Analyzing plant stress granules in response to plant viruses

植物病毒具有重定向宿主机制和过程以建立生产性感染的能力。病毒-宿主相互作用导致植物细胞周期和转录控制的重编程、细胞死亡途径的抑制、细胞信号传导和蛋白质周转的干扰以及抑制防御途径。本文以双生病毒属的Abutilon mosaic virus（AbMV）、Cleome leaf crumple virus（ClLCrV）和Potyvirus属的Turnip mosic virus（TuMV）为模型系统，详细研究了植物病毒与寄主的相互作用。TuMV是一种ssRNA病毒，其基因组编码一个大的多蛋白和一个额外的小蛋白。多聚蛋白被切割成几种蛋白质，N端蛋白质称为P1。P1通常作为蛋白酶发挥作用，但我们怀疑它还有一个额外的功能，即干扰应激颗粒（SGs）。AbMV和ClLCrV属于菜豆病毒属，它们的基因组由两个环状ssDNA分子：DNA A和DNA B组成。DNA A编码对复制和腺苷酸化重要的所有蛋白质，而DNA B编码病毒转运必需的蛋白质。两个DNA B编码的蛋白质，核穿梭蛋白（NSP）和运动蛋白（MP），介导病毒的运输过程。几项研究提供了证据表明，NSP促进了病毒DNA（vDNA）进出细胞核的运输。我们最近发现NSP与Ras-GAP SH 3结构域结合蛋白（G3 BP）相互作用，G3 BP是SGs的组成部分。SG是当施加非生物/生物胁迫时形成的细胞质定位的RNA-蛋白质复合物。在SGs中，预组装的翻译复合物停止下调基因表达以促进免疫应答，但当应激条件减弱时，基因表达也可以恢复。G3 BP是哺乳动物系统中SGs形成的关键因子，并且经常被病毒靶向。NSP与G3 BP相互作用，这表明在胁迫响应中控制和调节植物SG以优化用于病毒蛋白生产的细胞条件。植物SGs的特征及其在病毒感染中的作用研究很少。因此，本研究的总体目标是分析和表征植物SGs，并阐明它们在植物胁迫反应中的作用，特别强调病毒感染。G3 BP是SG功能中的关键酶，因此，我们将通过分析环境和胁迫条件下的G3 BP OEX和KO拟南芥植物来表征拟南芥G3 BP。我们将遵循蛋白质组学方法来分析SG的组成。我们将研究病毒蛋白质的功能作用，e。G. NSP和P1，了解植物病毒感染周期中病毒和SG的相互作用。综合起来，这将使我们不仅能够了解细胞复合体，而且能够了解植物病毒需要采用的策略和机制，并重新编程植物细胞。

Plant viruses have the ability to redirect host machineries and processes to establish a productive infection. Virus-host interactions lead to the reprogramming of the plant cell cycle and transcriptional controls, inhibition of cell death pathways, interference with cell signaling and protein turnover, and suppression defense pathways. We have focused on the geminiviruses Abutilon mosaic virus (AbMV), Cleome leaf crumple virus (ClLCrV) and potyvirus Turnip mosic virus (TuMV) as model systems to investigate plant virus host interactions in detail. TuMV is an ssRNA virus which genome encodes for a large polyprotein and an additional small protein. The polyprotein is cleaved into several proteins and the N-terminal protein is called P1. P1 generally functions as a protease, but we suspect an additional function, the interference with stress granules (SGs). AbMV and ClLCrV belong to the genus Begomovirus and their genome consists of two circular ssDNA molecules: DNA A and DNA B. DNA A encodes for all proteins important for replication and encapsidation, whereas DNA B encodes proteins essential for viral transport. The two DNA B-encoded proteins, the nuclear-shuttle protein (NSP) and movement protein (MP), mediate the viral transport processes. Several studies provide evidence that NSP facilitates trafficking of viral DNA (vDNA) into and out of the nucleus. We recently could show that NSP interacts with the Ras-GAP SH3 domain binding protein (G3BP), which is an integral part of SGs. SGs are cytoplasmic localized RNA-protein complexes that form when abiotic/biotic stresses are applied. In SGs preassembled translation complexes are stalled to down-regulate gene expression to promote immune response, but gene expression can also be resumed when stress conditions diminish. G3BP is a key factor for SGs formation in the mammalian system and often targeted by viruses. The NSP interacts with G3BP, which suggests control and regulation of plant SGs in stress response to optimize cellular conditions for the production of viral proteins. Characterization of plant SGs and their role in virus infection is poorly investigated. Thus, the overall aim of this study is to analyze and characterize plant SGs and to elucidate their role in plant stress response, with special emphasis on viral infections. The G3BP is the key enzyme in SG function, and therefore, we will characterize the Arabidopsis G3BP(s) by analyzing G3BP OEX and KO Arabidopsis plants under ambient and stress conditions. We will follow a proteomics approach to analyze the composition of SGs. We will investigate the functional role of virus proteins, e. g. NSP and P1, to understand the interplay of virus and SGs in the plant virus infection cycle. Taken together this will enable us to understand not only the cellular complexes but also the strategies and mechanisms a plant virus needs to employ and to re-program a plant cell to its favor.