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

我们的大部分粮食是植物繁殖的产物，肥力和结实对作物产量和粮食安全至关重要。基因控制的自交不亲和过程是开花植物阻止自交受精从而促进远交和物种适合度的主要机制。对SI的机理理解可以导致植物育种实践的改进，以生产出更好的作物。背景和理由机械地说，最好理解的SI系统之一是罂粟(罂粟)。在Papaver中，雌蕊分泌一种蛋白质(PRSS)，起到信号‘配体’的作用。这与“自我”花粉受体(PRPS)发生了特异性的相互作用，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间识别和排斥的关键步骤。这种相互作用触发了一个依赖于钙离子的信号网络，导致不亲和的花粉管生长和细胞程序性死亡(PCD)1，2。值得注意的是，PRPS和PRSS分别在拟南芥花粉和雌蕊中的表达，阻止了自交结实，有效地使拟南芥自交不亲和3，4。这表明，罂粟花SI决定因素可以在高度分化的植物之间进行功能转移。引人注目的是，最近的结果(在自然植物中综述)表明，PRPS和PRSS的异位表达在拟南芥体细胞中触发了PCD，这表明这个二分模块可以在营养组织中发挥生殖环境之外的功能，在那里它触发类似SI的反应导致异位PCD。因此，花粉PRPS的性质及其与PRSS的相互作用引起了人们的极大兴趣。目的：PRPS是一种质膜蛋白，在数据库中没有同源基因。有趣的是，结构预测，包括使用Liam McGuffin(Reding)博士开发的IntFOLD服务器，预测了参与离子运输的跨膜蛋白。基于这些预测和SI诱导的离子(Ca~(2+)、K~+和潜在的H~+)的内流，我们假设PRPS作为配体门控的离子通道发挥作用。利用重组同源PRSS蛋白处理表达拟南芥的PRPS诱导特异性反应的实验，该项目旨在鉴定PRPS的功能性质，并建立PRPS-PRSS受体-配体相互作用的动力学。1.我们将利用定点突变为PRPS-PRSS相互作用的特异性奠定基础。预测的配体结合残基和3D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蛋白质进行更详细的结构建模，并预测同源PRPS和PRSS蛋白质之间可能的相互作用。计算机研究中的这些将使我们能够确定特定的修改目标。2.我们将确定PRPS是否真的是一个离子通道。与加里·斯蒂芬斯教授(雷丁)合作，我们将使用在异种细胞系统(如HEK细胞)中表达的PRPS的电生理学研究来确定PRPS是否起到通道的作用。膜片钳电生理学还将使我们能够确定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特性。3.我们将确定PRPS是否形成多聚体复合体。PRPS很小(~20 kDa)，而离子通道受体通常是多聚体质膜蛋白，这表明PRPS可能是一个多聚体的亚单位。我们将使用蓝色天然PAGE来确定PRPS是否形成低聚物，齐聚程度，以及它的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)来可视化活细胞中PRPS与PRSS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.