The nature and biological significance of linear potato spindle tuber viroid molecules

Abstract

“Naturally occurring” linear potato spindle tuber viroid (PSTV L) was shown to be as infectious as circular PSTV (PSTV c). The occurrence of PSTV L was shown not to be (a) an artifact of the extraction procedure per se; (b) due to the presence of metal ions in extraction buffers; or (c) related to the host species used for propagation, to a particular PSTV strain, or to the duration of infection. From labeling and blot-hybridization experiments, it was concluded that in infected tissue PSTV c appears first followed by PSTV L, which is produced as the result of cleavage of PSTV c at specific sites. One of the sites of cleavage of PSTV c was delineated by 5′-end labeling PSTV L, digesting it with RNase T 1, separating the fragments by two-dimensional gel electrophoresis, and sequencing the major RNase T 1-resistant fragments. The major site of in vitro labeling was shown to be variable, but was confined to the right-hand loop of PSTV c, i.e., between nucleotides 177 and 182. Furthermore, the right-hand end stem and loop of PSTV c contains sequences which are similar to a rDNA gene promoter sequence, and thus we suggest that DNA-dependent RNA polymerase I may be involved in viroid biosynthesis. Other cleavage sites in PSTV were determined by primer extension cDNA synthesis and by dideoxynucleotide chain termination, and were shown to correspond to nucleotides 113–114 and 80–81 and to sequences in the region of 334–340, 300–312, and 271–275. The significance of these cleavage sites is discussed.