VIEW ARTICLE

Techniques

Diagnosis of Specific Viral RNA Sequences in Plant Extracts by Hybridization with a Polynucleotide Kinase-Mediated, ³²P-Labeled, Double-Stranded RNA Probe. A. Rosner, Virus Laboratory, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel; M. Bar-Joseph(2), M. Moscovitz(3), and M. Mevarech(4). (2)(3)Virus Laboratory, Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel; (4)Department of Microbiology, University of Tel Aviv, Israel. Phytopathology 73:699-702. Accepted for publication 26 November 1982. Copyright 1983 The American Phytopathological Society. DOI: 10.1094/Phyto-73-699.

A method was developed for screening of virus-related RNAs in plant extracts. The method is based on the hybridization of a polynucleotide kinase-mediated, ³²P-labeled, double-stranded (ds) RNA probe with RNA or sap extracts. The cucumber mosaic virus (CMV) and associated RNA-5(CARNA-5) was used as a model system. Naturally infected plants of Nicotiana glauca were screened for the presence of dsCARNA-5; dsRNA was purified on CF-11 columns and analyzed in polyacrylamide gels. Three types of low-molecular-weight CARNA-5-like dsRNA species, with estimated molecular weights of (type I) 0.27, (type II) 0.22, and (type III) 0.19 x 10⁶ daltons, were observed in plants of N. glauca that reacted with CMV antiserum. Types II and III were shown to contain CARNA-5 sequences by Northern blot hybridization of dsRNA patterns with ³²P-labeled dsCARNA-5. This labeled probe was further used to detect CARNA-5 infection in N. glauca and N. glutinosa plants by hybridization with dot-spots of dsRNA as well as plant saps. The applicability of ³²P-labeled dsRNA for diagnosing specific sequences of plant virus RNAs is discussed.