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 ≈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.

Genetic resistance has commonly been used to protect cultivated plants from viral infection. One major class of virus resistance genes is dominant, encodes proteins that contain nucleotide-binding sites and leucine-rich repeats, and elicits a hypersensitive reaction (HR) in response to infection (1). Another is recessive and encodes mutated host proteins that are primarily required for virus multiplication (1, 2).

The tomato Tm-1 gene is a semidominant trait that inhibits tomato mosaic virus (ToMV) multiplication in protoplasts (3, 4). Tm-1 was bred into cultivated tomato (Solanum lycopersicum) from the wild tomato Solanum habrochaites PI126445 (5) and has been used widely to protect tomato plants from ToMV infection. ToMV mutants that are able to multiply in Tm-1 tomato plants have been isolated; the Tm-1 resistance-breaking property of these mutants, including the LT1 strain, was correlated with mutations in the coding region of the replication proteins, i.e., the 130-kDa (130K) protein and its translational readthrough product, the 180-kDa (180K) protein (6–8).

Several dominant genes also inhibit virus multiplication in protoplasts (1). Examples include the potato Rx gene that confers resistance to potato virus X and encodes a protein that contains nucleotide-binding sites and leucine-rich repeats (9). In Rx protoplasts, the multiplication of Rx-insensitive virus is inhibited when coinoculated with the Rx-sensitive potato virus X strain, suggesting that the HR induced by Rx-sensitive virus inhibits even the multiplication of Rx-insensitive virus (10). In contrast, coinoculation with WT ToMV in Tm-1 protoplasts has no effect on LT1 multiplication (11), suggesting that Tm-1 does not mediate an inducible nonspecific resistance like an HR. Based on these characteristics, it was proposed that Tm-1 cells constitutively express an inhibitor of ToMV RNA replication (6, 12).

In this paper, we show that Tm-1 encodes a protein that binds to and inhibits the functioning of the replication proteins of ToMV. The findings indicate that Tm-1 has characteristics different from those of previously identified virus resistance genes in plants.

Results and Discussion

An Inhibitory Factor of ToMV RNA Replication Is Present in the Tm-1 Tomato Cell Extract.

Previously, we found that an extract isolated from evacuolated tobacco BY-2 protoplasts (BYL) is capable of translating ToMV RNA into the replication proteins, and that, if ribonucleoside triphosphates are added to the mixture after translation, ToMV RNA is replicated (13). Taking into consideration the possibility that Tm-1 cells express an inhibitor of ToMV RNA replication, we investigated whether in vitro ToMV RNA replication could be inhibited by the addition of an extract from evacuolated Tm-1 protoplasts. To this end, we established a suspension-cultured cell line from GCR237 tomato plants homozygous for Tm-1. Consistent with a previous report (14), protoplasts isolated from this cell line showed resistance to WT ToMV, whereas the Tm-1 resistance-breaking ToMV mutant LT1 multiplied efficiently (Fig. 1A). In contrast, protoplasts prepared from tobacco BY-2 cells or a suspension-cultured cell line derived from a near-isogenic Tm-1-lacking tomato, GCR26 (a generous gift from Tsubasa Takahashi), allowed WT ToMV to multiply at a level comparable to LT1 (Fig. 1A).

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.

We prepared evacuolated protoplast extracts from each cell line, added them to BYL, and performed the translation-replication reaction for WT ToMV and LT1 RNA. The level of WT ToMV RNA replication, represented by the incorporation of ³²P into genomic RNA, was significantly reduced upon the addition of the GCR237 (Tm-1) cell extract, compared with the level of LT1 RNA replication (Fig. 1B). In contrast, the levels of in vitro RNA replication were similar between WT ToMV and LT1 in the absence of tomato cell extract or in the presence of GCR26 cell extract. Assuming the difference between the replication of WT ToMV and LT1 RNA represents Tm-1 activity, we fractionated the GCR237 cell extracts (Fig. 1C). The sixth and seventh fractions from Mono Q column chromatography, which was the final purification step, showed high Tm-1 activity (Fig. 1D) and contained several specific protein bands (Fig. 1E), from which we identified an ≈80-kDa protein (p80) using LC–tandem MS (arrow in Fig. 1E).

