Author: Adam Richard Bentham
Bentham, Adam Richard, 2018 Plant NLR Signalling Domains Self-Associate Preceding Signal Transduction, Flinders University, School of Biological Sciences
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Plant nucleotide-binding [NB], leucine-rich repeat [LRR] receptors (NLRs) are critical components of the plant innate immune system. Plants have evolved NLRs to detect virulence proteins secreted by microbial pathogens, deemed effectors. Upon effector detection, NLRs signal a form of programmed cell death, called the hypersensitive response (HR), which isolates the pathogen, starving them of nutrients and providing the plant resistance to further colonisation and infection. The N-terminal domains of plant NLRs are the signal transduction domain of these proteins. Transient expression assays in planta have demonstrated these domains can signal autonomously of the rest of the protein, and in the absence of an effector. There are two major classes of NLR proteins, segregated by their N-terminal domains. The domains that occupy the N-terminus of a plant NLR can consist of either a Toll-interleukin-1 receptor (TIR) domain or a coiled-coil (CC) domain. While some progress has been made towards understanding the structure and function of TIR and CC domains, there are still many uncertainties concerning how cell death signalling is initiated. In the case of TIR domains, two separate interfaces have been identified to be important for self-association and signal transduction. These interfaces from the TIR domains of the flax NLR L6, and Arabidopsis NLR RPS4, share a conserved overall fold, however signal through different self-association interfaces. Why this is the case is unclear and highlights unknowns in TIR domain signalling. In regard to the CC domains from plant NLRs, only two structures have been determined. These CC domains belong to the barley NLR MLA10, and the potato NLR Rx. Despite these proteins belonging to the same subclass of CC domains, both have a significantly different fold, with MLA10 CC domain forming an obligate helix-loop-helix homodimer, and Rx forming a monomeric four-helix bundle. These differences raise questions about the true nature of CC domain structure and function. In this thesis, structural, biochemical, and biophysical techniques are used to examine the TIR domain of the Arabidopsis NLR, RPP1, and the CC domain of the wheat NLR, Sr33, in an attempt to contribute more to our understanding of N-terminal domain structure and signalling. The structure of the RPP1 TIR domain was resolved showing that both the previously identified L6 and RPS4 TIR domain interfaces are necessary for RPP1 TIR domain function, and are required simultaneously for effective signalling. The work presented here on the CC domain of Sr33 showed that this domain shares the same fold in solution to Rx, maintaining a monomeric four-helix bundle. Furthermore, a longer Sr33 CC construct than previously reported was shown to be required for the self-association of the Sr33 and MLA10 CC domains, which correlated with the ability of these CC domains to trigger cell death signalling in planta. This work on N-terminal domains has helped rectify some inconsistences in the literature surrounding both TIR domains and CC domains, and broaden our understanding of how NLRs signal in a more general sense.
Keywords: NLR, plant immunity, Toll/interleukin-1 receptor, coiled-coil, oligomerisation, self-association, structural biology, programmed cell death
Subject: Biology thesis
Thesis type: Doctor of Philosophy
Completed: 2018
School: School of Biological Sciences
Supervisor: Peter Anderson