Figure 1: Diagram of strand-specific RT-PCR for analyzing ASBVd replication in Nostoc. The pRLASBVd(-) and pRLASBVd(+) plasmids express the ASBVd(-) and ASBVd(+) DNA dimmers, respectively, from the petE promoter (grey boxes). HH: hammerhead ribozyme. During the first step, self-cleavage of the ASBVd dimeric form results in the linear monomeric form (lmASBVd) and the circular monomeric form (cmASBVd). During the second step, the RNA-dependent replication process generates linear oligomers (loASBVd), lmASBVd and cmASBVd having the opposite polarity. The primers used during the reverse transcription have a linker sequence at their 5’ extremity (LKASBV-O1 and LKASBV-O2). In order to prevent any amplification of DNA from the plasmid, the primers used during the PCR step are LK and ASBV-O2 for the replication of ASBVd(-) and LK and ASBV-O1 for ASBVd(+), respectively, which makes it possible to discriminate between the RNAs resulting from plasmid transcription and those resulting from RNA-RNA replication.