Characterization of Salicylic Acid-Binding Proteins in Plant Defense Responses

植物防御反应中水杨酸结合蛋白的表征

• 批准号: 9904660
• 负责人: Daniel Klessig
• 金额: $ 10.33万
• 依托单位: Rutgers University New Brunswick
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904660&HistoricalAwards=false
• 关键词: Characterization; Salicylic; Acid; Binding; Proteins

Plant disease has a major negative impact on food and fiber production. Currently, worldwide crop losses due to disease alone are estimated to exceed $100 billion annually, despite the extensive use of potentially toxic fungicides and pesticides. A variety of alternative strategies are being developed to protect plants against disease. One approach is to induce the plant's own natural defenses. This strategy is attractive since it can provide protection against a broad spectrum of pathogens. Broad-spectrum resistance to a wide array of pathogens, known as systemic acquired resistance (SAR), can be activated by infection with a given pathogen or treatment with SAR-inducing chemicals such as salicylic acid (SA) and its synthetic functional analogues, INA or BTH. In addition, while the initial perception and first few steps of the resistance signaling pathway(s) are probably distinct for each pathogen-plant interaction, these signals are thought to converge on a limited set of defense pathways. Thus, identification and characterization of the components involved in these common pathways should provide an opportunity to manipulate resistance to a broad spectrum of pathogens. Furthermore, the development of strategies that control disease by manipulating endogenous plant defense responses will be very important for sustaining agricultural production and improving our environment and health.It is now well established that SA plays a key role(s) in the development of resistance in many, but not all, plant-pathogen systems. While our understanding of SA function(s) in disease resistance is far from complete, it is becoming increasingly clear that its role(s) is complex. Initially, SA was shown to activate a subset of defense genes (such as the PR-1, PR-2, and PR-5) and enhance disease resistance. More recently, it has been shown to potentiate activation of a variety of defense responses by a second stimulus. Furthermore, there is mounting evidence for a self-amplification loop involving H2O2 accumulation, SA synthesis, and host cell death. This increase in the complexity of SA function(s) in defense is matched by the discovery in tobacco of multiple proteins with which SA interacts. An understanding of SA function(s) in defense against microbial pathogens will require the identification and characterization of these proteins.Characterization of SA effector proteins is likely to have implications beyond plant disease resistance. For example, it recently has been demonstrated that several plant genes controlling resistance to nematodes and insects share motifs, such as leucine rich repeats and nucleotide binding sites, with resistance genes for viral, bacterial and fungal pathogens. Thus, deciphering the SA-mediated signal transduction pathway(s) initiated by microbial pathogens is likely to impact studies on plant responses to nematodes and insects. Furthermore, there is increasing evidence that SAR shares many similarities to innate immunity in vertebrates and invertebrates and, thus, probably evolved from a primordial defense system present in the common ancestor to plants and animals. Elucidating the molecular mechanism(s) of disease resistance in plants therefore is likely to impact veterinary and human medicine.The major objective of this project is to complete purification of the chloroplastic SABP3 and the mitochondrial SABP4 and clone their corresponding genes. Characterization of these two proteins and their encoding genes will also be initiated. The properties and functions of these two SABPs will also be compared to those of SABP2, which is a lower abundance protein with high affinity for SA. These comparative analyses should enhance our understanding of how different SA effector proteins mediate SA's complex role in disease resistance.Standard biochemical, molecular and genetic approaches will be used. Protein purification will involve conventional ion exchange, reverse-phase and gel filtration chromatography as well as affinity chromatography with a SA analogue. Cloning will be based on peptide sequences of the purified proteins and will utilize degenerate oligonucleotide primers for PCR amplification. The copy number, distribution and expression pattern of genes for SABP3 and SABP4 will be determined as well as their subcellular location. A key aim is to determine function of the SABPs. The PVX-based gene silencing method pioneered by David Baulcombe's group will be employed for functional studies of both proteins. In addition, sense and antisense SABP3 and SABP4 transgenic tobacco will be constructed. Once the Arabidopsis SABP3 (or SABP4) ortholog has been obtained, it will be used not only to generate SABP3 (or SABP4) over- and underexpressing transgenic Arabidopsis but also to screen for SABP3 (or SABP4) mutants in libraries of insertion mutants of Arabidopsis using a PCR-based approach.

