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Title: Nuclear targeting by fragmentation of the Potato spindle tuber viroid genome

item Zhao, Yan
item Hammond, Rosemarie

Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2008
Publication Date: 4/11/2008
Citation: Abraitiene, A., Zhao, Y., Hammond, R. 2008. Nuclear targeting by fragmentation of the Potato spindle tuber viroid genome. Biochemical and Biophysical Research Communications. 368:470-475.

Interpretive Summary: Viroids are a distinct class of plant pathogens that cause a number of economically important diseases, however, no naturally occurring resistance to viroids is known. Viroids rely upon interactions with their host plant for multiplication and spread throughout the plant, and, as these interactions are poorly understood, attempts to control viroid diseases by novel strategies have not been successful. A key regulatory step in the ability of the Potato spindle tuber viroid (PSTVd) RNA to colonize its host is its movement from the cytoplasm into the nucleus, where it multiplies. We have identified a nucleic acid signature that functions in movement of PSTVd-containing RNAs into the nucleus. With this discovery arises the potential for disrupting the viroid-host interaction leading to nuclear import and perhaps subsequent disease control. The results will be of interest to scientists studying the regulation of intracellular transport of RNA molecules and those designing resistance strategies against viroid diseases.

Technical Abstract: The whole plant nuclear targeting assay is an experimental system that utilizes transient expression of engineered reporter RNAs encoding an intron-containing green fluorescent protein (GFP) from a vector derived from Potato virus X (PVX). The assay demonstrated the nuclear targeting capability of the full-length, 359 nucleotide, Potato spindle tuber viroid (PSTVd) RNA genome. In order to further delimit the putative nuclear-targeting signal, six PSTVd fragments, representing different portions of the RNA genome, were embedded within the intron, and the recombinant reporter RNAs were inoculated to Nicotiana benthamiana plants via the PVX-based vector. The 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. Intron removal was confirmed by RT-PCR assays. There was plant-to-plant variation in the timing of intron removal and subsequent GFP fluorescence; however we routinely observed that the earliest and most abundant GFP expression was obtained with two constructs that contained the conserved hairpin I palindrome structure and embedded upper central conserved region (CCR). Our results suggest that the evolutionarily-conserved hairpin I –CCR sequence and/or the stem-loop structure it forms is sufficient to direct import of PSTVd into the nucleus.