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

我们的大多数食物是植物繁殖的产物，其肥力和种子对作物产量至关重要，因此对粮食安全至关重要。自交不亲和的遗传控制过程是开花植物阻止自交受精从而促进植物物种远交和适应性的主要机制。对SI的机械理解可以导致植物育种实践的改进，以生产更好的作物。从机制上讲，最容易理解的SI系统之一是罂粟（罂粟）。在木瓜中，雌蕊分泌一种蛋白质（PrsS），作为信号“配体”。这与“自我”花粉受体（PrpS）特异性相互作用，使花粉能够区分“自我”和“非自我”伴侣。这种相互作用是决定细胞-细胞识别和排斥的关键步骤。相互作用触发Ca2+依赖的信号网络，导致抑制不相容的花粉管生长和程序性细胞死亡（PCD）1,2。值得注意的是，PrpS和PrsS在拟南芥花粉和雌蕊中的表达分别阻碍了自结实，有效地使拟南芥产生自不亲和性3,4。这表明Papaver SI决定因子可以在高度分化的植物物种之间进行功能转移。引人注目的是，最近的研究结果表明，PrpS和PrsS的异位表达会触发拟南芥体细胞中的PCD，这表明这个两部分模块可以在生殖环境之外发挥作用，在营养组织中，它会触发si样反应，导致异位PCD。因此，花粉PrpS的性质及其与PrpS的相互作用具有重要意义。目的PrpS是一种在数据库中没有同源物的质膜蛋白。有趣的是，结构预测，包括使用利亚姆·麦高芬博士开发的IntFOLD服务器，预测了一种参与离子运输的跨膜蛋白质。基于这些预测和si诱导的离子流入（Ca2+， K+和潜在的H+），我们假设PrpS作为配体门控离子通道起作用。利用重组同源PrsS蛋白处理诱导表达拟南芥PrpS的特异性反应的实验，该项目旨在确定PrpS的功能性质，并建立PrpS-PrsS受体-配体相互作用的动力学。1. 我们将利用靶向位点定向诱变技术建立PrpS-PrsS相互作用特异性的基础。预测配体结合残基和三维模型为这一目标提供了一个很好的起点。在Liam McGuffin博士的指导下，该学生将对PrpS蛋白进行更详细的结构建模，并预测同源PrpS和PrsS蛋白之间可能的相互作用。这些计算机研究将使我们能够确定修改的具体目标。2. 我们将确定PrpS是否确实是离子通道。我们将与Gary Stephens教授（Reading）合作，对异种细胞系统（如HEK细胞）中表达的PrpS进行电生理学研究，以确定PrpS是否作为通道。膜片钳电生理学也将使我们能够识别prss诱导的通道激活和动力学，通道电导和离子选择性以及药理学性质。3. 我们将确定PrpS是否形成多聚体复合物。PrpS很小（约20kDa），而离子通道受体通常是多聚体质膜蛋白，这表明PrpS可能是多聚体的亚基。我们将使用蓝色原生PAGE来确定PrpS是否形成低聚体，低聚化水平，以及其低聚状态是否依赖于与其同源配体的相互作用。我们将使用双分子荧光互补（BiFC）来可视化PrpS与PrsS在活细胞中的相互作用。

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.