[Studies of a plant antiviral defense system that inhibits viral RNA replication].

Abstract

Tm-1 is a semi-dominant resistance gene of tomato against tomato mosaic virus (ToMV). I identified the Tm-1 gene product through biochemical purification of an inhibitor of in vitro ToMV RNA replication from a tomato cell extract. 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. Through molecular evolutionary approach, I found that a small part of the Tm-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 a complex 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. 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 inhibits multiplication of tobacco green mild mosaic virus (TMGMV) and pepper mild mottle virus (PMMoV), which does not adapt to tomato. A TMGMV mutant that can escape the inhibition by tm-1 lost the ability to suppress RNA silencing. Therefore, the multifunctionality of replication proteins is an evolutionary constraint of tobamoviruses that restricts their host ranges.