Transcriptional regulation in effector-triggered immunity via NB-LRR resistance genes RPS4 & RRS1

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 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; more R genes are continuously needed by breeders, because pathogens can evolve to overcome them. R genes confer recognition of pathogen virulence-promoting molecules (so-called "effectors") that contribute to virulence. R gene durability varies; it is likely that more durable R genes recognize the most indispensable effectors for the pathogen. Plant resistance also involves responses to conserved pathogen molecular patterns- "pattern-triggered immunity (PTI)". Although there is overlap in PTI and R gene-mediated resistance responses, the mechanistic link between them is still completely obscure.
For durable resistance in our future crops, we need a deep understanding of pathogen virulence and host defence mechanisms. This requires knowledge of the spectrum of host targets of effectors, of how plant R proteins recognize (directly or indirectly) the presence of effectors, and of how upon recognition, defence mechanisms are activated that result in host plant immunity. Plant R proteins resemble mammalian Nod-like receptors (NLRs) that are involved in mammalian innate immunity; for example, Crohn's disease results from a defect in the human NOD2 gene. Mechanisms of NLR signalling are also incompletely understood. NOD2 and NB-LRR proteins carry similar protein modules, with an N-terminal signalling domain, a central nucleotide-binding (NB) domain and a C-terminal leucine-rich repeat (LRR) domain.
The proposed work will help us better understand mechanisms of plant resistance. We will study a fascinating disease resistance locus in the model plant Arabidopsis that confers resistance to two distinct bacterial species that cause either bacterial speck or bacterial wilt, and also confers resistance to a fungal pathogen. This locus contains two different NB-LRR R proteins, RRS1 and RPS4, that are transcribed away from each other, and both of which are required for resistance. RPS4 and RRS1 also carry a so-called TIR domain shared with immune receptors of humans and flies. RRS1 has an additional domain (a "WRKY" domain) that has been shown to bind DNA, and that could regulate expression of genes involved in plant defence. RPS4/RRS1 activates defence upon recognizing AvrRps4 effector protein from bacterial speck, or PopP2 effector protein from bacterial wilt, or the fungal pathogen Colletotrichum higginsianum. How this works is still a mystery.
- We wish to understand how RPS4/RRS1 activates expression of genes involved in defence upon recognition of AvrRps4 or PopP2. We will investigate the DNA sequences targeted by the DNA binding domain of RRS1, and compare the corresponding genes to the genes that are induced upon RPS4/RRS1 defence activation, and to genes induced during PTI. In this way we will define the direct targets of RPS4/RRS1 and the genes that when activated result in resistance. This will enable us to build up a picture of the chain of events triggered via R proteins that stop the pathogen from growing.
- Once we have all the data from these investigations we will be able to propose and test models for what takes place during defence activation, making this system one of the best understood R gene systems; this knowledge will inform approaches to attempting to broaden the recognition capacity of R genes, to thus enhance their utility and durability.

We will use gene expression profiling and chromatin immunoprecipitation (ChIP) to investigate how TIR-NB-LRR resistance proteins RPS4 and RRS1, after recognizing AvrRps4 and PopP2, activate defence gene expression and disease resistance.
1. Using an Illumina-based expression profiling method, we will identify genes most rapidly regulated by RPS4/RRS1 over a time course after delivery to Arabidopsis leaves of AvrRps4 and PopP2 from Pseudomonas. To improve the time course, we will use GRE-tagged AvrRps4 and PopP2 that can only enter the nucleus to activate defence after dexamethasone (DEX) treatment; we will allow 14 hrs overnight for effector-GRE delivery, and DEX induce and make mRNA from time-points at the beginning of the next light period. To define direct targets, we will identify the earliest induced/repressed genes that are still induced in the presence of translational inhibitor cycloheximide (CHX).
2. Inducible expression of the C- terminal DNA binding domain encoded by exons 5,6 and 7 (e567) of RRS1, also activates defence. We will expression profile e567-GRE induced genes after DEX provision, again prioritizing CHX-insensitive gene expression changes.
3. We will test inferred RRS1 promoter targets by ChIP and discover new targets of RRS1 by Illumina sequencing of e567- and RRS1-precipitated chromatin. Since WRKY transcription factors also play a role in basal defence and PTI triggered by flg22, we will look for overlap in flg22- and RRS1/RPS4- dependent gene expression changes.
4. We then aim to write up an analysis of RRS1/RPS4-dependent gene expression changes and infer models for RPS4/RRS1 activation of defence. We will also obtain knockout lines of likely key genes downstream of RPS4/RRS1, and test response phenotypes. We will use yeast 1-hybrid to identify all TFs that bind RRS1-targeted promoters. We will test PTI in double and triple mutants of RRS1 and its paralogs.

Our principal aim in this proposal is to carry out excellent science that will provide profound novel insights into mechanisms of plant innate immunity, particularly those that involve activation of defence via TIR-NB-LRR disease resistance proteins and WRKY transcription factors. Since disease resistance in crops is so important for sustainable yield, a profound understanding of these mechanisms is required for any future engineering of elevated resistance by, for example, extending the recognition capacity of a specific resistance gene. Details of such scenarios cannot be predicted in detail at this stage. Since the RPS4/RRS1 system we study can confer resistance to two different bacterial diseases and to a fungal disease, it has the potential to confer broad range resistance to multiple species if properly understood.
Because the TIR-NB-LRR proteins have counterparts in mammalian NLR proteins that are also involved in innate immunity, we anticipate a broad impact across the immunity field from plants to animals, especially if we can document a role of NB-LRR proteins in transcriptional regulation. Conceivably our insights may even impact human or veterinary health through generally applicable insights into innate immunity.
We will disseminate our discoveries by giving talks at scientific meetings- I regularly participate in such meetings- and by submitting manuscripts to high impact peer-reviewed journals.
Our work will also contribute impact by the elevation of skills of the appointed PDRA.
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 am a director of www.ISAAA.org.