Genetics of Disease Resistance in Arabidopsis thaliana

拟南芥抗病遗传学

• 批准号: 7523607
• 负责人: Roger W. Innes
• 金额: $ 34.36万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7523607
• 关键词: ATP Hydrolysis; Affect; Amino Acid Substitution; Animals; Arabidopsis; Arabidopsis Proteins; Arabidopsis RPS5 protein; Autoimmune Diseases; Binding; Biochemical; Biological; Breeding; Cell Nucleus; Cell Wall; Cell membrane; Cell surface; Cells; Chimeric Proteins; Class; Cleaved cell; Coiled-Coil Domain; Complex; Crohn' s disease; Cysteine Protease; Data; Detection; Disease; Disease Resistance; Event; Exclusion; Family; Fluorescence Resonance Energy Transfer; Fractionation; Genes; Genetic; Goals; Hereditary Disease; Human; Hydrolysis; Immune response; Immune system; In Vitro; Infection; Knowledge; Laboratories; Lead; Leucine-Rich Repeat; Life; Mass Spectrum Analysis; Mediating; Membrane; Modeling; Molecular; Molecular Genetics; Mouse-ear Cress; Mutation; N-terminal; Nuclear; Nucleotides; Organism; Pattern; Phosphotransferases; Plant Extracts; Plants; Play; Predisposition; Process; Proteasome Inhibitor; Protein Kinase; Proteins; Pseudomonas syringae; Public Health; Recombinant Proteins; Recombinants; Regulation; Relative (related person); Research; Resistance; Role; Signal Transduction; Signaling Protein; Structural Models; Structure; Syndrome; System; Testing; Translating; Walkers; Work; Yeasts; base; cellular imaging; defense response; insight; leucine-rich repeat protein; member; novel strategies; pathogen; pathogen exposure; prevent; protein function; protein purification; receptor; research study; sensor; yeast two hybrid system

DESCRIPTION (provided by applicant): Plants can detect thousands of different pathogens and actively respond. This detection system employs two major classes of receptors: transmembrane receptors on the cell surface that detect conserved pathogen associated molecular patterns (PAMPs), and intracellular receptors that detect specialized pathogen effector proteins. These intracellular receptors share a common structure consisting of a nucleotide binding (NB) domain and leucine rich repeats (LRR). NB-LRR genes are also found in humans, and mutations in several of these are associated with various autoinflammatory diseases, including Crohn's disease and Familial Cold-Induced Autoinflammatory Syndrome. Two human NB-LRR proteins, NOD1 and NOD2, have been shown to activate immune responses upon detection of bacterial cell wall fragments, thus NB-LRR proteins in plants and animals fulfill similar roles. However, the mechanisms by which NB-LRR proteins detect pathogen molecules and activate immune responses are poorly understood. This proposal focuses on the molecular mechanisms of NB-LRR activation, the role of subcellular localization of NB-LRR proteins, and the identification of downstream proteins engaged by activated NB-LRR proteins in plants. Specifically, this proposal focuses on the RPS5 protein from Arabidopsis. RPS5 mediates recognition of the AvrPphB protein secreted by the bacterial pathogen Pseudomonas syringae. A specific goal of the proposed research is to identify the mechanisms by which RPS5 detects AvrPphB and activates defense responses. Past work has shown that AvrPphB is a cysteine protease that targets the Arabidopsis PBS1 kinase. Recent work has demonstrated that PBS1 forms a complex with RPS5 and that cleavage of PBS1 is required for activation of RPS5. Activation appears to be dependent on ATP binding as mutations in the P- loop of the RPS5 NB domain block activation, while mutations in the Walker B motif that impair ATP hydrolysis cause RPS5 to be constitutively activated. Based on these and other data, it is hypothesized that PBS1 cleavage is detected by the LRR domain of RPS5, which causes a conformational change in RPS5 that enables exchange of ADP for ATP. Binding of ATP then releases the N-terminal coiled-coil domain of RPS5 from autoinhibition, freeing it to engage downstream signaling proteins. This model will be tested by pursuing three specific aims. First, an in vitro system containing soluble RPS5, PBS1 and AvrPphB proteins will be used to assess whether ATP binding, ATP hydrolysis and/or ADP/ATP exchange are regulated by PBS1 cleavage. Second, immunomicroscopy and live-cell imaging will be used to assess whether activation of downstream signaling events by RPS5 requires its subcellular relocalization, particularly to the nucleus. Finally, proteins that associate with activated forms of RPS5 will be identified using both yeast two-hybrid approaches and by purification of protein complexes from plant extracts. These experiments will provide key insights into the innate immune systems of both plants and humans. PUBLIC HEALTH RELEVANCE: The immune systems of plants and animals employ similar proteins to detect disease-causing organisms. Mutations in the genes encoding these proteins are associated with debilitating autoimmune diseases in humans, and susceptibility to disease in plants. By studying at a molecular level, how these proteins function, this research will lead to new treatments for autoimmune diseases, and new approaches to breeding disease resistant crops.

