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Phytopathology

M. Mevarech - Department of Microbiology, University of Tel Aviv, Israel

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.

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Diagnosis of Specific Viral RNA Sequences in Plant Extracts by Hybridization with a Polynucleotide Kinase-Mediated, 32P-Labeled, Double-Stranded RNA Probe
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M. Mevarech - Department of Microbiology, University of Tel Aviv, Israel

Diagnosis of Specific Viral RNA Sequences in Plant Extracts by Hybridization with a Polynucleotide Kinase-Mediated, 32P-Labeled, Double-Stranded RNA Probe

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.

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