A transmembrane domain determines the localization of rice stripe virus pc4 to plasmodesmata and is essential for its function as a movement protein

• A transmembrane domain is essential for localization of RSV pc4 to PD.
• PD localization is crucial for the function of pc4 as a movement protein.
• Other properties are needed for the movement function of pc4.

The pc4 protein encoded by rice stripe virus (RSV) is a viral movement protein (MP). A transmembrane (TM) domain spanning AAs 106-123 of pc4 was identified and shown to be essential for localization of pc4 to plasmodesmata (PD) (but not to chloroplasts) and for its ability to recover the movement of movement-deficient PVX. Analysis of alanine-scanning mutants showed that M116A and G120A had a similar localization to wild type pc4, being localized at PD and chloroplasts, but all other mutants were only localized at chloroplasts and not at PD. Mutants that could not be localized at PD had little (G123A) or no ability to recover PVX-GFPΔp25 movement, indicating that PD localization is crucial for the function of pc4 as a movement protein. Strangely, mutants M116A and G120A localized at PD and retained the ability to bind single-stranded RNA but did not support PVX-GFPΔp25 movement, indicating that properties other than PD localization and nucleotide binding ability may be needed for the function of pc4 as a movement protein.