We cloned the cDNA encoding p80 from GCR237 (p80^GCR237). The p80^GCR237 polypeptide had an amino acid sequence similar to that of uncharacterized proteins found in plants, fungi, and bacteria, yet showed no resemblance to any protein with known functions, including the plant resistance genes cloned and characterized to date [see supporting information (SI) Fig. 5]. The in vitro-translated p80^GCR237 protein inhibited the replication of WT ToMV RNA, but not LT1 RNA, suggesting that p80^GCR237 is a specific inhibitor of WT ToMV RNA replication (Fig. 2). The p80^GCR237 gene contains nine exons and eight introns and encodes a 754-aa polypeptide (SI Fig. 5). We also identified a splicing variant of p80^GCR237 mRNA that lacked the second exon, which encodes amino acid residues 46–263. The translation product from this shorter splicing variant from GCR237 did not inhibit WT ToMV RNA replication in vitro (Fig. 2).

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).

p80^GCR237 Is the Tm-1 Gene Product.

We found a p80-like gene in GCR26 (p80^GCR26). The coding region of p80^GCR26 differs from that of p80^GCR237 by 49 nucleotides, 25 of which result in amino acid substitutions (SI Fig. 5), although GCR26 is near-isogenic to GCR237. The in vitro translation products of the p80^GCR26 mRNA did not affect WT ToMV replication (Fig. 2). The mRNA sequence of the p80-coding region from S. habrochaites PI126445 was identical to that from GCR237, suggesting that p80^GCR237 was derived from S. habrochaites PI126445. To determine whether p80^GCR237 cosegregates with Tm-1, we designed sequence-characterized amplified region (SCAR) markers to independently detect the p80^GCR237 and p80^GCR26 genes. All of the F₂ plants derived from the crosses between GCR237 and GCR26 carried either or both SCAR markers, suggesting that p80^GCR237 and p80^GCR26 sequences are allelic to each other. Furthermore, 25 of the 95 F₂ plants tested were homozygous for p80^GCR26, allowing for high-level multiplication of ToMV (SI Fig. 6). The remaining plants were carriers of the p80^GCR237 gene, with reduced levels of ToMV multiplication, indicating that this designed sequence-characterized amplified region marker is linked to the Tm-1 locus (SI Fig. 6).

To determine whether the expression of p80^GCR237 confers resistance to ToMV in vivo, GCR26 tomato plants were transformed with a gene cassette containing the p80^GCR237 cDNA under the control of the cauliflower mosaic virus 35S RNA promoter. As expected from the semidominance of the Tm-1 gene, the T2 plants that expressed p80^GCR237 were resistant to WT ToMV (Fig. 3A). In transgenic line no. 24, in which the levels of p80^GCR237 mRNA were comparable to endogenous p80 mRNA, the level of LT1 multiplication was similar to that in nontransgenic GCR26 or GCR237 plants (Fig. 3A). In transgenic line no. 12, which overexpressed p80^GCR237 mRNA by >20 times, LT1 multiplication was reduced to significantly low levels, whereas the multiplication of cucumber mosaic virus (CMV) was unaffected (Fig. 3A).

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.

We next knocked down p80^GCR237 expression using the virus-induced gene silencing method with the tobacco rattle virus (TRV) vector (15), followed by challenge with WT ToMV. Significant levels of WT ToMV RNA accumulated in the uninoculated upper leaves of GCR237 plants that had been inoculated with TRV carrying the p80^GCR237 sequence (Fig. 3B; also see SI Text). Thus, we conclude that Tm-1 encodes p80^GCR237. Henceforth, we refer to p80^GCR237 and p80^GCR26 as Tm-1^GCR237 and tm-1^GCR26, respectively, based on their mode of inheritance. Because the knockdown of Tm-1^GCR237 sensitized Tm-1 tomato plants to WT ToMV, it is unlikely that Tm-1^GCR237 is a dominant-negative form of an essential factor for ToMV multiplication.

