Resistance to tobacco mosaic virus induced by the 54-kDa gene sequence requires expression of the 54-kDa protein.
Tobacco plants transformed with the sequence encoding the 54-kDa putative replicase protein of tobacco mosaic virus were resistant to systemic virus disease (D. B. Golemboski, G. P. Lomonossoff, and M. Zaitlin, Proc. Natl. Acad. Sci. USA 87:6311-6315, 1990). Resistance was due to a marked suppression of virus replication at the site of inoculation (J. P. Carr and M. Zaitlin, Mol. Plant-Microbe Interact. 4:579-585, 1991). Although RNA transcripts encoding the 54-kDa protein were present in resistant plants, the 54-kDa protein itself was not observed in vivo. We wished to assess the relative importance of the 54-kDa protein versus its RNA in mediating resistance. Further attempts to detect the 54-kDa protein in plant tissues were unsuccessful; therefore, an indirect approach was taken using a protoplast-based transient gene expression system. Electroporation of protoplasts with plasmids capable of expressing the wild-type 54-kDa protein gene sequence or a mutant lacking the first AUG initiation codon of the 54-kDa open reading frame and encoding a slightly truncated protein reduced virus replication in protoplasts. In contrast, a frameshift mutant that was capable of directing synthesis of a protein only 20% the size of the 54-kDa protein, did not produce resistance in protoplasts. These results show that expression of the 54-kDa protein gene sequence at the RNA level alone is insufficient for resistance, and they implicate the 54-kDa protein itself in mediating this resistance phenomenon.
D000595:Amino Acid Sequence; D001483:Base Sequence; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D005814:Genes, Viral; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D012329:RNA, Bacterial / Q000235:genetics; D014026:Nicotiana / Q000235:genetics / Q000382:microbiology; D014027:Tobacco Mosaic Virus / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D014170:Transformation, Genetic; D014764:Viral Proteins / Q000235:genetics*; D014774:Virulence / Q000235:genetics; D014779:Virus Replication / Q000235:genetics
D012329:RNA, Bacterial; D014764:Viral Proteins; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase