Effects of Sequence Variation on Symptom Induction by Citrus Viroid III

Abstract

Interest in the use of viroids as potential citrus dwarfing agents goes back more than 30 yr, and infection of Valencia sweet orange scions growing on trifoliate orange rootstocks by citrus viroids Ia, IIa, or IIIb was recently shown to result in a dwarfed phenotype and enhanced fruit production. Viroids exist in vivo as populations of closely related sequence vari- ants, and we are interested in developing variants of citrus viroid III as dwarfing agents specifi- cally adapted for use with certain citrus cultivars. Toward that goal, we have constructed an expression cassette that releases a precisely full length, potentially infectious CVd-III RNA of known sequence from an RNA transcript synthesized in vitro . The viroid RNA is released through the action of a ribozyme sequence located at the 3' terminus of the transcript. RNAs derived from wild-type CVd-IIIb, as well as four other naturally-occurring sequence variants, were infectious when slash-inoculated into Etrog citron, and most of the sequence changes present in these vari- ants were stably maintained in their respective progeny. In particular, two changes in the puta- tive pathogenicity domain of CVd-III were associated with a marked reduction in symptom expression in Etrog citron.