Tm-1 Inhibits the in vitro ToMV RNA Replication at a Step After Translation of ToMV RNA and Before the Formation of Replication Complexes on the Membranes.

ToMV RNA replication occurs in membrane-bound complexes (16–18). When ToMV RNA is translated in membrane-depleted BYL (mdBYL), the 130K and 180K replication proteins are produced, but ToMV-related RNA is not synthesized. However, ToMV RNA replication occurs when the BYL membranes are introduced back into the ToMV RNA-translated mdBYL (19). To elucidate the mechanism by which Tm-1^GCR237 inhibits ToMV RNA replication, we synthesized the Tm-1^GCR237 protein by translation in mdBYL and added it to the uncoupled in vitro translation and replication reaction for WT ToMV or LT1 RNA at one of three times: (i) before viral RNA translation in mdBYL; (ii) after viral RNA translation in mdBYL and before the addition of BYL membranes; or (iii) after incubation with BYL membranes (Fig. 4A). There was no inhibition of WT ToMV RNA replication upon addition of the Tm-1^GCR237 protein after incubation with BYL membranes, whereas WT ToMV-specific inhibition was observed in the other cases (Fig. 4A). The levels of the 130K protein accumulation were similar for WT ToMV and LT1 in all cases (Fig. 4A Lower). Thus, it appears that Tm-1^GCR237 inhibits ToMV RNA replication by affecting the function of ToMV replication proteins before, but not after, they make contact with the membranes. It is unlikely that Tm-1^GCR237 affects the synthesis or stability of the replication proteins.

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.

Tm-1 Binds to ToMV Replication Proteins to Inhibit Their Functions.

When the above results were considered in light of the fact that Tm-1 resistance-breaking ToMV strains carry mutations in the 130K and 180K replication protein-coding region (6–8), we were prompted to determine whether Tm-1^GCR237 interacts with these proteins. FLAG-tagged Tm-1^GCR237 and tm-1^GCR26 proteins were synthesized by in vitro translation in mdBYL. The Tm-1^GCR237-FLAG protein inhibited the in vitro replication of WT ToMV RNA, as did the nontagged Tm-1^GCR237 protein (data not shown). These Tm-1^GCR237-FLAG or tm-1^GCR26-FLAG preparations were incubated with WT ToMV RNA- or LT1 RNA-translated mdBYL, after which affinity purification was performed by using anti-FLAG antibody-conjugated agarose beads. The replication proteins of WT ToMV were detected in the Tm-1^GCR237-FLAG-purified fraction, whereas those of LT1 were not (Fig. 4B). In contrast, tm-1^GCR26-FLAG did not coprecipitate the replication proteins of either WT ToMV or LT1 (Fig. 4B). These results suggest that Tm-1^GCR237 inhibits WT ToMV RNA replication by binding, whether directly or indirectly, to the 130K and 180K proteins, and the LT1 mutation reduces the affinity of the replication proteins to Tm-1^GCR237. Although we were unable to detect the interaction in vitro, the replication proteins of LT1 should still have a weak affinity for the Tm-1^GCR237 protein, because the overexpression of Tm-1^GCR237 in GCR26 plants reduced the efficiency of LT1 multiplication (Fig. 3A, line no. 12).

Concluding Remarks.

Previous attempts to identify the Tm-1 gene using map-based cloning proved unsuccessful, because the Tm-1 locus is located near the centromere of chromosome 2, where the frequency of recombination is extremely low (20–22). We identified the Tm-1 gene by purifying its inhibitory activity toward ToMV RNA replication in vitro. We also showed that Tm-1^GCR237 binds directly or indirectly to ToMV replication proteins and inhibits their function only before they form active replication complexes on membranes. The Tm-1 protein has been predicted to have the TIM barrel structure (SI Fig. 5), which is found in a wide variety of enzymes (23). Currently, there are no clues to understanding the cellular functions of the Tm-1 protein.

