Deciphering the role of proteolysis in the activation and regulation of the RPM1 plant disease resistance protein

NLR plant disease resistance genes are a central component of plant innate immunity. They provide the "second line of defense" when basal immunity is breached, by actively monitoring perturbation of cellular homeostasis by pathogen effectors delivered into the plant cell. The identification and characterization of cytosolic NLRs has significantly enhanced molecular assisted plant breeding approaches. While effective, NLR resistance is often overcome resulting in >25% of the worlds crops being lost annually to plant pathogens, either in the field or post-harvest.
The "NLR" architecture comprises an amino terminal signalling domain, a central nucleotide-binding (NB-ARC) domain containing a P-loop motif involved in binding ATP or ADP, and a carboxyl terminal leucine-rich repeat (LRR) domain. Using the NB-ARC domain of RPM1 (Resistance to Pseudomonas syringae pv. maculicola 1) we identified two NB-ARC domain interactors implicated in proteolytic regulation of the RPM1 complex. RIN12 encodes a small protease inhibitor of the potato PIN1 class, which binds RPM1 within a defined region of the NB-ARC domain which includes the highly conserved ARC2 domain implicated in RPM1 autoinhibition. RIN13 binds within part of the NB-ARC region comprising the P-loop and conserved Walkers A and B kinase domains. Our data supports a role for RIN12 as a gatekeeper, maintaining RPM1 in an inactive configuration, whereas RIN13 directly or indirectly targets RPM1 for degradation.
We have generated a range of transgenic lines, mutants and other biological tools which facilitate the aim of structurally and biochemically characterising these two proteins and their role in activated RPM1 signalling using conventional assays, targeted mutagenesis, proteomics and structural biology approaches
Through this study we aim to demonstrate, for the first time, a role for targeted proteolysis in regulation of NLR signalling and identify the structure of a protease inhibitor...