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

我们以前已经鉴定了cerk1-4，它是拟南芥LysM-RLK CERK1(几丁质激发子受体激酶1)的一个点突变，它导致夸大的细胞死亡反应和对白粉病的抗性。在正向遗传筛查中，我们发现隐性突变NOLE1(无病变1)是cerk1-4细胞死亡表型的完全抑制者。作图和互补显示NOLE1在XLG2(超大G蛋白2)中携带突变。XLG蛋白含有一个C-末端结构域，与异三聚体G-蛋白的α-亚基(G-α)有很大的相似性，但其功能及其与其他亚基G-β和G-伽马的相互作用尚不清楚。G-β和伽马(而不是G-α)在PAMP和细胞死亡信号中作用于RLKs的下游。因此，我们计划研究XLG蛋白是否作为PAMP信号的中介并控制与RLKs相关的细胞死亡，而不是CERK1。XLG2与G-β和伽马的相互作用以及PAMP-、病原体或细胞死亡刺激也将被讨论。我们将特别强调亚细胞定位分析，因为我们有初步证据表明XLG2的核积累依赖于刺激。到目前为止，还没有关于XLG N-末端结构域功能的任何信息。此外，目前还不清楚体外证明的依赖于钙的GTPase活性在体内是否起作用。为了解决这些问题并对XLG2进行功能鉴定，我们将利用在我们的抑制子屏幕上发现的突变体(cerk1-4nole1-1)。我们将通过正向遗传方法和定向突变产生一组XLG2突变等位基因。这些在cerk1-4nole1-1背景中的表达将允许根据转化植物的细胞死亡表型(nole1-1互补)来评估XLG2等位基因的功能。通过这些突变研究获得的信息将被深入的细胞生物学和生化分析所跟踪，以阐明单个XLG2特征和结构域的功能。总之，我们的研究将为XLGs在植物天然免疫中的结构-功能关系、作用模式和亚细胞行为提供新的见解。

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.