The mechanistic basis of plant NLR signalling in effector triggered immunity

进化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，以阻止细胞保护性免疫反应。植物通过一种特定的蛋白质家族的进化来做出反应，NLR蛋白质(首字母缩写的意思是核苷酸结合和富含亮氨酸的重复结构域)可以检测这些细菌蛋白质并抑制它们的活性。NLR蛋白触发强大的先天免疫反应，包括遗传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，并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，这是提高作物生产力和养活世界日益增长的人口的战略的一部分。尽管NLR在保护植物免受病原体入侵方面发挥了关键作用，但人们对NLR靶标分子的特异性了解相对较少，使它们能够执行免疫功能。这在一定程度上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对它们的功能有新的了解。我们在试管中对NLR蛋白质功能的分析得出了一个令人惊讶的结果。我们已经证明了NLR蛋白能够与DNA结合并局部扭曲DNA。平行实验证明，DNA结合也发生在植物中，并具体依赖于免疫系统的激活。这一结果更加令人兴奋，因为多名独立研究人员越来越清楚地表明，许多NLR在细胞核中具有功能。这些多种多样的证据让我们提出了一个支撑这项工作的中心假设。我们认为，NLR蛋白与辅助蛋白一起特异性地靶向于基因组DNA，参与免疫激活，NLR蛋白促进这些位点的转录启动。在这里，我们进行实验来研究这一假说的各个方面。首先，我们将研究NLR与植物细胞中DNA结合对免疫系统激活的要求。为了实现这一点，我们将利用植物遗传学和病理学的丰富历史，这些历史产生了令人难以置信的一系列定义良好的NLR变体，这些变体既能激活免疫，也能使免疫失活，改变与附属蛋白的相互作用，并调节蛋白质本身的接触。这些重要的实验将把NLRs结合DNA的能力置于NLR功能的现有知识库的背景下。第二，我们将研究NLRs如何通过与蛋白质配对来靶向特定的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可以与这种靶向蛋白相关联，我们已经确定了一些正在为这一提议而工作的NLR。我们认为，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NLRs如何激活特定的免疫反应提供新的见解。第三，我们将建立NLRs如何在DNA上调节转录反应。我们认为NLRs针对特定的DNA序列，一旦被靶向，就会激活改变DNA结构的蛋白质，从而允许转录。这些实验意义重大，因为它们将揭示NLR在复合体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.