CAREER: Genetic and Proteomic Analyses of MAP Kinase Pathways in Plant Disease Resistance

该职业计划旨在将研究融入教学，并促进生物化学/蛋白质组学方法纳入学生和博士后的分子/遗传学研究。 越来越多的生物化学和分子生物学研究表明，烟草丝裂原活化蛋白激酶（MAPK）SIPK和WIPK参与植物抗病信号转导。 然而，缺乏遗传学证据。 通过表达其上游激酶的活性突变体NtMEK 2DD来激活内源性SIPK和WIPK，导致诱导防御基因和过敏反应（HR）样细胞死亡。 拟南芥中同源途径AtMEK 4/AtMEK 5-AtMPK 6/AtMPK 3的鉴定允许对该MAPK途径的修饰剂和下游组分进行遗传筛选，这是该项目的主要目标之一。 激活标签，T-DNA，和EMS突变群体已产生类固醇诱导的转基因拟南芥。 将从T1、T2或M2群体中分离在类固醇处理后丧失细胞死亡表型的突变体，MAPK-活化-无死亡（mand）。将克隆负责表型改变的基因，其可以是：1）AtMPK 6和AtMPK 3本身; 2）AtMPK 6和/或AtMPK 3的底物; 3）AtMPK 6/AtMPK 3途径的基因外抑制剂和负调节剂，例如MAPK磷酸酶和支架蛋白;和4）参与HR细胞死亡的调节和执行的其他组分。 此外，蛋白质组学方法将被用来确定这两个MAPK的体内底物。 该项目的另一个主要目标是将研究融入课堂教学，并促进使用多维方法研究生理/形态表型，这对后基因组生物学的发展至关重要。 本科生，特别是那些来自代表性不足的群体的学生，将通过参加接触少数民族的机构方案而被积极招募。蛋白质组学的概念和技术将被纳入先进的生物化学实验室课程在此计划的任期内。在一个技术进步显著的时代，课堂教学必须吸收这些进步，并为学生提供最新的信息。 MAPK底物和HR的调节/执行者的鉴定将对植物细胞程序性死亡的研究做出重大贡献。 这项工作的一个实际延伸是，鉴定植物防御信号通路中的重要调控组分可能允许产生具有增强的抗病性的作物，这对于维持农业生产和改善我们的环境是重要的。

This CAREER program is designed to integrate research into teaching and to promote the incorporation of biochemical/proteomic methods into molecular/genetic research among students and post-docs. Increasing evidence from biochemical and molecular studies implicated SIPK and WIPK, two tobacco mitogen-activated protein kinases (MAPKs) in plant disease resistance signaling. However, genetic evidence is lacking. Activation of endogenous SIPK and WIPK by expressing the active mutant of their upstream kinase, NtMEK2DD leads to the induction of defense genes and hypersensitive response (HR)-like cell death. The identification of the homologous pathway in Arabidopsis, AtMEK4/AtMEK5-AtMPK6/AtMPK3 permitted genetic screens for modifiers and downstream components of this MAPK pathway, which is one of the major goals of this project. Activation tagging, T-DNA, and EMS mutant populations have been generated in steroid-inducible transgenic Arabidopsis. Mutants that lose cell death phenotype after steroid treatment, MAPK-activation-no-death (mand) will be isolated from T1, T2, or M2 population. Genes responsible for the phenotypic alterations will be cloned, which could be: 1) AtMPK6 and AtMPK3 themselves; 2) the substrate(s) of AtMPK6 and/or AtMPK3; 3) extragenic suppressors and negative regulators of AtMPK6/AtMPK3 pathway such as MAPK phosphatases and scaffold proteins; and 4) other components involved in the regulation and execution of HR cell death. In addition, proteomic approaches will be employed to identify the in vivo substrates of these two MAPKs. The other major goal of this project is to integrate research into classroom teaching and to promote the use of multi-dimensional approach to study a physiological/morphological phenotype, which is critical for the advance of post-genome biology. Undergraduate students, especially those from under represented groups will be actively recruited by participating in institutional programs that reach out to minorities. The concept and techniques of proteomics will be incorporated into the Advanced Biochemistry Laboratory course during the tenure of this program. In an era of significant technological advances, it is important for classroom teaching to absorb these advances and to prepare students with the most updated information. The identification of MAPK substrates and the regulators/executioners of HR would be a significant contribution to the study of plant programmed cell death. One practical extension of this work is that the identification of important regulatory components in plant defense signaling pathway may allow the generation of crops with enhanced disease resistance, which is important for sustaining agricultural production and improving our environment.