Although most of the dominant traits that confer resistance against viruses elicit an HR (1), several others may encode inhibitory factors of viral functions like Tm-1^GCR237. The systemic spread of tobacco etch virus is inhibited in some ecotypes of Arabidopsis thaliana without elicitation of an HR (24); the RTM1 and RTM2 genes are required for this resistance. Positional cloning revealed that these genes encode a lectin-like protein (25) and a small heat-shock-like protein (26), respectively. Although the mechanisms of resistance are not well understood, one possibility is that the RTM system inhibits viral movement functions. In cowpea, a dominant trait that inhibits the multiplication of cowpea mosaic virus in protoplasts is suggested to be associated with the inhibition of viral polyprotein processing, although the corresponding resistance gene has not been identified (27).

Essentially, the key to successful viral adaptation within its host lies not just in the use of its host factors but in evading inhibition. In the natural hosts that permit the multiplication of a virus, the virus must have already evolved to escape the inhibitory interactions between viral and host factors. Considering that most studies of virus multiplication have been performed in the natural host, the ubiquitousness of the inhibitory interactions may have been underestimated. Recent reports have shown that the overexpression of natural host-derived factors that bind to viral molecules can inhibit virus multiplication (28–30). These examples suggest that, even in the natural hosts, the inhibitory interactions between host and viral factors occur, although the inhibition is weak enough to allow virus multiplication. Future studies should explore how widely such inhibitory interactions contribute to determine the host range and multiplication efficiency of viruses.

Materials and Methods

Viruses.

WT (Tm-1-sensitive) ToMV (ToMV-L; ref. 31), the Tm-1-resistance-breaking mutant of ToMV (LT1, designated as T1 in ref. 6), and the Y strain of CMV (32) were propagated and purified, and their virion RNA was purified as described (33, 34). TLIle (35) was used in several experiments as the Tm-1-sensitive ToMV strain to assay for Tm-1 activity (Fig. 1D). Purified virions (100 μg/ml) were mechanically inoculated, and the viral RNA that accumulated in inoculated leaves at 4 days postinoculation (dpi; for ToMV in Fig. 3A) or in upper uninoculated leaves at 10 dpi (for CMV in Fig. 3A and for ToMV in Fig. 3B) were analyzed by Northern blot hybridization (34, 36).

Plants and Cells.

GCR237 and GCR26 are near-isogenic lines of tomato (Solanum lycopersicum cv. Craigella), with the exception that GCR237 is homozygous for Tm-1 and GCR26 lacks Tm-1 (37). A suspension-cultured cell line was established from GCR237 seedlings in Murashige–Skoog medium supplemented with 2 mg/liter 1-naphthaleneacetic acid/0.2 mg/liter 2,4-dichlorophenoxy acetic acid/0.2 mg/liter zeatin. The suspension-cultured cell line of GCR26 was obtained from Tsubasa Takahashi and maintained in the same medium. Nicotiana tabacum BY-2 cell lines were maintained as described (38). Protoplasts were isolated from 3-day-cultured cell suspension and inoculated with ToMV RNA by electroporation (14). Transformation of GCR26 tomato plants using Agrobacterium tumefaciens was performed as described (39). S. habrochaites PI126445 was obtained from the Germplasm Resources Information Network.

Detection of Tm-1 Activity in GCR237 Cell Extracts.

