Proposed PLMVd secondary structures. (A) Compilation of the mapping results on the proposed secondary structure of the minus polarity PLMVd transcripts. The red boxes represent single-stranded nucleotides, while the blue ones designate the double-stranded nucleotides. The dark red boxes indicate intense cleavage by single-stranded RNases, while the dark blue boxes indicate unambiguous double-stranded nucleotides. The black and white boxes represent the conserved nucleotides that form the plus and minus hammerhead structures, respectively. The oligonucleotides used in the EMSA and RNase H hydrolysis analyses are illustrated using either full or dotted lines in order to differentiate the accessible (loops and junctions) and unaccessible regions (stems), respectively. The nucleotide numbers of the viroid to which each oligonucleotide is complementary are indicated in italics above each oligonucleotide. All stems, loops, and junctions are identified. (B) Schematic representation of the proposed secondary structure for the PLMVd transcripts of plus polarity. All stems are identified. The nucleotides involved in the formation of the P8 pseudoknot are also indicated. (C) Autoradiogram of a EMSA performed in order to investigate the presence of the P8 pseudoknot. Lanes 1 and 2 are the experiments performed with the oligonucleotides complementary to sequence of the region of the L7 loop of plus polarity either in the absence or presence of the PLMVd transcripts of plus polarity. Lanes 3 and 4 are the experiments performed with the oligonucleotides complementary to sequence of the region of the L7 loop of minus polarity either in the absence or presence of the PLMVd transcripts of minus polarity.
