A plant-specific syntaxin-6 protein contributes to the intracytoplasmic route for begomoviruses

Abstract

2526 Due to limited free diffusion in the cytoplasm, viruses must use active transport mechanisms to 27 move intracellularly. Nevertheless, how the plant ssDNA begomovirus hijacks the host 28 intracytoplasmic transport machinery to move from the nucleus to the plasmodesma remains 29 enigmatic. Here, we identified nuclear shuttle protein (NSP)-interacting proteins by probing a 30 protein microarray and demonstrated that viral NSP, a facilitator of the nucleocytoplasmic 31 trafficking of viral (v)DNA, interacts with a new endosomal vesicle-localized plant-specific 32 syntaxin-6 protein, designated NISP in planta. We also showed that begomovirus infection 33 requires the NISP-NSP interaction; NISP displays a pro-viral function, but not the syntaxin-6 34 paralog AT2G18860 that failed to interact with NSP. Consistent with these findings, nisp-1 mutant 35 plants were less susceptible to begomovirus infection, a phenotype reversed by NISP 36 complementation, whereas overexpressing lines accumulated higher levels of viral DNA than 37 wild-type. Furthermore, NISP interacts with NIG, another pro-viral factor that accessorizes the 38 NSP-vDNA nucleocytoplasmic translocation. Additionally, the NISP-NIG interaction is enhanced 39 by NSP. We also showed that NISP associates with vDNA and might assemble a NISP-NIG-NSP-40 vDNA-complex. NISP may function as a docking site for recruiting NIG and NSP into trafficking 41 vesicles, providing a mechanism for the intracytoplasmic translocation of the NSP-vDNA complex 42 towards the cell periphery. 43 44 Author Summary 45 As viruses must use an active and directed intracellular movement, they hijack the intracellular 46 host transport system for their benefit. Therefore, the identification of interactions between host 3 47 proteins and begomovirus movement proteins should target the intracellular transport machinery.48 This work focused on the identification of these protein-protein interactions; it addressed the 49 molecular bases for the intracellular transport of begomoviruses. We used a protein microarray to 50 identify cellular partners for the movement protein (MP) and the viral nuclear shuttle protein 51 (NSP), which is a facilitator of the nucleocytoplasmic trafficking of viral (v)DNA. We identified 52 relevant protein-protein interaction (PPI) hubs connecting host and viral proteins. We revealed a 53 novel NSP-interacting protein, which functions in the intracytoplasmic transport of proteins and 54 DNA from begomoviruses and was designated NSP-interacting syntaxin domain-containing 55 protein (NISP). Our data suggest an intracellular route connecting the release of newly-synthesized 56 begomoviral DNA in the cytosol with the cell surface. Resolving viral DNA-host protein 57 complexes led to the identification of a novel class of components of the cell machinery and a 58 representative member, NISP, that functions as a susceptibility gene against begomoviruses. As 59 geminiviruses pose a severe threat to agriculture and food security, this recessive gene can now be 60 exploited ...