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

该职业计划旨在将研究与教学相结合，并促进学生和博士后将生化/蛋白质组学方法纳入分子/遗传研究。越来越多的生化和分子研究证据表明，烟草丝裂原活化蛋白激酶（MAPKs） SIPK和WIPK参与植物抗病信号传导。然而，缺乏遗传证据。通过表达上游激酶NtMEK2DD的活性突变体激活内源性SIPK和WIPK，可诱导防御基因和超敏反应（hypersensitive response， HR）样细胞死亡。在拟南芥中鉴定同源通路AtMEK4/AtMEK5-AtMPK6/AtMPK3为该MAPK通路的修饰子和下游组分的遗传筛选提供了可能，这是本项目的主要目标之一。激活标记、T-DNA和EMS突变群体已经在类固醇诱导转基因拟南芥中产生。在类固醇治疗后失去细胞死亡表型的突变体，mapk激活-无死亡（mand）将从T1， T2或M2群体中分离出来。引起表型改变的基因将被克隆，可能是：1)AtMPK6和AtMPK3本身；2) AtMPK6和/或AtMPK3的底物；3) AtMPK6/AtMPK3通路的基因外抑制因子和负调节因子，如MAPK磷酸酶和支架蛋白；4)参与HR细胞死亡调控和执行的其他成分。此外，蛋白质组学方法将用于鉴定这两个MAPKs的体内底物。该项目的另一个主要目标是将研究整合到课堂教学中，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群体的本科生，将通过参与面向少数族裔的机构项目积极招收。蛋白质组学的概念和技术将在本课程期间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，并为学生准备最新的信息是很重要的。MAPK底物和HR调控因子/执行者的鉴定将对植物程序性细胞死亡的研究做出重大贡献。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使作物具有增强的抗病性，这对维持农业生产和改善我们的环境至关重要。

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.