BYL (extract of evacuolated BY-2 protoplasts) was prepared as described (38). To obtain mdBYL, BYL (135 μl) was centrifuged at 30,000 × g for 15 min, and the supernatant was recovered. The pellet was resuspended in 40 μl of TR buffer [30 mM Hepes-KOH (pH 7.4)/80 mM KOAc/1.8 mM Mg(OAc)₂/2 mM DTT/one tablet of Complete EDTA-free (Roche, Indianapolis, IN) per 50 ml] to obtain P30BYL membrane suspension. Evacuolated tomato protoplast extracts were prepared by using the protocol for the tobacco BY-2 cells (38) with slight modifications. Twenty nanograms (10 fmol) of WT ToMV (or TLIle in Fig. 1D) or LT1 RNA was translated in 10 μl of mdBYL-based reaction mixture at 23°C for 1 h (38). The translation mixture (10 μl) was incubated at 23°C for 20 min with 5 μl of a test sample, followed by incubation at 15°C for 2 h with 5 μl of P30BYL and further incubation at 23°C for 1 h with 5 μl of a ribonucleoside triphosphate mixture [5 mM ATP/5 mM GTP/5 mM UTP/125 μM CTP/50 mM DTT/500 μg/ml actinomycin D/150 mM creatine phosphate/17 mM Mg(OAc)₂] containing 10 μCi [α-³²P]CTP (1 Ci = 37 GBq). The reaction was terminated by phenol extraction, and the RNA products were purified and analyzed by electrophoresis in 8 M urea–2.4% polyacrylamide gel and autoradiography. The original evacuolated tomato cell extracts (Fig. 1B) or fractions of the GCR237 cell extract (see below) for which the solvent had been exchanged to TR buffer using ultrafiltration (VIVA SPIN MWCO 10,000 PES; Sartorius, Goettingen, Germany) or mdBYL-based in vitro translation mixture of Tm-1^GCR237, Tm-1_sv^GCR237, or tm-1^GCR26 mRNA (0.5 pmol of mRNA per 5-μl reaction; Fig. 2) were used as test samples.

Fractionation of GCR237 Cell Extracts for Purification of Tm-1.

For purification of Tm-1 activity, protoplasts (not evacuolated) were isolated from ≈10 liters of 3-day-cultured GCR237 tomato cell suspension and homogenized at 4°C in buffer H [25 mM Tris·HCl (pH 8.0)/10 mM KOAc/1.8 mM Mg(OAc)₂/2 mM DTT/100 mM NaCl/one tablet of Complete EDTA-free per 50 ml] using a Dounce homogenizer. All manipulations were performed at 4°C. The homogenate was clarified by centrifugation at 800 × g for 10 min followed by centrifugation at 24,000 rpm for 30 min in a Beckman SW28Ti rotor, and the supernatant (S100) was collected (≈150 ml). An 18-ml aliquot of the S100 fraction was loaded onto a HiTrap ANX anion exchange column (column volume 10 ml; GE Healthcare, Little Chalfont, U.K.) equilibrated with buffer A [20 mM Tris·HCl (pH 8.0)/10 mM KOAc/1.8 mM Mg(OAc)₂/0.5 mM DTT/5% (vol/vol) glycerol] containing 100 mM NaCl. The column was washed with buffer A containing 250 mM NaCl, and the fraction with Tm-1 activity was eluted with buffer A containing 300 mM NaCl (10 ml). ANX column chromatography was performed eight times for one aliquot after the other, and all of the eluates were pooled (total 80 ml), from which the Tm-1-active, ANX-purified proteins were precipitated with ammonium sulfate (40% saturation). The precipitate was dissolved in 2 ml of buffer G [10 mM K-Pi buffer (pH 6.8)/10 mM KOAc/1.8 mM Mg(OAc)₂/150 mM NaCl/0.5 mM DTT/10% (vol/vol) glycerol]. A 0.3-ml aliquot of the sample was subjected to gel filtration using a Superdex 200 10/300 GL column (GE Healthcare) in buffer G, and the Tm-1-active fractions (2 ml) were collected (approximate molecular mass of 600–700 kDa). Gel-filtration chromatography was performed seven times for one aliquot after the other, and all of the Tm-1-active fractions were pooled (total 14 ml). The pooled Tm-1-active fractions were diluted with 56 ml of buffer C [10 mM K-Pi buffer (pH 6.8)/10 mM KOAc/1.8 mM Mg(OAc)₂/0.5 mM DTT/10% (vol/vol) glycerol], and loaded onto a buffer C-equilibrated hydroxyapatite column (Bio-Scale CHT2-I; Bio-Rad, Hercules, CA). The sample was eluted by using a linear gradient (12 ml) with a Pi concentration from 10 to 300 mM, and the Tm-1-active fractions (total 2 ml) were pooled. Onto four 10-ml 10–40% glycerol gradients, the pooled Tm-1-active fractions (500 μl each) were applied and subjected to centrifugation at 40,000 rpm for 16 h in a Beckman SW40Ti rotor. The active fractions (the third 2-ml fraction from the top of the gradient) were pooled (total 8 ml) and diluted with 10 ml of buffer M [20 mM Tris·HCl (pH 8.0)/10 mM KOAc/1.8 mM Mg(OAc)₂/200 mM NaCl/0.5 mM DTT/10% (vol/vol) glycerol] and loaded onto a Mono Q 5/50 GL column (GE Healthcare) equilibrated with buffer M. The Tm-1-active fraction was eluted with 10 ml of 200-350-mM NaCl in a linear gradient. Fractions (1 ml each, the first to tenth from the start of the gradient) were collected and assayed for Tm-1 activity. The sixth and seventh fractions showed high Tm-1 activity (Fig. 1D).

