Replication of Potato spindle tuber viroid in cultured cells of tobacco and Nicotiana benthamiana: the role of specific nucleotides in determining replication levels for host adaptation.

We have developed an electroporation protocol to inoculate cultured cells of tobacco and Nicotiana benthamiana with in vitro transcripts of Potato spindle tuber viroid (PSTVd) to characterize viroid structural features that determine replication efficiency at the cellular level. Both (+)- and (-)-strands of PSTVd were detected by Northern blots as early as 6 h postinoculation (h.p.i.). Accumulation of the (+)-circular PSTVd increased very rapidly starting at 24 h.p.i. and continued beyond 6 days postinoculation. Viroid accumulation in individual cells was visualized by in situ hybridization, which showed that 60-70% of the cells were infected. Previous work showed that C259 --> U substitution converted tomato isolate PSTVd(KF440-2) into a strain that is infectious on tobacco (M. Wassenegger, R. L. Spieker, S. Thalmeir, F.-U. Gast, L. Riedel, and H. L. Sänger, 1996. Virology 226, 191-197). Similarly, C259 --> U or U257 --> A substitution in the Intermediate strain (PSTVd(Int)) conferred infectivity in tobacco (Y. Zhu, Y. Qi, Y. Xun, R. Owens, and B. Ding, 2002. Plant Physiol. 130, 138-146). Our replication assays in tobacco-cultured cells demonstrated that U257 --> A and C259 --> U substitutions each enhanced PSTVd replication by 5- to 10-fold. Replacement of U257 with C, but not with G, also led to enhanced replication in tobacco cells. Replacement of C259 with nucleotide A or G did not enhance replication. Elevated accumulation of the (-)- and (+)-strands of these mutants was in part due to enhanced transcription. Interestingly, all of the nucleotide changes did not alter PSTVd replication levels in N. benthamiana cells. These results provide insights about PSTVd structures that modulate replication efficiency in adapting to a specific host.