| Viroids are the smallest known agents of infectious disease--small, highly structured, single-stranded RNA molecules which lack both a protein capsid and detectable mRNA activity, yet are able to replicate autonomously. Four related molecular features of potato spindle tuber viroid (PSTVd) replication; temperature sensitivity, the possible involvement of circular ({dollar}-{dollar})PSTVd RNA, the stability of "lethal" mutants, and the ability of such mutants to recombine in vivo, were examined. Using the polymerase chain reaction and an overlap extension strategy, seven mutant PSTVd cDNAs were produced, and the properties of their respective RNA transcripts were studied. Quantitative bioassays of selected mutants showed a correlation between structural stability and rate of replication in vivo. Higher temperatures were able to partially overcome the inhibition of replication associated with a more stable secondary structure, but numerous spontaneous mutations appeared in the progeny, and no well-defined temperature-sensitive mutants were identified. Depending on the exact nature and location of the mutation, selection of the stable mutants may occur at the level of either the (+) or the ({dollar}-{dollar}) strand.; Although PSTVd-infected tissue contains multimeric linear ({dollar}-{dollar})RNAs, previous attempts to initiate infection with multimeric ({dollar}-{dollar})RNA transcripts have failed. Transgenic Nicotiana benthamiana plants which constitutively express either (+) or ({dollar}-{dollar})PSTVd RNA were shown to contain high levels of (+)PSTVd progeny. Only the plants expressing ({dollar}-{dollar})PSTVd RNA contained monomeric linear and circular forms of this RNA. Although not a normal intermediate in PSTVd replication, circularized ({dollar}-{dollar})PSTVd is able to initiate viroid replication.; Introduction of three nucleotide substitutions into the left terminal loop of PSTVd abolished infectivity. Northern hybridization and RT-PCR were used to analyze RNA transcripts isolated from transgenic plants expressing this mutant RNA. Most plants contained only non-replicating monomeric linear RNAs, but sequence analysis revealed that a spontaneous mutation at one of these three positions allowed the mutant to resume replication. The noninfectious nature of this mutant seems to be due to its instability in vivo.; Viroid recombination was studied by challenge-inoculating transgenic plants expressing a non-replicating PSTVd RNA with several different PSTVd mutants. No evidence of viroid recombination in vivo, was obtained. |
