[Intellectual Contribution]

Structural basis for the coevolution of Tomato mosaic virus and the resistance protein Tm-1

Kazuhiro Ishibashi1, Chihoko Kobayashi2, Masahiko Kato2, Masayuki Ishikawa1, Etsuko Katoh3
1 Plant-Microbe Interactions Research Unit, 2 Biomolecular Research Unit
We determined the crystal structures for the complex between the N-terminal inhibitory domain of Tm-1 and helicase domain of tomato mosaic virus replication proteins (ToMV-Hel). The complex contains a Tm-1 dimer and two ToMV-Hel monomers, with the Tm-1-ToMV-Hel interface bridged by an ATPγS. Residues in ToMV-Hel and Tm-1 involved in antagonistic coevolution are also found at the interface. The crystal structures provide an atomic view of step-by-step coevolutionary arms race between a plant resistance protein and a viral protein.
Keywords: tomato mosaic virus, crystal structure, protein complex, arms race, coevolution


Viruses evolve so rapidly that they can escape host defense systems. As a counter strategy, the sequences of many host restriction factor genes are subject to positive selection and, consequently, evolve rapidly. Molecular evolutionary approaches in conjunction with the tertiary structures of related proteins may provide useful information on virus–host evolutionary arms races. We previously found that the resistance protein Tm-1 binds the tomato mosaic virus (ToMV) replication proteins thereby inhibiting RNA replication, and that a part of the Tm-1 gene has been under positive selection. In this study, we aimed to clarify the atomic details of the coevolutionary arms race between them through crystal structure determination and molecular dynamics simulation.
[Results and Discussion]
  1. The ToMV replication proteins are involved in RNA replication and harbor a superfamily 1 (SF1) helicase-like domain (ToMV-Hel). Determination of the crystal structure of ToMV-Hel revealed a novel N-terminal domain tightly associated with a helicase core. Prediction of secondary structures in other viral SF1 helicases and comparison of those structures with that of the ToMV-Hel suggested that many viral SF1 helicases have a similar fold.
  2. We determined a crystal structure of a complex of an N-terminal fragment of Tm-1 (residues 1–431:referred to herein as Tm-1 (431)), which is sufficient for the inhibitory activity, and ToMV-Hel. The structure of Tm-1 (431) and ToMV-Hel complex shows a tetrameric complex, comprised of a Tm-1 (431) dimer and two monomeric ToMV-Hel. Notably, an ATPγS molecule is found in each ToMV-Hel–Tm-1 (431) interface and ATP is required for the complex formation.
  3. The residues in ToMV-Hel that are changed in the resistance-breaking mutant LT1, which has Q979 to E and H984 to Y substitutions, are directly involved in the interaction. The positively selected region of Tm-1 forms the binding surface with ToMV-Hel.
  4. A naturally occurring amino acid change (I91 to T) in Tm-1 makes it a stronger inhibitor of ToMV RNA replication, which enables it to inhibit the replication of LT1. We also solved the structure of the ToMV-Hel–Tm-1 (431/I91T) complex. The overall structure of this complex is very similar to that of the ToMV-Hel–Tm-1 (431) complex. In the ToMV-Hel–Tm-1 (431/I91T) structure, T91 is located at the center of the interface with ToMV-Hel, and is involved in a hydrogen bond network containing water molecules. The structural information reasonably explains how the I91 to T substitution strengthens the inhibitory activity of Tm-1.
  5. Based on the crystal structure, we simulated how the resistance-breaking mutations in ToMV-Hel affect the interaction with Tm-1. Together with all above results, an atomic view of the step-by-step coevolutionary arms race between a plant resistance protein and a viral protein emerged.
[Future prospects]
  1. The structures revealed here provide useful information in developing new anti-viral drugs.
  2. Although co-evolution between ToMV-Hel and Tm-1 has been described based on the structure, there are many unclear points for ToMV replication. We are going to obtain the structural information of the full length replication protein

Fig. 1. ToMV-inoculated nontransgenic (left) and transgenic tomato expressing the Tm-1 gene (right).


Fig. 2. Crystal structure of the ToMV-Hel and Tm-1 (431) complex. Tm-1 (431) molecules are shown in blue and cyan, and ToMV-Hel molecules shown in violet and light pink.


Fig. 3. The arms race between ToMV-Hel and Tm-1. Left:Tm-1 binds ToMV-Hel and thereby inhibits RNA replication. I91 of Tm-1 makes hydrophobic interaction with Q979 and D1097 of ToMV-Hel. Center:When Q979 is replaced by E, Tm-1 cannot bind ToMV-Hel and viral replication is allowed. Right:A naturally occurring amino acid change (I91 to T) in Tm-1 renders the ability to bind ToMV-Hel with the Q979E substitution.



Yuichiro Kezuka, Takamasa Nonaka (Iwate Medical University), Tsuyoshi Inoue, Hiroyoshi Matsumura (Osaka University)


  1. Ishibashi K, Kezuka Y, Kobayashi C, Kato M, Inoue T, Nonaka T, Ishikawa M, Matsumura H, Katoh E (2014) Structural basis for the recognition-evasion arms race between Tomato mosaic virus and the resistance gene Tm-1 Proceedings of the National Academy of Sciences of the United States of America 111 (33):E3486-E3495
  2. Kato M, Kezuka Y, Kobayashi C, Ishibashi K, Nonaka T, Ishikawa M, Katoh E (2013) Crystallization and preliminary X-ray crystallographic analysis of the inhibitory domain of the tomato mosaic virus resistance protein Tm-1 Acta Crystallographica Section F 69 (12):1411-1414
  3. Nishikiori M, Sugiyama S, Xiang H, Niiyama M, Ishibashi K, Inoue T, Ishikawa M, Matsumura H, Katoh E (2012) Crystal structure of the superfamily 1 helicase from Tomato mosaic virus Journal of Virology 86 (14):7565-7576
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