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

世界人口的增加和气候变化的影响对世界粮食供应提出了越来越大的需求。全球粮食安全的一个主要制约因素是植物病虫害造成的农作物损失。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、持久的和可持续的手段来防治作物病害。这种新方法的发展需要深入了解植物的免疫系统，它是如何调节的，以及病原体如何能够克服它。植物用可诱导的免疫系统来防御自己。免疫是通过识别基本的、广泛保守的分子(称为PAMP)来激活的，这些分子在感染期间被病原体暴露。成功的(适应的)病原体分泌和传递被称为效应器的蛋白质来抑制这些防御。植物免疫涉及一个相互关联的信号和调控过程的复杂网络。调控发生在许多层面，其中一个主要组成部分涉及蛋白质修饰和周转。泛素化是植物免疫的一个关键的蛋白质修饰，它通常导致26S蛋白酶体介导的泛素化蛋白质的降解。在过去的十年里，我们和其他人已经揭示了泛素E3连接酶这一关键酶，它可以积极或消极地调节植物免疫。在揭示抑制免疫的E3连接酶的作用模式方面已经取得了相当大的进展，方法是鉴定其泛素化的蛋白质底物，标记它们的蛋白酶体介导的降解。积极调节免疫的E3连接酶了解较少，其泛素化的底物也不清楚。这项建议将解决这一关键的知识差距。我们将集中在3个主要的E3连接酶，CMPG1，PUB17和UBK，其中两个是晚疫病病原菌Phytophthora infestans的效应物的靶标，强调它们作为中枢免疫调节因子的重要性，必须被这种病原菌修饰以抑制免疫。我们提供了重要的初步证据，证明我们的方法和途径揭示了激活免疫的E3连接酶底物。具体地说，我们的初步工作揭示了一种KH RNA结合蛋白(KH17)，我们发现它是PUB17泛素化的底物，目标是降解它。我们的目标是将这项工作扩展到识别所有三种E3连接酶的底物，这三种连接酶被预测为免疫的负面调节因子。一个具体的结果将是确定底物是否作为[S]易感因素(即感染所必需的)，因为这些因素提供了通过有条件沉默来消除以增强免疫力和提供抗病能力的靶标。另一个目的是研究致病疫霉的效应物在抑制E3连接酶中的作用。我们将利用这一知识来产生两个E3连接酶的突变形式，以便相应的效应器不再抑制它们的活性，从而恢复抗病能力。值得注意的是，尽管泛素化已经成为植物生长、发育和免疫的中央调节因子，但人们对它如何控制免疫知之甚少。E3连接酶底物和调节因子在控制免疫中的鉴定和功能特征，从而改变了我们对植物防御如何受这一关键的翻译后修饰控制的理解。

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.