Circular double-stranded RNA in potato spindle tuber viroid-infected tomatoes

Abstract

Viroids are circular molecules of single-stranded RNA consisting of ∼360 nucleotides^1,2, and the mechanism by which these smallest of all known infectious agents replicate in host cells has been extensively studied. Earlier work suggested that viroids replicate using DNA intermediates³, but rigorous hybridization experiments^4,5 failed to detect DNA complementary to potato spindle tuber viroid (PSTV) in infected plants, and there is no specific inhibition of viroid synthesis⁶ by actinomycin D. There is strong evidence that the replication of viroids involves RNA intermediates. Citrus exocortis viroid (CEV)^7,8-infected and PSTV-infected^4,9,10 tissues have been shown to contain RNA that is complementary to the infecting viroid, and recently it was shown that DNA-dependent RNA polymerase II, isolated from tomato, will produce full-length complementary copies of purified viroids in vitro¹¹. Because much of the RNA complementary to CEV was shown to be double stranded, it could be anticipated that viroid-infected tissue contains unique species of double-stranded RNA (dsRNA). Using serologically specific electron microscopy¹², we have now detected molecules of circular dsRNA in crude extracts of PSTV-infected tomato. These molecules have contour lengths that would be expected for a double-stranded intermediate of PSTV.