Using a rice blast effector target to engineer NLR immune receptors with novel recognition specificities

Maidment, Josephine (2020) Using a rice blast effector target to engineer NLR immune receptors with novel recognition specificities. Doctoral thesis, University of East Anglia.

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Arable farmers across the world are engaged in a constant battle with plant pathogens. The rice blast fungus Magnaporthe oryzae is a threat to global rice production. During infection, M. oryzae secretes effector proteins which interact with host targets to promote pathogen virulence. Specific effectors can also be recognised by intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptor proteins, activating host defences.

Recognition of the rice blast effector AVR-Pik is mediated by the paired rice NLR proteins Pik-1/Pik-2. An integrated heavy metal-associated (HMA) domain was previously identified in Pik-1, to which certain variants of AVR-Pik directly bind to trigger resistance. Multiple Pik-1 alleles have evolved in rice and exhibit differential responses to different AVR-Pik effector variants. AVR-PikC and AVR-PikF do not interact with any known Pik-1 alleles and thus evade plant defences.

In this work, I used biochemical and structural techniques to characterise the interaction between AVR-Pik and the HMA domain of its putative virulence target, OsHIPP19. I demonstrated that a second rice blast effector, AVR-Pia, also interacts with OsHIPP19-HMA. By modifying the HMA domain of Pik-1 to resemble OsHIPP19, I engineered two Pik-1 variants (Pikp-1SNK-EKE and Pikp-1HIPP19_mbl7) which trigger HR-like cell death in response to AVR-PikC/AVR-PikF in a model system. Pikp-1SNK-EKE was engineered through targeted mutagenesis of Pik-1 guided by the crystal structure of AVR-PikF in complex with OsHIPP19-HMA. Exchanging the HMA domain of Pikp-1 for that of OsHIPP19 resulted in an autoactive chimera, however subsequent modifications produced a second Pik-1 variant, Pikp-1HIPP19_mbl7, which was not autoactive and could respond to AVR-PikC/AVR-PikF in a model plant.

The engineered Pik-1 NLR proteins are being incorporated into transgenic rice and barley to investigate whether they confer resistance to Magnaporthe oryzae. Rationally engineered immune receptors offer opportunities to control emerging and rapidly evolving pathogens, contributing to global food security.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Chris White
Date Deposited: 20 Oct 2022 09:39
Last Modified: 28 Feb 2023 01:38

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