Mechanisms of nuclear suppression of host immunity by Arabidopsis downy mildew effectors

Crop plants are subject to diseases caused by various microbes that can cause substantial yield losses. Farmers spray agrochemicals to control disease; this is expensive, requires fuel and labour, and leads to CO2 emissions and soil compaction. It would be preferable for crops to be disease resistant, so that no fungicide applications are required. More knowledge is required to achieve this goal. Breeders use disease resistance (R) genes for crop improvement. R genes confer recognition of pathogen virulence-promoting molecules (so-called "effectors") that contribute to virulence. In order to be able to predict and (in future) design the most durable R genes, we need to fully understand what pathogen effectors do, and the host processes that they target.

We work with a well-characterized model host/pathogen system, downy mildew of Arabidopsis; this is a close relative the potato late blight pathogen Phytophthora infestans. The genome sequence of this downy mildew has been determined, and over 100 likely effectors have been predicted. We have tested many of these and shown that they suppress host defences. Many of them are predominantly targeted to the host cell nucleus, where they could interfere with host gene expression. We are focusing this project on nuclear localized effectors. Thanks to two excellent European funded postdoctoral fellows, analysis of two effectors is already well advanced, and reagents to study many others are also well advanced.
Effector HaRxL106 interacts with host components that carry proteins from the cytoplasm to the nucleus, and with a transcriptional regulator that responds to redox stress. Arabidopsis lines that express this protein also show developmental changes in light responses, suggesting that this effector targets some fundamental host mechanisms. Arabidopsis is well defined genetically, and using this deep knowledge of Arabidopsis biology, we expect to be able to reveal exactly how HaRxL106 interactions with plant components results in enhanced susceptibility to disease.
HaRxL44 interacts with components of the Mediator complex, which plays an important fundamental role linking DNA-binding proteins that alter gene expression, to the transcription machinery itself. HaRxL44 expressing Arabidopsis lines show enhanced disease susceptibility and curly leaves, again consistent with a profound effect on host gene regulation. This curly leaf phenotype is often seen in plants that are altered in their regulation by small RNA molecules. For both of these effectors we aim to fully understand how they suppress host defence.
We are analysing additional nuclear effectors and aim to advance these studies too. HaRxL62 is the Hpa effector that most profoundly suppresses immunity, but we have so far not identified interacting host proteins; we aim to do so. HaRxL2, HaRxL73 and HaRxL3 also show profound effects on host immunity and cause developmental perturbations when expressed in planta; we aim to advance our understanding of their mechanisms of action by identifying proteins with which they interact in planta.
How will this knowledge be useful? By understanding how effectors bind to and interfere with host components, we should be able to design forms of these host components that are refractory to the action of the effectors; plants carrying these novel forms should exhibit enhanced disease resistance.

We will discover mechanisms by which multiple Hpa effectors suppress host defence via protein-protein interaction studies, expression profiling, genetic analysis and proteomics.

For HaRxL106 we will address:
1a) Is the interaction with alpha-importins solely in order to enable HaRxL106 to enter the nucleus, or does interference with alpha-importin function contribute to HaRxL106's virulence activity?
1b) Why does the C-terminal domain of HaRxL106 attenuate light signalling and does this effect on light signal transduction contribute to its virulence function?
1c) Why do mutations in RCD1 impair NPR1 signalling? What is the function of RCD1 in plant defence?
1d) How does HaRxL106 bind to RCD1 and what are the molecular consequences of this interaction?
1e) Is HaRxL106 associated with specific defence promoters of genes that are down-regulated in HaRxL106 overexpressors?

For HaRxL44 we will ask:
Is HaRxL44 associated with the Mediator complex? Does HaRxL44 expression influence known defence processes that require Mediator, such as jasmonate signalling? Do alterations in JA signalling also compromise salicylic acid signalling for biotroph resistance? What is the mechanism by which HaRxL44 destabilizes MED19 subunit; does it promote ubiquitination of Med19 via E3 ligase BOI or At1g17970? Or does it compete with MED19 for assembly in the Mediator complex? Since HaRxL44 overexpressors resemble some mutants altered in small RNA-mediated regulation, we will use small RNA profiling to ask if overexpression alters the abundance of particular small RNAs

For the less well-characterized effectors HaRxL62, 2, 3 and 73, and 8 others, we will complete generation of tagged lines that will enable us to directly search for interacting proteins using forward proteomics, and further characterize phenotypes of mutations in host genes that encode interacting proteins, to build models for effector action.

Our principal aim in this proposal is to carry out excellent science that will provide profound novel insights into how pathogens impose disease susceptibility on their hosts.

We will disseminate our discoveries by giving talks at scientific meetings and at University departmental talks - I regularly participate in such events- and by submitting manuscripts to high impact peer-reviewed journals.
Our work will also contribute impact by very significantly contributing to the experience, qualifications and publications of the two designated PDRAs, Marie-Cecile Caillaud and Lennart Wirtmueller. Their data so far, though already encouraging and interesting, need considerable addition and consolidation for a high impact journal. The proposed work is likely to make profound contributions to understanding regulation of nucleocytoplasmic trafficking, plant immunity and small RNA regulation.

We hope that once we have revealed effector targets, we will be able generate alleles of those targets that are still functional but are refractory to the action of the effectors. Lines carrying such alleles should be more resistant to the pathogen than wild type.
Our philosophy on impact is that "fortune favours the prepared mind", and we are fully prepared to identify and act efficiently to bring to public use any discoveries we make that could elevate disease resistance in crops. If we discover new ways to elevate crop disease resistance, we will file for intellectual property protection either with Plant Biosciences Ltd at JIC or with the 2Blades foundation (www.2blades.org) which is a charity dedicated to delivering knowledge-based solutions to the most important crop disease problems. As a member of the 2Blades science advisory board, I am well placed to identify new opportunities and create a path to deployment in collaboration with 2Blades.
We are thus in a good position to ensure that any discoveries are translated as rapidly as possible to transgenic plant lines with elevated disease resistance, prior to market acceptance and commercialization. As a first step, I am leading a GM potato blight resistance field trial initiated summer 2010 at the JIC site, and we hope this will be first of many technologies we will be able to test in the field. Achievement of durable disease resistance while minimizing the need for agrichemicals will benefit farmers, breeders and consumers.

I engage in multiple outreach activities and will use these opportunities to deliver improved public understanding of our research. For example, I spoke at a Norwich Science Café event in June 2010 in a talk entitled "Why GM crops". I have been an outspoken advocate of GM solutions to crop problems (see http://www.guardian.co.uk/environment/2011/jul/21/gm-debate, http://news.bbc.co.uk/1/hi/sci/tech/8789279.stm, http://www.speakerscornertrust.org/forum/forum-for-debate/). I on the board of European Plant Science Organization and I am a director of the GM advocacy organization www.ISAAA.org. I regularly comment to the media on GM issues, and also I regularly engage with farmers groups (most recently on an NFU-sponsored visit by farmers to JIC on 19th April). I am lead organizer on an EMBO course on plant/microbe interactions at JIC/TSL in June 2012.