Functional analyses of NPR1 in plant defense

描述（由申请人提供）：先天免疫机制在进化中是保守的。拟南芥的NPR 1蛋白是植物防御中基因表达的主要调节因子。与哺乳动物免疫调节剂NF-κ B类似，NPR 1蛋白在诱导后发生核转位。在静息状态下，NPR 1通过二硫键与细胞器翻译延伸因子EF-Tu形成复合物存在于细胞质中。病原体攻击后水杨酸的增加导致双相氧化还原变化和NPR 1单体释放进入细胞核。NPR 1蛋白水平通过蛋白酶体介导的细胞核降解而振荡。这种动态变化是NPR 1功能所需的，并且通过NPR 1中发现的DSXXXS（“IkB位点”）的磷酸化来促进。NPR 1作为TGA转录因子的辅因子控制基因表达。基因组学和遗传学研究鉴定了HSF 4和几种DNA损伤修复蛋白，如BRCA 2A和RAD 51 D，作为参与NPR 1介导的基因表达的额外核因子。在目的1中，我们将揭示在细胞质中发现的NPR 1-EF-Tu复合物的生物学意义。NPR 1-EF-Tu相互作用将通过诱变来破坏，以确定EF-Tu是否仅是NPR 1寡聚体的一部分或也将防御与细胞器活性联系起来。由于EF-Tu是一种已知的植物防御激发子，我们还将研究假单胞菌EF-Tu干扰NPR 1寡聚体形成的可能性。在目的2中，我们将研究NPR 1单体的振荡。IkB位点突变体将被进一步研究，以了解磷酸化在NPR 1功能中的调节作用。将鉴定相应的蛋白激酶。在目标3中，我们将使用TAP标记的NPR 1分离和表征NPR 1核复合物。NPR 1，转录因子和染色质重塑蛋白之间的物理相互作用将被检查和表征的时间方式。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非常新颖的研究领域。第二，人类健康直接受到环境健康的影响。利用植物天然免疫控制疾病，将有助于减少农药污染，增加食物营养，节约自然和人力资源。

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.