Nicotiana tabacum cv. Xanthi nn plants were transformed with nucleotides 3472-4916 of tobacco mosaic virus (TMV) strain U1. This sequence contains all but the three 3' terminal nucleotides ofthe TMV 54-kDa gene, which encodes a putative component of the replicase complex. These plants were resistant to infection when challenged with either TMV U1 virions or TMV U1 RNA at concentrations of up to 500 ,ug/ml or 300 ,ug/ml, respectively, the highest concentrations tested. Resistance was also exhibited when plants were inoculated at 100 ,g/ml with the closely related TMV mutant YSI/1 but was not shown in plants challenged at the same concentrations with the more distantly related TMV strains U2 or L or cucumber mosaic virus. Although the copy number of the 54-kDa gene sequence varied in individual transformants from 1 to S5, the level of resistance in plants was not dependent on the number of copies of the 54-kDa gene sequence integrated. The transformed plants accumulated a 54-kDa gene sequencespecific RNA transcript of the expected size, but no protein product was detected.The organization of the tobacco mosaic virus (TMV) genome is fairly well understood. However, one aspect of the genome strategy that has not been fully elucidated is the exact nature of the replicase enzyme responsible for the synthesis of the genomic and subgenomic RNAs. It is generally accepted that the virus codes for four proteins, two of which are translated from the genomic RNA and two from individual subgenomic RNAs (see review in ref. 1). The 5' proximal region of the genomic RNA encodes two coinitiated proteins, the 126-kDa and 183-kDa proteins, considered to be components of the replicase (2). The 183-kDa protein is generated by a readthrough of the UAG stop codon of the 126-kDa protein. (8), although on occasion we have seen inconclusive faint bands in the region of the gel where such a protein would be expected. This is in spite of the fact that our antiserum is capable of precipitating the 54-kDa protein generated from in vitro translation products of either TMV RNA or T7 transcripts of the 54-kDa gene.In an effort to attribute a function to the 54-kDa protein, we have transformed tobacco with the coding sequence for this nonstructural viral protein. Unexpectedly, these plants showed a complete resistance to replication of the Ul strain of TMV from which the 54-kDa gene sequence was derived. These findings and their implications for the production of virus-resistant plants are discussed. MATERIALS AND METHODSPlants and Virus Strains. TMV strain U1 was purified from infected Nicotiana tabacum cv. Turkish Samsun as described by Asselin and Zaitlin (9). Unless otherwise indicated, subsequent use of the term TMV implies strain U1. Before use, preparations of TMV strain U2 (10) and L (11), which had been stored as laboratory stocks at 40C, were pelleted at 75,000 X g for 25 min and resuspended in water to enrich for full-length virions. A preparation of freshly prepared cucumber mosaic virus (CMV) strain Fny was a gift of M. J. R...

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