Optimization of RNAscope™ assays for the in-situ localization of viroid RNA in plant tissue.

35 Due to their small size, locating pathogenic RNAs, such as viroids, in plant tissue and cell 36 organelles has been difficult. Viroids are small circular single-stranded RNA plant 37 pathogens that reduce plant growth, vigor, and yield in economically important crops 38 such as potato, tomato, hops and citrus. Viroid infections in plants are largely diagnosed 39 by dot blot hybridization, polyacrylamide gel electrophoresis (PAGE) or gels, or real-time 40 quantitative polymerase chain reaction (qPCR). Because traditional plant in situ 41 hybridization studies for viroids are often limited by the lack of signal amplification and 42 binding specificity due to the small target sequence, we examined the use of RNAscope™ 43 (Advanced Cell Diagnostics Inc., Newark, CA). This in situ hybridization method increases 44 the detection by amplifying the signal of a single target, to detect the cellular distribution 45 of citrus exocortis viroid (CEVd) with higher sensitivity and specificity. We found that 46 after optimization, CEVd was localized in nuclei of infected cells as clearly distinguishable 47 punctate red dots visible with light microscopy at low magnification, suggesting that the 48 RNAscope™ can be used to study viroids in situ . 49 50