Replacement of Gln-979 by Glu in the putative replicase of tomato mosaic tobamovirus is sufficient to overcome Tm-1 resistance in tomato. It has been suggested that this change decreases the local net charge of the protein which is important in overcoming the resistance. In this study, we constructed five mutants, designated TLAsn, TLAsp, TLHis, TLLys and TLArg, in which Gln-979 was replaced by Asn, Asp, His, Lys and Arg, respectively, and analysed their abilities to overcome Tm-1 resistance. Unexpectedly, not only TLAsp, but also TLLys multiplied in tomato cells with the Tm-1 gene. TLAsn and TLArg multiplied at a reduced level. Multiplication of TLHis was virtually almost inhibited. From these results, it is unlikely that a decrease in the local net charge is the major reason for overcoming Tm-1 resistance.

Tomato mosaic virus (ToMV) causes a serious loss of yield and fruit quality in tomato crops. To control ToMV, three resistance genes, Tm-1, Tm-2, and Tm-2(2) from wild tomato species were introduced into commercial tomato cultivars. Soon after, however, single and, rarely, double-resistance-breaking virus strains for Tm-1 and Tm-2 emerged. Sequence analysis of a Tm-1/Tm-2 double-resistance-breaking virus, designated ToMV1-2, revealed 30 nucleotide substitutions compared with wild-type ToMV. Of these, six substitutions result in amino acid exchanges. Two exchanges are in the methyl transferase/helicase domain of the ToMV1-2 130/180-kDa proteins (D-1097 to V, R-1100 to Q), two exchanges are found in the 30-kDa movement protein (MP; E-52 to K, E-133 to K), and two exchanges are in the coat protein (I-22 to V, A-87 to S). Construction of chimeric full-length viral cDNA clones containing the base substitutions resulting in altered amino acids, either in the 130/180 kDa proteins or the MP, revealed that the amino acid exchanges in the 130/180-kDa proteins enable ToMV1-2 to break the Tm-1 resistance, while those in the MP enable ToMV1-2 to overcome the Tm-2 resistance. Furthermore, a novel Tm-1/Tm-2 double-resistance-breaking virus was generated by combining the Tm-1-breaking domain of ToMV1-2 and the MP of a new virus, ToMV2, containing the amino acid exchanges E-133 to K and N-135 to S. Together, the data suggest that double-resistance-breaking ToMV1-2 may have resulted from recombination between Tm-1 and Tm-2 single resistance-breaking virus strains.

The tomato Tm-1 gene confers resistance to tomato mosaic virus (ToMV). Here, we report that the extracts of Tm-1 tomato cells (GCR237) have properties that inhibit the in vitro RNA replication of WT ToMV more strongly than that of the Tm-1-resistance-breaking mutant of ToMV, LT1. We purified this inhibitory activity and identified a polypeptide of approximately 80 kDa (p80(GCR237)) using LC-tandem MS. The amino acid sequence of p80(GCR237) had no similarity to any characterized proteins. The p80(GCR237) gene cosegregated with Tm-1; transgenic expression of p80(GCR237) conferred resistance to ToMV within tomato plants; and the knockdown of p80(GCR237) sensitized Tm-1 tomato plants to ToMV, indicating that Tm-1 encodes p80(GCR237) itself. We further show that in vitro-synthesized Tm-1 (p80(GCR237)) protein binds to the replication proteins of WT ToMV and inhibits their function at a step before, but not after, the viral replication complex is formed on the membrane surfaces. Such binding was not observed for the replication proteins of LT1. These results suggest that Tm-1 (p80(GCR237)) inhibits the replication of WT ToMV RNA through binding to the replication proteins.

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.