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

植物不断地对环境中的非生物和生物压力做出反应，以确保生存。多聚ADP核糖聚合酶（PARP）作为多种应激信号（包括遗传毒性和病原体应激）的信号整合剂发挥作用。PARP催化ADP-核糖部分在其自身上和靶蛋白上的共价翻译后连接。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主要作为DNA损伤的传感器和信号放大器发挥作用，但在应激反应中的转录调控中的其他功能正在出现。病原体对植物的感染会引发DNA损伤。parp突变体对DNA损伤剂博来霉素不敏感，我们发现博来霉素引发拟南芥中水杨酸依赖性防御基因的转录激活。我们将检验PARP在植物免疫中作为病原诱导的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和靶蛋白的哪些氨基酸在胁迫下会发生ADP-核糖基化。该PARP相关蛋白家族以拟南芥自由基诱导的细胞死亡1（RCD 1）为创始成员，影响植物对盐和渗透胁迫的响应。RCD 1型蛋白是否具有酶活性仍有争议。我们已经解决了RCD 1 PARP结构域的晶体结构，并提供了分子和结构的证据，RCD 1型蛋白已经失去了ADP-核糖基化活性，因此可以被归类为假PARP。使用RCD 1伪PARP结构域结构作为基础，我们将定义区分活性PARP与伪PARP的分子特征。我们将测试RCD 1的假定活性位点是否为RCD 1在非生物胁迫反应中的功能所需。我们发现，RCD 1及其与RCD 11相似的paraminase（SRO 1）形成同源和异源聚体，可能由它们的N-末端WWE结构域介导，并且SRO 1稳定植物细胞中的RCD 1。我们将定义RCD 1和SRO 1同源和异源聚体形成的结构基础，并测试这些相互作用在拟南芥中的生物相关性。此外，我们确定MUT 9样激酶（MLK）作为RCD 1的第一个植物相互作用因子。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。我们将测试是否RCD 1与MLKs一起影响组蛋白H3修饰的RCD 1依赖的非生物胁迫反应基因。

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.