A point-mutation of Coleus blumei viroid 1 switches the potential to transmit through seed

Viroids are highly structured, single-stranded, non-protein-coding circular RNA pathogens that replicate, spread and elicit severe to mild disease symptoms in sensitive host species. The functions of viroids are thought to be due to a molecular element (or elements) embedded within the small RNA molecule that recruits the host factors responsible for transcription, RNA transportation and regulation of gene expression. Coleus blumei viroid 1 (CbVd-1) is distributed worldwide and is known for its characteristic property of having an extremely high frequency of seed transmission. During our analysis of CbVd-1 seed transmission, two variants, CbVd-1/25A and CbVd-1/25UU, were shown to have distinct seed-transmission frequencies: 30 and 0 %, respectively. Seven infectious dimeric forms of CbVd-1 cDNA clones were created based on the sequences of CbVd-1/25A,CbVd-1/25UU and an additional five variants with unique loop structures in other portion(s) of the molecule, and in vitro transcripts were inoculated into viroid-free coleus seedlings. All seven CbVd-1 variants showed infectivity. Nucleotide sequence analysis of the progeny revealed that four of the five additional mutants changed to either CbVd-1/25A or CbVd-1/25UU, while, CbVd-1/25A, CbVd-1/25UU and one of the five additional mutants (CbVd-1/I2) replicated stably. As expected, CbVd-1/25A and CbVd-1/I2 were transmitted through seeds, but CbVd-1/25UU was not. CbVd-1/25A and CbVd-1/I2 shared the same nucleotide at position 25 in loop five but are different from CbVd-1/25UU at that position. Therefore, nucleotide 25 in loop five was identified as a determinant for seed transmission of CbVd-1.