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

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.

Ubiquitination is emerging as a central post-translational modification (PTM) regulating plant immunity. Ubiquitin E3 ligases represent the critical step in substrate specification for ubiquitination and subsequent 26S proteasome-mediated degradation. In contrast to mammalian genomes, plants possess thousands of potential E3 ligases, emphasising the central role of this PTM in regulatory processes. So far only targets for two E3 ligases that negatively regulate plant immunity have been found. As yet, substrates of three key E3 ligases, CMPG1, PUB17 and UBK, which activate immunity, are unknown. This proposal will address this critical gap in our understanding by identifying and functionally characterising the substrates of these E3 ligases. Two of these E3 ligases are targeted by RXLR effectors from the late blight pathogen, Phytophthora infestans, emphasising their importance to immunity.
This proposal is underpinned by significant preliminary data indicating that our approaches work. Our methods identify a substrate for ubiquitination by PUB17, a KH RNA binding (KH17). We will extend this work to identify substrates of all three E3 ligases. Using methods well-established in our laboratories, we will characterise the functions of these substrates in governing specific immune responses. Identifying substrates of CMPG1, PUB17 and UBK will be a critical translational outcome, as these are likely to be negative immune regulators (susceptibility [S] factors); their targeted removal would thus enhance disease resistance.
Further key translational outcomes of this work will be to generate mutant forms of two E3 ligases that positively regulate immunity, so that corresponding effectors can no longer inhibit their activity. This proposal thus generates fundamental knowledge about the role of ubiquitination in promoting immunity which can be exploited to develop novel approaches to undermine one of the major diseases threatening food security.

Who will benefit from this research?
Pests and diseases are a major constraint to achieving food security. Up to 50% of crop losses in developing nations are due to pests and diseases. A deep understanding of how the plant immune system is regulated by ubiquitination will lead to new, durable and sustainable means of combating crop disease, offering a long-term opportunity to make a significant impact on food security across the world. The proposed research is expected to benefit:
1. Ag-Biotechnology industry. This project will identify substrates for ubiquitination and degradation of E3 ligases that activate immunity, which will themselves be negative regulators of immunity. The expression of such proteins may be manipulated to provide durable resistance. In addition, we will mutate E3 ligases so that they can no longer be targeted by pathogen effectors, thus restoring immunity. Both will be of benefit to the AgBiotech companies with whom we interact.
2. Researchers investigating crop diseases and disease resistance;
3. The environment, by reducing the amount of chemical sprays required for crop protection.
4. The public, with whom regular engagement will be sought each year to communicate our science and the underlying reasons for it.
5. Staff working on the project, who will receive a broad experimental training and experience communicating their research to the public.

How will they benefit from this research?
As yet, substrates of three key ubiquitin E3 ligases, CMPG1, PUB17 and UBK, which activate immunity, are not known. This proposal will address this critical gap in our understanding. Two of these E3 ligases are targeted by RXLR effectors from the late blight pathogen, Phytophthora infestans, emphasising their importance to immunity. A key outcome of the project will be demonstrating that substrates of CMPG1, PUB17 and UBK act as negative immune regulators (susceptibility [S] factors). S factors act to suppress immunity in the plant, leading to more rapid colonisation by pathogens such as P. infestans. S factors present attractive opportunities to undermine infection as effectively and durably as nonhost resistance. For example, whereas a resistance protein imposes a selection pressure on a pathogen to alter or lose the cognate avirulence gene to evade detection, modification or conditional removal of an S factor imposes a far more challenging selection pressure, in that the associated host protein activity is required to suppress immunity. In addition, we will use random mutagenesis and shuffling of CMPG1, and targeted mutagenesis of UBK, to modify these E3 ligases so that they can evade interaction with corresponding effectors, thus restoring immunity in the presence of the pathogen. We will thus engage AgBiotech partners with whom we collaborate to promote the take-up of findings from this work.
These approaches to providing durable late blight resistance will have benefits to the environment, as less pesticide will be needed to prevent this disease, and benefits to growers and consumers, as increased yields are likely to translate to decreased costs.
Knowledge of how the E3 ligases function will reveal novel insights into the regulation of plant immunity, of benefit to the research community. The breadth of molecular and cell biological techniques and their application to strategic and applied outcomes, will benefit the career development of PDRAs employed on the project.
Finally, we will interact with the public to describe and discuss the issues underlying food security, environmental change, and genetic modification.