Identification of p80.

The proteins contained in the Mono Q fractions were concentrated by acetone precipitation, separated by SDS/PAGE (NuPAGE 4–12% Bis-Tris gel; Invitrogen, Carlsbad, CA) and visualized by silver staining (Silver Staining MS Kit; Wako Pure Chemical Industries, Osaka, Japan). The p80 band was excised from the gel. The gel piece was treated with trypsin, and the resultant peptides were subjected to LC–tandem MS analysis (LC-MS/MS; this part was performed by APLO Life Science Institute, Tokushima, Japan). A Mascot search suggested that the tryptic digest contained two polypeptides VVLSNAGAAFAGMVIGR and EVAVLAGVCATDPFR, each representing amino acid sequences derived from ESTs from grape (gi 30136942) and potato (gi 42510913), respectively. A BLAST search indicated that the ESTs had sequence similarity to distinct regions of the same A. thaliana mRNA (At5g66420). We designed PCR primers corresponding to regions where the nucleotide sequences of At5g66420 and the potato EST were identical, i.e., 5′-ccctttgctgatgcaaatgctattg-3′ and 5′-tatagggcctccatggcagagcac-3′, and then amplified and isolated the tomato cDNA fragment by RT-PCR. We used the 5′- and 3′-RACE method to determine the entire mRNA sequence of the corresponding gene (p80^GCR237) in GCR237 tomato. Evidence from LC-MS/MS analysis indicated that the deduced amino acid sequence contained three additional tryptic peptides (VGVTVVDVSTSWK, ALETFLSIANDEQNLAGVIGLGGSGGTSLLSSAFR, and VLPYHINDAEFANALVDSFLEISPK) in the tryptic digest of p80 (SI Fig. 5C).

Plasmid Construction and RNA Synthesis.

The cDNA fragments encompassing the p80-coding regions of GCR237 and GCR26 were amplified by RT-PCR by using the primers 5′-ggacgtcgctagctccattttgaaatctcgattgt-3′ and 5′-ggagctcaccatacatataggttcggacattt-3′, digested with AatII and SacI (recognition sequences in the primers underlined), and cloned into the pGEM^(R)-7Zf(+) vector (Promega, Madison, WI). For the transformation of plants, p80^GCR237 cDNA was excised by digestion with NheI (recognition sequence in the primer italicized) and SacI from the pGEM-based p80^GCR237-containing plasmid and then recloned between the XbaI and SacI sites of the binary vector pBI121 (Clontech, Mountain View, CA). For virus-induced gene silencing of p80^GCR237, the p80^GCR237 cDNA was excised by digestion with BamHI and SacI from the pGEM-based p80^GCR237-containing plasmid and recloned in the TRV-RNA2 vector pYL156 (ref. 15; provided by S. P. Dinesh-Kumar, Yale University, New Haven, CT). To construct the plasmid encoding FLAG-tagged p80^GCR237 and p80^GCR26, the termination codon of p80 was modified by PCR by using the primers 5′-ggacgtcgctagctccattttgaaatctcgattgt-3′ (AatII site underlined) and 5′-aaccaaaatccactgcatcgagCTCcataga-3′ (SacI site underlined; the last codon of p80 in upper case). The amplified fragments were digested with AatII and SacI, ligated to the SacI-XmaI fragment of pTL180SF containing the FLAG tag (19), and XmaI- and AatII-digested pGEM^(R)-7Zf(+). In vitro transcripts were synthesized from linearized plasmids by using an AmpliCap T7 High Yield Message Maker kit (Epicentre Technologies, Madison, WI). The p80 mRNA was detected by Northern blot hybridization (Fig. 3A) by using a ³²P-labeled in vitro transcript synthesized from a SmaI-linearized pGEM-based p80^GCR237-containing plasmid with SP6 RNA polymerase as a probe.

