Interactions of blast fungus effectors with small HMAs and the paired rice NLR Pik

Stone, Caroline Eleanor (2024) Interactions of blast fungus effectors with small HMAs and the paired rice NLR Pik. Doctoral thesis, University of East Anglia.

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Abstract

Plant pathogens can cause extreme crop losses. The blast fungus, Magnaporthe oryzae, is a leading threat to rice production. One significant mechanism of immunity to M. oryzae is through intracellular nucleotide binding leucine-rich repeat receptors (NLRs), which can detect intracellular translocated pathogen effectors.

In rice, NLRs can function in pairs - for example, the Pik-1/Pik-2 pair. The Pik-1 “sensor” NLR contains an integrated heavy metal associated (HMA) domain which recognises the M. oryzae effector AVR-Pik, and the Pik-2 “helper” NLR is required for signal transduction to produce the immune response. A conserved feature of NLR activation is oligomerisation into an immune complex called the resistosome, however, to date there is no structure of a paired NLR with an integrated domain.

Therefore, the first objective of this thesis was to express and purify the Pik resistosome for structural and functional studies. I present progress in expression and purification of Pik NLRs using insect cell culture, and preliminary work towards understanding the oligomeric status and subcellular localisation of the Pik pair in Nicotiana benthamiana.

The second part of my work was to investigate the novel M. oryzae effector AVR-Mgk1 and its interactions with HMA domain containing proteins of rice. I present a crystal structure of AVR-Mgk1 with the small HMA OsHPP02 and used surface plasmon resonance to quantify the interaction. Finally, I attempted a proof-of-concept engineering using the Pik-chassis by exchanging the HMA of Pikm-1 with OsHPP02.

The molecular characterisation of the interaction of AVR-Mgk1 with HMAs adds to our understanding of the different effector binding modes of HMA domains, ultimately contributing towards efforts to engineer immune receptors with integrated domains. Meanwhile, understanding the physical arrangement of the Pik resistosome before and during activation would aid future immune receptor engineering efforts, which may ultimately contribute to securing future crop health and productivity.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Science > School of Biological Sciences
Depositing User:	Chris White
Date Deposited:	18 Mar 2025 09:01
Last Modified:	18 Mar 2025 09:01
URI:	https://ueaeprints.uea.ac.uk/id/eprint/98769
DOI:

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