Analysis of the EDRI MAPKKK Pathway in Plants

植物中 EDRI MAPKKK 途径的分析

基本信息

批准号：
6880066
负责人：
Roger W. Innes
金额：
$ 26.08万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-04-01 至 2006-03-31
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant):The EDR1 gene of Arabidopsis encodes a MAP Kinase Kinase Kinase that functions as a negative regulator of defense responses and leaf senescence. An overlap between plant defense responses and senescence at the molecular level has been well documented; however, the mechanistic basis of this overlap is not understood. The research proposed here is aimed at answering three fundamental questions regarding the EDR1 signal transduction pathway: 1) What environmental and cellular signals regulate activity of the EDR1 MAPKKK? 2) What MAP kinase(s) are activated by EDR1? and 3) What plant responses are directly regulated by this (these) MAP kinases? Because EDR1 is homologous to CTR1, which is regulated by histidine kinase response regulators, we predict that EDR1 may also be regulated by a histidine kinase. The recent identification of a cytokinin receptor that belongs to this family, along with the classic observation that cytokinins inhibit leaf senescence, has led us to hypothesize that EDR1 may function in a cytokinin signal transduction pathway that negatively regulates both age-induced and pathogen-induced leaf senescence. Five specific aims will be pursued to test this hypothesis. First, the edr1 mutant will be assessed for resistance to cytokinins, and cytokinin receptor mutants will be assessed for resistance to pathogens and to dark-induced senescence. Second, a genetic screen will be performed to identify mutations that suppress or enhance the edr1 mutant phenotype. These screens will uncover additional genes in the EDR1 pathway, as well as identify other pathways that interact with the EDR1 pathway. Third, we will evaluate global gene expression in Arabidopsis plants expressing constitutively activated forms of EDR1. The phenotypic responses of these plants to pathogens will also be investigated. These analyses will reveal specific genes regulated by EDR1. Fourth, proteins that physically interact with EDR1 will be identified using yeast two-hybrid analyses and in vitro kinase assays. Fifth, Arabidopsis MAP kinases that are activated by EDR1 will be identified using a myelin basic protein (MBP) phosphorylation assay. This assay involves coexpression of EDR1 and candidate MAP kinases in Arabidopsis protoplasts. The candidate MAPKs are then immunoprecipitated and assayed for their ability to phosphorylate MBP, a universal substrate of MAPKs. These analyses, together, will enable us to assemble the EDR1 MAPKKK pathway and identify signal inputs and outputs. This information, in turn, may provide new insights into the regulation of programmed cell death in plants and the overlap between normal senescence processes and pathogen defense.

描述（申请人提供）：拟南芥的EDR1基因编码一个MAP 激酶激酶发挥防御负调节作用的激酶反应和叶片衰老。植物防御反应和植物防御反应之间的重叠分子水平上的衰老已有充分记录；然而，这种重叠的机制基础尚不清楚。这里提出的研究旨在回答有关 EDR1 信号的三个基本问题转导途径：1）环境和细胞信号调节什么 EDR1 MAPKKK 的活性？ 2) EDR1 激活哪些 MAP 激酶？和 3) 哪些植物反应是由这些 MAP 激酶直接调节的？因为EDR1与CTR1同源，受组氨酸激酶调节反应调节剂，我们预测 EDR1 也可能受到组氨酸的调节激酶。最近鉴定出属于此的细胞分裂素受体家族，以及细胞分裂素抑制叶的经典观察衰老，使我们推测 EDR1 可能在细胞分裂素中发挥作用信号转导途径对年龄诱导的和病原体引起的叶片衰老。将测试五个具体目标这个假设。首先，将评估 edr1 突变体的抗性将评估细胞分裂素和细胞分裂素受体突变体的抗性病原体和黑暗诱导的衰老。其次，将进行基因筛查进行以确定抑制或增强 edr1 突变体的突变表型。这些筛选将发现 EDR1 通路中的其他基因，如以及识别与 EDR1 通路相互作用的其他通路。第三，我们将评估拟南芥植物中的全局基因表达 EDR1 的组成型激活形式。这些的表型反应植物对病原体的影响也将受到调查。这些分析将揭示受 EDR1 调节的特定基因。第四，物理相互作用的蛋白质带有 EDR1 的病毒将通过酵母双杂交分析和体外鉴定激酶测定。第五，被 EDR1 激活的拟南芥 MAP 激酶将使用髓磷脂碱性蛋白 (MBP) 磷酸化测定进行鉴定。这测定涉及 EDR1 和候选 MAP 激酶在拟南芥中的共表达原生质体。然后对候选 MAPK 进行免疫沉淀并进行检测它们磷酸化 MBP（MAPK 的通用底物）的能力。这些综合分析将使我们能够组装 EDR1 MAPKKK 通路并识别信号输入和输出。这些信息反过来可能会提供新的对植物程序性细胞死亡调控及其重叠的见解正常衰老过程和病原体防御之间的关系。