A New Method to Isolate Total dsRNA
When a diseased plant is suspected to be infected with unknown viruses, the approach of isolating double-stranded RNA (dsRNA) from diseased tissues and analyzing the sequence has been useful for detecting the viruses. This procedure owes its success to the majority of plant pathogenic viruses being RNA viruses, which accumulate dsRNAs as copies of their genome or as a replicative intermediate in infected cells. Conventional dsRNA isolation methods (e.g., chromatography using CF-11 cellulose) require a significant amount of plant material and are laborious and time consuming. Therefore, it has been impractical to isolate dsRNA from many samples at the same time. To overcome these problems, we developed a novel dsRNA isolation method involving a recombinant dsRNA-binding protein. Using this method, we can readily isolate viral dsRNA from a small amount of plant material, and can process numerous samples simultaneously. Purified dsRNA can be used as a template for cDNA synthesis and sequencing, enabling detection of both known and unknown viruses.
Key wordsDiagnosis RNA virus Replicative intermediate dsRNA isolation dsRNA-binding protein
We thank Reiko Tomita for technical advice.
- 4.Fauquet CM, Mayo M, Maniloff J, Desselberger U, Ball LA (2005) Virus taxonomy: VIIIth report of the International Committee on Taxonomy of Viruses. Online via Elsevier, AccessGoogle Scholar
- 10.Eamens AL, Curtin SJ, Waterhouse PM (2011) The Arabidopsis thaliana double-stranded RNA binding (DRB) domain protein family. In: Erdmann VA and Barciszewski J (eds) Non coding RNAs in plants. Springer-Verlag, Berlin, Heidelberg, pp 385–406Google Scholar
- 13.Kobayashi K, Atsumi G, Iwadate Y, Tomita R, Chiba K, Akasaka S, Nishihara M, Takahashi H, Yamaoka N, Nishiguchi M, Sekine KT (2013) Gentian Kobu-sho-associated virus: a tentative, novel double-stranded RNA virus that is relevant to gentian Kobu-sho syndrome. J Gen Plant Pathol 79:56–63CrossRefGoogle Scholar