P3N-PIPO interacts with P3 via the shared N-Terminal domain to recruit viral replication vesicles for cell-to-cell movement

P3N-PIPO, the only dedicated movement protein (MP) of potyviruses, directs cylindrical inclusion (CI) protein from the cytoplasm to the plasmodesma (PD), where CI forms conical structures for intercellular movement. To better understand potyviral cell-to-cell movement, we further characterized P3N-PIPO using Turnip mosaic virus (TuMV) as a model virus. We found that P3N-PIPO interacts with P3 via the shared P3N domain and that TuMV mutants lacking the P3N domain of either P3N-PIPO or P3 are defective in cell-to-cell movement. Moreover, we found that the PIPO domain of P3N-PIPO is sufficient to direct CI to the PD, whereas the P3N domain is necessary for localization of P3N-PIPO to 6K2-labeled vesicles or aggregates. Finally, we discovered that the interaction between P3 and P3N-PIPO is essential for the recruitment of CI to cytoplasmic 6K2-containing structures and the association of 6K2-containing structures with PD-located CI inclusions. These data suggest that both P3N and PIPO domains are indispensable for potyviral cell-to-cell movement and that the 6K2 vesicles in proximity to PDs resulting from multipartite interactions among 6K2, P3, P3N-PIPO, and CI may also play an essential role in this process. IMPORTANCE Potyviruses include numerous economically important viruses that represent approximately 30% of known plant viruses. However, there is still limited information about the mechanism of potyviral cell-to-cell movement. Here, we show that P3N-PIPO interacts with and recruits CI to the PD via the PIPO domain and interacts with P3 via the shared P3N domain. We further report that the interaction of P3N-PIPO and P3 is associated with 6K2 vesicles and brings the 6K2 vesicles into proximity with PD-located CI structures. These results support the notion that the replication and cell-to-cell movement of potyviruses are processes coupled by anchoring viral replication complexes at the entrance of PDs, which greatly increase our knowledge of the intercellular movement of potyviruses.