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Coevolution and hierarchical interactions of Tomato mosaic virus and the resistance gene Tm-1.

Ishibashi K, Mawatari N, Miyashita S, Kishino H, Meshi T, Ishikawa M - PLoS Pathog. (2012)

Bottom Line: The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra.However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1.Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Japan. bashi@affrc.go.jp

ABSTRACT
During antagonistic coevolution between viruses and their hosts, viruses have a major advantage by evolving more rapidly. Nevertheless, viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato mosaic virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild tomato species Solanum habrochaites into the cultivated tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.

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Inhibition of in vitro RNA replication of ToMV mutants by Tm-1 variants.The genomic RNA of TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y and the mRNA for tm-1GCR26, Tm-1GCR237, or Tm-1I91T proteins were translated in mdBYL. The translation mixtures of the Tm-1 variants were mixed with the viral RNA-translated mixtures, followed by RNA replication reaction as described in the Materials and Methods section. The amount of added Tm-1 mRNA were approximately 9 (lanes 2, 5, 8), 42 (lanes 3, 6, 9), or 126 (lanes 4, 7, 10) times as much as viral RNA on a molar basis. Mock-translated mixture was added as a control (lane 1). The positions of the genomic RNA (G) and the replicative form RNA (RF) are indicated. Asterisks represent the background signals.
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ppat-1002975-g006: Inhibition of in vitro RNA replication of ToMV mutants by Tm-1 variants.The genomic RNA of TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y and the mRNA for tm-1GCR26, Tm-1GCR237, or Tm-1I91T proteins were translated in mdBYL. The translation mixtures of the Tm-1 variants were mixed with the viral RNA-translated mixtures, followed by RNA replication reaction as described in the Materials and Methods section. The amount of added Tm-1 mRNA were approximately 9 (lanes 2, 5, 8), 42 (lanes 3, 6, 9), or 126 (lanes 4, 7, 10) times as much as viral RNA on a molar basis. Mock-translated mixture was added as a control (lane 1). The positions of the genomic RNA (G) and the replicative form RNA (RF) are indicated. Asterisks represent the background signals.

Mentions: We further analyzed the inhibitory effect of Tm-1 proteins on ToMV RNA replication using an in vitro ToMV RNA replication system [37], [38]. Briefly, ToMV RNA was translated in mdBYL. Tm-1 proteins were separately synthesized by in vitro translation with mdBYL, mixed with ToMV RNA-translated mdBYL, and incubated with BYL membranes and α-32P-labeled ribonucleoside triphosphates, followed by analysis of 32P-labeled RNA. Using this assay, we observed inhibition of RNA replication of the ToMV derivatives in a pattern consistent with the results of the protoplast experiment (Figure 6). Note that the inhibitory effect of tm-1GCR26 to TLIle RNA replication in vitro is weak [30]. Moreover, the in vitro experiment showed that (i) the inhibitory effect of Tm-1 variants is dose-dependent, (ii) TLIle is more sensitive to Tm-1GCR237 than ToMV-L (Figure 6, lanes 5–7), (iii) ToMV-L is more sensitive to Tm-1I91T than LT1 (Figure 6, lanes 8–10), (iv) Tm-1GCR237 and Tm-1I91T inhibit TLIle RNA replication more strongly than tm-1GCR26, and (v) Tm-1I91T inhibits ToMV-L RNA replication more strongly than Tm-1GCR237 (Figure 6). These results suggest that I91T substitution in the Tm-1 protein strengthens its inhibitory activity enough to inhibit LT1 RNA replication, thus extending the antiviral spectrum.


Coevolution and hierarchical interactions of Tomato mosaic virus and the resistance gene Tm-1.

Ishibashi K, Mawatari N, Miyashita S, Kishino H, Meshi T, Ishikawa M - PLoS Pathog. (2012)

