Any individual virus can infect only a limited range of hosts, and most plant species are "nonhosts" to a given virus; i.e., all members of the species are insusceptible to the virus. In nonhost plants, the factors that control virus resistance are not genetically tractable, and how the host range of a virus is determined remains poorly understood. Tomato (Solanum lycopersicum) is a nonhost species for Tobacco mild green mosaic virus (TMGMV) and Pepper mild mottle virus (PMMoV), members of the genus Tobamovirus. Previously, we identified Tm-1, a resistance gene of tomato to another tobamovirus, Tomato mosaic virus (ToMV), and found that Tm-1 binds to ToMV replication proteins to inhibit RNA replication. Tm-1 is derived from a wild tomato species, S. habrochaites, and ToMV-susceptible tomato cultivars have the allelic gene tm-1. The tm-1 protein can neither bind to ToMV replication proteins nor inhibit ToMV multiplication. Here, we show that transgenic tobacco plants expressing tm-1 exhibit resistance to TMGMV and PMMoV. The tm-1 protein bound to the replication proteins of TMGMV and PMMoV and inhibited their RNA replication in vitro. In one of the tm-1-expressing tobacco plants, a tm-1-insensitive TMGMV mutant emerged. In tomato protoplasts, this mutant TMGMV multiplied as efficiently as ToMV. However, in tomato plants, the mutant TMGMV multiplied with lower efficiency compared to ToMV and caused systemic necrosis. These results suggest that an inhibitory interaction between the replication proteins and tm-1 underlies a multilayered resistance mechanism to TMGMV in tomato.