The Resistance Protein Tm-1 Inhibits Formation of a Tomato Mosaic Virus Replication Protein-Host Membrane Protein Complex

The Tm-1 gene of tomato confers resistance to Tomato mosaic virus (ToMV). Tm-1 encodes a protein that binds ToMV replication proteins and inhibits the RNA-dependent RNA replication of ToMV. The replication proteins of resistance-breaking mutants of ToMV do not bind Tm-1, indicating that the binding is important for inhibition. In this study, we analyzed how Tm-1 inhibits ToMV RNA replication in a cell-free system using evacuolated tobacco protoplast extracts. In this system, ToMV RNA replication is catalyzed by replication proteins bound to membranes, and the RNA polymerase activity is unaffected by treatment with 0.5 M NaCl-containing buffer and remains associated with membranes. We show that in the presence of Tm-1, negative-strand RNA synthesis is inhibited; the replication proteins associate with membranes with binding that is sensitive to 0.5 M NaCl; the viral genomic RNA used as a translation template is not protected from nuclease digestion; and host membrane proteins TOM1, TOM2A, and ARL8 are not copurified with the membrane-bound 130K replication protein. Deletion of the polymerase read-through domain or of the 3′ untranslated region (UTR) of the genome did not prevent the formation of complexes between the 130K protein and the host membrane proteins, the 0.5 M NaCl-resistant binding of the replication proteins to membranes, and the protection of the genomic RNA from nucleases. These results indicate that Tm-1 binds ToMV replication proteins to inhibit key events in replication complex formation on membranes that precede negative-strand RNA synthesis.