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. 2009 May 26;106(21):8778-83.
doi: 10.1073/pnas.0809105106. Epub 2009 May 7.

An inhibitory interaction between viral and cellular proteins underlies the resistance of tomato to nonadapted tobamoviruses

Kazuhiro Ishibashi  1 , Satoshi Naito, Tetsuo Meshi, Masayuki Ishikawa
Affiliations

An inhibitory interaction between viral and cellular proteins underlies the resistance of tomato to nonadapted tobamoviruses

Kazuhiro Ishibashi et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Transgenic tobacco plants that express either tm-1 or Tm-1 show resistance to tobamoviruses that cannot infect tomato. Nontransgenic tobacco (Samsun) (A) and tomato (GCR26) (B) plants and the T2 plants of transgenic tobacco expressing tm-1 (C) or Tm-1 (D) were inoculated with tobamoviruses indicated at the top. At 4 days postinoculation (dpi) (A, C, and D) or 6 dpi (B), whole leaf extracts were prepared from inoculated leaves and analyzed by SDS/PAGE and Coomassie blue staining. The lanes represent single individual plants. In C and D, the plants were derived from single tm-1-expressing and Tm-1-expressing T1 plants, respectively. Consistent results were obtained from another transgenic line for C and D each. CP: coat protein.
Fig. 2.
Fig. 2.
The tm-1/Tm-1 proteins bind to the replication proteins of tobamoviruses that cannot multiply in tm-1/Tm-1-expressing transgenic tobacco plants and inhibit their RNA replication in vitro. (A) Inhibition of in vitro replication of tobamovirus RNAs by tm-1 or Tm-1. In vitro RNA replication reaction of tobamovirus RNA was performed in the presence of tm-1 or Tm-1 as described in Materials and Methods. 32P-labeled RNA products were separated by PAGE followed by autoradiography. The positions of genomic RNA (G) and replicative form RNA (RF) are indicated. Asterisks show the positions of tm-1 or Tm-1 mRNAs that were labeled with 32P by an unknown mechanism independent of viral factors. We confirmed that similar amounts of the tm-1 and Tm-1 proteins were added to the reaction mixtures (data not shown). (B) Interaction between tobamovirus replication proteins and tm-1 or Tm-1. The genomic RNA of the tobamoviruses and mRNA for tm-1-FLAG and Tm-1-FLAG were translated in mdBYL in the presence of [35S]methionine. Viral RNA-translated mixtures were mixed with mock- (for the panel indicated by a hyphen), tm-1-FLAG-, or Tm-1-FLAG-translated mixtures followed by purification with anti-FLAG antibody-conjugated agarose beads. Protein samples before (Input) or after (IP: anti-FLAG) FLAG purification were analyzed by SDS/PAGE and autoradiography. The positions of the replication proteins of tobamoviruses (Rep.), tm-1-FLAG, and Tm-1-FLAG are indicated.
Fig. 3.
Fig. 3.
Analyses of a TMGMV mutant that escaped from the tm-1-mediated inhibition of viral RNA replication. (A) Multiplication of TMGMV-T894M,F970Y in tobacco leaves. Nontransgenic tobacco (WT) and the T2 plants of a tm-1-expressing transgenic tobacco plant were inoculated with TMGMV-T894M,F970Y, and CP accumulation was analyzed as in Fig. 1. (B) The effect of adding tm-1 protein on in vitro replication of TMGMV-T894M,F970Y RNA. The analysis was performed as in Fig. 2A. (C) The ability of TMGMV-T894M,F970Y replication proteins to bind tm-1. The analysis was performed as in Fig. 2B. For lanes indicated by a hyphen, a mock-translated mixture was added instead of a tm-1-FLAG-translated mixture. (D) Multiplication of TMGMV-T894M,F970Y in protoplasts. Protoplasts from suspension-cultured tobacco (BY-2) or tomato (GCR26) cells were inoculated with WT ToMV, WT TMGMV, or TMGMV-T894M,F970Y RNA or mock-inoculated and cultured for 22 h. CP accumulation was analyzed by SDS/PAGE and Coomassie blue staining. (E) Multiplication of TMGMV-T894M,F970Y in inoculated leaves of tomato plants. The accumulation of WT TMGMV and TMGMV-T894M,F970Y CPs in inoculated leaves of tomato plants (GCR26) was analyzed as in Fig. 1. (F) Photographs of WT TMGMV-inoculated (right) and TMGMV-T894M,F970Y-inoculated (left) tomato plants at 16 dpi. Inset: close-up photograph of a TMGMV-T894M,F970Y-inoculated tomato plant showing stem necrosis. (Scale bars, 3 cm.) (G) Accumulation of TMGMV-T894M,F970Y RNA in the upper uninoculated leaves of tomato plants. Total RNA was extracted from the upper uninoculated leaves of tomato plants at 8 dpi. TMGMV-related RNAs (G, genomic RNA; CPsg, subgenomic RNA for the coat protein) and 28S ribosomal RNA (loading control) were detected by Northern blot hybridization and methylene blue staining, respectively.
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