A single amino acid mutation in the Plum pox virus HC-Pro gene abolishes both synergistic and RNA silencing suppression activities

Abstract

The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-protein- ase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PV X recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombi- nant virus expressing a variant of PPV HC-Pro containing a single point mutation (HCL 134 H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL 134 H mu- tant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. ben- thamiana plants infected with either the PVX recombinant constructs or with native viruses in double infect ion experiments. PVX (+) and (–) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an in crease in PVX pathology, compared with plants infected with PVX alone . This finding confirms that the en- hancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.