The Interaction Network of NSm and Its Role as a Movement Protein in the Tomato Zonate Spot Virus

The tomato zonate spot virus (TZSV) poses a significant threat to agriculture. Therefore, the elucidation of the functional roles and interactions of its encoded proteins is crucial for the development of effective control strategies. The aim of this study was to investigate the interaction network between the TZSV nucleocapsid (N), the non-structural M-segment (NSm) and the non-structural S-segment (NSs) proteins, with a focus on the functional characterization of the NSm protein. Yeast two-hybrid (Y2H) analysis indicated that both the N protein (N-N) and the NSm protein (NSm-NSm) exhibit self-interaction in vitro, with successful expression of all fusion proteins confirmed by Western blotting. Subsequently, we used bimolecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI) assays in epidermal cells of Nicotiana benthamiana to confirm that N and NSm proteins self-interact. In addition, heterologous interactions between NSs-N, N-NSm and NSs-NSm were also detected. BiFC and co-localization experiments with fusion proteins elucidated the interaction place of the cell: N-N and NSm-N interactions occurred in both the cytoplasm and nucleus, with NSm-NSm interaction occurring in the nucleus, whereas NSs-N and NSs-NSm interactions only occurred in the cytoplasm. Subcellular localization studies showed that the N protein is distributed in both the cytoplasm and the nucleus, whereas the NSm and NSs proteins are predominantly localized in the cytoplasm. In particular, NSm was found to specifically target plasmodesmata (PD) and co-localize with the known PD marker protein PDLP8. Interestingly, TZSV NSm was demonstrated to mediate the cell-to-cell movement of a cucumber mosaic virus mutant (ΔCMV-GFP) lacking its native movement protein (3a). This was evidenced by the spread of approximately 50 fluorescent foci to neighboring cells observed at 6 dpi. This study comprehensively describes the intricate interaction network between the N, NSm and NSs proteins of TZSV and clarifies their subcellular localizations within plant cells. Crucially, we provide conclusive evidence that the NSm protein of TZSV is a functional movement protein essential for facilitating viral intercellular transport which promotes viral spread within the host during systemic infection. These findings offer important insights into the infection mechanism of TZSV and provide potential targets for the control of TZSV.