Study on the Plant Resistance Mechanisms against Viruses
Kazuhiro Ishibashi (Institute of Agrobiological Sciences, NARO)
bashi@affrc.go.jp
Plants evolved defense mechanisms to fight viruses. Many genes that confer resistance to specificviruses,

namely,resistancegenes,

encodenucleotide-binding

siteandleucine-richrepeat(NB-LRR)-containing proteins. These proteins elicit a defense reaction upon recognition of viral infection.
Tm-1 is a semi-dominant resistance gene of tomato against tomato mosaic virus (ToMV). Before I started
this study, Tm-1 was suggested to inhibit ToMV multiplication in a unique mechanism, however, positional
cloning has been hampered by the extremely low frequency of recombination around the Tm-1 locus.
I identified the Tm-1 gene product through biochemical purification of an inhibitor of in vitro ToMVRNA replication from a tomato cell extract [1]. The amino acid sequence of the polypeptide had nosimilarity to any characterized proteins. I characterized the mechanisms of action of Tm-1 and found that
the Tm-1 protein binds ToMV replication proteins and inhibits formation of ToMV replication complex.
Replication proteins of resistance-breaking ToMV mutants did not bind Tm-1, suggesting that ToMV
mutants break the resistance by escaping the inhibitory interaction.
Tm-1 was derived from a wild relative of tomato, Solanum habrochaites. To explore the evolutionary
history of Tm-1, I sequenced the Tm-1 gene of S. habrochaites accessions and found that a small part of theTm-1 gene is under positive selection, suggesting that this region underwent rapid amino acid changes
against selective pressure by ToMV infection. Crystal structures of a fragment of the Tm-1 protein and acomplex between the Tm-1 fragment and a ToMV helicase domain fragment of replication proteins
revealed that Tm-1 and ToMV have coevolved by changing both sides of the interaction interface [2].
Viruses infect a specific range of hosts, and thus most plant species are nonhosts of a virus. However,why viruses cannot infect nonhost plants had remained unknown because of the inapplicability of classicalgenetics approaches. Tomato is a nonhost of tobacco green mild mosaic virus (TMGMV) and pepper mildmottle virus (PMMoV), relatives of ToMV. ToMV-susceptible tomato cultivars have a Tm-1 allele, tm-1,
whose product neither binds to ToMV replication proteins nor inhibits RNA replication. I found that tm-1 isa functional inhibitor of TMGMV and PMMoV replication proteins [3]. Replication proteins of theseviruses (tobamoviruses) are multifunctional that also act as an RNA silencing suppressor. A TMGMV
mutant that can escape the inhibition by tm-1 lost the ability to suppress RNA silencing. Therefore, themultifunctionality of replication proteins is an evolutionary constraint of tobamoviruses that restricts their
host ranges.
References[1] Ishibashi K., Masuda K., Naito S., Meshi T. and Ishikawa M.: Proc Natl Acad Sci USA104:13833-13838 (2007).
[2] Ishibashi K., Kezuka Y., Kobayashi C., Kato M., Inoue T., Nonaka T., Ishikawa M., Matsumura
H. and Katoh E.: Proc Natl Acad Sci USA 111(33):E3486-E3495 (2014).
[3] Ishibashi K., Naito S., Meshi T. and Ishikawa M.: Proc Natl Acad Sci USA 106:8778-8783(2009).