Study of chloroplast stromules during PCD and inter-organellar communication

项目摘要 该项目的重点是叶绿体作为产生免疫信号的核心参与者， 调节针对病原体感染的先天免疫应答所需的程序性细胞死亡（PCD）。 虽然线粒体在哺乳动物PCD过程中起着核心作用，但新出现的证据表明，在植物中， 叶绿体在执行限制病原体传播的局部PCD中具有关键功能。叶绿体 除了参与产生免疫信号如活性氧（ROS）和 防御激素水杨酸（SA）也直接参与病原菌的识别。有趣的是 叶绿体在免疫应答过程中动态地改变其形态，并发出管状突起 叫做基质。这些被诱导的基质利用细胞骨架延伸然后锚到细胞核， 其促进叶绿体的核周聚集并运输叶绿体产生的ROS和防御 蛋白质进入细胞核。本申请的总体目标是使用新的细胞生物学、遗传学 蛋白质组学和计算方法，以解开小孢子驱动的半透明体的机制基础 叶绿体聚类，并确定从叶绿体释放的免疫信号的身份和功能， 原子核。具体来说，目标1将研究植物先天免疫过程中的细胞器和细胞骨架动态。 将进一步开发新的计算方法来量化小孢子特征和动态， 应用于了解叶绿体，它们的基质，和细胞骨架动态在先天免疫。两 植物和动物病原体的目标是细胞骨架，这一目标将研究病原体效应如何改变细胞骨架。 细胞骨架和相关的细胞器动力学作为毒力策略。目标2将研究免疫信号 所需的小孢子诱导和叶绿体信号的释放。不同活性氧之间的关系 免疫应答期间的细胞器运动和PCD将使用以下组合进行检查： 目标1中开发的遗传工具和计算方法。在目标3中，已知的叶绿体衍生的免疫 信号将被表征，并且新的蛋白质信号将被鉴定。H_2O_2的下游靶点 核将被阐明和研究。质谱法将被用来识别蛋白质， 从叶绿体到细胞核。遗传学方法将被用来研究它们在PCD过程中的作用。最后 将研究先天免疫期间叶绿体释放的机制，以确定它是否类似于 在哺乳动物细胞凋亡过程中线粒体释放。了解不同细胞器在PCD过程中的作用 和先天免疫将提供细胞死亡和细胞存活过程的统一机制基础， 对感染性病原体的反应。我们的模型系统的结果将广泛影响对 细胞器到核的通讯，影响对传染病的先天免疫。

Project Summary This project focuses on chloroplasts as central player in the generation of immune signals and the regulation of programmed cell death (PCD) required for innate immune responses against pathogen infection. Although mitochondria play a central role during mammalian PCD, emerging evidence suggests that in plants chloroplasts have a critical function in executing localized PCD that limits pathogen spread. Chloroplasts in addition to being involved in the generation of immune signals such as reactive oxygen species (ROS) and defense hormone salicylic acid (SA), also participate directly in the recognition of pathogen. Interestingly, the chloroplasts dynamically change their morphology during immune responses and send out tubular projections called stromules. These induced stromules use the cytoskeleton to extend and then anchor to the nucleus, which facilitate perinuclear clustering of chloroplasts and transport chloroplast-generated ROS and defense proteins to the nucleus. The overall goal of this application is to use combination of novel cell biology, genetics, proteomics and computational approaches to unravel the mechanistic basis of stromule driven periunclear chloroplast clustering and determine the identity and function of immune signals released from chloroplasts to nuclei. Specifically, Aim 1 will examine organelle and cytoskeleton dynamics during plant innate immunity. Novel computational methods quantifying stromule features and dynamics will be further developed and applied to understand chloroplasts, their stromules, and cytoskeleton dynamics during innate immunity. Both plant and animal pathogens target the cytoskeleton, and this aim will examine how pathogen effectors alter the cytoskeleton and related organelle dynamics as a virulence strategy. Aim 2 will investigate immune signals required for stromule induction and the release of chloroplast signals. The relationship of different ROS sources, organelle movement and PCD during immune responses will be examined using a combination of genetic tools and the computational methods developed in Aim 1. In Aim 3, known chloroplast-derived immune signals will be characterized and novel protein signals will be identified. The downstream targets of H2O2 in the nucleus will be elucidated and studied. Mass spectrometry will be used to identify proteins that are transported from chloroplasts to nuclei. Genetic approaches will be employed to examine their role during PCD. Lastly, the mechanism of release from chloroplasts during innate immunity will be studied to determine if it is akin to mitochondrial release during apoptosis in mammals. Understanding the role of different organelles during PCD and innate immunity will provide a unified mechanistic basis of cell death and cell survival process that occur in response to infectious pathogens. The results from our model systems will impact broadly on understanding of organelle-to-nuclear communication that influence innate immunity against infectious diseases.