Coevolution and hierarchical interactions of Tomato mosaic virus and the resistance gene Tm-1.

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.

Identification of a ribonucleoprotein intermediate of tomato mosaic virus RNA replication complex formation.

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.