Functional and structural analyses of plant ATG8-RabGAP interactions

由于缺乏循环系统和可移动的免疫细胞，植物免疫依赖于细胞自主免疫。这种免疫的一个关键组成部分是自噬的分解代谢过程，曾经被认为是一种非特异性降解途径。病原体特异性植物货物受体的发现提示自噬在免疫中的作用。与许多植物病原体一样，马铃薯晚疫病的卵菌疫霉（Phytophthora infestans）依靠宿主通过蛋白质效应器调节和抑制免疫来感染宿主。最近的一项研究表明，P. infestans分泌的效应物perrd54通过它们共同的ATG8相互作用基序（AIM）与宿主货物受体Joka2结合核心自噬蛋白ATG8CL。效应器和货物受体都以AIM依赖的方式触发自噬体的形成。由于这样一个重要的途径被单独的结合激活，这表明可能存在一个负调节因子。质谱分析显示，ATG8CL与RabGTPase激活蛋白Rab3GAP有很强的相互作用。生物信息学分析表明Rab3GAP携带一个假定的AIM，指出在ATG8CL自噬途径中具有调节作用。先前的研究表明，Rab3GAP不仅在体内与ATG8CL相互作用，而且还以AIM依赖的方式减少ATG8CL自噬体。此外，初步数据显示Rab3GAP增加了对P. infeans的易感性。本研究的主要目的是在细胞、分子和结构水平上分析Rab3GAP在ATG8CL自噬中的作用。此外，我的目的是阐明Rab3GAP在对P. Infestans的免疫中的作用，并在更广泛的范围内阐明ATG8CL自噬的作用。我打算用烟叶作为感染、蛋白质表达和细胞生物学的模型系统。我还打算广泛使用农杆菌转化系统进行瞬时表达试验。这将为创造CRISPR/Cas9转基因品系铺平道路。为了实现这一点，我将重点关注这个项目的三个目标：1。阐明Rab3GAP在ATG8CL通路中的作用。为了研究Rab3GAP在自噬中的作用，我打算采用细胞和分子生物学的多方位方法。首先，我的目的是通过体外相互作用实验来证实Rab3GAP与ATG8CL的aim的相互作用。此外，我将采用Co-IP，然后采用质谱分析来确定与ATG8CL途径相关的Rab3GAP的相互作用伙伴。我将通过监测Rab3GAP相互作用子沉默或过表达后的自噬活性来研究Rab3GAP相互作用子在自噬中的作用。剖析Rab3GAP在感染大肠杆菌免疫中的作用。我们的目的是阐明Rab3GAP引起的对P. Infestans易感性增加的机制。第一步将是确定对免疫的影响是否是由于ATG8CL途径。其次，我们打算研究Rab3GAP的其他领域。GAP活性位点特别有趣，确定相关的Rab(s)将使我们深入了解Rab3GAP在膜交通通路网络中的作用。Rab3GAP及其合作伙伴在自噬中的结构和功能分析我们方法的最后一个方面是在结构上表征Rab3GAP的相互作用，并深入了解其在自噬体形成中的抑制作用。了解Rab3GAP与ATG8CL自噬通路其他成员的结合动力学可以帮助我们了解这些通路中的相互作用网络。我们打算通过修饰GAP活性位点的催化精氨酸和gtp锁定任何潜在的Rab3GAP来稳定相互作用，从而确定与Rab3GAP相关的Rabs。将结构观察与蛋白质功能联系起来，将使我们能够描述Rab3GAP的基本基序，甚至可能描述Rab3GAP超家族其他成员的基本基序。

Due to the lack of circulatory system and mobile immune cells, plant immunity hinges on cell autonomous immunity. A key component of this immunity is the catabolic process of autophagy, once thought to be a non-specific degradation pathway. The discovery of pathogen-specific plant cargo receptors suggested a role for autophagy in immunity. Like many plant pathogens, Phytophthora infestans, the oomycete responsible for late blight disease in potato, relies on host modulation and suppression of immunity through proteinaceous effectors to infect its host. A recent study has shown that the P. infestans secreted effector PexRD54 outcompetes the host cargo receptor Joka2 for binding of the core autophagy protein ATG8CL through their common ATG8 interacting motif (AIM). Both the effector and cargo receptor trigger autophagosome formation in an AIM dependent manner. With such an essential pathway being activated by binding alone, it was suggested that there could exist a negative regulator. Mass spectrometry analysis of ATG8CL revealed a strong interaction with a RabGTPase activating protein, Rab3GAP. Bioinformatic analysis has shown that Rab3GAP carries a putative AIM, pointing towards a regulatory role in the ATG8CL autophagy pathway. Previous work showed that not only does Rab3GAP interact with ATG8CL in vivo but also reduces ATG8CL autophagosomes in an AIM dependent manner. Furthermore, preliminary data shows that Rab3GAP increases susceptibility to P. infestans.The primary objective of this study is to analyse Rab3GAP's role in the ATG8CL autophagy at the cellular, molecular and structural level. Furthermore, I aim to elucidate Rab3GAP's role in immunity against P. Infestans and to a wider extent, the role of the ATG8CL autophagy. I intend to use Nicotiana Benthamiana as a model system for infections, expression of proteins and cell biology. I also intend to make extensive use of the Agrobacterium transformation system for transient expression assays. This will pave the way for the creation of CRISPR/Cas9 transgenic lines. To achieve this, I will focus on three aims for this project:1. Elucidating Rab3GAP's role in the ATG8CL pathway.In order to investigate Rab3GAP's role in autophagy, I intend to use a multi-faceted approach of cellular and molecular biology. First, I aim to confirm Rab3GAP's interaction with ATG8CL's AIM through in vitro interaction assays. Furthermore, I will employ Co-IP followed by mass spectrometry analysis to determine the interacting partners of Rab3GAP that are associated to the ATG8CL pathway. I will investigate the role of candiate Rab3GAP interactors in autophagy via monitoring autophagic activity upon silencing or overexpression of the Rab3GAP interactors.2. Dissecting Rab3GAP's role in immunity against P. Infestans.We aim to elucidate the mechanisms behind the increased suscepbility to P. Infestans caused by Rab3GAP. The first step would be to determine if the effect on immunity is due to the ATG8CL pathway. Secondly, we intend to look into the other domains of Rab3GAP. The GAP active site is of particular interest and indentifying the associated Rab(s) would give us insight into Rab3GAP's role in the network of membrane traffic pathways.3. Structural and functional analyses of Rab3GAP and its partners in autophagyThe final facet of our approach is to structurally characterise Rab3GAP's interactions and gain insights into its suppresive role in autophagosome formation. Understanding Rab3GAP's binding dynamics with other members of the ATG8CL autophagic pathway could help us understand the network of interactions in the pathways. We intend to identify the Rab(s) associated with Rab3GAP by modifying the catalytic arginine in the GAP active site and GTP-locking any potential Rabs to stabilise the interaction. Linking structural observations with protein function would allow us to describe essential motifs in Rab3GAP and maybe even in other members of the Rab superfamily.