Vesicle-associated double-stranded ribonucleic acid genetic elements in Agaricus bisporus

Double-stranded ribonucleic acids (dsRNAs) were isolated from fruit bodies of commercial strains of the cultivated mushroom (Agaricus bisporus) by polyethylene glycol-NaCl precipitation, differential centrifugation, rate-zonal centrifugation in sucrose, and equilibrium centrifugation in cesium sulphate. In all seven of the mushroom isolates examined, three dsRNAs were identified: two major dsRNA segments of > 13.1-kb (L-RNA) and 2.4-kb (S-RNA) and a minor segment of 5.2-kb (M-RNA). L-, M-, and S-RNAs co-purified with spherical fungal vesicles measuring approximately 75 nm in diameter. The three dsRNAs were intimately associated with the vesicles as suggested by their lower buoyant density in cesium sulphate (1.27 g/cc) compared to that of phenol-extracted dsRNAs (1.42 g/cc) and by their resistance to hydrolysis by ribonuclease at low ionic strength. Using a variety of conditions during purification, no virus-like particles were found to be associated with the dsRNAs. In Northern analysis, L-, M-, and S-RNAs failed to cross-hybridize with the genomic dsRNAs of La France isometric virus. We report here the first description of non-encapsidated, vesicle-associated, dsRNA genetic elements in the common cultivated mushroom.