ALT Figure 3 Network visualization of the integrated interactome. Cytoscape visualization of the N-related SnRK1 and TOR interactome combining all Affinity Purification coupled to Mass Spectrometry (AP-MS) and Proximity Labeling coupled to Mass Spectrometry (PL-MS) experiments. Integrated node and edge attributes (Supplemental Table S3C-D) are shown next to the network. The network was divided in a subnetwork covering interactions found in prior corresponding networks related to C signaling (upper), and a subnetwork covering all novel and/or N-specific interactions (bottom). Overlap with prior C-dependent TOR and SnRK1 interactome networks is shown in the Venn diagram. Nodes were circled as N related on the condition that the proteins were present in either N-related transcriptome or phosphoproteome datasets, a Yeast-one-Hybrid (Y1H) network for nitrogen-associated metabolism, or are known to be involved in N transportation (Supplemental Table S3G).
ALT Figure 4. Validation of a selection of novel N-related interactions. (A-B) Autoradiograms and Coomassie brilliant blue R loading controls of in vitro kinase assays, confirming phosphorylation of recombinant PANK2 (142 kDa), NLP8 (146 kDa), RAB18 (65 kDa) and SEC12 (84 kDa) proteins N-terminally fused to a HisMBP tag by SnRK1α1 in the presence of ATP-γ-32P.The negative controls with kinase-dead SnRK1α1 K48M are shown in (B). (C) In vivo SPPIER-dependent validation of protein–protein interactions (PPIs) upon transient co-expression in N. benthamiana leaves of SnRK1α1-mScarlet-HOTag3 with IRE1A_cyt-eGFP-HOTag6, HOTag6-eGFP-BSK4, or NLP8-eGFP-HOTag6, as visualized by dual-colored phase-separated droplets. Scale bar, 50 μm. (D) IRE1 activity assay. Five-day old seedlings were transferred to ½ MS medium containing either full N or no N and continued growing for the times indicated. RNA samples were analyzed for the presence of spliced bZIP60 and BiP3 mRNA by RT-PCR.
ALT Meme abstract of the following paper: Persyn, F., Smagghe, W., Eeckhout, D., Mertens, T., Smorscek, T., De Winne, N., ... & De Jaeger, G. (2024). A Nitrogen-Specific Interactome Analysis Sheds Light on the Role of the SnRK1 and TOR Kinases in Plant Nitrogen Signaling. Molecular & Cellular Proteomics, 100842. DOI: 10.1016/j.mcpro.2024.100842
ALT First Author Wouter Smagghe. Phase separation-based visualization of protein-protein interactions and kinase activities in plants. PhD student. Msc Bioscience engineering cell and gene biotechnology. RESEARCH INTERESTS:How sucrose and nutrient signaling drives source-sink communication, steering plant growth according to the environmental changes.PERSONAL INTERESTS: Competitive cycling, hiking, gardening. Image: Person smiling with arms crossed.
