The mechanistic basis of plant NLR signalling in effector triggered immunity

进化已经见证了植物和入侵病原体之间的军备竞赛。病原体向植物细胞注入蛋白质，阻断细胞保护性免疫反应。植物通过进化出一种特殊的蛋白质家族来做出反应，即NLR蛋白质（核苷酸结合和富含亮氨酸的重复结构域的缩写），它可以检测这些细菌蛋白质并对抗它们的活性。NLR蛋白触发强大的先天免疫反应，包括基因编码的细胞死亡反应，从而限制病原体的进一步传播。了解NLR蛋白的功能，重要的是，利用我们从他们的分析中获得的知识平台，最终可以提高作物对病原体的防御能力，作为提高作物生产力和养活世界不断增长的人口的战略的一部分。尽管它们在保护植物免受入侵病原体的侵害方面发挥着关键作用，但人们对nlr靶向分子的具体特征知之甚少，这些分子使它们能够发挥免疫功能。这部分是由于在试管中产生用于分析的NLR蛋白的困难。我们已经开发了用于试管分析的NLR蛋白的生产方法。这使我们能够利用丰富的生化和生物物理技术工具箱来获得对其功能的新见解。我们在试管中对NLR蛋白功能的分析得出了一个惊人的结果。我们已经证明NLR蛋白能够结合并局部扭曲DNA。类似的实验表明，DNA结合也发生在植物中，并且特别依赖于免疫系统的激活。这一结果更加令人兴奋，因为多个独立研究人员越来越清楚地表明，许多nlr在细胞核中起作用。这些多重证据使我们提出了一个支撑这项工作的中心假设。我们提出NLR蛋白在免疫激活上特异性靶向基因组DNA和辅助蛋白，并且NLR蛋白增强了这些位点的转录起始。在这里，我们进行实验来研究这一假设的各个方面。首先，我们将研究NLR在激活免疫系统时与植物细胞DNA结合的要求。为了实现这一目标，我们将利用植物遗传学和病理学的丰富历史，这些历史已经产生了一系列令人难以置信的定义良好的NLR变异体，这些变异体可以激活和灭活免疫，改变与辅助蛋白的相互作用，并调节蛋白质本身的接触。这些重要的实验将至关重要地将NLR结合DNA的能力置于NLR功能的现有知识库的背景下。其次，我们将研究nlr如何通过配对蛋白质靶向特定的DNA序列，从而为免疫反应提供特异性。越来越多的证据表明，许多nlr可以与这样的靶向蛋白相关联，我们已经确定了一些朝着这一建议的工作。我们建议建立nlr与特定DNA序列的关联是建立保护性免疫反应的关键阶段。因此，这些实验将为nlr如何激活特异性免疫反应提供新的见解。第三，我们将建立NLRs如何在DNA上发挥作用来调节转录反应。我们建议nlr靶向特定的DNA序列，一旦靶向，激活改变DNA结构的蛋白质，从而允许转录。这些实验具有重要意义，因为它们将揭示nlr在复合物中发挥作用以促进保护性转录反应的一般机制。

Evolution has seen an arms race between plants and invading pathogens. Pathogens inject plant cells with proteins to block cell protective immune responses. Plants have responded through the evolution of a specific family of proteins, the NLR proteins (an acronym meaning nucleotide-binding and leucine-rich repeat domains) that detect these bacterial proteins and counter their activity. The NLR proteins trigger powerful innate immune responses including a genetically encoded cell death response thus restricting further spread of the pathogen. Understanding NLR protein function and, importantly, exploiting the knowledge platform that we gain from their analysis can ultimately be developed to enhance crop defences to pathogens as part of a strategy to improve crop productivity and feed the world's growing population.Despite their key role in protecting plants from invading pathogens, relatively little is known of the specifics of molecules targeted by NLRs to enable them to perform their immune function. This has been partly due to difficulties in generating NLR proteins for analysis in the test tube. We have developed methods for the production of NLR proteins for test tube based analysis. This has enabled us to exploit the rich toolbox of biochemical and biophysical techniques to gain new insight into their function.Our analysis of NLR protein function in the test tube has thrown up a surprising result. We have demonstrated that NLR proteins are able to bind to and locally distort DNA. Parallel experiments have demonstrated that DNA binding also occurs in plants and is specifically dependent on activation of the immune system. This result is all the more exciting as it is becoming clear from multiple independent researchers that many NLRs are functional in the nucleus. These multiple lines of evidence lead us to posit a central hypothesis that underpins this work. We propose that NLR proteins are specifically targeted to genomic DNA, with accessory proteins, on immune activation and that NLR proteins enhance transcription initiation at these sites.Here we perform experiments to study various aspects of this hypothesis.First, we will investigate the requirements for an NLR to bind to DNA in the plant cell on activation of the immune system. To achieve this we will exploit the rich history of plant genetics and pathology that has produced an incredible array of well defined NLR variants that both activate and inactivate immunity, alter interactions with accessory proteins, and modulate contacts within the protein itself. These important experiments will crucially place the capacity of NLRs to bind DNA within the context of the pre-existing knowledge base for NLR function.Second, we will investigate how NLRs can be targeted to particular DNA sequences by partnering proteins and thus provide specificity for the immune response. There is growing evidence that many NLRs can associate with such targeting proteins and we have identified some in work towards this proposal. We propose to establish that the association of NLRs with particular DNA sequences is a key stage in establishing the protective immune response. Together these experiments will therefore provide new insight into how NLRs activate specific immune responses.Third, we will establish how NLRs function at DNA to regulate a transcriptional response. We propose that NLRs are targeted to specific DNA sequences and once targeted, activate proteins that alter DNA structure thus permitting transcription. These experiments are significant, as they will reveal general mechanisms by which NLRs can function in complexes to promote protective transcriptional responses.