Pepper mild mottle virus pathogenicity determinants and cross protection effect of attenuated mutants in pepper

To determine the pathogenicity domain and to apply cross protection, Pepper mild mottle virus (PMMoV) point-mutations in the replicase (REP) gene between the methyl-transferase and helicase domains, and deletions truncating pseudoknots in the 3′ non-coding region (NCR), were constructed. Some mutants substituting a single amino acid in REP residue 348 exhibited mild symptoms in Nicotiana benthamiana or pepper plants. Accumulation of these mutants was higher than that of other REP mutants or wild-type PMMoV. Deletion mutants in the 3′ NCR pseudoknot showed the lowest virus replication and accumulation among the mutants tested. Six attenuated mutants, which combined 3′ NCR deletions and single or double REP substitution mutations were constructed to investigate cross protection effects on pepper plants. All six of the attenuated mutants showed milder symptom development than wild-type virus. These results suggest that REP and the pseudoknot in the 3′ NCR are major pathogenicity determinants of the virus, and engineered PMMoV attenuated mutants can be useful for protection against the virus in pepper plants.