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

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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.