Roles of protein phosphorylation during effector-triggered immunity

Johnson, Sophie (2023) Roles of protein phosphorylation during effector-triggered immunity. Doctoral thesis, University of East Anglia.

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Plants have intracellular nucleotide-binding leucine-rich repeat (NLR) immune receptors. NLRs recognize pathogen-derived effectors and activate effector-triggered immunity (ETI). This work aimed to investigate protein phosphorylation events that occur during the early activation of ETI using Arabidopsis lines carrying estradiol-inducible AvrRps4. We used a quantitative phospho-proteomics approach and generated a comprehensive phospho-peptide library from the nuclear fraction. We used parallel reaction monitoring (PRM) to target and quantify several hundred phospho-peptides. We identified proteins with differential phosphorylation upon ETI activation, some of which are known transcriptional regulators, suggesting a role of phosphorylation in activating ETI-induced transcriptional changes. To understand the biological significance of phosphorylation changes in these proteins, we assessed the impact of amino-acid substitutions at the phosphorylated residues on transcriptional regulation and protein interactions.

The signalling pathways downstream of helper NLR activation remain elusive. Therefore, we used proximity labelling and identified novel protein interactors of SARD1, TPR1 and EDS1. We identified an overlap between proteins phosphorylated during ETI and those interacting with SARD1, suggesting these proteins may be involved in ETI signal transduction. We also revealed a WRKY18 EAR domain is important for TPR1 nuclear puncta co-localisation. This study provides an important protein interaction database for which further work can utilise to enhance our understanding of protein complex formation to regulate transcriptional changes.

Previous work has revealed that PTI and ETI are required for NRG1 oligomerisation. In the third section of this study, we aimed to identify phosphorylation changes of NRG1 during PTI and ETI activation using Phos-tag gels and IP-MS. We revealed two residues of NRG1 important for HR activation in N.benthamiana. Further investigations could reveal if these phosphorylation sites are required for NRG1 oligomerisation, enhancing our understanding of helper NLR signalling mechanisms.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Nicola Veasy
Date Deposited: 27 Jun 2023 13:31
Last Modified: 31 Mar 2024 01:38


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