Characterization of Salicylic Acid-Binding Proteins in Plant Defense Responses

植物病害对粮食和纤维生产有重大负面影响。目前，尽管广泛使用了潜在的有毒杀菌剂和杀虫剂，但仅疾病造成的全球农作物损失估计每年就超过1000亿美元。人们正在开发各种替代策略来保护植物免受疾病侵袭。一种方法是诱导植物自身的自然防御。这一策略很有吸引力，因为它可以提供对广泛病原体的保护。对一系列病原体的广谱抗性，称为系统获得性抗性(SAR)，可通过感染特定病原体或使用诱导SAR的化学物质如水杨酸(SA)及其合成功能类似物INA或BTH来激活。此外，虽然抗性信号通路(S)的最初感知和最初几步可能在每个病原体与植物的相互作用中是不同的，但这些信号被认为聚集在一组有限的防御途径上。因此，鉴定和表征这些共同途径中涉及的成分应该提供一个机会来操纵对广泛病原体的抗性。此外，开发通过操纵内源植物防御反应来控制疾病的策略对于维持农业生产、改善我们的环境和健康将是非常重要的。现已证实，SA在许多(但不是所有)植物-病原菌系统的抗性发展中发挥关键作用(S)。虽然我们对SA在抗病中的作用(S)的认识还很不完整，但我们越来越清楚它(S)在抗病中的作用是复杂的。最初，SA被证明激活了防御基因的一个子集(如PR-1、PR-2和PR-5)，并增强了抗病能力。最近，它被证明通过第二次刺激增强了各种防御反应的激活。此外，越来越多的证据表明，一个涉及过氧化氢积累、SA合成和宿主细胞死亡的自我扩增环。在烟草中发现了与SA相互作用的多种蛋白质，这与SA在防御中功能复杂性的增加(S)相匹配。要理解SA在防御微生物病原体中的功能(S)，需要对这些蛋白进行鉴定和特性分析。SA效应蛋白的特性可能具有超越植物抗病能力的意义。例如，最近已经证明，一些控制线虫和昆虫抗性的植物基因具有相同的基序，如富含亮氨酸的重复序列和核苷酸结合位点，以及病毒、细菌和真菌病原体的抗性基因。因此，破译微生物病原菌启动的SA介导的信号转导途径(S)可能会影响植物对线虫和昆虫的反应研究。此外，越来越多的证据表明，SAR与脊椎动物和无脊椎动物的天然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动植物。因此，阐明植物抗病的分子机制(S)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，并克隆它们相应的基因。这两种蛋白质及其编码基因的特性也将启动。这两个SABP的性质和功能也将与SABP2的性质和功能进行比较，SABP2是一种与SA具有高亲和力的低丰度蛋白质。这些比较分析将加强我们对不同SA效应蛋白如何介导SA在抗病中的复杂作用的理解。将使用标准的生化、分子和遗传学方法。蛋白质的纯化将包括传统的离子交换、反相和凝胶过滤层析，以及带有SA类似物的亲和层析。克隆将基于纯化蛋白质的肽序列，并将使用简并的寡核苷酸引物进行PCR扩增。SABP3和SABP4基因的拷贝数、分布和表达模式以及它们的亚细胞位置将被确定。一个关键目标是确定SABP的功能。David Baulcombe团队首创的基于PVX的基因沉默方法将被用于这两种蛋白质的功能研究。此外，还将构建正、反义SABP3和SABP4转基因烟草。一旦获得了拟南芥SABP3(或SABP4)的同源基因，它不仅将被用来产生SABP3(或SABP4)高表达和低表达的转基因拟南芥，而且还将利用基于PCR的方法在拟南芥的插入突变体文库中筛选SABP3(或SABP4)突变体。

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.