Host and Tissue Selection, Virion Purification, RNA Characterization and Serological Studies of Barley Yellow Dwarf Virus-Rpv-Il
Murphy, John Francis
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https://hdl.handle.net/2142/70616
Description
Title
Host and Tissue Selection, Virion Purification, RNA Characterization and Serological Studies of Barley Yellow Dwarf Virus-Rpv-Il
Author(s)
Murphy, John Francis
Issue Date
1988
Department of Study
Plant Pathology
Discipline
Plant Pathology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Agriculture, Plant Pathology
Abstract
A purification scheme was improved for an isolate of barley yellow dwarf virus transmitted specifically by Rhopalosiphum padi L. (RPV-IL). RPV-IL yields average 1.60 mg virus/kg 'Coast Black' oat root tissue when extraction involves grinding tissue in liquid nitrogen and overnight incubation in a pectinolytic enzyme. No virus is obtained from roots of 'Clintland 64' or 'California Red' oats. Coast Black, Clintland 64 and California Red shoot tissues average 0.79, 0.78 and 0.54 mg versus/kg tissue, respectively. Enzyme-linked immunosorbent assay (ELISA) detects RPV-IL in all tissues and therefore is an inaccurate measure of extractable virus concentration in plants.
The 5$\sp\prime$ terminus of RPV-IL RNA does not become labeled with $\sp{32}$P after dephosphorylation with phosphatase and subsequent treatment with ($\gamma - \sp{32}$P) ATP and kinase and therefore is some derivative other than phosphate(s). An $\sp{125}$I-labeled 17,000 d protein is detected after treatment of RPV-IL RNA with $\sp{125}$I-Bolten-Hunter reagent and analyses by acid precipitation and polyacrylamide gel electrophoresis. This 17,000 d protein is not BYDV coat protein, as determined by immunoprecipitation assays, and therefore is a 17,000 d BYDV VPg (virion protein-genome linked).
The serological relationships among five luteoviruses were compared using three variations of ELISA. Serological relationships are detected among RPV-IL, RPV-NY and beet western yellows virus (BWYV) in direct DAS ELISA which detects common epitopes on the surface of the virus particles. Serological relationships are detected among RPV-IL, RPV-NY, PAV and soybean dwarf virus (SDV) in indirect and direct ELISA with ELISA plates coated with purified virus and therefore share common internal epitopes. No relationship is detected between potato leafroll virus (PLRV) and the other luteoviruses used in this study when sap from infected plants was tested in direct DAS ELISA. Monoclonal antibodies made to RPV-IL detect the presence of common epitopes among the RPV isolates, PAV and SDV when the virus particles are dissociated and among the RPV isolates, BWYV and PLRV when from sap from infected tissues is tested in indirect ELISA.
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