Meeting Abstract
Viroids are small circular single-stranded RNA virus-like organisms that cause disease on economically important crops such as potato, tomato, hops and citrus, reducing plant growth, vigor, and yield. Viroid infections in plants are largely diagnosed by RT-PCR, by dot blot hybridization, or PAGE-gels. Citrus exocortis viroid (CEVd) was one of the first viroids characterized and the causal agent was shown to be a short 371 nucleotide single-stranded RNA molecule. Citrus cachexia viroid (CVd II) is a similar viroid discovered 10 years later that has been shown to be 300 nucleotides long and is the causal agent of some cases of xyloporosis. Due to their small size locating these RNAs in plant tissue has been problematic. Traditional plant in situ hybridization studies are often limited by specificity of binding, limited target signal amplification. Additionally, traditional in situ hybridization methods are limited to a single specific probe designed from a known viroid sequence. RNAscope (Advanced Cell Diagnostics Inc., Newark, CA), however is a novel in situ hybridization method that has been developed to hybridize double Z probes along an RNA sequence up to 1kb long to which a scaffold is built to allow for greater amplification of signal for each Z probe. The scaffold contains multiple binding sites for the detection enzyme when bind to a colorimetric or fluorometric substrate. Here we present data showing that RNAscope can be used to detect CEVd and CVd-II in citrus petioles, stem, and root tissues. Additionally, a RNAscope duplex assay can be used to detect co-infection of viroids. RNAscope has broad application in plant research including the detection of plant RNAs and RNAs of Plant pathogens.
