Mapping nucleotides in the 126-kDa protein gene that control the differential symptoms induced by two strains of tobacco mosaic virus

The differential symptom determinants of the Holmes' masked (M) and U1 strains of tobacco mosaic virus previously were mapped to the 5'-coterminal open reading frame (ORF) encoding the 126-kDa protein and the N-terminal two-thirds of the 183-kDa protein. Both proteins influence viral RNA accumulation, but the function of, and impact on, symptom formation by large domains within the 126-kDa gene, which are not conserved with sequences in analogous ORFs from other related viruses, are unknown. In the current study, cDNA clones representing each strain (i.e,, M(IC)-TMV and UI-TMV) were mutated in these nonconserved domains to further define the nucleotides responsible for mosaic symptom induction on Nicotiana tabacum. Progeny virus of a mutant containing only eight nucleotide substitutions from the M(IC)-TMV sequence to the U1-TMV sequence within the 126-kDa protein ORF of M(IC)-TMV induced U1-TMV-like symptoms. Single or multiple substitutions among these eight nucleotides further defined residues critical for symptom modulation. Complementary substitutions in the M(IC)-TMV and U1-TMV sequences did not always yield progeny virus that induced complementary visual symptoms. Progeny of some mutants contained second-site spontaneous mutations at specific positions shown to influence symptom phenotype. For a subset of the stable site-directed mutants, there was no correlation between severity of systemic symptoms and chlorotic lesion size or virus accumulation in these chlorotic lesions on inoculated leaves. (C) 1996 Academic Press, Inc