An inhibitor of viral RNA replication is encoded by a plant resistance gene

Abstract

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.

Fig. 1.

Detection and purification of the activity to inhibit ToMV RNA replication in vitro from evacuolated Tm-1 tomato protoplast extracts. (A) ToMV multiplication in protoplasts. Protoplasts from suspension-cultured Tm-1 tomato (GCR237), ToMV-susceptible tomato (GCR26), and tobacco (BY-2) cells were mock-inoculated or inoculated with WT (wt) ToMV (Tm-1-sensitive strain) RNA or LT1 (Tm-1-resistance-breaking strain) RNA by electroporation and cultured for 24 h. ToMV genomic RNA (G) and 28S rRNA (loading control) were detected by using Northern blot hybridization and methylene blue staining, respectively. (B) The effect of adding tomato cell extracts on ToMV RNA replication. The evacuolated GCR237 or GCR26 protoplast extracts were added to the uncoupled translation and replication reactions for WT ToMV RNA or LT1 RNA, as described in Materials and Methods. ³²P-labeled RNA products were separated by PAGE followed by autoradiography. The positions of ToMV genomic RNA (G) and the replicative-form RNA (RF) are indicated. Asterisks represent background signals. (C) Purification of Tm-1 activity. (D) Detection of Tm-1 activity in the Mono Q fractions. The Mono Q fractions were added to the uncoupled translation and replication reactions for WT ToMV RNA or LT1 RNA as described in Materials and Methods. ³²P-labeled RNA products were analyzed as in B (see above for abbreviations). (E) Proteins contained in the Mono Q fractions. The Mono Q fractions were subjected to SDS/PAGE, followed by silver staining. Arrow indicates the p80 band.

Fig. 2.

In vitro-synthesized p80^GCR237 inhibits ToMV RNA replication. Messenger RNAs for p80^GCR237, p80_sv^GCR237 (the product of the splicing variant mRNA lacking the second exon), and p80^GCR26 were translated by using mdBYL at 23°C for 1 h (0.5 pmol of mRNA/5 μl of translation reaction mixture). The translation mixtures containing p80-related proteins (or a mock-translated mixture as a control) were added to the uncoupled translation and replication reactions for WT (wt) ToMV RNA or LT1 RNA, as described in Materials and Methods. ³²P-labeled RNA products were analyzed as in Fig. 1B; see Fig. 1B legend for abbreviations. In this condition, the p80^GCR237 protein is produced ≈20 times as much as the 130K protein on a molar basis (data not shown).

Fig. 3.

The p80^GCR237 gene confers ToMV resistance to tomato plants. (A) Inhibition of ToMV multiplication in GCR26 tomato plants expressing p80^GCR237. The T2 plants of transgenic line nos. 24 or 12, or nontransgenic GCR26 or GCR237 plants were mechanically inoculated with WT (wt) ToMV, LT1, or CMV. GCR26 plants were also mock-inoculated. Total RNA was prepared separately from the ToMV-inoculated leaves at 4 dpi, from the uninoculated upper leaves of CMV-inoculated plants at 10 dpi, and from healthy plants (for p80 mRNA detection). Equal amounts of RNA from three individuals were mixed for each plant genotype–virus combination, and the mixture was analyzed by Northern blotting and hybridization. (B) Multiplication of WT ToMV in p80-silenced Tm-1 tomato. GCR237 plants were inoculated with A. tumefaciens strains harboring the pTRV vector with (pTRV-p80) or without (pTRV) the fragment of the p80^GCR237 cDNA. After 3 weeks, WT ToMV or LT1 were challenge-inoculated within young leaves. Total RNA was extracted from the upper uninoculated leaves 10 days after challenge inoculation. ToMV RNA and 28S rRNA were detected as in Fig. 1A. Each lane represents a sample from one plant. Typically, the accumulation of WT ToMV genomic RNA was <1% of that of LT1 genomic RNA in pTRV-inoculated plants. High levels of WT ToMV RNA accumulation comparable to those for LT1 RNA were observed in 32 of 47 pTRV-p80-inoculated GCR237 plants, but were not observed in any of the 22 pTRV-inoculated GCR237 plants.

Fig. 4.

Tm-1^GCR237 (p80^GCR237) binds to ToMV replication proteins and inhibits ToMV RNA synthesis if added before the formation of the replication complex on the membrane surface. (A) The effect of the timing of the addition of Tm-1^GCR237 on ToMV RNA replication. Tm-1^GCR237 was synthesized in mdBYL and added to the uncoupled translation and replication reaction mixture for WT (wt) ToMV or LT1 RNA (i) before the translation in mdBYL, (ii) after termination of translation with puromycin and before the addition of membranes (BYL P30), or (iii) after incubation with membranes and before addition of rNTPs. As a control, mock-translated mdBYL was added. ³²P-labeled RNA products were analyzed as in Fig. 1B; see Fig. 1B legend for abbreviations. The 130K protein was detected by Western blotting after incubation with the membranes. (B) Interaction between Tm-1^GCR237 and ToMV replication proteins. FLAG-tagged Tm-1^GCR237 or FLAG-tagged tm-1^GCR26 proteins and the replication proteins of WT ToMV or LT1 were synthesized by translation in mdBYL, mixed, and purified by using anti-FLAG antibody-conjugated agarose beads. Protein samples before (input) or after (FLAG-IP) FLAG purification were analyzed by Western blotting using anti-130K protein or anti-FLAG antibodies. Positions for the 130K, 180K, and FLAG-tagged Tm-1^GCR237 (tm-1^GCR26) protein bands are indicated. The asterisk represents the background signal derived from a host (BYL) protein.