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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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Fitness costs to the ToMV mutants in the absence of Tm-1.(A) Competition of two ToMV derivatives in BY-2 protoplasts. Protoplasts isolated from non-transgenic BY-2 cells were co-inoculated with two of the six ToMV derivatives used in this study. As a control, individual derivatives were separately inoculated and the protoplasts were cocultured. At 20 hpi, RNA was extracted, and amplified cDNA was sequenced using the GS-FLX titanium. Ratios of the viral count (virus A/virus B) normalized to the respective control experiment (individual infection) are shown. *: p<0.05, **: p<0.01 based on a chi-square test for the ratio of the two derivatives in the coinfection experiment against the ratio expected from the control experiment. A result of each competition is represented twice so that each virus A histogram shows the results of competition against all the other derivatives (virus B). (B) Competition of LT1E979K with ToMV-L or LT1 in tomato plants. Mixtures of viral RNA were mechanically inoculated onto the leaves of 16 tomato (GCR26: tm-1) plants. RNA was extracted from the inoculated leaves (IL) and upper non-inoculated leaves (UL) at 10 and 42 dpi, respectively. At least four young leaflets of each co-inoculated plant at 46 dpi were homogenized; each homogenate was inoculated onto a healthy plant, and RNA was extracted from upper non-inoculated leaves at 42 dpi (Passage). RT-PCR-amplified cDNA fragments were directly sequenced and the numbers of plants accumulating either both or one of the co-inoculated derivatives are shown.
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ppat-1002975-g007: Fitness costs to the ToMV mutants in the absence of Tm-1.(A) Competition of two ToMV derivatives in BY-2 protoplasts. Protoplasts isolated from non-transgenic BY-2 cells were co-inoculated with two of the six ToMV derivatives used in this study. As a control, individual derivatives were separately inoculated and the protoplasts were cocultured. At 20 hpi, RNA was extracted, and amplified cDNA was sequenced using the GS-FLX titanium. Ratios of the viral count (virus A/virus B) normalized to the respective control experiment (individual infection) are shown. *: p<0.05, **: p<0.01 based on a chi-square test for the ratio of the two derivatives in the coinfection experiment against the ratio expected from the control experiment. A result of each competition is represented twice so that each virus A histogram shows the results of competition against all the other derivatives (virus B). (B) Competition of LT1E979K with ToMV-L or LT1 in tomato plants. Mixtures of viral RNA were mechanically inoculated onto the leaves of 16 tomato (GCR26: tm-1) plants. RNA was extracted from the inoculated leaves (IL) and upper non-inoculated leaves (UL) at 10 and 42 dpi, respectively. At least four young leaflets of each co-inoculated plant at 46 dpi were homogenized; each homogenate was inoculated onto a healthy plant, and RNA was extracted from upper non-inoculated leaves at 42 dpi (Passage). RT-PCR-amplified cDNA fragments were directly sequenced and the numbers of plants accumulating either both or one of the co-inoculated derivatives are shown.

Mentions: Although CP accumulation levels were not significantly different among the ToMV derivatives when they were individually inoculated into non-transgenic BY2 protoplasts (Figure 4), we examined the relative fitness between the ToMV derivatives by co-inoculation of BY2 protoplasts with a 1∶1 mixture of two ToMV derivative RNAs. As we had six ToMV derivatives, 15 combinations were tested. As a control, individual derivative RNAs were separately inoculated and the protoplasts were cocultured. At 20 hpi, RNA was extracted from the protoplasts and RT-PCR-amplified cDNA fragments of progeny viruses were sequenced by GS-FLX titanium (Roche, Basel, Switzerland). The ratio of the two strains in the progeny of the co-inoculation experiment was normalized to the control (individual) infection, and dominance by one of the two strains was examined using a chi-square test (Table S1). LT1D1097Y was less competitive than the other five variants, as was T21 (excluding LT1D1097Y) (Figure 7A). Having amino acid substitutions at the same residue (D1097V for T21 and D1097Y for LT1D1097Y; Figure 1), the replication proteins of LT1D1097Y and T21 would be disadvantageous with regard to multiplication within protoplasts, probably replicating the viral RNA. Similarly, LT1 RNA accumulation was lower than TLIle or ToMV-L RNA when co-inoculated (Figure 7A). Thus, LT1 is less competitive than ToMV-L and TLIle in the absence of Tm-1.


