The double-stranded RNA associated with the ?447? cytoplasmic male sterility in Vicia faba is packaged together with its replicase in cytoplasmic membranous vesicles

Abstract

The 447 male sterility trait in Vicia faba is strictly correlated with the presence of well-defined membranous vesicles or 'cytoplasmic spherical bodies' not found in fertile isogenic maintainer plants, and by the occurrence of a discrete high molecular weight double-stranded RNA. We have purified these cytoplasmic membranous vesicles and find that they contain the dsRNA together with an RNA-dependent RNA polymerase whose activity depends upon the presence of Mg2+, requires the four-nucleoside triphosphates and is unaffected by inhibitors of cellular transcriptases, e.g. alpha-amanitin and Actinomycin D. The dsRNA can be labelled in vitro by incubating the cytoplasmic vesicles with radioactive NTPs, and the RNA synthesized in vitro is also in a double-stranded form as judged by its resistance to RNase digestion at high salt and its behaviour upon CF-11 chromatography. Treatment of the vesicles with a non-ionic detergent releases the dsRNA in the form of a complex with the RNA-dependent RNA polymerase. The enzyme can still carry out the specific synthesis of dsRNA in these solubilized complexes. The cytoplasmic vesicles therefore isolate this vertically transmitted, self-replicating dsRNA from the cellular milieu: the possible mode of action and relevance of this novel genetic element to the 447 cytoplasmic male sterility trait are discussed.