The mechanistic basis of plant NLR signalling in effector triggered immunity

Evolution has seen an arms race between plants and invading pathogens. Pathogens inject plant cells with proteins to block cell protective immune responses. Plants have responded through the evolution of a specific family of proteins, the NLR proteins (an acronym meaning nucleotide-binding and leucine-rich repeat domains) that detect these bacterial proteins and counter their activity. The NLR proteins trigger powerful innate immune responses including a genetically encoded cell death response thus restricting further spread of the pathogen. Understanding NLR protein function and, importantly, exploiting the knowledge platform that we gain from their analysis can ultimately be developed to enhance crop defences to pathogens as part of a strategy to improve crop productivity and feed the world's growing population.

Despite their key role in protecting plants from invading pathogens, relatively little is known of the specifics of molecules targeted by NLRs to enable them to perform their immune function. This has been partly due to difficulties in generating NLR proteins for analysis in the test tube. We have developed methods for the production of NLR proteins for test tube based analysis. This has enabled us to exploit the rich toolbox of biochemical and biophysical techniques to gain new insight into their function.

Our analysis of NLR protein function in the test tube has thrown up a surprising result. We have demonstrated that NLR proteins are able to bind to and locally distort DNA. Parallel experiments have demonstrated that DNA binding also occurs in plants and is specifically dependent on activation of the immune system. This result is all the more exciting as it is becoming clear from multiple independent researchers that many NLRs are functional in the nucleus. These multiple lines of evidence lead us to posit a central hypothesis that underpins this work. We propose that NLR proteins are specifically targeted to genomic DNA, with accessory proteins, on immune activation and that NLR proteins enhance transcription initiation at these sites.

Here we perform experiments to study various aspects of this hypothesis.

First, we will investigate the requirements for an NLR to bind to DNA in the plant cell on activation of the immune system. To achieve this we will exploit the rich history of plant genetics and pathology that has produced an incredible array of well defined NLR variants that both activate and inactivate immunity, alter interactions with accessory proteins, and modulate contacts within the protein itself. These important experiments will crucially place the capacity of NLRs to bind DNA within the context of the pre-existing knowledge base for NLR function.

Second, we will investigate how NLRs can be targeted to particular DNA sequences by partnering proteins and thus provide specificity for the immune response. There is growing evidence that many NLRs can associate with such targeting proteins and we have identified some in work towards this proposal. We propose to establish that the association of NLRs with particular DNA sequences is a key stage in establishing the protective immune response. Together these experiments will therefore provide new insight into how NLRs activate specific immune responses.

Third, we will establish how NLRs function at DNA to regulate a transcriptional response. We propose that NLRs are targeted to specific DNA sequences and once targeted, activate proteins that alter DNA structure thus permitting transcription. These experiments are significant, as they will reveal general mechanisms by which NLRs can function in complexes to promote protective transcriptional responses.

Plant NLR proteins trigger disease resistance in response to pathogen effectors. Pathogen effector detection via the NLR leucine rich repeat is signalled to an N-terminal domain that forms protein interactions crucial for immunity. The intramolecular signal is also transmitted to a central nucleotide binding (NB) domain whose function in signalling is ambiguous. Specific mechanistic knowledge for NLR function is a major gap in our knowledge of plant immunity.

We have developed methodology for the production of recombinant NLR proteins. These have been used to demonstrate that the NB domain is an immune dependent DNA binding domain. We hypothesize that NLR proteins can function as bridging molecules that respond to pathogen effectors by distorting DNA and activating a protective transcriptional response. In support of this hypothesis we have demonstrated that a model NLR interacts with a transcription factor and a chromatin remodelling protein.

We will use in vitro analysis, with supporting experiments in plants, to develop a model for NLR DNA binding in immune signalling.

First, we will use fluorescence lifetime techniques to examine how DNA binding is altered in NLR proteins with altered spatial localization, inter- and intramolecular contacts, and immune signalling function. We will thus place DNA binding within the context of our knowledge of NLR signalling.

Second, we will use mass spectrometric and gene silencing techniques to identify NLR interacting proteins that target NLRs to specific DNA and provide a mechanistic basis for NLRs in transcription initiation. We will thus provide a clear link between immune activation, NLRs, and transcriptional reprogramming.

Third, we will use biochemical assays to identify how an interacting transcription factor targets an NLR to specific DNA sequences to permit subsequent chromatin remodelling. We will thus provide a clear link between immune activation and processes required for transcriptional activation.

How will Impact arise from this work?

Our overall Impact goal is the production of excellent quality science that will both provide foundational insights into NLR biology and enhance the future development of NLR proteins as targets for genetic engineering in plants. The future development of sustainable increases in crop yield through enhanced disease resistance via engineered NLRs will require a deep and thorough understanding of NLR function. Impact development therefore requires a mechanistic investigation of NLR activity as it relates to the transcriptional responses that underpin many aspects of immunity. The knowledge obtained from our work can offer routes to impact through the engineering of crops with bespoke transcriptional responses to specific molecular cues. We will use the knowledge platform from this work to initiate this process. We will utilise a pre-existing relationship between the Durham Business and Innovation Services, the PI, and intellectual property consultancy company IP Pragmatics to open routes to commercial development.

How will the impact agenda be initialized?

We will perform the following initial consultancy work with IP Pragmatics toward the close of year 1.

1. Patent Analysis: A formal assessment outlining the patent landscape surrounding the initial findings.
2. Partner and Competitor Analysis: An analysis of the potential collaborator/licensing landscape for the initial findings.
3. Market Analysis: A market analysis overview including competitors and a commercialisation route map. This will include direct contact with relevant agri-business companies to validate the proposition.

Based on feedback from companies we will initialize an experimental stream whose sole purpose is to realise key proof of concept data to validate commercialization potential. This experimental plan will run alongside and be timed to coincide with the proposed experiments of the Case for Support (see Work Plan). On the basis of these findings we will initiate a second round of consultancy with IP Pragmatics towards the start of year 3. This second round of consultancy will include an updated patent, partner, and market analysis. This further consultancy will also be oriented toward partnering support and agreed routes to commercial development with an identified agri-business company. Partnering support will consist of the compilation of a data package and marketing material for the findings, identification and introduction to suitable agri-business companies as commercial partners, support for evaluation and (if appropriate) deal negotiation, and advice on the value of the technology and suitable partnership structures.

Who will benefit from the research?

The long-term goal of a resulting commercialization venture is to enhance pathogen resistance in crops either through GM modification of plants or the development of biologics to be used in sprays or seed coatings. If such a goal is realised then the benefits will be realised by partnering agri-business, the farming industry, and ultimately the public as end user.