A receptor-ligand module that triggers cell death in plants: a killer in disguise

Most of our food is a product of plant reproduction with fertility and seed set critical for crop yield and thus food security. The genetically controlled process of self-incompatibility (SI) is the predominant mechanism used by flowering plants to prevent self-fertilization and thus promote outbreeding and fitness of plant species. Mechanistic understanding of SI can lead to improvements of plant breeding practises to produce better crops. Background & Justification Mechanistically, one of the best-understood SI systems is that of Papaver rhoeas (poppy). In Papaver, the pistil secretes a protein (PrsS) which acts as a signalling 'ligand'. This interacts specifically with a 'self' pollen receptor (PrpS), allowing pollen to distinguish between 'self' and 'non-self' partners. This interaction is the critical step in determining cell-cell recognition and rejection. The interaction triggers a Ca2+ -dependent signalling network resulting in inhibition of incompatible pollen tube growth and programmed cell death (PCD)1,2. Remarkably, the expression of PrpS and PrsS in Arabidopsis pollen and pistil,respectively, prevents self-seed set, effectively rendering Arabidopsis self-incompatible3,4. This demonstrates that the Papaver SI determinants can be functionally transferred between highly diverged plant species. Strikingly, recent results (under review in Nature Plants) show that ectopic expression of PrpS and PrsS triggers PCD in Arabidopsis somatic cells, demonstrating that this bipartite module can function outside the reproductive context, in vegetative tissues where it triggers an SI-like response leading to ectopic PCD. Therefore, the nature of pollen PrpS and its interaction with PrsS is of considerable interest. Aim and Objectives PrpS is a plasma-membrane protein with no homologues in databases. Interestingly, structural predictions, including using the IntFOLD server developed by Dr Liam McGuffin (Reading), predict a transmembrane protein involved in ion transport. Based on these predictions and the SI-induced influx of ions (Ca2+, K+ and potentially H+ ) we hypothesise that PrpS functions as a ligand-gated ion channel. Benefitting from assays that induce a specific response in PrpS expressing Arabidopsis by treatment with recombinant cognate PrsS proteins, the project aims to identify the functional nature of PrpS and establish the dynamics of PrpS-PrsS receptor-ligand interactions. 1. We will establish the basis for the specificity of the PrpS-PrsS interaction using targeted site-directed mutagenesis. Predicted ligand binding residues and 3D models present a good starting point for this aim. Supervised by Dr Liam McGuffin, the student will perform more detailed structural modelling of PrpS proteins as well as predicting the likely interactions between cognate PrpS and PrsS proteins. These in silico studies will allow us to identify specific targets for modification. 2. We will determine if PrpS is indeed an ion channel. In collaboration with Prof Gary Stephens (Reading), we will use electrophysiological studies of PrpS expressed in a heterologous cell system such as HEK cells to establish if PrpS acts as a channel. Patch-clamp electrophysiology will also allow us to identify PrsS-induced channel activation and kinetics, channel conductance and ion selectivity and pharmacological properties. 3. We will establish if PrpS forms a multimeric complex. PrpS is small (~20kDa), whereas ion channel receptors are usually multimeric plasma membrane proteins, this suggests that PrpS may be a subunit that multimerizes. We will use blue native PAGE to determine if PrpS forms oligomers, the level of oligomerization, and if its oligomeric state is dependent on interaction with its cognate ligand. We will use Bimolecular Fluorescence Complementation (BiFC) to visualize PrpS interacting with PrsS in live cells.