COMPLEX INTERACTIONS AMONG SEVERAL NUCLEOTIDE POSITIONS DETERMINE PHENOTYPES DEFECTIVE FOR LONG-DISTANCE TRANSPORT IN THE SATELLITE RNA OF CUCUMBER MOSAIC-VIRUS

Ix-satRNA is a CMV-satRNA necrogenic on tomato that is defective for long-distance systemic movement when coinoculated in tobacco with V-TAV. To analyze the determinants for this defective phenotype, full-length cDNA clones of Ix-satRNA and of the closely related, nondefective I17N-satRNA were obtained. Infectious transcripts from these clones (Ix5-satRNA and I(17)N1-satRNA) had the same phenotypes as the original satRNAs and differed only in four positions: positions 20 (Ix5, C; I(17)N1, U), 102 (Ix5, C; I(17)N1, U), 224 (Ix5, deleted; I(17)N1, A), 327 (Ix5, G; I(17)N1, A). By site-directed mutagenesis at positions 224 and 327 and by recombination using two common restriction sites, satRNAs in which the bases at these four positions were changed from the composition at Ix5-satRNA to the composition at I(17)N1-satRNA were obtained. A comparison of the phenotypes of the 13 single, double, and triple mutants (respective to Ix5-satRNA) showed that the defective phenotype of Ix5-satRNA is determined by the nature of the four positions analyzed; mutants at any of the positions 102, 224, and 327 accumulated as efficiently as I(17)N1-satRNA in systemically infected tobacco leaves when coinoculated with V-TAV. The change C20 --> U also restored systemic movement, albeit imperfectly, giving rise to a phenotype that moved systemically less efficiently than I(17)N1-satRNA. This phenotype determined by U20 is expressed irrespective of the base at position 102, indicating an epistatic interaction between determinants 20 and 102; this interaction does not occur with position 224 or 327. That differences in the analyzed satRNAs are due to their being able, or not, to move systemically is shown by the fact that all of them (including Ix5-satRNA) accumulated to the same high level in directly inoculated leaves. The similarity in the sequences of the analyzed satRNAs and the complexity of the interactions among the effects of base changes at four positions scattered over the satRNA molecule suggest that the observed movement phenotypes may depend on conformational changes in the satRNAs. (C) 1994 Academic Press, Inc.