Functional Analysis of NPR1 in SAR Signal Transduction

9728111本项目的长期目标是了解植物系统性获得性抗性（SAR）的诱导途径和分子机制。SAR对广泛的病原体具有免疫力，通常在初次接触无毒病原体后形成。利用遗传方法，研究人员从拟南芥中分离出npr1突变体，并证实其能够控制SAR的发生。npr1缺陷突变体对各种SAR诱导信号（如水杨酸（SA）和无毒病原体）没有反应，其致病相关基因（PR）表达很少，对感染的易感性增加。最近，利用基于图谱的方法克隆了NPR1，发现该基因编码一种含有锚蛋白重复序列的新蛋白质。锚蛋白重复序列已在多种蛋白质中被发现，并被认为介导蛋白质之间的相互作用。npr1突变等位基因的损伤破坏了锚蛋白一致序列，表明这些重复序列对npr1功能很重要。将克隆的野生型NPR1基因转化为NPR1基因，不仅补充了突变型表型，而且使转基因植株更能抵抗丁香假单胞菌和Peronospora寄生虫的感染。此外，对携带NPR1- gfp融合的转基因系的研究表明，NPR1在诱导后定位于细胞核。这些发现，以及NPR1过表达植物完全健康的事实表明，NPR1基因是SAR的正调节因子，可能作为基因工程抗病植物的靶基因。NPR1在SA信号转导和PR基因表达调控中的生物学作用已在以往的研究中得到证实。本项目将重点阐述NPR1分子功能的两个具体目标：(1)确定NPR1核定位的功能重要性和调控：将开展诱变研究，确定NPR1蛋白的核定位对于激活PR基因表达和SAR是否必要和充分，以及定位过程是如何调控的。(2)确定NPR1在调控PR基因表达中的作用：将分析NPR1调控的PR基因的启动子，并进行凝胶迁移位移试验，以确定NPR1是直接调控PR基因启动子还是间接影响控制这些基因表达的转录因子的活性。这项研究的结果将对基础研究和社会产生有益的影响。利用抗病机制知识培育出的基因工程抗病植物可以减少有害农药的使用。研究还开始比较植物与动物系统中对微生物感染的防御机制，以确定这一基本生物学功能的进化是否存在任何守恒。

9728111 The long-term goal of this project is to understand the induction pathway and molecular mechanisms that confer systemic acquired resistance (SAR) to plants. SAR confers immunity to a broad spectrum of pathogens and is normally established after a primary exposure to avirulent pathogens. Using a genetic approach, the npr1 mutant in Arabidopsis was isolated and demonstrated to control the onset of SAR. Mutants with defects in NPR1 fail to respond to various SAR-inducing signals, such as salicyclic acid (SA) and avirulent pathogens, display little expression of pathogenesis-related (PR) genes and exhibit increased susceptibility to infections. Recently NPR1 was cloned using a map-based approach and the gene was found to encode a novel protein containing ankyrin repeats. Ankyrin repeats have been identified in a variety of proteins and are thought to mediate protein-protein interactions. Lesions in the npr1 mutant alleles disrupt the ankyrin consensus sequence, suggesting that these repeats are important for NPR1 function. Transformation of the cloned wild-type NPR1 gene into npr1 not only complemented the mutant phenotypes but also rendered the transgenic plants more resistant to infections by Pseudomonas syringae and Peronospora parasitica. Furthermore, studies of transgenic lines carrying the NPR1-GFP fusion showed that NPR1 is localized to the nucleus upon induction. These findings, along with the fact that NPR1-overexpressing plants are perfectly healthy, suggests that the NPR1 gene is a positive regulator of SAR and may serve as a target gene for genetically engineering disease-resistant plants. The biological role of NPR1 in transducing the SA signal and in regulating PR gene expression has been demonstrated in previous studies. This project will focus on two specific aims to elucidate the molecular function of NPR1: (1) Determining the functional importance and the regulation of NPR1 nuclear localization: Mutagenesis studies will be carried out to determine whether nucle ar localization of the NPR1 protein is necessary and sufficient for activating PR gene expression and SAR, and how the localization process is regulated. (2) Determining the role of NPR1 in regulating PR gene expression: Promoters of the NPR1-regulated PR genes will be analyzed and gel mobility shift assays will be performed to determine whether NPR1 directly regulates the PR gene promoters or indirectly affects the activity of a transcription factor(s) which controls the expression of these genes. The outcome of this research will have a beneficial impact on basic research and on society. Genetically engineered disease-resistant plants generated with the knowledge of disease resistance mechanisms can lead to reduction in the usage of harmful pesticides. Studies have also begun to compare the plant defense mechanisms against microbial infection with those in animal systems to determine whether there is any conservation in the evolution of this fundamental biological function.