Functional analyses of NPR1 in plant defense

描述(申请人提供)：先天免疫机制在进化中是保守的。拟南芥NPR1蛋白是植物防卫反应中基因表达的主要调节因子。与哺乳动物免疫调节因子NF-kB类似，NPR1蛋白在诱导后发生核转位。在静息状态下，NPR1通过二硫键与细胞器翻译延伸因子EF-Tu形成复合体存在于细胞质中。病原体攻击时增加水杨酸导致两相氧化还原变化和NPR1单体释放进入细胞核。NPR1蛋白水平通过蛋白酶体在细胞核内的降解而振荡。这种动态变化是NPR1功能所必需的，并通过在NPR1中发现的DSXXXS(“IKB位点”)的磷酸化来促进。NPR1作为TGA转录因子的辅助因子控制基因表达。基因组和遗传学研究导致HSF4和几种DNA损伤修复蛋白，如BRCA2A和RAD51D，被确定为参与NPR1介导的基因表达的额外核因子。在目标1中，我们将揭示在细胞质中发现的NPR1-EF-Tu复合体的生物学意义。NPR1-EF-Tu的相互作用将通过突变被破坏，以确定EF-Tu是否只是NPR1寡聚体的一部分，或者也将防御与细胞器活动联系在一起。由于Ef-Tu是已知的植物防御激发子，我们还将研究假单胞菌Ef-Tu在感染过程中干扰NPR1寡聚体形成的可能性。在目标2中，我们将研究NPR1单体的振荡。将进一步研究IKB位点突变，以了解磷酸化在NPR1功能中的调节作用。相应的蛋白激酶将被识别出来。在目标3中，我们将使用TAP标记的NPR1分离和表征NPR1核复合体。NPR1、转录因子和染色质重塑蛋白之间的物理相互作用将以时间的方式进行检测和表征。该项目对人类健康的意义是双重的：首先，了解防御和细胞器活动之间的相互作用，转录调节中的振荡，以及防御相关转录和DNA重组之间的可能联系是与所有真核生物相关的高度新颖的研究领域。第二，环境健康直接影响人类健康。利用植物先天免疫控制疾病，有助于减少农药污染，增加食品营养，节约自然资源和人力资源。

DESCRIPTION (provided by applicant): Innate immune mechanisms are conserved in evolution. The NPR1 protein of Arabidopsis is a master regulator of gene expression in plant defense. Similar to the mammalian immunoregulator NF-kB, the NPR1 protein is nuclear translocated upon induction. In the resting state, NPR1 is present in the cytoplasm as a complex with the organelle translation elongation factor EF-Tu through disulfide bonds. An increase in salicylic acid upon pathogen challenge results in a biphasic redox change and the release of NPR1 monomer to enter the nucleus. The NPR1 protein levels oscillate through proteasome-mediated degradation in the nucleus. This dynamic change is required for NPR1 function and is facilitated by phosphorylation of DSXXXS ("IkB site") found in NPR1. NPR1 controls gene expression as a cofactor for the TGA transcription factors. Genomic and genetic studies led to the identification of HSF4 and several DNA damage repair proteins such as BRCA2A and RAD51D as additional nuclear factors involved in NPR1-mediated gene expression. In aim 1, we will reveal the biological significance of the NPR1-EF-Tu complex found in the cytoplasm. The NPR1-EF-Tu interaction will be disrupted through mutagenesis to determine whether EF-Tu is only part of the NPR1 oligomer or also links defense with organelle activities. Since EF-Tu is a known elicitor of plant defense, we will also investigate the possibility that Pseudomonas EF-Tu interferes with NPR1 oligomer formation during infection. In aim 2, we will study the oscillation of NPR1 monomer. The IkB site mutants will be further studied to understand the regulatory role of phosphorylation in NPR1 function. The corresponding protein kinase will be identified. In aim 3, we will isolate and characterize the NPR1 nuclear complex using TAP-tagged NPR1. Physical interactions between NPR1, transcription factors and chromatin remodeling proteins will be examined and characterized in a temporal fashion. The significance of the project to human health is two-fold: First, understanding the interplay between defense and organelle activity, the oscillation in transcription regulation, and a possible link between defense-associated transcription and DNA recombination are highly novel research areas relevant to all eukaryotic organisms. Second, human health is directly impacted by environmental health. Using plant innate immunity to control diseases will help reduce pesticide pollution, increase food nutrition, and save natural and human resources.