Virus-Induced Gene Silencing.

GCR237 plants were grown at 24°C under long-day (16 h of light) conditions. A. tumefaciens were cultured as described (15) and inoculated to the cotyledons of young GCR237 tomato seedlings by using sterile toothpicks. ToMV was inoculated in young true leaves 3 weeks after the initial inoculation with A. tumefaciens.

Inhibition of ToMV RNA Replication by in Vitro-Synthesized Tm-1^GCR237 Protein in the Uncoupled in vitro Translation-Replication System (Fig. 4A).

Tm-1^GCR237 mRNA (4 pmol) was translated in 40 μl of mdBYL reaction mixture at 23°C for 1 h. A mock-translated mdBYL reaction mixture was also prepared as a control. WT ToMV or LT1 RNA (20 ng) was translated in 5 μl of fresh mdBYL-based reaction mixture supplemented with 5 μl of Tm-1^GCR237- or mock-translated mdBYL mixtures at 23°C for 1 h, followed by the addition of 0.5 μl of 6 mM puromycin to terminate translation. Then, 5 μl of Tm-1^GCR237- or mock-translated mdBYL mixture was added to the mixture for a total sample volume of 15.5 μl. The mixture was incubated at 23°C for 5 min, followed by the addition of 5 μl of P30BYL membrane suspension and further incubation at 15°C for 2 h. Tm-1^GCR237- or mock-translated mdBYL mixture (5 μl) was added to each mixture for a total sample volume of 25.5 μl and incubated at 15°C for 5 min. A portion of each mixture (20 μl) was used for the RNA replication reaction (38) and the remainder for Western blotting. Tm-1^GCR237-translated mdBYL was added only once.

FLAG Immunoprecipitation Experiment (Fig. 4B).

Tm-1^GCR237-FLAG or tm-1^GCR26-FLAG mRNA (6 μg) or WT ToMV RNA or LT1 RNA (3 μg) was translated in mdBYL-based reaction mixture (80 μl) at 23°C for 1 h. Mock translation was also performed as a control. The Tm-1^GCR237-FLAG-, tm-1^GCR26-FLAG-, or mock-translated reaction mixtures (25 μl) were mixed with WT ToMV RNA-, LT1 RNA-, or mock-translated reaction mixtures (25 μl), incubated at 23°C for 20 min, and 1 μl of each mixture was analyzed as an input sample. The remainder of each sample was diluted by 10 times with TR buffer and incubated with 20 μl of anti-FLAG antibody-conjugated agarose beads (Sigma, St. Louis, MO; 50% slurry) at 4°C for 1 h. The beads were washed with 1 ml of TR buffer four times and then eluted in 40 μl of TR buffer containing 100 ng/μl FLAG peptide (Sigma). The eluted protein samples (10 μl) were analyzed by Western blotting to detect FLAG epitope and ToMV replication proteins. Anti-FLAG antibody was purchased from Sigma and the anti-130K antibody has been described (17).

Abbreviations

ToMV

Tomato mosaic virus

HR

hypersensitive reaction

BYL

evacuolated BY-2 protoplast lysate

CMV

cucumber mosaic virus

TRV

tobacco rattle virus

mdBYL

membrane-depleted BYL

dpi

days postinoculation.