植物病害对粮食和纤维生产具有重大的负面影响。 目前，尽管广泛使用潜在有毒的杀真菌剂和杀虫剂，但仅因疾病造成的全球作物损失估计每年就超过1000亿美元。 正在开发各种替代策略来保护植物免受疾病的侵害。 一种方法是诱导植物自身的自然防御。 这种策略是有吸引力的，因为它可以提供针对广谱病原体的保护。 对多种病原体的广谱抗性，称为系统获得性抗性（SAR），可以通过感染给定的病原体或用SAR诱导化学品如水杨酸（SA）及其合成功能类似物、INA或BTH处理来激活。 此外，虽然抗性信号传导途径的最初感知和最初几个步骤对于每种病原体-植物相互作用可能是不同的，但这些信号被认为集中在有限的一组防御途径上。 因此，鉴定和表征这些共同途径中涉及的组分应该提供操纵对广谱病原体的抗性的机会。 此外，通过操纵内源植物防御反应来控制疾病的策略的发展对于维持农业生产和改善我们的环境和健康将是非常重要的。 虽然我们对SA在抗病性中的功能的理解还远未完成，但越来越清楚的是，它的作用是复杂的。 最初，SA被证明可以激活一组防御基因（如PR-1，PR-2和PR-5）并增强抗病性。 最近，它已被证明可以通过第二刺激增强各种防御反应的激活。 此外，有越来越多的证据表明，涉及过氧化氢积累，SA合成和宿主细胞死亡的自我放大循环。 SA防御功能复杂性的增加与烟草中发现的SA相互作用的多种蛋白质相匹配。 了解SA在防御微生物病原体中的功能需要鉴定和表征这些蛋白质。SA效应蛋白的表征可能具有超出植物抗病性的意义。 例如，最近已经证明，控制对线虫和昆虫的抗性的几种植物基因与对病毒、细菌和真菌病原体的抗性基因共享基序，例如富含亮氨酸的重复序列和核苷酸结合位点。 因此，破译SA介导的信号转导途径（S）启动的微生物病原体可能会影响植物对线虫和昆虫的反应的研究。 此外，越来越多的证据表明，SAR与脊椎动物和无脊椎动物中的先天免疫有许多相似之处，因此，可能是从共同祖先中存在的原始防御系统进化到植物和动物。 因此，阐明植物抗病性的分子机制可能会对兽医和人类医学产生影响。本项目的主要目标是完成叶绿体SABP 3和线粒体SABP 4的纯化并克隆其相应的基因。 这两种蛋白质及其编码基因的表征也将开始。 这两种SABP的性质和功能也将与SABP 2的性质和功能进行比较，SABP 2是对SA具有高亲和力的较低丰度蛋白质。 这些比较分析将提高我们对不同SA效应蛋白如何介导SA在抗病性中的复杂作用的理解。 蛋白质纯化将涉及常规的离子交换、反相和凝胶过滤层析以及用SA类似物的亲和层析。 克隆将基于纯化蛋白的肽序列，并将利用简并寡核苷酸引物进行PCR扩增。 将确定SABP 3和SABP 4基因的拷贝数、分布和表达模式以及它们的亚细胞位置。 一个关键目标是确定SABP的功能。 由大卫鲍尔科姆小组开创的基于PVX的基因沉默方法将用于这两种蛋白质的功能研究。 此外，还将构建SABP 3和SABP 4的正义和反义转基因烟草。 一旦获得拟南芥SABP 3（或SABP 4）直系同源物，其将不仅用于产生SABP 3（或SABP 4）过表达和低表达的转基因拟南芥，而且用于使用基于PCR的方法在拟南芥插入突变体文库中筛选SABP 3（或SABP 4）突变体。