Figure 1

Analysis of PLMVd replication and sequences. (A) Agarose gel electrophoresis of RT-PCR amplifications performed in order to detect the presence of PLMVd in the RNA isolated from both the healthy and the infected leaf RNA samples. Lane 1 is a 1 kb-plus ladder that serves as a size standard. Lanes 2 and 3 are negative control reactions for the reverse transcription and PCR amplification, respectively performed in the presence of water alone. Lane 4 is a positive control PCR reaction performed using pPD1 plasmid that contains a dimer of PLMVd sequences as template. Finally, lanes 5 and 6 are the RT-PCR reactions performed in the presence of the RNA samples isolated from both the healthy and the PLMVd-infected samples, respectively. (B) Secondary structure according to previous studies [18] and nucleotide sequence of PLMVd novel variants. The (+) polarity sequence used to fold the above structure is the one that was detected twice following sequencing [NCBI accession number GQ499310]. All of the mutations found in the novel sequences are represented by the boxes around the structure. The mutations are identified by squares, the deletions by triangles and the insertions by diamonds. The alternative P6 stem found in one clone [NCBI accession number GQ499317] is illustrated in the inset. The arrows juxtaposed to the + and - signs identify the hammerhead self-cleavage sites of each polarity, while the boxes identify the conserved nucleotides that form the hammerhead motifs. The white objects indicate minus (-) polarity, while those in black denote the plus (+) polarity.