In silico analysis and molecular investigation of the nuclear shuttle protein-interacting kinase 1 (NIK1) in six Solanaceous species

The first layer of innate immunity in plants is initiated through the perception of microbe-associated molecular patterns (MAMPs) or damage-associated molecular patterns (DAMPs) by pattern recognition receptors (PRRs). MAMP/DAMP perception initiates downstream defense responses, a process which ultimately leads to pattern triggered immunity (PTI). In Arabidopsis, the nuclear shuttle protein-interacting kinase 1 (NIK1), among other PRRs, is one of the most important central components of PTI signaling and kinase signaling cascade, since it is involved in the plant antiviral response against geminiviruses. Despite the characterization of the structure and function of the NIK1 receptors made by some groups, studies related to NIK1 importance in the current gene-editing era are missing. By simple in silico analysis, in this study we investigated the NIK1 homologues from six Solanaceous plant species including: tomato (Solanum lycopersicum), potato (Solanum tuberosum), Solanum pennellii, eggplant (Solanum melongena), pepper (Capsicum annum), and Nicotiana benthamiana. The phylogenetic analyses of different NIK1 proteins from Arabidopsis and six Solanaceous plants revealed nine different clades. As expected, we found that these NIK1 orthologs have similar genomic structures suggesting a similar function. We could identify that SotubNIK1, SolyNIK1, SopenNIK1, CANIK1, NibenNIK1, SmeNIK1 have the highest sequence homology with AtNIK1. Additionally, the conserved protein kinase domain (PKD) that is present in NIK1 from Arabidopsis thaliana was bioinformatically analyzed and found in other species. As this highly conserved NIK1 region is present in several crops of economic importance, its potential is highlighted as a possible target site for gene editing, to develop crops tolerant to geminiviral infections.

Co-Acquired Nanovirus and Geminivirus Exhibit a Contrasted Localization within Their Common Aphid Vector

Single-stranded DNA (ssDNA) plant viruses belong to the families Geminiviridae and Nanoviridae. They are transmitted by Hemipteran insects in a circulative, mostly non-propagative, manner. While geminiviruses are transmitted by leafhoppers, treehoppers, whiteflies and aphids, nanoviruses are transmitted exclusively by aphids. Circulative transmission involves complex virus–vector interactions in which epithelial cells have to be crossed and defense mechanisms counteracted. Vector taxa are considered a relevant taxonomic criterion for virus classification, indicating that viruses can evolve specific interactions with their vectors. Thus, we predicted that, although nanoviruses and geminiviruses represent related viral families, they have evolved distinct interactions with their vector. This prediction is also supported by the non-structural Nuclear Shuttle Protein (NSP) that is involved in vector transmission in nanoviruses but has no similar function in geminiviruses. Thanks to the recent discovery of aphid-transmitted geminiviruses, this prediction could be tested for the geminivirus alfalfa leaf curl virus (ALCV) and the nanovirus faba bean necrotic stunt virus (FBNSV) in their common vector, Aphis craccivora. Estimations of viral load in midgut and head of aphids, precise localization of viral DNA in cells of insect vectors and host plants, and virus transmission tests revealed that the pathway of the two viruses across the body of their common vector differs both quantitatively and qualitatively.

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

Due to limited free diffusion in the cytoplasm, viruses must use active transport mechanisms to move intracellularly. Nevertheless, how the plant ssDNA begomoviruses hijacks 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 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 with an endosomal vesicle-localized plant-specific syntaxin-6 protein, designated NSP-interacting syntaxin-6 domain-containing protein (NISP) in planta. NISP displays a pro-viral function, but not 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 NISP associates with vDNA and might assemble a NISP-NIG-NSP-vDNA-complex. 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 to and from the cell periphery.Author SummaryAs viruses must use an active and directed intracellular movement, they hijack the intracellular host transport system for their own benefits. Therefore, the identification of interactions between host proteins and begomovirus movement proteins should target the intracellular transport machinery. This work focused on the identification of these protein-protein interactions; it addressed the molecular bases for the intracellular transport of begomoviruses. We used a protein microarray to identify cellular partners for the movement protein (MP) and the viral nuclear shuttle protein (NSP), which is a facilitator of the nucleocytoplasmic trafficking of viral (v)DNA. We identified relevant protein-protein interaction (PPI) hubs connecting host and viral proteins. We revealed a novel NSP-interacting protein, which functions in the intracytoplasmic transport of proteins and DNA from begomoviruses and was designated NSP-interacting syntaxin domain-containing protein (NISP). Our data suggest an intracellular route connecting the release of newly synthesized begomoviral DNA in the cytosol with the cell surface. Resolving viral DNA-host protein complexes led to the identification of a novel class of components of the cell machinery and a representative member, NISP, that functions as a susceptibility gene against begomoviruses. As geminiviruses pose a severe threat to agriculture and food security, this recessive gene can now be exploited as a target for engineering resistance by gene editing in crops.