Functional characterization of Arabidopsis Extra Large G proteins (XLGs) and their role as signal transducers downstream of receptor kinases

拟南芥超大 G 蛋白 (XLG) 的功能特征及其作为受体激酶下游信号转导器的作用

• 批准号: 278618894
• 负责人: Professor Dr. Volker Lipka
• 金额: --
• 依托单位: Abteilung Zellbiologie der Pflanze
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2019-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/278618894?language=en
• 关键词: Functional; characterization; Arabidopsis; Extra; Large

We have previously characterized cerk1-4, a point mutant of the Arabidopsis LysM-RLK CERK1 (Chitin Elicitor Receptor Kinase 1), which causes exaggerated cell death responses and resistance to powdery mildews. In a forward genetic screen, we identified the recessive mutant nole1 (No Lesions 1) as a complete suppressor of the cerk1-4 cell death phenotype. Mapping and complementation revealed nole1 to carry a mutation in XLG2 (Extra Large G-Protein 2). XLG proteins contain a C-terminal domain with significant similarity to the alpha-subunit of heterotrimeric G-proteins (G-alpha), but their function and interaction with the other subunits, G-beta and G-gamma, is poorly understood. G-beta and gamma (but not G-alpha) have been shown to act downstream of RLKs in PAMP and cell death signaling. Thus, we plan to investigate if XLG proteins act as mediators of PAMP signaling and control cell death associated with RLKs other than CERK1. XLG2 interaction with G-beta and gamma in conjunction with PAMP-, pathogen or cell death stimuli will also be addressed. Particular emphasis will be placed on subcellular localization analyses, as we have preliminary evidence for stimulus-dependent nuclear accumulation of XLG2. To date, no information at all is available on the function of XLG N-terminal domains. Also, it is not known if the Ca2+-dependent GTPase activity that has been demonstrated in vitro plays any role in vivo. To address these questions and to functionally characterize XLG2, we will make use of the mutant identified in our suppressor screen (cerk1-4 nole1-1). We will generate a collection of XLG2 mutant alleles by forward genetic approaches as well as targeted mutagenesis. Expression of these in the cerk1-4 nole1-1 background will allow assessment of functionality of the XLG2 alleles based on the cell death phenotype of the transformed plant (nole1-1 complementation). The information gained by these mutational studies will be followed up with in-depth cell biological and biochemical analyses to elucidate the function of the individual XLG2 features and domains. Together, our research will provide novel insights into structure-function relationships, mode of action and subcellular behavior of XLGs within plant innate immunity.

我们以前的特点cerk 1 -4，拟南芥LysM-RLK CERK 1（几丁质激发子受体激酶1）的点突变体，导致夸大的细胞死亡反应和抗白粉病。在正向遗传筛选中，我们确定了隐性突变体nole 1（无病变1）作为cerk 1 -4细胞死亡表型的完全抑制因子。定位和互补显示nole 1携带XLG 2（特大G蛋白2）突变。XLG蛋白含有与异源三聚体G蛋白（G-α）的α亚基具有显著相似性的C-末端结构域，但其功能和与其他亚基G-β和G-γ的相互作用知之甚少。G-β和γ（但不是G-α）已被证明在PAMP和细胞死亡信号传导中作用于RLK的下游。因此，我们计划研究XLG蛋白是否作为PAMP信号传导的介质并控制与ERK 1以外的RLK相关的细胞死亡。XLG 2与G-β和γ的相互作用以及PAMP-、病原体或细胞死亡刺激也将被解决。特别强调将放在亚细胞定位分析，因为我们有刺激依赖的XLG 2核积累的初步证据。迄今为止，没有任何关于XLG N-末端结构域功能的信息。此外，尚不清楚体外证实的Ca 2+依赖性GT3活性是否在体内发挥任何作用。 为了解决这些问题，并在功能上表征XLG 2，我们将利用我们的抑制筛选（cerk 1 -4 nole 1 -1）中确定的突变体。 我们将通过正向遗传学方法以及靶向诱变产生XLG 2突变等位基因的集合。这些在cerk 1 -4 nole 1 -1背景中的表达将允许基于转化植物的细胞死亡表型（nole 1 -1互补）评估XLG 2等位基因的功能性。通过这些突变研究获得的信息将通过深入的细胞生物学和生物化学分析来跟进，以阐明单个XLG 2特征和结构域的功能。总之，我们的研究将为植物先天免疫中XLG的结构-功能关系、作用模式和亚细胞行为提供新的见解。