Viability and genetic stability of potato spindle tuber viroid mutants with indels in specific loops of the rod-like secondary structure

- Six indel mutants of PSTVd were created and characterized.
- Mutants in the lower part of the V domain were viable but genetically unstable.
- Repair of 3 nucleotides indel mutations was observed.
- A haplotype network of mutant progeny from leaves and roots is presented.

Maintenance of the rod-like structure of potato spindle tuber viroid (PSTVd), which contains over 20 loops and bulges between double-stranded helices, is important for viroid biology. To study tolerance to modifications of the stem-loop structures and PSTVd capacity for mutation repair, we have created 6 mutants carrying 3-4 nucleotides deletions or insertions at three unique restriction sites, EagI, StyI and AvaII. Differences in the infectivity of these in vitro generated PSTVd mutants can result from where the mutations map, as well as from the extent to which the secondary structure of the molecule is affected. Deletion or insertion of 4 nucleotides at the EagI and StyI sites led to loss of infectivity. However, mutants with deletion (PSTVd-Ava-del) or insertion (PSTVd-Ava-in) of 3 nucleotides (221GAC223), at the AvaII site (loop 20) were viable but not genetically stable. In all analyzed plants, reversion to the wild type PSTVd-S23 sequence was observed for the PSTVd-Ava-in mutant a few weeks after agroinfiltration. Analysis of PSTVd-Ava-del progeny allowed the identification of 10 new sequence variants carrying various modifications, some of them having retained the original three nucleotide deletion at the AvaII site. Interestingly, other variants gained three nucleotides in the deletion site but did not revert to the original wild type sequence. The genetic stability of the progeny PSTVd-Ava-del sequence variants was evaluated in tomato leaves (early infection) and in both leaves and roots (late infection), respectively.