Undermining effector-targeted susceptibility factors to provide late blight resistance

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. Furthermore, as introgressed host resistance genes are rapidly defeated by pathogens and pests in the field, there is an urgent need to explore the development of novel, durable and sustainable means to combat crop diseases.
Plants face a barrage of microbial threats and defend themselves by employing two layers of inducible defence responses. The first involves recognition by cell surface receptors 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. The second defence layer involves recognition of effectors by immune receptors, resistance proteins, to evoke effector-triggered immunity.
This project focusses on the late blight pathogen Phytophthora infestans, the major pathogen of potato and tomato, two of the world's main food crops. Amongst the weapons P. infestans has to subjugate its hosts are RXLR effectors which are delivered inside plant cells to promote susceptibility. RXLR effectors have been shown to target and suppress positive regulators of immunity. In contrast, here we present considerable unpublished information revealing the discovery of two RXLR effectors that target host susceptibility (S) factors; proteins whose activity promotes disease. One effector interacts with isoforms of plant protein phosphatase 1 (PP1), forming holoenzymes that may dephosphorylate host proteins to activate or inactivate them. The other effector interacts with NRL, member of a family of plasma membrane-associated regulators of light signalling. NRL promotes susceptibility by suppressing immunity triggered by perception of a P. infestans PAMP.
S factors present very attractive opportunities to undermine infection effectively and durably. 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 for infection. Critically, reducing endogenous levels of these S factors (PP1c isoforms and NRL) attenuates P. infestans infection, suggesting that combined reduction of both S factors may provide a durable strategy to combat late blight.
Little is understood about how S factors promote disease. It is an emerging, exciting area of plant disease research, making our proposal timely. We will provide detailed understanding of 1) the virulence targets dephosphorylated by effector-PP1c holoenzymes, and the mechanistic consequences of dephosphorylation (objective 1); and 2) the means by which NRL suppresses PAMP-triggered immunity (objective 2). It will also reveal whether natural variants exist of these S factors that cannot be targeted by the associated effectors (objective 3C), implicating evolution of S factors to evade effector-mediated exploitation. These studies will provide novel insights into negative regulation of plant immunity. They also provide novel means with which to combat plant disease, and potato blight specifically.
Industry is keen to exploit understanding of S factors as a durable means to provide disease resistance. This project, with industrial partner JR Simplot, will deliver novel approaches for pathogen-inducible removal of S factors to provide late blight resistance (objectives 3A; 3B).

The late blight pathogen Phytophthora infestans is the major pathogen of potato and tomato, two of the world's main food crops, making it a threat to global food security. It delivers RXLR effectors inside host cells to suppress PAMP-triggered immunity (PTI) and promote disease. So far, RXLR effectors have been shown to target positive regulators of immunity.
We have discovered that two RXLR effectors from P. infestans target host susceptibility (S) factors; proteins whose activity enhances disease. This provides a unique opportunity to prevent late blight, as our unpublished data reveals that silencing these S factors significantly attenuates late blight infection.
The first overall aim of this protect is to understand how P. infestans effectors target S factors, and how the activity of these S factors is used to support infection. One effector acts as a surrogate regulatory subunit of PP1c isoforms, hijacking their activities to promote late blight disease. We aim to discover the host proteins dephosphorylated by the effector-PP1c holoenzymes, and investigate the roles of these substrates in positively or negatively regulating immunity. The other effector interacts with NRL, a member of a plasma membrane-associated family that is known to regulate light signalling by interaction with phototropin receptors and the ubiquitin E3 ligase Cullin 3. NRL enhances P. infestans infection by suppressing PTI. We will investigate its activity in planta and the mechanism by which NRL increases plant susceptibility.
This proposal is supported by industrial partner, JR Simplot (see pathways to impact) and a second overall aim is thus to develop novel approaches to specifically undermine the utilisation of these S factors by the pathogen during infection. This will provide effective and potentially durable disease resistance.

Who will benefit from this research?
Solanaceous crops form an essential component of the world's food supply with potato ranking as the most important global non-cereal food crop. 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. New, durable and sustainable means of combating crop disease therefore offer an opportunity to make a significant impact on food security across the world. The proposed research is expected to benefit:
1. Growers worldwide, especially those in developing countries;
2. Consumers, as our research may contribute to increased potato/tomato production;
3. Biotechnology and industry. Specifically, this is an IPA proposal, supported by cash and in-kind contributions by an industrial partner, JR Simplot.
4. Breeders who will receive gene information providing markers for introgression of durable resistance traits
5. Researchers investigating crop diseases and disease resistance;
6. The environment, by reducing the amount of chemical sprays required for crop protection.
7. Staff working on the project, who will receive a broad experimental training and experience the interface between academia and industry

How will they benefit from this research?
The research focusses on host susceptibility (S) factors that act to promote infection and disease. Specifically, these S factors are targeted and utilised by effectors from the late blight pathogen, Phytophthora infestans. Removal of the S factors considerably attenuates late blight. These observations reveal points of considerable vulnerability for the pathogen, as S factors present very attractive opportunities to undermine infection as effectively and durably as nonhost resistance. Whereas introduction of 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 for infection.
The proposal has specific objectives to develop approaches for conditional removal or modification of the S factors to prevent late blight disease. The findings will be immediately taken up for exploitation by the industrial partner JR Simplot, who has received USDA and FDA approval for modified potato crops in the US. They will also transform constructs from the project into the major UK variety Maris Piper for future deregulation and potential use in the UK.
Specific objectives also seek, within late blight resistant wild potato species in the Commonwealth Potato Collection, housed in Dundee, natural S factor variants that 'evade' interaction with P. infestans effectors. These will provide molecular markers to introgress such genes into the cultivated potato as part of the commercially-funded potato breeding programme at JHI.
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 S factors 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, their application to strategic and applied outcomes, and direct interaction with industry and the commercial sector, 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, through our ongoing interactions with Dundee Botanic Gardens, through 'Skeptics in the Pub' and 'Cafe Science' discussions and through annual open days, such as 'Potatoes in Practice'.