiene
Asta
A
Eukaryote Genetic Engineering Laboratory, Institute of Biotechnology, Vilnius, Lithuania.
Zhao
Yan
Y
Hammond
Rosemarie
R
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D002467:Cell Nucleus / Q000235:genetics* / Q000821:virology*; D053938:DNA Fragmentation; D018390:Gene Targeting / Q000379:methods*; D016679:Genome, Viral / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics* / Q000648:ultrastructure / Q000821:virology*; D014162:Transfection / Q000379:methods; D014772:Viroids / Q000235:genetics*