Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity

The P19 protein of Tomato bushy stunt virus is a potent suppressor of RNA silencing and, depending on the host species, is required for short- and long-distance virus movement and symptom production. P19 interacts with plant ALY proteins and relocalizes a subset of these proteins from the nucleus to the cytoplasm. Here we showed that coexpression by agroinfiltration in Nicotiana benthamiana of P19 and the subset of ALY proteins that are not relocalized from the nucleus interfered with the ability of P19 to suppress RNA silencing. We demonstrated that this interference correlates with the relocation of P19 from the cytoplasm into the nucleus, and by constructing and analyzing chimeric ALY genes, we showed that the C-terminal part of the central, RNA recognition motif of ALY is responsible for interaction with P19, relocalization or nonrelocalization of ALY, and inhibition of silencing suppression by P19. We studied the interaction of ALY and P19 by using the technique of bimolecular fluorescence complementation to show that these proteins associate physically in the nucleus but not detectably in the cytoplasm, and we present a model to explain the dynamics of this interaction.