In vitro-generated 'inverse' chimeric Coleus blumei viroids evolve in vivo into infectious RNA replicons

The three Coleus blumei viroids (CbVd) CbVd 1-RL, CbVd 2-RL and CbVd 3-RL occur naturally in plants of Coleus blumei cultivar (cv.) 'Ruhm von Luxemburg'. CbVd 2-RL is a viroid chimera made up of the right half of CbVd 1-RL and the left half of CbVd 3-RL. Using the cDNAs of the left half of CbVd 1-RL and the right half of CbVd 3-RL, 'inverse' viroid chimeras (CbVd-Ch1 and CbVd-Ch2) were constructed in vitro as dimeric cDNA units. The in vitro-generated cDNA-transcripts of CbVd-Ch1 and CbVd-Ch2 were used to inoculate plants of Coleus blumei cv. 'Ruhm von Luxemburg' harbouring CbVd 1-RL and CbVd 3-RL, leading to the emergence of new viroid-like RNA replicons, named CbVd A-1 and CbVd A-2, that hitherto have not been found in Coleus plants. The sequences of CbVd A-1 and CbVd A-2 are not identical to the corresponding sequences of the cDNA-transcripts of CbVd-Ch1 and CbVd-Ch2, but show a deletion of a segment at the border of the two original halves as well as two U to A transitions. When the cDNA-transcripts of CbVd-Ch1 and CbVd-Ch2 were used to inoculate plants of Coleus blumei cv. 'Scarlet Dragonfly' that were either viroid-free or infected with CbVd 1-RL and CbVd 3-RL, no new viroid-like RNAs emerged. However, the cDNA-transcripts of CbVd A-1 and CbVd A-2 are infectious by mechanical inoculation of viroid-free plants of cv. 'Scarlet Dragonfly', thus proving their viroid nature.