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)

Fitness costs to the ToMV mutants in the absence of Tm-1.(A) Competition of two ToMV derivatives in BY-2 protoplasts. Protoplasts isolated from non-transgenic BY-2 cells were co-inoculated with two of the six ToMV derivatives used in this study. As a control, individual derivatives were separately inoculated and the protoplasts were cocultured. At 20 hpi, RNA was extracted, and amplified cDNA was sequenced using the GS-FLX titanium. Ratios of the viral count (virus A/virus B) normalized to the respective control experiment (individual infection) are shown. *: p<0.05, **: p<0.01 based on a chi-square test for the ratio of the two derivatives in the coinfection experiment against the ratio expected from the control experiment. A result of each competition is represented twice so that each virus A histogram shows the results of competition against all the other derivatives (virus B). (B) Competition of LT1E979K with ToMV-L or LT1 in tomato plants. Mixtures of viral RNA were mechanically inoculated onto the leaves of 16 tomato (GCR26: tm-1) plants. RNA was extracted from the inoculated leaves (IL) and upper non-inoculated leaves (UL) at 10 and 42 dpi, respectively. At least four young leaflets of each co-inoculated plant at 46 dpi were homogenized; each homogenate was inoculated onto a healthy plant, and RNA was extracted from upper non-inoculated leaves at 42 dpi (Passage). RT-PCR-amplified cDNA fragments were directly sequenced and the numbers of plants accumulating either both or one of the co-inoculated derivatives are shown.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1002975-g007: Fitness costs to the ToMV mutants in the absence of Tm-1.(A) Competition of two ToMV derivatives in BY-2 protoplasts. Protoplasts isolated from non-transgenic BY-2 cells were co-inoculated with two of the six ToMV derivatives used in this study. As a control, individual derivatives were separately inoculated and the protoplasts were cocultured. At 20 hpi, RNA was extracted, and amplified cDNA was sequenced using the GS-FLX titanium. Ratios of the viral count (virus A/virus B) normalized to the respective control experiment (individual infection) are shown. *: p<0.05, **: p<0.01 based on a chi-square test for the ratio of the two derivatives in the coinfection experiment against the ratio expected from the control experiment. A result of each competition is represented twice so that each virus A histogram shows the results of competition against all the other derivatives (virus B). (B) Competition of LT1E979K with ToMV-L or LT1 in tomato plants. Mixtures of viral RNA were mechanically inoculated onto the leaves of 16 tomato (GCR26: tm-1) plants. RNA was extracted from the inoculated leaves (IL) and upper non-inoculated leaves (UL) at 10 and 42 dpi, respectively. At least four young leaflets of each co-inoculated plant at 46 dpi were homogenized; each homogenate was inoculated onto a healthy plant, and RNA was extracted from upper non-inoculated leaves at 42 dpi (Passage). RT-PCR-amplified cDNA fragments were directly sequenced and the numbers of plants accumulating either both or one of the co-inoculated derivatives are shown.
Mentions: Although CP accumulation levels were not significantly different among the ToMV derivatives when they were individually inoculated into non-transgenic BY2 protoplasts (Figure 4), we examined the relative fitness between the ToMV derivatives by co-inoculation of BY2 protoplasts with a 1∶1 mixture of two ToMV derivative RNAs. As we had six ToMV derivatives, 15 combinations were tested. As a control, individual derivative RNAs were separately inoculated and the protoplasts were cocultured. At 20 hpi, RNA was extracted from the protoplasts and RT-PCR-amplified cDNA fragments of progeny viruses were sequenced by GS-FLX titanium (Roche, Basel, Switzerland). The ratio of the two strains in the progeny of the co-inoculation experiment was normalized to the control (individual) infection, and dominance by one of the two strains was examined using a chi-square test (Table S1). LT1D1097Y was less competitive than the other five variants, as was T21 (excluding LT1D1097Y) (Figure 7A). Having amino acid substitutions at the same residue (D1097V for T21 and D1097Y for LT1D1097Y; Figure 1), the replication proteins of LT1D1097Y and T21 would be disadvantageous with regard to multiplication within protoplasts, probably replicating the viral RNA. Similarly, LT1 RNA accumulation was lower than TLIle or ToMV-L RNA when co-inoculated (Figure 7A). Thus, LT1 is less competitive than ToMV-L and TLIle in the absence of Tm-1.

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