Biochemical characterisation of PARP and pseudo-PARP function in plant responses to biotic and abiotic stress

植物响应生物和非生物胁迫中 PARP 和假 PARP 功能的生化特征

• 批准号: 321374930
• 负责人: Dr. Lennart Wirthmüller
• 金额: --
• 依托单位: Abteilung Biochemie pflanzlicher Interaktionen
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/321374930?language=en
• 关键词: Biochemical; characterisation; PARP; pseudo; function

Plants constantly respond to abiotic and biotic stressors in their environment to ensure survival. Poly-ADP-ribose-polymerases (PARPs) function as signal integrators of multiple stress signals including genotoxic and pathogen stress. PARPs catalyse the covalent post-translational attachment of ADP-ribose moieties onto themselves and onto target proteins. However, target proteins of plant PARPs remain unknown hampering our understanding of how PARPs influence plant stress responses. PARPs primarily function as sensors and signal amplifiers of DNA damage but additional functions in transcriptional regulation in response to stress are emerging. Plant infection by pathogens triggers DNA damage. parp mutants are insensitive to the DNA damaging agent bleomycin and we found that bleomycin primes transcriptional activation of salicylic acid-dependent defence genes in Arabidopsis. We will test the hypothesis that PARPs function as sensors and signal amplifiers of pathogen-induced DNA damage in plant immunity. To gain a better understanding of PARP function we will identify PARP target proteins and determine which amino acids of PARPs and target proteins are subject to ADP-ribosylation under stress.In addition to PARPs, plant genomes encode an additional protein family with a predicted PARP domain. This PARP-related protein family with Arabidopsis RADICAL INDUCED CELL DEATH 1 (RCD1) as founding member influences plant responses to salt and osmotic stress. Whether RCD1-type proteins are enzymatically active is debated. We have solved the crystal structure of the RCD1 PARP domain and provide molecular and structural evidence that RCD1-type proteins have lost ADP-ribosylation activity and can therefore be classified as pseudo-PARPs. Using the RCD1 pseudo-PARP domain structure as foundation we will define molecular features that distinguish active PARPs from pseudo-PARPs. We will test if the postulated active site of RCD1 is required for RCD1 function in abiotic stress responses. We found that RCD1 and its paralogue SIMILAR TO RCD1 1 (SRO1) form homo- and heteromers, likely mediated by their N-terminal WWE domains, and that SRO1 stabilises RCD1 in plant cells. We will define the structural basis for RCD1 and SRO1 homo- and heteromer formation and test the biological relevance of these interactions in Arabidopsis. In addition, we identified MUT9-like kinases (MLKs) as the first in planta interactors of RCD1. MLKs phosphorylate histone H3 thereby mediating transcriptional stress responses to salt and osmotic stress. We will test whether RCD1 together with MLKs influences histone H3 modifications of RCD1-dependent abiotic stress response genes.

植物不断地对环境中的非生物和生物应激源做出反应，以确保生存。聚腺苷二磷酸核糖聚合酶(PARP)是多种胁迫信号的信号整合因子，包括遗传毒性胁迫和病原菌胁迫。PARP催化ADP-核糖部分与自身和靶蛋白的共价翻译后连接。然而，植物PARP的靶蛋白仍然未知，阻碍了我们对PARP如何影响植物胁迫反应的理解。PARP的主要功能是作为DNA损伤的传感器和信号放大器，但在应对压力的转录调控中也出现了额外的功能。植物感染病原体会引发DNA损伤。PARP突变体对DNA损伤剂博莱霉素不敏感，我们发现博莱霉素启动了拟南芥中水杨酸依赖的防御基因的转录激活。我们将验证PARP在植物免疫中作为病原体诱导的DNA损伤的传感器和信号放大器的假设。为了更好地了解PARP的功能，我们将鉴定PARP靶蛋白，并确定PARP和靶蛋白中的哪些氨基酸在胁迫下受到ADP核糖化。除了PARP之外，植物基因组还编码一个额外的蛋白家族，其中包含一个预测的PARP结构域。这个PARP相关蛋白家族以拟南芥自由基诱导的细胞死亡1(RCD1)为创始成员，影响植物对盐和渗透胁迫的反应。RCD1类蛋白是否具有酶活性还存在争议。我们已经解决了RCD1PARP结构域的晶体结构，并提供了RCD1型蛋白失去ADP核糖基化活性的分子和结构证据，因此可以归类为伪PARP。以RCD1伪PARP结构域结构为基础，我们将定义区分活性PARP和伪PARP的分子特征。我们将测试Rcd1在非生物胁迫反应中的功能是否需要Rcd1的假设活性部位。我们发现Rcd1及其类似物Rcd1 1(SRO1)形成同源和异构体，可能是由它们的N端WWE结构域介导的，并且SRO1在植物细胞中稳定Rcd1。我们将定义RCD1和SRO1同源和异构体形成的结构基础，并测试这些相互作用在拟南芥中的生物学相关性。此外，我们还发现MUT9样蛋白(MUT9-like kinase，MLK)是RCD1的第一个相互作用蛋白。MLKs磷酸化组蛋白H3，从而介导对盐和渗透胁迫的转录应激反应。我们将测试Rcd1和MLKs是否一起影响依赖Rcd1的非生物应激反应基因的组蛋白H3修饰。