Post-translational Modifications as Modulators of Crop Plant Defense Signaling: a Systems Approach

Pi：Jean T.Greenberg(芝加哥大学)Copis：Stephen J.Kron(芝加哥大学)和Howard C.Hang(洛克菲勒大学)病原微生物与植物的相互作用引发的信号通路可以决定感染的结果。在微生物感知之后发展起来的翻译后修饰(PTM)使植物能够激活促进寄主抗性的信号事件网络的灵活机制。然而，通过微生物注入植物体内的蛋白质，如乙酰基转移酶(AC-TR)效应器，可以修饰宿主蛋白，然后由于折叠、细胞位置、酶活性、结合其他蛋白的能力和/或稳定性的改变，宿主蛋白的信号功能受到影响。经济上重要的植物和新技术的基因组序列数据的最近的可获得性使得在系统水平上对光敏核不育的全球分析和进一步剖析它们对抗病的影响成为可能。这个项目的目标是剖析植物蛋白在感染过程中的PTM如何调节作物抵御病原菌的能力。该项目将(1)利用来自细菌和寄主底物的AC-tr效应器优化研究蛋白质乙酰化的体外方法；(2)使用AC-tr效应器对番茄寄主蛋白的乙酰化进行全局分析，并测试选定的乙酰化靶标在植物防御中的作用；以及(3)通过分析全球变化对防御信号的影响，评估乙酰化对防御信号的影响。该项目将开发用于(并向社区传播)的试剂，以定量研究信号动力学，并将乙酰化引起的动力学变化与感染结果联系起来。在这项研究中对光温敏核不育进行的全球分析将提高基因组研究的价值，并提供对防御信号的系统水平的理解，这将扩大作物改良的机会。该项目将整合研究和教学，为项目人员提供蛋白质组学、化学生物学、生物化学、分子遗传学和植物-微生物相互作用方面的跨学科培训。它还将产生广泛的影响，向社区提供分析防御信号的新工具和试剂，并通过举办关于分析PTMS的研讨会，使其他大学的学生有机会接受实践培训。吸纳代表人数不足的群体的学生也将对扩大不同群体对研究的参与产生影响。该项目的一个主要创新是开发和应用了新的方法，用于在全球范围内分析蛋白质中不同的乙酰化位点与其他潜在的竞争性修饰的关系。该方法将对与农作物中其他蛋白质修饰相关的乙酰化研究具有变革性，并适用于广泛种类的植物和其他生物体。一个关键的外展部分将是为来自其他机构的学生举办为期三天的讲习班，以学习检测植物蛋白质修饰的原理。学生将获得使用质谱学生成和分析翻译后修饰数据的实践经验。更多的外联工作将包括参加关于该项目的讨论和与参观芝加哥大学和洛克菲勒大学的学校团体进行示威，与教师协调以使科学课程现代化，以及在社区集市上与公众讨论研究问题。该项目将为项目人员提供重要的跨学科培训，包括化学生物学、蛋白质组学、生物化学、分子遗传学和植物病理学，包括博士后学者和研究生、学士学位后、本科生和高中阶段的学生。项目人员将来自不同的群体，以扩大对研究科学的参与。最后，该项目将开发有用的试剂(抗体)和方案来检测番茄蛋白质的修饰。公众将可以通过同行评议的出版物和项目网站获得该项目产生的信息，如需要修改的特定蛋白质和监测修改的方法。公众还可以根据要求获得抗体和西红柿种子等试剂以及初级MS/MS光谱。蛋白质组学数据集还将存储在一个或多个数据库中，如P3DB(http://p3db.org/).

PI: Jean T. Greenberg (University of Chicago)CoPIs: Stephen J. Kron (University of Chicago) and Howard C. Hang (Rockefeller University)The interaction of pathogenic microbes with plants elicits signaling pathways that can dictate the outcome of infection. Post-translational modifications (PTMs) elaborated after microbial perception enable a flexible mechanism for plants to activate networks of signaling events that promote host resistance. However, proteins such as acetyltransferase (AC-TR) effectors that are injected into plants by microbes can modify host proteins, which are then affected in their signaling functions due to alterations in folding, cellular location, enzymatic activity, ability to bind other proteins and/or stability. The recent availability of genomic sequence data of economically important plants and new technologies now makes it feasible to do systems-level global analysis of PTMs and to further dissect their impact on disease resistance. The goal of this project is to dissect how PTMs of plant proteins during infection modulates the ability of crop plants to defend against pathogenic bacteria. The project will (1) optimize in vitro methods for studying protein acetylation using AC-TR effectors from bacteria and host substrates; (2) perform global analyses of host protein acetylation in tomato using AC-TR effectors and test the role of select acetylated targets in plant defense; and (3) evaluate the impact of acetylation on defense signaling by analyzing global changes host protein phosphorylation, another PTM that is potentially competitive with acetylation. The project will develop reagents that will be used (and disseminated to the community) to quantitatively study signaling dynamics and relate changes in dynamics due to acetylation to infection outcomes. The global analysis of PTMs performed during this research will enhance the value of genomic studies and give a systems-level understanding of defense signaling that will expand the opportunities for crop improvements. The project will integrate research and teaching to provide interdisciplinary training to project personnel in proteomics, chemical biology, biochemistry, molecular genetics and plant-microbe interactions. It will also have broad impact by providing new tools and reagents to the community for analyzing defense signaling, and giving access to students from other universities to practical training through workshops on the analysis of PTMs. Inclusion of students from underrepresented groups will also have an impact on broadening participation in research of diverse groups. A major innovation of the project is the development and application of new methods for analyzing different acetylation sites in proteins at a global scale in relation to other potentially competitive modifications. The approach will be transformative for the study of acetylation in relation to other protein modifications in crop plants and applicable to a broad variety of plants and other organisms. A key outreach component will be three-day workshops for students from other institutions to learn the principles of detecting modifications of plant proteins. Students will gain hands-on experience on the generation and analysis of data on post-translational modifications using mass spectrometry. Additional outreach efforts will include participation in discussions about this project and demonstrations with school groups that visit the University of Chicago and Rockefeller University, coordination with teachers to modernize science curricula and discussions about research with the public at community fairs. This project will provide important interdisciplinary training in chemical biology, proteomics, biochemistry, molecular genetics and plant pathology to project personnel, including postdoctoral scholars and students at the graduate, post-baccalaureate, undergraduate and high school levels. Project personnel will be drawn from diverse groups to broaden participation in research science. Finally, the project will develop useful reagents (antibodies) and protocols to detect modifications in tomato proteins. The public will have access to the information generated by the project such as specific proteins that are subject to modification and approaches to monitor modifications through peer-reviewed publications and the project website. The public will also have access to reagents generated such as antibodies and tomato seeds as well as primary MS/MS spectra upon request. Proteomics datasets will also be deposited in one or more databases such as P3DB (http://p3db.org/).