New approaches to undermine late blight disease by exploiting an understanding of ubiquitin E3 ligases that positively regulate immunity

世界人口的不断增长和气候变化的影响对世界粮食供应提出了更高的要求。全球粮食安全的一个主要制约因素是植物病虫害造成的作物损失。随着批准用于农业的化学品的条件越来越严格，在不久的将来，有效的杀真菌剂和杀虫剂的选择将变得更加有限。迫切需要探索开发新的、持久的和可持续的方法来防治作物病害。这种新方法的发展需要深入了解植物免疫系统，它是如何调节的，以及病原体是如何克服它的。植物用诱导型免疫系统来保护自己。免疫是通过识别在感染过程中被病原体暴露的基本的、广泛保守的分子（称为PAMP）来激活的。成功的（适应）病原体分泌和传递称为效应子的蛋白质来抑制这些防御。植物免疫涉及一个复杂的网络相互联系的信号和调节过程。调节发生在许多水平上，主要成分涉及蛋白质修饰和周转。一个关键的蛋白质修饰，正在成为一个中央调节植物免疫是泛素化，这往往会导致26 S蛋白酶体介导的降解泛素化蛋白。在过去的十年中，我们和其他人已经发现了关键酶，泛素E3连接酶，它可以积极或消极地调节植物免疫。已经取得了相当大的进展，以揭示E3连接酶抑制免疫的作用模式，通过鉴定其蛋白底物的泛素化，标记它们的蛋白酶体介导的降解。E3连接酶积极调节免疫力的了解较少，其底物的泛素化是未知的。我们将重点关注3种积极调节免疫力的主要E3连接酶，CMPG1，PUB17和UBK，其中两种被晚疫病病原体致病疫霉的效应子靶向，强调它们作为中央免疫调节因子的重要性，必须被病原体修饰以抑制免疫力。我们提供了关键的初步证据，表明我们的方法和途径揭示了激活免疫的E3连接酶的底物。具体来说，我们的初步工作揭示了一种KH RNA结合蛋白（KH 17），我们表明它是PUB 17泛素化的底物，靶向它进行降解。我们的目标是扩展这项工作，以确定所有三个E3连接酶，这是预测免疫力的负调节底物。一个具体的结果将是鉴定底物是否作为易感性[S]因子（即感染所需的），因为这些因子提供了通过条件沉默去除的靶标，以增强免疫力并提供抗病性。进一步的目的是研究致病疫霉效应子在抑制E3连接酶中的作用。我们将利用这些知识来产生两种E3连接酶的突变形式，使相应的效应子不再抑制它们的活性，从而恢复抗病性。值得注意的是，尽管泛素化已成为植物生长，发育和免疫的中心调节因子，但人们对它如何控制免疫知之甚少。因此，E3连接酶底物和调节剂在管理免疫的识别和功能特性提供了一个步骤的变化，我们的理解如何植物防御控制这一关键的翻译后修饰。

An increasing world population and impacts of climate change place ever-greater demands on the world food supply. A major constraint to global food security is crop loss due to plant pests and diseases. With the increasing stringency of conditions under which chemicals are approved for agriculture, the choice of effective fungicides and pesticides will become more limited in the near future. There is an urgent need to explore the development of novel, durable and sustainable means to combat crop diseases. The development of such new approaches requires a deep understanding of the plant immune system, how it is regulated, and how pathogens are able to overcome it.Plants defend themselves with an inducible immune system. Immunity is activated by recognition of essential, widely conserved molecules (called PAMPs) that are exposed by pathogens during infection. Successful (adapted) pathogens secrete and deliver proteins called effectors to suppress these defences. Plant immunity involves a complex network of inter-linked signalling and regulatory processes. Regulation occurs at many levels, and a major component involves protein modification and turnover. A key protein modification that is emerging as a central regulator of plant immunity is ubiquitination, which often results in 26S proteasome-mediated degradation of ubiquitinated proteins. In the past decade we and others have revealed key enzymes, ubiquitin E3 ligases, which either positively or negatively regulate plant immunity. Considerable advances have been made to reveal the modes-of-action of E3 ligases that suppress immunity, by identifying their protein substrates for ubiquitination, marking them for proteasome-mediated degradation. E3 ligases that positively regulate immunity are less well understood and their substrates for ubiquitination are unknown. This proposal will address this critical knowledge gap.We will focus on 3 major E3 ligases that positively regulate immunity, CMPG1, PUB17 and UBK, two of which are targeted by effectors from the late blight pathogen Phytophthora infestans, emphasising their importance as central immune regulators that must be modified by this pathogen to suppress immunity. We provide crucial preliminary evidence that our methods and approaches reveal substrates of E3 ligases that activate immunity. Specifically, our preliminary work has revealed a KH RNA binding protein (KH17) which we show is a substrate for ubiquitination by PUB17, targeting it for degradation. We aim to extend this work to identify substrates of all three E3 ligases, which are predicted to be negative regulators of immunity. A specific outcome will be the identification of whether the substrates act as susceptibility [S] factors (i.e. are required for infection), as these provide targets to remove, by conditional silencing, to enhance immunity and provide disease resistance. A further aim is to investigate the roles of P. infestans effectors in inhibiting the E3 ligases. We will exploit this knowledge to generate mutant forms of two E3 ligases so that corresponding effectors can no longer inhibit their activity, thus restoring disease resistance.Remarkably, although ubiquitination has emerged as a central regulator of growth, development and immunity in plants, little is known about how it controls immunity. The identification and functional characterisation of E3 ligase substrates and regulators in governing immunity thereby provide a step-change in our understanding of how plant defence is controlled by this critical post-translational modification.