PS Webinar Series: PhD Exit Webinar - A new method to visualize CEP peptide-CEP receptor interactions in vascular tissue in vivo
Abstract - The interactions of peptide ligands with leucine-rich repeat receptor-like kinases (LRR-RLKs) coordinate multiple plant biochemical pathways. Thus, there is a need for a simple method that identifies and validates peptide hormone-receptor pairings in vivo without disturbing native receptor complexes. Here we use formaldehyde or photo-activation to cross-link fluorescently tagged group 1 or group 2 C-TERMINALLY ENCODED PEPTIDEs (CEPs) to receptors in semi-purified Medicago truncatula or Arabidopsis thaliana leaf vascular tissue. Formaldehyde cross-linked FITC-tagged Medicago group 1 CEP (MtCEP1) to a specific subset of vascular tissue cells in wild type leaves but not those from Arabidopsis or Medicago CEP receptor mutants. Binding competition showed that unlabeled MtCEP1 displaced the FITC-MtCEP1 from the CEP receptor in vivo. By contrast, the group 2 CEP, FITC-AtCEP12, which has divergent N-terminal amino acids to group 1 CEPs, bound to vascular tissue cells independently of CEPR1 or CRA2. The crosslinking of photo-activated FITC-MtCEP1 to the periphery of vascular tissue cells suggested that the putative Medicago CEP receptor, COMPACT ROOT ARCHITECTURE 2 (CRA2), localized to the plasma membrane. We visualized the photo-cross-linking of FITC-MtCEP1 to CRA2 using SDS-PAGE and fluorescence-detection. Mass spectrometry analysis identified CRA2-specific peptides in this band. Collectively these results indicate that FITC-MtCEP1 binds to CRA2, and that MtCRA2 and AtCEPR1 are functionally orthologous. The use of formaldehyde or photo-activation to crosslink biologically-active, fluorescently-tagged ligands to receptors is a simple technique that may find wide utility to demonstrate ligand receptor pairings in specific tissues, augment genetic evidence supporting ligand-receptor pairing, or identify ligand binding sites.
Biography - Han is a PhD candidate in Michael Djordjevic’s lab at the ANU. Prior to his PhD he studied for his master degree in biochemistry at Georgetown University and had his research in NHLBI, NIH.