Characterization of local and systemic RNA silencing by replication deficient Potyvirus rapae

To deal with viral infection, plants take advantage of the conserved RNA silencing pathway to stop the spread of the virus and protect against future infection by the same virus. To counteract this, viruses encode silencing suppressors to disrupt the silencing pathway. It is known that RNA silencing is triggered by the presence of double-stranded viral RNA in the cytoplasm. However, the source of this double-stranded RNA has not been confirmed. In the case of single-stranded RNA viruses, the current model suggests that this double-stranded RNA may either be from a replication intermediate or a hairpin loop formed when viral RNA anneals to itself. Understanding the trigger of antiviral RNA silencing has important ramifications in biotechnology and plant protection. Thus, we sought to characterize the contribution of hairpin loops and replication intermediates in the initiation of silencing. We generated mutants of Potyvirus rapae deficient in replication and suppressor activity to test infection and antiviral silencing initiation in Nicotiana benthamiana. We found that a mutant of P. rapae without a replicase or a functioning silencing suppressor could not establish infection but was still sufficient to trigger local silencing. After two weeks, systemic silencing failed to establish in the replication deficient mutant. These findings suggest that P. rapae hairpin loops are sufficient for local silencing, but viral replication may be required to properly initiate systemic silencing in plants.