Location: Molecular Plant Pathology LaboratoryTitle: Extraction and purification of viroids from Herbaceous hosts
Submitted to: Methods in Molecular Biology
Publication Type: Book / Chapter
Publication Acceptance Date: 7/9/2020
Publication Date: 11/30/2021
Citation: Hammond, R. 2021. Extraction and purification of viroids from Herbaceous hosts. Methods in Molecular Biology. 2316:65-70. https://doi.org/10.1007/978-1-0716-1464-8_6.
Interpretive Summary: Viroids are the smallest known infectious agents of plant diseases and are pathogens of food, industrial, and ornamental plants. Yield losses caused by viroids (small noncoding circular infectious RNAs) can reach 17-64% depending on the viroid strain and plant crop species. Characterization of known and newly discovered viroids relies on highly sensitive and specific detection methods based on purified nucleic acid preparations from infected plant tissue. The unique structural properties of viroid RNAs has allowed development of extraction and purification protocols that enrich these unique molecules for downstream molecular characterization. In this chapter, a specific method routinely used for viroid purification from herbaceous hosts, and problems that may be encountered, is described and is intended as a reference for beginners in the field. This work will be of value to an international audience of researchers in industry, academia, and government organizations with an interest in plant pathology and virology and the detection and control of plant diseases.
Technical Abstract: Protocols for extraction and purification of viroid RNAs from the tissues of infected herbaceous plant hosts are numerous and range from lengthy, traditional protocols that require large amounts of starting infectious tissue and take several days to perform to those based on column chromatography, are more efficient and do not require handling large amounts of infected tissue. The goal of all protocols is to enrich for RNA fractions that contain viroid RNAs, and the RNA extraction procedure is chosen and adjusted for the downstream method used for detection and characterization. Removal of inhibitors/impurities is generally not an issue for herbaceous hosts unless they contain and inordinate amounts of polysaccharides, tannins, acids, and phenols. Subsequent purification of viroid circular and linear RNAs is performed using various forms of denaturing polyacrylamide gel electrophoresis. In this chapter, protocols to achieve both purposes are described.