Site-directed mutagenesis of a plant viral satellite RNA changes its phenotype from ameliorative to necrogenic.

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Abstract
Comparison of the nucleotide sequences of cucumber mosaic virus (CMV) satellite RNAs, which induce necrosis on tomatoes, reveals a highly conserved region within their 3' halves. The sequence of WL1 satellite (WL1-sat) RNA, which attenuates CMV symptoms on tomatoes, differs from all necrogenic satellite RNAs at three nucleotide positions within this conserved region. These nucleotides were progressively mutated to determine what sequence is required for the induction of necrosis in tomatoes. Infectious transcripts from a cDNA clone of WL1-sat RNA, and its mutated derivatives, were assayed on tomato. Three of the four mutant satellite RNAs, in which two of the three nucleotides correspond to those present in necrogenic satellite RNAs, and the rest of the molecule corresponded to WL1-sat RNA, attenuated CMV symptoms on tomatoes, and were phenotypically identical to the parental WL1-sat RNA. One other mutant satellite RNA, in which all three mutated nucleotides corresponded to the sequence of necrogenic satellite RNAs and the rest of the molecule corresponded to WL1-sat RNA, induced a lethal necrosis on tomatoes. Necrosis was also observed when the same mutant satellite RNA was associated with a second CMV strain as helper virus. These results indicate that a single nucleotide change at any of the three nucleotides examined distinguishes necrogenic from nonnecrogenic satellite RNAs. This necrosis was similar to that induced by a naturally necrogenic satellite RNA. The various mutations did not modify the effect of WL1-sat RNA on the symptoms induced by CMV on tobacco or squash.