Co-inoculation with two non-infectious cDNA copies of potato spindle tuber viroid (PSTVd) leads to the appearance of novel fully infectious variants

Potato spindle tuber viroid (PSTVd) is one of the smallest (about 360 nt) infectious plant agents. It is composed of a single-stranded circular non-coding RNA molecule. In the course of previous passage experiments with two intermediate PSTVd variants 12 and 14, three non-infectious clones (12-50, I4-37 and 14 VI-17) were found. When inoculated separately as cDNAs on tomato "Rutgers" test plants these variants did not induce any visible disease symptoms and did not produce progeny. The presence of such non-infectious variants raises several questions about their origin and biology and to answer them, mixed co-infections with cDNA copies of two non-infectious variants (12-50,14-37) were performed. PSTVd infection was observed in seven out of 30 inoculated plants. The progeny isolated from three separate plants contained novel variants, together with the parental 12 and 14 sequences. It is conceivable that the appearance of repaired PSTVd molecules, clearly capable of cell-to-cell movement leading to the systemic infection, results from recombination events. An analysis of the recombinant molecules and comparison with databases identified the specific sites responsible for the restricted infectivity of the 12-50 and 14-37 PSTVd variants. In parallel experiments in which (+) strand PSTVd infectious transcripts were used, no recombinants were observed, and the original 12-50 and 14-37 non-infectious sequences were not detected in the progeny.