Search

Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1 -suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1 -suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1 -suppressed plants is likely the result of cell-autonomous events.

Autosomal dominant retinitis pigmentosa caused by the frequent rhodopsin P23H mutation is characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Considering the gain-of-function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable. We have designed small interfering RNAs (siRNA) to silence specifically the P23H rhodopsin allele expressed by a transgenic rat model of the disease. We have selected in vitro one siRNA and generated an adeno-associated viral (AAV) vector expressing the short hairpin RNA (shRNA) based on the selected siRNA. In vitro the shRNA significantly inhibits the expression of the P23H but not the wild-type rhodopsin allele. Subretinal administration of the AAV2/5 vector encoding the shRNA in P23H transgenic rats results in inhibition of rhodopsin P23H expression that is not able to prevent or block photoreceptor degeneration. Since rhodopsin is the most abundant rod photoreceptor protein, systems resulting in more robust shRNA expression in the retina may be required to achieve therapeutic efficacy in vivo.