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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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Tm-1I91T binds LT1 replication proteins, but not LT1E979K or LT1D1097Y.The genomic RNA of TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y were translated in mdBYL; mixed with mdBYL in which tm-1GCR26-FLAG, Tm-1GCR237-FLAG, or Tm-1I91T-FLAG mRNA were translated; and immunoprecipitated using anti-FLAG antibody-conjugated agarose. Mock-translation was performed as controls and indicated as no viral RNA or no FLAG RNA. Protein samples before (Input) or after (IP: anti-FLAG) FLAG purification were analyzed by Western blotting using anti-130K protein or anti-FLAG antibodies. Positions of the replication proteins (130K and 180K proteins) and FLAG-tagged tm-1GCR26, Tm-1GCR237, or Tm-1I91T proteins are indicated.
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ppat-1002975-g005: Tm-1I91T binds LT1 replication proteins, but not LT1E979K or LT1D1097Y.The genomic RNA of TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y were translated in mdBYL; mixed with mdBYL in which tm-1GCR26-FLAG, Tm-1GCR237-FLAG, or Tm-1I91T-FLAG mRNA were translated; and immunoprecipitated using anti-FLAG antibody-conjugated agarose. Mock-translation was performed as controls and indicated as no viral RNA or no FLAG RNA. Protein samples before (Input) or after (IP: anti-FLAG) FLAG purification were analyzed by Western blotting using anti-130K protein or anti-FLAG antibodies. Positions of the replication proteins (130K and 180K proteins) and FLAG-tagged tm-1GCR26, Tm-1GCR237, or Tm-1I91T proteins are indicated.

Mentions: Tm-1 inhibits ToMV RNA replication by binding to the replication proteins [15]. Therefore, we examined the ability of Tm-1I91T to bind LT1 replication proteins. FLAG-tagged tm-1GCR26, Tm-1GCR237, and Tm-1I91T proteins were synthesized by in vitro translation using evacuolated tobacco BY-2 protoplast extracts from which membranes were removed by centrifugation (membrane-depleted BYL: mdBYL). The translation mixtures were mixed with mdBYL, in which TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y RNA was translated or mock-translated, and immunoprecipitation using anti-FLAG antibody-conjugated agarose was performed. As expected, the LT1, TLIle, and ToMV-L replication proteins coprecipitated with Tm-1I91T-FLAG, while the LT1E979K or LT1D1097Y replication proteins did not (Figure 5). Also, the replication proteins of ToMV mutants whose multiplication was inhibited coprecipitated with the Tm-1 variants (Figure 5).


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)

Tm-1I91T binds LT1 replication proteins, but not LT1E979K or LT1D1097Y.The genomic RNA of TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y were translated in mdBYL; mixed with mdBYL in which tm-1GCR26-FLAG, Tm-1GCR237-FLAG, or Tm-1I91T-FLAG mRNA were translated; and immunoprecipitated using anti-FLAG antibody-conjugated agarose. Mock-translation was performed as controls and indicated as no viral RNA or no FLAG RNA. Protein samples before (Input) or after (IP: anti-FLAG) FLAG purification were analyzed by Western blotting using anti-130K protein or anti-FLAG antibodies. Positions of the replication proteins (130K and 180K proteins) and FLAG-tagged tm-1GCR26, Tm-1GCR237, or Tm-1I91T proteins are indicated.
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Related In: Results  -  Collection

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

ppat-1002975-g005: Tm-1I91T binds LT1 replication proteins, but not LT1E979K or LT1D1097Y.The genomic RNA of TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y were translated in mdBYL; mixed with mdBYL in which tm-1GCR26-FLAG, Tm-1GCR237-FLAG, or Tm-1I91T-FLAG mRNA were translated; and immunoprecipitated using anti-FLAG antibody-conjugated agarose. Mock-translation was performed as controls and indicated as no viral RNA or no FLAG RNA. Protein samples before (Input) or after (IP: anti-FLAG) FLAG purification were analyzed by Western blotting using anti-130K protein or anti-FLAG antibodies. Positions of the replication proteins (130K and 180K proteins) and FLAG-tagged tm-1GCR26, Tm-1GCR237, or Tm-1I91T proteins are indicated.
Mentions: Tm-1 inhibits ToMV RNA replication by binding to the replication proteins [15]. Therefore, we examined the ability of Tm-1I91T to bind LT1 replication proteins. FLAG-tagged tm-1GCR26, Tm-1GCR237, and Tm-1I91T proteins were synthesized by in vitro translation using evacuolated tobacco BY-2 protoplast extracts from which membranes were removed by centrifugation (membrane-depleted BYL: mdBYL). The translation mixtures were mixed with mdBYL, in which TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y RNA was translated or mock-translated, and immunoprecipitation using anti-FLAG antibody-conjugated agarose was performed. As expected, the LT1, TLIle, and ToMV-L replication proteins coprecipitated with Tm-1I91T-FLAG, while the LT1E979K or LT1D1097Y replication proteins did not (Figure 5). Also, the replication proteins of ToMV mutants whose multiplication was inhibited coprecipitated with the Tm-1 variants (Figure 5).

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