Genetic variability of potato spindle tuber viroid RNA replicon

The genetic continuity of the potato spindle tuber viroid (PSTVd) genome was analysed after infection of tomato plants with cloned cDNAs of parental strains. During the six weeks of the experiment, several new sequence variants appeared. The sequence variants detected in the progeny population induced sequence-specific disease symptoms. The PSTVd genome therefore follows the pattern expected for typical pseudo-strains propagating in plants as a population of similar sequences. Assessing further the replicon continuity, a PSTVd cDNA mutant with a deletion in the central conserved region was constructed and proven to be non-infectious. Surprisingly, in a sub-population of potato transformants expressing the same deleted PSTVd RNA an infectious viroid was detected. This suggests specific transcript conversion followed by recovery of the full-length pathogen genome

Use of randomly mutagenized genomic cDNA banks of Potato spindle tuber viroid to screen for viable versions of the viroid genome

In an effort to study sequence space allowing the recovery of viable Potato spindle tuber viroid (PSTVd) variants we have developed an in vivo selection (Selex) method to produce and bulk-inoculate by agroinfiltration large PSTVd cDNA banks in which a short stretch of the genome is mutagenized to saturation. This technique was applied to two highly conserved six nucleotide-long regions of the PSTVd genome, the left terminal loop (TL bank) and part of the polypurine stretch in the upper strand of pre-melting loop 1 (PM1 bank). In each case, PSTVd accumulation was observed in a large fraction of bank-inoculated tomato plants. Characterization of the progeny molecules showed the recovery of the parental PSTVd sequence in 89% (TL bank) and 18% (PM1 bank) of the analyzed plants. In addition, viable and genetically stable PSTVd variants with mutations outside of the known natural variability of PSTVd were recovered in both cases, although at different rates. In the case of the TL region, mutations were recovered at five of the six mutagenized positions (357, 358, 359, 1 and 3 of the genome) while for the PM1 regions mutations were recovered at all six targeted positions (50-55), providing significant new insight on the plasticity of the PSTVd genome

Saturation mutagenesis of short regions of the P and TL domains of the PSTVd molecule yields novel viable mutants

International audienc

Using of a random-mutated genomic bank of the Potato spindle tuber viroid to screen viable versions of viroid genome

International audienc

Restoration of secondary hairpin II is associated with restoration of infectivity of a non-viable recombinant viroid

International audienc

The genetic stability of potato spindle tuber viroid (PSTVd) molecular variants

International audienc

Construction of random-mutated genomic banks of Potato spindle tuber viroid (PSTVd)

National audienc

Construction of two genomic banks of Potato spindle tuber viroid carrying random mutated short region of the P and TL domain

International audienc

Expression of RNA in transgenic plants carrying a mutated cDNA copy of the PSTVd genome

National audienc