Agrobacterium-mediated inoculation of PSTVd cDNAs onto tomato reveals the biological effect of apparently lethal mutations.

Potato spindle tuber viroid (PSTVd) mutants which contain alterations in the terminal loops of the rod-like native structure have previously been reported from our laboratory. PSTVd-P contains mutations at positions 2, 4, and 6 in the left terminal loop; PSTVd-R+, a sequence permutation of PSTVd-R, contains the same mutations at positions 177 and 178 in the right terminal loop as PSTVd-R and contains in addition a 1-nucleotide G insertion at position 176. PSTVd-P, PSTVd-R, and PSTV-R+ were noninfectious when either cDNA or SP6-generated RNA transcripts were used as inoculum onto tomato cotyledons. In the current study, mutant and wild-type PSTVd constructs were mobilized into Agrobacterium tumefaciens and used for stem inoculation of tomato plants. Agrobacterium-mediated inoculation of the mutant and wild-type constructs has confirmed the inability of the PSTVd-P mutant to establish an infection. The PSTV-R+ mutant and/or sequence variants derived in vivo can establish an infection, although PSTVd-R+ progeny and replicative intermediates appear to be primarily restricted to the gall and root tissues of the plant and only occasionally are progeny detectable in the newly developing leaves. The reduced level of viroid accumulation from the PSTVd-R+ mutant appears to be consistent with the mutant viroid replicating/accumulating only in a limited number of cells or cell types. The mutations in the right terminal loop may alter interactions with specific host components and thereby disrupt the normal pattern of intercellular transport of the viroid or limit its replication to a cell type but not abolish replication per se.