Abstract
In Nicotiana sylvestris, the N′ gene confers hypersensitive resistance to some strains of tobacco mosaic virus (TMV) but not to the common strain. TMV sequences responsible for inducing local lesion formation in this host were identified by using cDNA clones to construct genomic recombinants between the common strain genome and a local-lesion-inducing mutant. To assay for sequences conferring the mutant phenotype, in vitro transcripts of recombinants were inoculated onto leaves of N. sylvestris and observed for the formation of either local lesions or a systemic infection. Sequences from the mutant that converted the hybrid genome to the mutant phenotype were located between nucleotides 5972 and 6206. Sequence analysis of this region revealed point mutations in the mutant at nucleotides 6157 (cytosine to uracil) and 6199 (adenine to guanine). The mutation at 6157 changes the capsid protein gene to specify phenylalanine rather than serine at position 148; nucleotide 6199 occurs in the 3′ nontranslated region. When each point mutation was individually substituted into the wild-type background, transcripts containing only the alteration at 6157 produced local lesions on N. sylvestris, whereas transcripts containing only the alteration at 6199 produced systemic mosaic symptoms. The frequency of mutation was examined by partially sequencing virion RNA from six additional independent local-lesion mutants. Five mutants had the same alteration at 6157 as the original mutant and none had the alteration at 6199. This work demonstrates that the capsid protein gene of TMV is multifunctional, both encoding the virion structural protein and mediating the outcome of infection in N. sylvestris.
Keywords: in vitro transcription, recombinant DNA, genetic mapping, N′ gene
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