Roles of the helper NLR NRG1 in effector-triggered immunity

Feehan, Joanna (2020) Roles of the helper NLR NRG1 in effector-triggered immunity. Doctoral thesis, University of East Anglia.

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Plants encode intracellular immune receptors that detect pathogen effectors and activate defence mechanisms. These receptors carry nucleotide-binding and leucinerich repeat (NLR) domains. NLR receptors may directly bind pathogen molecules (effectors) as sensor NLRs, or act as helper NLRs in signalling pathways initiated by sensor NLRs. NRG1 is a conserved helper NLR that is required for function of sensor NLRs with N-terminal TIR domains (TNLs). NRG1 co-functions with the lipase-like EDS1 and SAG101 immune signalling proteins to mediate cell death in Arabidopsis upon TNL-dependent effector-triggered immunity (ETI).

The initial goal of this research project was to uncover novel ETI signalling components. However, a forward-genetics screen revealed numerous false-positive mutant candidates, so my research focus shifted towards the signalling mechanisms of NRG1. Using Arabidopsis complementation lines, the association of NRG1 with EDS1 and SAG101 was investigated in pre- and post-immune activation contexts. In addition, large-scale purification of full-length NRG1 after transient expression in N. benthamiana was optimized for structural investigations by cryo-electron microscopy.

This thesis describes an optimized protocol for recovery of highly pure, though low yield, NRG1 protein after transient expression in N. benthamiana and immunoprecipitation. This resulted in a low-resolution map of NRG1 reconstructed from negative stain electron microscopy data. Co-immunoprecipitation assays in Arabidopsis revealed that NRG1 associates with EDS1 and SAG101 upon delivery of a TNL-recognized effector. Blue native PAGE assays indicated that higher-order states of NRG1 are formed upon PAMP-triggered immunity in the absence of effectors. Therefore, PAMP-triggered immunity may prime NRG1 for associations with EDS1 and SAG101 upon ETI initiation.

The research presented in this thesis provides a foundation for future purifications of NRG1 or other NLRs after transient expression in N. benthamiana for structural investigations. Additionally, this research has revealed the effector-dependent association of NRG1 with EDS1 and SAG101. Future investigations into the dynamics of these associations upon PAMP- and effector-triggered immunity should further resolve the mechanisms by which NRG1 functions with EDS1 and SAG101 to mediate cell death.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 24 Oct 2022 14:37
Last Modified: 30 Nov 2022 01:38


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