Identification of the CEP1 peptide receptor in Medicago truncatula

Abstract

Signalling peptides can mediate communication between plant cells by binding their corresponding plasma membrane-localised receptor and triggering downstream responses. In Medicago truncatula, the receptor that physically interacts with CEPs remains unknown, although, during this thesis, genetic evidence emerged that suggested that the plasma membrane localised, COMPACT ROOT ARCHITECTURE2 (CRA2), was a strong candidate. Thus, this thesis aimed to identify which Medicago protein is the CEP receptor using direct bottom-up proteomics approaches and in vivo ligand-receptor binding assays. In this thesis, bottom-up proteomic strategies aimed at identifying CRA2 directly in membrane enriched fractions from Medicago were developed and trialled for their suitability using novel approaches, however, the low natural abundance of CRA2 and other LRR-RLKs prevented any meaningful characterisation of these types of proteins. Therefore the techniques developed enabled a more effective characterisation of TMPs than currently-used bottom-up proteomics approaches and the utility of this approach was explored further. Mass spectrometry was used to compare the effectiveness of two solubilisation and protein cleavage methods to identify shoot-derived TMPs from the legume Medicago. We compared urea solubilisation, trypsin Lys-C (UR-TLC) cleavage method to a formic acid solubilisation, cyanogen bromide, and trypsin Lys-C (FA-CTLC) cleavage method. We identified 8993 protein groups including 3289 TMPs in young shoot tissues, which accounts for around 36 % of the predicted Medicago TMPs. The results suggest that combining plant membrane purification techniques with both the FA-CTLC and UR-TLC methods will achieve a more complete identification and coverage of TMPs. Therefore, even though the approach used to investigate CRA2 and other LRR-RLKs was not suitable, it may find wider utility in studying other plant transmembrane proteins (TMPs). More direct approaches were developed to investigate if CEPs bound to CRA2. Initially, we used formaldehyde to cross-link fluorescently tagged group 1 or group 2 CEPs to receptors in semi-purified Medicago truncatula or Arabidopsis thaliana leaf vascular tissue isolated from wild type and their corresponding CEP receptor mutants. Formaldehyde was found to cross-link FITC-tagged Medicago group 1 CEP (MtCEP1) to a specific subset of semi-purified vascular tissue cells in wild type leaves from Medicago and Arabidopsis, but not those from a bona fide Arabidopsis CEP receptor mutant (cepr1) or Medicago cra2 leaves. By contrast, the group 2 CEP, FITC-AtCEP14, which has divergent N-terminal amino acids to group 1 CEPs, bound to semi purified vascular tissue cells independently of CEPR1 or CRA2. These results suggested that CRA2 was functionally similar to the bona fide CEP receptor, CEPR1, and bound group 1, but not group 2 CEPs. The results also suggested that group 2 CEPs bind a different receptor. To investigate in more depth the association of group 1 CEPs with CRA2, we used an alternative crosslinking approach utilizing photo-activated crosslinking. The crosslinking of photo-activated FITC-MtCEP1 to the periphery of wild type vascular tissue cells suggested that CRA2 is localised to the plasma membrane. We visualized the photo-cross-linking of FITC-MtCEP1 to CRA2 using SDS-PAGE and fluorescence-detection aimed at detecting the FITC fluorescence. A single band was identified, which corresponded to the predicted molecular mass of CRA2. Mass spectrometry analysis identified CRA2-specific peptides in this band. These results indicate that FITC-MtCEP1 binds to CRA2 and that MtCRA2 and AtCEPR1 have similar functions. The use of this approach is a simple technique that may find a wide utility to demonstrate ligand-receptor pairings in specific tissues, augment genetic evidence supporting ligand-receptor pairing, or identify ligand binding sites. Show more