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

这一职业计划旨在将研究整合到教学中，并促进在学生和博士后中将生化/蛋白质组学方法纳入分子/遗传学研究。越来越多的生化和分子研究证据表明，SIPK和WIPK这两个烟草丝裂原活化蛋白激酶(MAPK)参与了植物抗病信号转导。然而，缺乏遗传证据。内源性SIPK和WIPK通过表达其上游活性突变体NtMEK2DD而被激活，导致防御基因的诱导和类似超敏反应(HR)的细胞死亡。在拟南芥中鉴定同源途径AtMEK4/AtMEK5-AtMPK6/AtMPK3允许对该MAPK途径的修饰物和下游成分进行遗传筛选，这是本项目的主要目标之一。在类固醇诱导的转基因拟南芥中已经产生了激活标签、T-DNA和EMS突变群体。在类固醇治疗后失去细胞死亡表型、MAPK激活无死亡(MAND)的突变株将从T1、T2或M2群体中分离出来。导致表型改变的基因将被克隆，这些基因可能是：1)AtMPK6和AtMPK3本身；2)AtMPK6和/或AtMPK3的底物(S)；3)AtMPK6/AtMPK3途径的基因外抑制和负调控因子，如MAPK磷酸酶和支架蛋白；以及4)参与调节和执行HR细胞死亡的其他成分。此外，蛋白质组学方法将被用来鉴定这两个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来研究生理/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人数不足的群体的学生，将通过参加面向少数族裔的机构项目来积极招募。蛋白质组学的概念和技术将在本课程期间被纳入高级生物化学实验室课程。在一个技术进步显著的时代，课堂教学吸收这些进步并让学生准备好掌握最新的信息是很重要的。MAPK底物和HR调节子/执行子的识别将对植物细胞程序性死亡的研究做出重要贡献。这项工作的一个实际扩展是，识别植物防御信号通路中的重要调控成分可能会产生抗病能力增强的作物，这对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.