Precision guidance: Mechanisms driving targeted secretion in response to invasive microbes

植物细胞被一层横跨数万平方微米的质膜（PM）所包围。PM中的模式识别受体检测微生物分子和与细胞壁损伤相关的分子，这些分子可能表明病原体的存在。这些信息被转导以激活防御过程，包括产生新的蛋白质和化合物，然后被运送回接触部位。最近发现，分子模式足以有效地指导货物的精确递送，从而使植物细胞在植物病原体物理穿透宿主细胞壁之前数小时改变植物病原体接触部位的PM和细胞壁的含量。该提案旨在回答一个基本问题，即这些货物如何被运送到病原体附近的几个特定平方微米的PM中。我们的初步数据表明，真菌植物病原体相关分子诱导胞吐（向PM和细胞外部运输和传递物质的过程）是一种“实时”监测植物胞吐的优秀系统。我们已经建立了一种使用全内反射荧光（TIRF）显微镜的检测方法，可以控制胞吐的刺激，并可视化囊泡（膜和内部货物的小“包”）与PM的相互作用。我们已经证明，PEN3和其他感兴趣的蛋白质聚集在稳定的“纳米结构域”中：纳米尺度的质膜子结构域。这些纳米畴太小，无法用标准光学显微镜完全分辨。我们还观察到移动囊泡“巡回”PM纳米结构域。本提案旨在了解这些行为如何导致微生物接触部位下方出现专门的PM区域。为此，我们将结合两个专业领域：植物细胞生物学和光学物理学。我们的团队包括一个实验室，负责“超”分辨率显微镜领域的几项创新。这些超分辨率技术，dSTORM和PALM可以将光学分辨率提高到比标准荧光显微镜更高的数量级。这将能够准确评估蛋白质共定位和与PM纳米结构域的相互作用。在我们研究的第一部分，我们将使用TIRF显微镜和超分辨率显微镜来研究囊泡的“旅行”行为及其在纳米域的停顿是否会导致货物转移到（或从）纳米域。我们还将询问细胞骨架是否锚定在纳米结构域上，以促进囊泡的游动/暂停行为。在第二部分中，我们将分离我们正在观察的囊泡的未知货物。这将确定新的抗微生物因子和以前未表征的蛋白质，有助于囊泡靶向。最后，我们将与从囊泡分离实验中产生的候选蛋白质并行，对称为囊泡的蛋白质复合物进行功能分析。在动物和真菌系统中，囊泡起着囊泡-系带复合物的作用，但在植物中具有根本的行为差异。因此，我们将验证外囊是分子机制的一部分的假设，该分子机制将囊泡系在PM响应微生物的特定位点上。通过完成这个跨学科项目，我们将确定货物选择性沉积在与微生物接触的PM位点的机制。我们将测试保守因子和新因子的作用，我们已经分离出作为这种靶向机制的候选成分，为植物的靶向胞吐提供基本的见解。此外，我们将利用我们的工具建立一种将酶货物运送到植物病原体界面的方法。这将支持未来应用生物技术解决方案预防植物病害的努力。

Plant cells are surrounded by a plasma membrane (PM) that spans tens of thousands of square microns. Pattern recognition receptors in the PM detect microbial molecules and molecules associated with cell wall damage that may indicate the presence of a pathogen. This information is transduced to activate defensive processes including the production of new proteins and compounds that are then transported back to the site of contact. Recently it was discovered that molecular patterns are sufficient to guide the precise delivery of cargoes effectively allowing the plant cell to change the content of the PM and the cell wall at the site of phytopathogen contact hours before a phytopathogen physically penetrates the host cell wall. This proposal aims to answer the fundamental question of how these cargoes are delivered to only a few specific square microns of PM in the vicinity of the pathogen. Our preliminary data show that the induction of exocytosis (the process of transporting and delivering material to the PM and cell exterior) by fungal phytopathogen-associated molecules is an excellent system for monitoring plant exocytosis in 'real time'. We have established an assay using Total Internal Reflection Fluorescence (TIRF) microscopy that allows controlled stimulation of exocytosis and visualisation of the interaction of vesicles (small 'packets' of membrane and internal cargo) with the PM. We have shown that PEN3 and other proteins of interest are clustered into stable 'nano domains': sub-domains of the plasma membrane at the nano-metre scale. These nano domains are too small to be fully resolved by standard light microscopy. We also observe mobile vesicles 'touring' PM nano domains. This proposal seeks to understand how these behaviours lead to the emergence of specialised PM zones precisely beneath the site of microbial contact.To do this we will combine two areas of expertise: plant cell biology and optical physics. Our team includes a laboratory responsible for several innovations in the field of 'super' resolution microscopy. These super resolution techniques, dSTORM and PALM can increase optical resolution to an order of magnitude greater than standard fluorescence microscopy. This will enable accurate assessment of protein co-localisation and interactions with PM nano domains. In the first part of our study we will use TIRF microscopy and super resolution microscopy to ask whether the 'touring' behaviours of vesicles and their pausing at nano domains results in the transfer of cargo to (or from) nano domains. We will also ask whether the cytoskeleton is anchored to nano domains in order to promote vesicle touring/pausing behaviour. In the second part we will isolate the unknown cargoes of the vesicles we are observing. This will identify novel anti-microbial factors and previously uncharacterised proteins that contribute to vesicle targeting. Finally, we will perform a functional analysis of a protein complex known as the exocyst in parallel with candidate proteins emerging from the vesicle isolation experiments. The exocyst acts as a vesicle-tethering complex in animal and fungal systems but has fundamental behavioural differences in plants. We will therefore test the hypothesis that the exocyst is part of the molecular machinery that tethers vesicles to specific sites at the PM responding to microbes. By the completion of this inter disciplinary project we will have identified the mechanism by which cargoes are deposited selectively at PM sites in contact with microbes. We will have tested the role of both conserved and novel factors that we have isolated as candidate components of this targeting machinery, giving fundamental insights into targeted exocytosis in plants. Moreover, we will have used our tools to establish a means to deliver enzyme cargoes to the plant pathogen interface. This will underpin future efforts to apply biotechnology solutions to preventing plant disease.