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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 inhibits the multiplication of LT1, but not LT1E979K or LT1D1097Y.Protoplasts isolated from transgenic BY-2 cells expressing tm-1GCR26, Tm-1GCR237, or Tm-1I91T, or non-transgenic BY-2 cells were inoculated with TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y by electroporation. At 20 hpi, protoplasts were harvested and coat protein (CP) accumulation was analyzed by SDS-PAGE and Coomassie blue staining.
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ppat-1002975-g004: Tm-1I91T inhibits the multiplication of LT1, but not LT1E979K or LT1D1097Y.Protoplasts isolated from transgenic BY-2 cells expressing tm-1GCR26, Tm-1GCR237, or Tm-1I91T, or non-transgenic BY-2 cells were inoculated with TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y by electroporation. At 20 hpi, protoplasts were harvested and coat protein (CP) accumulation was analyzed by SDS-PAGE and Coomassie blue staining.

Mentions: To determine whether the Thr residue at position 91 is important for LT1 resistance, we prepared transgenic tobacco BY-2 cell lines, which constitutively expressed tm-1GCR26 protein, Tm-1GCR237 protein, or Tm-1 protein with the I91T substitution (Tm-1I91T). ToMV-LT1 cDNA was also mutagenized to encode an E979K (LT1E979K) or D1097Y (LT1D1097Y) substitution in the replication proteins to determine whether these mutations are responsible for overcoming the resistance by Tm-1I91T (Figure 1). A ToMV-L mutant that has the same mutations as another Tm-1-resistance-breaking mutant (ToMV1-2) [28] was also constructed and named T21 (Figure 1). Protoplasts isolated from the transgenic BY-2 cells expressing tm-1GCR26, Tm-1GCR237, or Tm-1I91T, or non-transgenic BY-2 cells were inoculated with TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y RNA by electroporation, or mock-inoculated, and CP accumulation was analyzed at 20 hours postinoculation (hpi). In non-transgenic BY-2 cells, the CP of these viruses accumulated to similar levels (Figure 4). In tm-1GCR26-expressing cells, multiplication of TLIle was inhibited (Figure 4). In Tm-1GCR237-expressing cells, multiplication of TLIle and ToMV-L was inhibited (Figure 4). In Tm-1I91T-expressing cells, multiplication of TLIle, ToMV-L, and LT1 was inhibited (Figure 4). Multiplication of T21, LT1E979K, and LT1D1097Y was not inhibited by any of the Tm-1 variants (Figure 4). These results indicate that the I91T substitution in the Tm-1 protein confers the ability to inhibit the multiplication of LT1, while LT1E979K and LT1D1097Y emerged in LT1-resistant S. habrochaites plants by escaping from the I91T-type Tm-1 alleles. Remarkably, sensitivity of ToMV mutants to Tm-1 variants was hierarchical; a virus that was unable to overcome tm-1GCR26 was also unable to overcome Tm-1GCR237 and Tm-1I91T, and viruses that were unable to overcome Tm-1GCR237 were also unable to overcome Tm-1I91T.


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 inhibits the multiplication of LT1, but not LT1E979K or LT1D1097Y.Protoplasts isolated from transgenic BY-2 cells expressing tm-1GCR26, Tm-1GCR237, or Tm-1I91T, or non-transgenic BY-2 cells were inoculated with TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y by electroporation. At 20 hpi, protoplasts were harvested and coat protein (CP) accumulation was analyzed by SDS-PAGE and Coomassie blue staining.
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Related In: Results  -  Collection

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ppat-1002975-g004: Tm-1I91T inhibits the multiplication of LT1, but not LT1E979K or LT1D1097Y.Protoplasts isolated from transgenic BY-2 cells expressing tm-1GCR26, Tm-1GCR237, or Tm-1I91T, or non-transgenic BY-2 cells were inoculated with TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y by electroporation. At 20 hpi, protoplasts were harvested and coat protein (CP) accumulation was analyzed by SDS-PAGE and Coomassie blue staining.
Mentions: To determine whether the Thr residue at position 91 is important for LT1 resistance, we prepared transgenic tobacco BY-2 cell lines, which constitutively expressed tm-1GCR26 protein, Tm-1GCR237 protein, or Tm-1 protein with the I91T substitution (Tm-1I91T). ToMV-LT1 cDNA was also mutagenized to encode an E979K (LT1E979K) or D1097Y (LT1D1097Y) substitution in the replication proteins to determine whether these mutations are responsible for overcoming the resistance by Tm-1I91T (Figure 1). A ToMV-L mutant that has the same mutations as another Tm-1-resistance-breaking mutant (ToMV1-2) [28] was also constructed and named T21 (Figure 1). Protoplasts isolated from the transgenic BY-2 cells expressing tm-1GCR26, Tm-1GCR237, or Tm-1I91T, or non-transgenic BY-2 cells were inoculated with TLIle, ToMV-L, LT1, T21, LT1E979K, or LT1D1097Y RNA by electroporation, or mock-inoculated, and CP accumulation was analyzed at 20 hours postinoculation (hpi). In non-transgenic BY-2 cells, the CP of these viruses accumulated to similar levels (Figure 4). In tm-1GCR26-expressing cells, multiplication of TLIle was inhibited (Figure 4). In Tm-1GCR237-expressing cells, multiplication of TLIle and ToMV-L was inhibited (Figure 4). In Tm-1I91T-expressing cells, multiplication of TLIle, ToMV-L, and LT1 was inhibited (Figure 4). Multiplication of T21, LT1E979K, and LT1D1097Y was not inhibited by any of the Tm-1 variants (Figure 4). These results indicate that the I91T substitution in the Tm-1 protein confers the ability to inhibit the multiplication of LT1, while LT1E979K and LT1D1097Y emerged in LT1-resistant S. habrochaites plants by escaping from the I91T-type Tm-1 alleles. Remarkably, sensitivity of ToMV mutants to Tm-1 variants was hierarchical; a virus that was unable to overcome tm-1GCR26 was also unable to overcome Tm-1GCR237 and Tm-1I91T, and viruses that were unable to overcome Tm-1GCR237 were also unable to overcome Tm-1I91T.

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