Plants deploy various immune receptors to recognize pathogens and defend themselves. Crosstalk may happen among receptor-mediated signal transduction pathways in the same host during simultaneous infection of different pathogens. However, the related function of the receptor-like kinases (RLKs) in thwarting different pathogens remains elusive. Here, we report that NIK1, which positively regulates plant antiviral immunity, acts as an important negative regulator of antibacterial immunity. nik1 plants exhibit dwarfed morphology, enhanced disease resistance to bacteria and increased PAMP-triggered immunity (PTI) responses, which are restored by NIK1 reintroduction. Additionally, NIK1 negatively regulates the formation of the FLS2/BAK1 complex. The interaction between NIK1 and FLS2/BAK1 is enhanced upon flg22 perception, revealing a novel PTI regulatory mechanism by an RLK. Furthermore, flg22 perception induces NIK1 and RPL10A phosphorylation in vivo, activating antiviral signalling. The NIK1-mediated inverse modulation of antiviral and antibacterial immunity may allow bacteria and viruses to activate host immune responses against each other.

The bipartite begomoviruses (Geminiviridae family), which are DNA viruses that replicate in the nucleus of infected cells, encode the nuclear shuttle protein (NSP) to facilitate the translocation of viral DNA from the nucleus to the cytoplasm via nuclear pores. This intracellular trafficking of NSP-DNA complexes is accessorized by the NSP-interacting guanosine triphosphatase (NIG) at the cytosolic side. Here, we report the nuclear redistribution of NIG by AtWWP1, a WW domain-containing protein that forms immune nuclear bodies (NBs) against begomoviruses. We demonstrated that AtWWP1 relocates NIG from the cytoplasm to the nucleus where it is confined to AtWWP1-NBs, suggesting that the NIG-AtWWP1 interaction may interfere with the NIG pro-viral function associated with its cytosolic localization. Consistent with this assumption, loss of AtWWP1 function cuased plants more susceptible to begomovirus infection, whereas overexpression of AtWWP1 enhanced plant resistance to begomovirus. Furthermore, we found that a mutant version of AtWWP1 defective for NB formation was no longer capable of interacting with and relocating NIG to the nucleus and lost its immune function against begomovirus. The antiviral function of AtWWP1-NBs, however, could be antagonized by viral infection that induced either the disruption or a decrease in the number of AtWWP1-NBs. Collectively, these results led us to propose that AtWWP1 organizes nuclear structures into nuclear foci, which provide intrinsic immunity against begomovirus infection.

Due to limited free diffusion in the cytoplasm, viruses must use active transport mechanisms to move intracellularly. Nevertheless, how the plant ssDNA begomoviruses hijack the host intracytoplasmic transport machinery to move from the nucleus to the plasmodesmata remains enigmatic. Here, we identified nuclear shuttle protein (NSP)-interacting proteins from Arabidopsis (Arabidopsis thaliana) by probing a protein microarray and demonstrated that the cabbage leaf curl virus (CabLCV) NSP, a facilitator of the nucleocytoplasmic trafficking of viral (v)DNA, interacts in planta with an endosomal vesicle-localized, plant-specific syntaxin-6 protein, designated NSP-interacting syntaxin-6 domain-containing protein (NISP). NISP displays a pro-viral function, unlike the syntaxin-6 paralog AT2G18860 that failed to interact with NSP. Consistent with these findings, nisp-1 mutant plants were less susceptible to begomovirus infection, a phenotype reversed by NISP complementation. NISP-overexpressing lines accumulated higher levels of viral DNA than wild-type. Furthermore, NISP interacted with NIG, an NSP-interacting GTPase involved in NSP-vDNA nucleocytoplasmic translocation. The NISP-NIG interaction was enhanced by NSP. We also showed that endosomal NISP associates with vDNA. NISP may function as a docking site for recruiting NIG and NSP into endosomes, providing a mechanism for the intracytoplasmic translocation of the NSP-vDNA complex towards and from the cell periphery.

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 ...