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

PI: Jean T. Greenberg (University of Chicago)CoPIs: Stephen J. Kron （University of Chicago）和Howard C. Hang （University of Rockefeller）病原微生物与植物的相互作用引发了可以决定感染结果的信号通路。在微生物感知后阐述的翻译后修饰（PTMs）为植物激活促进宿主抗性的信号事件网络提供了一种灵活的机制。然而，微生物注入植物体内的乙酰转移酶（AC-TR）效应物等蛋白质可以修饰宿主蛋白质，然后由于折叠、细胞位置、酶活性、结合其他蛋白质的能力和/或稳定性的改变，宿主蛋白质的信号功能受到影响。最近经济上重要植物的基因组序列数据和新技术的可用性使得对PTMs进行系统级全球分析并进一步剖析其对抗病性的影响成为可能。本项目的目标是剖析植物蛋白在感染期间的ptm如何调节作物植物抵御致病菌的能力。该项目将(1)优化利用细菌和宿主底物中的AC-TR效应物研究蛋白质乙酰化的体外方法；(2)利用AC-TR效应物对番茄宿主蛋白乙酰化进行全局分析，并测试选定乙酰化靶点在植物防御中的作用；(3)通过分析宿主蛋白磷酸化的全局变化来评估乙酰化对防御信号的影响，宿主蛋白磷酸化是另一个可能与乙酰化竞争的PTM。该项目将开发试剂，用于（并向社区传播）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PTMs进行的全球分析将提高基因组研究的价值，并对防御信号提供系统级的理解，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、分子遗传学和植物与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，为社区提供新的工具和试剂，用于分析防御信号，并通过ptm分析研讨会为其他大学的学生提供实际培训。纳入来自代表性不足群体的学生也将对扩大不同群体的研究参与产生影响。该项目的一个主要创新是开发和应用新的方法来分析蛋白质中不同的乙酰化位点在全球范围内与其他潜在的竞争性修饰的关系。该方法将对与作物植物中其他蛋白质修饰相关的乙酰化研究具有变革性，并适用于各种各样的植物和其他生物。一个重要的外展组成部分将是为来自其他机构的学生提供为期三天的讲习班，以学习检测植物蛋白修饰的原理。学生将获得使用质谱法生成和分析翻译后修饰数据的实践经验。其他的外联工作将包括参与关于这个项目的讨论，并与访问芝加哥大学和洛克菲勒大学的学校团体进行示范，与教师协调使科学课程现代化，并在社区博览会上与公众讨论研究。本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、学士后、本科和高中阶段的学生。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。公众将可以获得项目产生的信息，例如需要修改的特定蛋白质，以及通过同行评审出版物和项目网站监测修改的方法。公众还可以根据要求获得抗体和番茄种子等生成的试剂以及初级质谱/质谱。蛋白质组学数据集也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/).