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

精准引导：针对入侵微生物驱动定向分泌的机制

• 批准号: BB/M024172/1
• 负责人: Michael Deeks
• 金额: $ 56.84万
• 依托单位: UNIVERSITY OF EXETER
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM024172%2F1
• 关键词: Precision; guidance; Mechanisms; driving; targeted

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.

植物细胞被一个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可能指示病原体存在的细胞壁损伤相关的分子。这些信息被转换以激活防御过程，包括产生新的蛋白质和化合物，然后将其运送回接触部位。最近发现，分子模式足以有效地引导货物的精确递送，允许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位点处的PM和细胞壁的含量。该提案旨在回答这些货物如何被运送到病原体附近的仅几个特定平方微米的PM的基本问题。我们的初步数据表明，真菌植物病原体相关分子诱导的胞吐作用（运输和交付材料到PM和细胞外部的过程）是一个很好的系统，用于监测植物胞吐作用在“真实的时间”。我们已经建立了一种使用全内反射荧光（TIRF）显微镜的测定方法，该方法允许胞吐的受控刺激和囊泡（膜和内部货物的小“包”）与PM的相互作用的可视化。我们已经表明，PEN 3和其他感兴趣的蛋白质聚集成稳定的“纳米结构域”：纳米尺度的质膜子域。这些纳米结构域太小，无法通过标准光学显微镜完全分辨。我们还观察到移动的囊泡“旅游”PM纳米域。该建议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，我们将联合收割机两个专业领域结合起来：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大一个数量级。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。在我们的研究的第一部分，我们将使用TIRF显微镜和超分辨率显微镜，以询问是否“旅游”行为的囊泡和他们在纳米域的暂停结果在货物转移到（或从）纳米域。我们还将询问细胞骨架是否锚定到纳米结构域，以促进囊泡旅游/暂停行为。在第二部分中，我们将分离我们观察到的囊泡的未知货物。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，我们将进行一个蛋白质复合物的功能分析称为外囊在平行的候选蛋白质出现的囊泡分离实验。外囊在动物和真菌系统中作为囊泡束缚复合物，但在植物中具有根本的行为差异。因此，我们将测试的假设，外囊是分子机制的一部分，拴囊泡在PM响应微生物的特定网站。通过完成这个跨学科的项目，我们将确定货物选择性地存放在与微生物接触的PM站点的机制。我们将测试保守和新的因素，我们已经分离出的候选成分，这种靶向机制的作用，在植物中有针对性的胞吐作用的基本见解。此外，我们将使用我们的工具来建立一种将酶货物传递到植物病原体界面的方法。这将为今后应用生物技术解决办法预防植物病害的努力奠定基础。

项目成果

Making microscopy count: quantitative light microscopy of dynamic processes in living plants.

让显微镜发挥作用：活体植物动态过程的定量光学显微镜。

• DOI: 10.1111/jmi.12403
• 发表时间: 2016
• 期刊: Journal of microscopy
• 影响因子: 2
• 作者: Fricker MD

Plant biology: Plant formins roll out the welcome wagon for microbes.

植物生物学：植物形态为微生物带来了欢迎。

• DOI: 10.1016/j.cub.2021.04.081
• 发表时间: 2021
• 期刊: CB
• 作者: Deeks MJ

The Exocyst Complex in Health and Disease.

• DOI: 10.3389/fcell.2016.00024
• 发表时间: 2016
• 期刊: Frontiers in cell and developmental biology
• 影响因子: 5.5
• 作者: Martin-Urdiroz M;Deeks MJ;Horton CG;Dawe HR;Jourdain I
• 通讯作者: Jourdain I

Live-Cell Imaging of Cytoskeletal Responses and Trafficking During Fungal Elicitation.

真菌诱导过程中细胞骨架反应和运输的活细胞成像。

• DOI: 10.1007/978-1-0716-2867-6_22
• 发表时间: 2023
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Connerton AJ

An Immune-Responsive Cytoskeletal-Plasma Membrane Feedback Loop in Plants.

• DOI: 10.1016/j.cub.2018.05.014
• 发表时间: 2018-07-09
• 期刊: Current biology : CB
• 作者: Sassmann S;Rodrigues C;Milne SW;Nenninger A;Allwood E;Littlejohn GR;Talbot NJ;Soeller C;Davies B;Hussey PJ;Deeks MJ
• 通讯作者: Deeks MJ