Potato spindle tube̊r viroid (PSTV) RNA oligomers of (+) and (−) polarity are synthesized in potato protoplasts after liposome-mediated infection with PSTV

Abstract

Potato protoplasts prepared from suspension culture cells were inoculated with potato spindle tuber viroid (PSTV) and with cloned PSTVcDNA, respectively, encapsulated in reverse-phase evaporation vesicle (REV) liposomes. PSTV-infection was achieved by incubation of 10 6 protoplasts with 100 ng of encapsulated PSTV. The replication of PSTV in protoplasts was analyzed by monitoring the synthesis of PSTV(+) and (−)RNA by Northern blot hybridization. In addition to monomeric circular PSTV(+)RNA, i.e. the viroid proper as the main product of replication, linear oligomers of PSTV(+) and (−)RNA could be detected in infected protoplasts. Time course experiments revealed that the synthesis of oligomeric linear PSTV(−)RNA preceded that of the accumulating circular PSTV(+)RNA, suggesting that the oligomeric PSTV(−)RNA molecules represent the transient linear intermediates of replication. A normal PSTV replication and accumulation was also initiated, when liposomes were used to deliver cloned double-stranded DNA containing an infectious multimeric PSTVcDNA-sequence to potato protoplasts.