Molecular characterization of the interaction between the N-terminal region of Potato virus X (PVX) coat protein (CP) and Nicotiana benthamiana PVX CP-interacting protein, NbPCIP1.

Using yeast two-hybrid assays and a Nicotiana benthamiana cDNA library, we previously identified an N. benthamiana protein, NbPCIP1, that interacts with Potato virus X (PVX) coat protein (CP). We also previously determined that NbPCIP1 enhances PVX replication in plants. To determine the domains and/or amino acid residues required for PVX CP and NbPCIP1 interaction, here we used yeast two-hybrid and β-galactosidase filter assays to test the effects of deletion and site-directed mutations on the interaction. Truncation analysis revealed that the N-terminal region of PVX CP interacts with NbPCIP1. To identify which N-terminal region PVX CP amino acid(s) interact with NbPCIP1, we substituted the 12 charged amino acids on the PVX CP N-terminal region to alanine. Yeast two-hybrid, β-galactosidase filter, and bimolecular fluorescence complementation (BiFC) assays confirmed that ten of the 12 alanine-substituted mutations blocked the interaction with NbPCIP1. The results suggest that the N-terminal region of PVX CP including its helical structure is important for interaction with NbPCIP1.