描述（由申请人提供）：植物可以检测数千种不同的病原体并积极做出反应。该检测系统采用两大类受体：细胞表面的跨膜受体，用于检测保守的病原体相关分子模式 (PAMP)；以及细胞内受体，用于检测专门的病原体效应蛋白。这些细胞内受体具有共同的结构，由核苷酸结合 (NB) 结构域和富含亮氨酸的重复序列 (LRR) 组成。 NB-LRR 基因也存在于人类中，其中一些基因的突变与各种自身炎症性疾病相关，包括克罗恩病和家族性寒冷诱发的自身炎症综合症。两种人类 NB-LRR 蛋白 NOD1 和 NOD2 已被证明在检测到细菌细胞壁碎片时会激活免疫反应，因此植物和动物中的 NB-LRR 蛋白发挥相似的作用。然而，人们对 NB-LRR 蛋白检测病原体分子和激活免疫反应的机制知之甚少。该提案重点关注 NB-LRR 激活的分子机制、NB-LRR 蛋白亚细胞定位的作用，以及植物中激活 NB-LRR 蛋白参与的下游蛋白的鉴定。具体来说，该提案重点关注来自拟南芥的 RPS5 蛋白。 RPS5 介导对细菌病原体丁香假单胞菌分泌的 AvrPphB 蛋白的识别。本研究的具体目标是确定 RPS5 检测 AvrPphB 并激活防御反应的机制。过去的研究表明，AvrPphB 是一种针对拟南芥 PBS1 激酶的半胱氨酸蛋白酶。最近的研究表明，PBS1 与 RPS5 形成复合物，并且 PBS1 的裂解是 RPS5 激活所必需的。激活似乎依赖于 ATP 结合，因为 RPS5 NB 结构域 P 环中的突变会阻止激活，而损害 ATP 水解的 Walker B 基序中的突变会导致 RPS5 被组成型激活。基于这些和其他数据，假设 PBS1 裂解是由 RPS5 的 LRR 结构域检测到的，这会导致 RPS5 发生构象变化，从而实现 ADP 与 ATP 的交换。然后，ATP 的结合将 RPS5 的 N 端卷曲螺旋结构域从自身抑制中释放出来，使其能够与下游信号蛋白结合。该模型将通过追求三个具体目标进行测试。首先，将使用含有可溶性 RPS5、PBS1 和 AvrPphB 蛋白的体外系统来评估 ATP 结合、ATP 水解和/或 ADP/ATP 交换是否受 PBS1 裂解的调节。其次，免疫显微镜和活细胞成像将用于评估 RPS5 下游信号传导事件的激活是否需要其亚细胞重新定位，特别是细胞核。最后，将使用酵母双杂交方法和从植物提取物中纯化蛋白质复合物来鉴定与激活形式的 RPS5 相关的蛋白质。这些实验将为植物和人类的先天免疫系统提供重要的见解。公共卫生相关性：植物和动物的免疫系统利用类似的蛋白质来检测致病生物体。编码这些蛋白质的基因突变与人类衰弱的自身免疫性疾病以及植物对疾病的易感性有关。通过在分子水平上研究这些蛋白质如何发挥作用，这项研究将带来治疗自身免疫性疾病的新方法，以及培育抗病作物的新方法。