Inhibition of in vitro RNA replication of ToMV mutants by Tm-1 variants.The genomic RNA of TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y and the mRNA for tm-1GCR26, Tm-1GCR237, or Tm-1I91T proteins were translated in mdBYL. The translation mixtures of the Tm-1 variants were mixed with the viral RNA-translated mixtures, followed by RNA replication reaction as described in the Materials and Methods section. The amount of added Tm-1 mRNA were approximately 9 (lanes 2, 5, 8), 42 (lanes 3, 6, 9), or 126 (lanes 4, 7, 10) times as much as viral RNA on a molar basis. Mock-translated mixture was added as a control (lane 1). The positions of the genomic RNA (G) and the replicative form RNA (RF) are indicated. Asterisks represent the background signals.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475678&req=5

ppat-1002975-g006: Inhibition of in vitro RNA replication of ToMV mutants by Tm-1 variants.The genomic RNA of TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y and the mRNA for tm-1GCR26, Tm-1GCR237, or Tm-1I91T proteins were translated in mdBYL. The translation mixtures of the Tm-1 variants were mixed with the viral RNA-translated mixtures, followed by RNA replication reaction as described in the Materials and Methods section. The amount of added Tm-1 mRNA were approximately 9 (lanes 2, 5, 8), 42 (lanes 3, 6, 9), or 126 (lanes 4, 7, 10) times as much as viral RNA on a molar basis. Mock-translated mixture was added as a control (lane 1). The positions of the genomic RNA (G) and the replicative form RNA (RF) are indicated. Asterisks represent the background signals.
Mentions: We further analyzed the inhibitory effect of Tm-1 proteins on ToMV RNA replication using an in vitro ToMV RNA replication system [37], [38]. Briefly, ToMV RNA was translated in mdBYL. Tm-1 proteins were separately synthesized by in vitro translation with mdBYL, mixed with ToMV RNA-translated mdBYL, and incubated with BYL membranes and α-32P-labeled ribonucleoside triphosphates, followed by analysis of 32P-labeled RNA. Using this assay, we observed inhibition of RNA replication of the ToMV derivatives in a pattern consistent with the results of the protoplast experiment (Figure 6). Note that the inhibitory effect of tm-1GCR26 to TLIle RNA replication in vitro is weak [30]. Moreover, the in vitro experiment showed that (i) the inhibitory effect of Tm-1 variants is dose-dependent, (ii) TLIle is more sensitive to Tm-1GCR237 than ToMV-L (Figure 6, lanes 5–7), (iii) ToMV-L is more sensitive to Tm-1I91T than LT1 (Figure 6, lanes 8–10), (iv) Tm-1GCR237 and Tm-1I91T inhibit TLIle RNA replication more strongly than tm-1GCR26, and (v) Tm-1I91T inhibits ToMV-L RNA replication more strongly than Tm-1GCR237 (Figure 6). These results suggest that I91T substitution in the Tm-1 protein strengthens its inhibitory activity enough to inhibit LT1 RNA replication, thus extending the antiviral spectrum.

Bottom Line: The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra.However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1.Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.

View Article: PubMed Central - PubMed

Affiliation: Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Japan. bashi@affrc.go.jp

ABSTRACT
During antagonistic coevolution between viruses and their hosts, viruses have a major advantage by evolving more rapidly. Nevertheless, viruses and their hosts coexist and have coevolved, although the processes remain largely unknown. We previously identified Tm-1 that confers resistance to Tomato mosaic virus (ToMV), and revealed that it encodes a protein that binds ToMV replication proteins and inhibits RNA replication. Tm-1 was introgressed from a wild tomato species Solanum habrochaites into the cultivated tomato species Solanum lycopersicum. In this study, we analyzed Tm-1 alleles in S. habrochaites. Although most part of this gene was under purifying selection, a cluster of nonsynonymous substitutions in a small region important for inhibitory activity was identified, suggesting that the region is under positive selection. We then examined the resistance of S. habrochaites plants to ToMV. Approximately 60% of 149 individuals from 24 accessions were resistant to ToMV, while the others accumulated detectable levels of coat protein after inoculation. Unexpectedly, many S. habrochaites plants were observed in which even multiplication of the Tm-1-resistance-breaking ToMV mutant LT1 was inhibited. An amino acid change in the positively selected region of the Tm-1 protein was responsible for the inhibition of LT1 multiplication. This amino acid change allowed Tm-1 to bind LT1 replication proteins without losing the ability to bind replication proteins of wild-type ToMV. The antiviral spectra and biochemical properties suggest that Tm-1 has evolved by changing the strengths of its inhibitory activity rather than diversifying the recognition spectra. In the LT1-resistant S. habrochaites plants inoculated with LT1, mutant viruses emerged whose multiplication was not inhibited by the Tm-1 allele that confers resistance to LT1. However, the resistance-breaking mutants were less competitive than the parental strains in the absence of Tm-1. Based on these results, we discuss possible coevolutionary processes of ToMV and Tm-1.

Show MeSH
Related in: MedlinePlus