Domain/domain interactions in RPS4/RRS1 immune complex activation by bacterial effectors

Plant disease can cause big crop losses, which is costly to farmers. For example, control of potato late blight costs UK farmers ~ £60M/year. Resistance (R) genes enable plants to detect and resist pathogens, but how R proteins work is very poorly understood. Plant pathogens deliver molecules called effectors into host cells to interfere with host immune mechanisms. R genes enable plants to recognize such effectors and then activate immunity. To overcome R genes, pathogens must evade detection by mutations in recognized effectors.

We aim to understand R protein molecular mechanisms in such detail that we can design new R genes to recognize effectors from any pathogen. A shorter-term goal is generate intelligently designed libraries of R protein variants that can be screened for recognition of previously unrecognized effectors. Before we can do either, we need to thoroughly understand how R proteins convert recognition of effectors into activation of defense.

We study an R gene locus in the model plant Arabidopsis that confers resistance to two different bacteria, and to a fungus. The locus comprises two R genes, RPS4 and RRS1, which encode two proteins that associate to form a receptor complex that recognizes AvrRps4 and PopP2 bacterial effectors. A similar, linked R gene pair, RPS4B and RRS1B, also recognizes AvrRps4, but not PopP2. Although RPS4 and RPS4B, and RRS1 and RRS1B, are closely related (each pair ~ 60% identical), RPS4 and RRS1B associate with each other but do not function; this also true of RPS4B and RRS1. We aim to define the specific interactions between protein domains of RPS4 and RRS1, or of RPS4B and RRS1B, that enable formation of a functional complex that (i) perceives effectors and (ii) then activates signaling. We also aim to understand why non-authentic combinations fail to function.

RPS4 & RPS4B comprise 4 domains (AAAA or BBBB), and RRS1 & RRS1B comprise 5 domains (AAAAA or BBBBB). We will exchange domains between homologs, and test, for example, whether RPS4/RPS4B domain swaps BAAA, ABAA, AABA or AAAB function in combination with RRS1, and ABBB, BABB, BBAB and BBBA function in combination with RRS1B. We will also test RRS1/RRS1B swaps such as BAAAA, ABAAA etc in combination with RPS4. We can test function of RPS4 and RRS1 domain swap variants, within 3 days after transient expression of these genes in tobacco leaf tissue using Agrobacterium ("agroinfiltration").
We will also investigate domain/domain interactions by expressing each of the domains of these 4 proteins and looking for domain/domain interactions, both in yeast and after co-expression in plants and coimmunoprecipitation.

By removing a domain from the end of RRS1, or by introducing mutations into this domain, we create an RPS4/RRS1 complex that constitutively activates defense. We will investigate the domain requirements of this constitutive activity. Wild type RRS1 suppresses this constitutive defence activation triggered by RPS4 and constitutive forms of RRS1; we will investigate the mechanisms of this suppression.

RRS1 can form higher order associations with itself, so we will investigate RRS1 domain/domain interactions that might explain this multimerisation. Furthermore, the two ends of RRS1 appear to be able to interact with each other; we will investigate this in more detail.

We can monitor protein/protein or domain/domain interactions in living cells using split fluorescent proteins. These do not fluoresce separately. When attached to proteins of interest, they only become active when brought together by protein/protein interactions of the domains to which they are attached. Using this method, we have found that the N terminal domains of RPS4 and RRS1 become closely associated only upon perception of AvrRps4 or PopP2 effectors. We will investigate the temporal and genetic requirements for this association, which we believe to be key to the reconfiguration of the protein complex that leads to activation

We will investigate the Arabidopsis RPS4/RRS1 and RPS4B/RRS1B immune receptor complexes to define intra- and inter-protein domain interactions required for recognition of effectors AvrRps4 and PopP2, and for constitutive defence signaling of mutant or truncated RRS1 mutant alleles.
We will use GoldenGate cloning to make subclones of modules of each of the 4 domains of RPS4 or RPS4B, and the 5 domains of RRS1 or RRS1B. The junctions are in short regions of homology between A and B pairs, enabling easy exchange with the corresponding homolog, for facile creation of domain swap alleles of RPS4/B or RRS1/B. Constructs will be transiently coexpressed in tobacco leaves with the other pair member and effector, and tested for function. For coimmunoprecipitation (coIP), proteins will be coexpressed in Nicotiana benthamiana leaves.
To understand domain/domain interactions, individual domains (or non-functional mutant alleles thereof) will be coexpressed in N. benthamiana with other domains for which association will be tested by coIP. We will also use yeast 2 hybrid to investigate domain-domain affinities, and if affinity is detected, use E. coli or insect cell protein expression for in vitro study of domain/domain interactions.
To study domain/domain interactions in vivo, we will use split fluorescent proteins (FPs) in which a C-terminal CFP domain (cCFP) can interact with either the N terminus of Cerulean FP (nCer) or of Venus FP (nVen). Proximity of cCFP and nCer gives blue fluorescence, and cCFP and nVen yellow fluorescence. We see constitutive association of RRS1 C- and N- termini, and Avr-induced proximity of RPS4 and RRS1 N-termini. We will define the genetic and temporal requirements for Avr-dependent RPS4/RRS1 TIR proximity after AvrRps4 provision. We will use similar methods to understand effector independent defence activation by constitutive alleles of RRS1 with RPS4.

The PI and Dr Panos Sarris (PS), the proposed PDRA, will jointly manage the pathways to impact.

Our principal aim in this proposal is to carry out excellent science that will provide profound novel insights into mechanisms of plant innate immunity that involve activation of defence via TIR-NB-LRR resistance (R) proteins. Since disease resistance in crops is so important for sustainable yield, a profound understanding of these mechanisms is required for engineering of elevated resistance by, for example, extending the recognition capacity of a specific R gene. Details of such scenarios cannot be predicted in detail at this stage. Since RPS4/RRS1 confers resistance to two different bacterial diseases and a fungal disease, it could 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. 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.

Exploitation and Application
Our philosophy on impact is that "fortune favors 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 crop disease resistance. We will file for intellectual property protection of such discoveries, 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 for important crop diseases. As a member of the 2Blades science advisory board, I am well placed to bring new opportunities to their attention.
We are thus in a good position to ensure that any discoveries are translated rapidly to transgenic plant lines with elevated disease resistance, prior to market acceptance and commercialization. I led a GM potato blight resistance field trial initiated summer 2010 at the JIC site, and I 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.

Communications and Public Engagement
The output of this proposal will be disseminated to a broad audience through primary research articles and review articles in academic journals, and at international and UK conferences.

I already engage broadly with the public around the value of GM field trials for potato late blight resistance. I am an outspoken advocate of GM solutions to crop problems (see http://www.guardian.co.uk/environment/2011/jul/21/gm-debate , Twitter @jonathandgjones, and http://news.bbc.co.uk/1/hi/sci/tech/8789279.stm ) and I am a director of www.ISAAA.org. This project will provide an additional basis for public engagement.

Training
Our work will also contribute impact by the elevation of skills of Dr Sarris. TSL/JIC/UEA offers extensive opportunities for professional and scientific training. PS will use these resources to facilitate professional and technical development, and will also be trained in drafting manuscripts for publication. We aim to submit at least two manuscripts to international journals based on the outcomes of this research.

JJ and PS will participate in both formal and informal seminar series within TSL/JIC/UEA, as well as lab meetings. The PDRA will be given opportunities to attend national and international conferences and present his work via poster and oral presentations. He also will continue to help supervise PhD students and Masters or rotation student participants in the project.