About the Project
Two interconnecting stories underpinning this project. First, from a high-resolution microarray experiment comparing innate immunity and its suppression by pathogens (Lewis et al. 2015) we identified a cluster of truncated Toll Interleukin Receptor disease resistance genes (tTIRs) that specifically induced by pathogen effectors. This was puzzling – why does a pathogen induce plant disease resistance genes? There are two possibilities:
- This induction is a failed host response to the effectors
- The pathogen induces these to interfere with a “normal” TIR disease resistance signaling pathway.
Recent publications showed that TIR domains under proximity induced dimerization (homo- and hetero) to regulate signaling so we hypothesized that these tTIRs interfere with defence signaling. Validation would provide a new paradigm for pathogen virulence strategies.
In parallel to this work untargeted metabolite profiling of disease development identified a unique dinucleoside molecule of MW 542.
In August, two Science papers demonstrated that self-associated TNLR TIR domains form a complex with NADase activity which is (i) essential for hypersensitive cell death and (ii) produces a “variant cyclic ADP ribose (v-cADPR)”. Remarkably v-cADPR is identical to 542, which we have characterised by accurate mass ToF/MS as a novel cyclic nucleoside.
So why is this exciting? First, plant TIR-domain resistant proteins confer resistance against a vast array of plant pathogens including bacteria, fungi, oomycetes and viruses. Moreover, the pathogen, Pseudomonas syringae contains within its complement of 28 effectors, 4 annotated as ribosyl-transferases providing a mechanistic insight into 542 formation.
This project will extend these exciting discoveries in two inter-related work programmes as follows.
First we will address the question, do pathogen induced truncated TIRs homo- and heterodimerise to form functional NADase? If so, do they function to sequester NADP+? This involves expressing TIR domains from the tTIRs and a function TIR resistant protein within E. coli and assaying for NADase activity to determine if they indeed deplete cellular NAD.
In parallel, with Chemistry, we will develop techniques to purify 542 (and v-cADPR from activated TIRs) for definitive structure determination by NMR. 542 is really rapidly induced in a susceptible interactions, accumulating to high levels within 18h of infection.
The project suits an enthusiastic and motivated student with an interest in cutting edge discovery work which could make a real difference to future Food Security. Along the journey, the project will provide a training in a wide range of techniques – molecular biology, protein expression, biochemical assays, microbiology (infection assays), chemistry (HPLC. Mass spectrometry, NMR) providing an ideal foundation for future career opportunities.
Techniques that will be undertaken during the project:
- Plant molecular biology, including generation of transgenic lines.
- Whole plant imaging for infection phenotypes.
- Biochemistry - protein expression, purification and enzyme and inhibitor assays.
- Analytical chemistry involving HPLC, mass spectrometry and NMR techniques.
- Plant pathology, encompassing infection assays and modifying pathogens.
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