Structural and biochemical investigation of the RRS1/RPS4 immune receptor pair

Brown, Hannah (2019) Structural and biochemical investigation of the RRS1/RPS4 immune receptor pair. Doctoral thesis, University of East Anglia.

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Pathogens secrete an arsenal of effectors to facilitate infection, however these effectors are themselves susceptible to molecular detection by plant immune receptors known as NLRs (nucleotide-binding, leucine-rich repeat proteins). Our understanding of how NLRs function mechanistically still remains relatively rudimentary. Mechanistic studies of NLRs have largely been hampered by difficulties in producing sufficient quantities of soluble NLR protein for biochemical and structural investigation. A major goal in the field is to expand our structural understanding of how effectors activate NLRs and to increase the variety and number of protein structures of these receptors.

Using the example of the Arabidopsis paired TIR-NLRs RRS1 and RPS4, this work aimed to evaluate a diverse range of protein expression systems for their suitability for NLR protein production. This study investigated the use of classical heterologous expression systems of E. coli and insect cells as well as plant-based systems such as cell-free wheat germ, transgenic lines of Arabidopsis thaliana and agroinfiltration of Nicotiana benthamiana. Soluble full-length RRS1 protein could be purified from plant-based systems, though protein yield issues have hampered current efforts to gain structural information on this protein. The insights presented in this multi-system screening process should provide a valuable foundation for future studies into NLR purification.

In the second part of this work, I set out to examine the structural basis of the recognition of Pseudomonas syringae effector AvrRps4 by RRS1 via RRS1’s integrated WRKY transcription factor domain. Previous work has shown that binding of the effector AvrRps4 to RRS1 is required but not sufficient to activate a defence response. I therefore set out to gain a structural insight into this NLR-effector interface to help guide and support our biological understanding of the inter- and intra-molecular interactions involved in the activation of this receptor complex. Through the use of quantitative biochemical techniques including surface plasmon resonance and analytical gel filtration, we gained important insights into the molecular basis of this interaction.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 11 Feb 2021 14:25
Last Modified: 11 Feb 2021 14:25


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