In situ localization of citrus exocortis viroid RNA using an optimized RNAscope™ assay

Abstract

Due to their small size, locating pathogenic RNAs, such as viroids, in plant tissue and cell organelles has been difficult. Viroids are small circular single-stranded RNA plant pathogens that reduce plant growth, vigor, and yield in economically important crops such as potato, tomato, hops and citrus. Viroid infections in plants are largely diagnosed by dot blot hybridization, polyacrylamide gel electrophoresis (PAGE) or gels, or real-time quantitative polymerase chain reaction (qPCR). Because traditional plant in situ hybridization studies for viroids are often limited by the lack of signal amplification and binding specificity due to the small target sequence, we examined the use of RNAscope™ (Advanced Cell Diagnostics Inc., Newark, CA). This in situ hybridization method increases the detection by amplifying the signal of a single target, to detect the cellular distribution of citrus exocortis viroid (CEVd) with higher sensitivity and specificity. We found that after optimization, CEVd was localized in nuclei of infected cells as clearly distinguishable punctate red dots visible with light microscopy at low magnification, suggesting that the RNAscope™ can be used to study viroids in situ .