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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Effector target-guided engineering of an integrated domain expands the disease resistance profile of a rice NLR immune receptor Josephine H. R. Maidment , Motoki Shimizu , Adam R. Bentham , Sham Vera , Marina Franceschetti , Apinya Longya , Clare E. M. Stevenson , Juan Carlos De La Concepcion , Aleksandra Bialas , Sophien Kamoun , Ryohei Terauchi , Mark J. Banfield Elife 12 DOI: 10.7554/eLife.81123 Diamond Proposal Number(s): [13467, 18565] Open Access Show Abstract ▼ Abstract: A subset of plant intracellular NLR immune receptors detect effector proteins, secreted by phytopathogens to promote infection, through unconventional integrated domains which resemble the effector’s host targets. Direct binding of effectors to these integrated domains activates plant defenses. The rice NLR receptor Pik-1 binds the Magnaporthe oryzae effector AVR-Pik through an integrated heavy metal-associated (HMA) domain. However, the stealthy alleles AVR-PikC and AVR-PikF avoid interaction with Pik-HMA and evade host defenses. Here, we exploited knowledge of the biochemical interactions between AVR-Pik and its host target, OsHIPP19, to engineer novel Pik-1 variants that respond to AVR-PikC/F. First, we exchanged the HMA domain of Pikp-1 for OsHIPP19-HMA, demonstrating that effector targets can be incorporated into NLR receptors to provide novel recognition profiles. Second, we used the structure of OsHIPP19-HMA to guide the mutagenesis of Pikp-HMA to expand its recognition profile. We demonstrate that the extended recognition profiles of engineered Pikp-1 variants correlate with effector binding in planta and in vitro, and with the gain of new contacts across the effector/HMA interface. Crucially, transgenic rice producing the engineered Pikp-1 variants was resistant to blast fungus isolates carrying AVR-PikC or AVR-PikF. These results demonstrate that effector target-guided engineering of NLR receptors can provide new-to-nature disease resistance in crops.	May 2023
I03-Macromolecular Crystallography	A blast fungus zinc-finger fold effector binds to a hydrophobic pocket in host Exo70 proteins to modulate immune recognition in rice Juan Carlos De La Concepcion , Koki Fujisaki , Adam R. Bentham , Neftaly Cruz Mireles , Victor Sanchez De Medina Hernandez , Motoki Shimizu , David M. Lawson , Sophien Kamoun , Ryohei Terauchi , Mark J. Banfield Proceedings Of The National Academy Of Sciences 119 DOI: 10.1073/pnas.2210559119 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: Exocytosis plays an important role in plant–microbe interactions, in both pathogenesis and symbiosis. Exo70 proteins are integral components of the exocyst, an octameric complex that mediates tethering of vesicles to membranes in eukaryotes. Although plant Exo70s are known to be targeted by pathogen effectors, the underpinning molecular mechanisms and the impact of this interaction on infection are poorly understood. Here, we show the molecular basis of the association between the effector AVR-Pii of the blast fungus Maganaporthe oryzae and rice Exo70 alleles OsExo70F2 and OsExo70F3, which is sensed by the immune receptor pair Pii via an integrated RIN4/NOI domain. The crystal structure of AVR-Pii in complex with OsExo70F2 reveals that the effector binds to a conserved hydrophobic pocket in Exo70, defining an effector/target binding interface. Structure-guided and random mutagenesis validates the importance of AVR-Pii residues at the Exo70 binding interface to sustain protein association and disease resistance in rice when challenged with fungal strains expressing effector mutants. Furthermore, the structure of AVR-Pii defines a zinc-finger effector fold (ZiF) distinct from the MAX (Magnaporthe Avrs and ToxB-like) fold previously described for a majority of characterized M. oryzae effectors. Our data suggest that blast fungus ZiF effectors bind a conserved Exo70 interface to manipulate plant exocytosis and that these effectors are also baited by plant immune receptors, pointing to new opportunities for engineering disease resistance.	Oct 2022
I04-Macromolecular Crystallography	The allelic rice immune receptor Pikh confers extended resistance to strains of the blast fungus through a single polymorphism in the effector binding interface Juan Carlos De La Concepcion , Josephine H. R. Maidment , Apinya Longya , Gui Xiao , Marina Franceschetti , Mark J. Banfield Plos Pathogens 17 DOI: 10.1371/journal.ppat.1009368 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Arms race co-evolution drives rapid adaptive changes in pathogens and in the immune systems of their hosts. Plant intracellular NLR immune receptors detect effectors delivered by pathogens to promote susceptibility, activating an immune response that halts colonization. As a consequence, pathogen effectors evolve to escape immune recognition and are highly variable. In turn, NLR receptors are one of the most diverse protein families in plants, and this variability underpins differential recognition of effector variants. The molecular mechanisms underlying natural variation in effector recognition by NLRs are starting to be elucidated. The rice NLR pair Pik-1/Pik-2 recognizes AVR-Pik effectors from the blast fungus Magnaporthe oryzae, triggering immune responses that limit rice blast infection. Allelic variation in a heavy metal associated (HMA) domain integrated in the receptor Pik-1 confers differential binding to AVR-Pik variants, determining resistance specificity. Previous mechanistic studies uncovered how a Pik allele, Pikm, has extended recognition to effector variants through a specialized HMA/AVR-Pik binding interface. Here, we reveal the mechanistic basis of extended recognition specificity conferred by another Pik allele, Pikh. A single residue in Pikh-HMA increases binding to AVR-Pik variants, leading to an extended effector response in planta. The crystal structure of Pikh-HMA in complex with an AVR-Pik variant confirmed that Pikh and Pikm use a similar molecular mechanism to extend their pathogen recognition profile. This study shows how different NLR receptor alleles functionally converge to extend recognition specificity to pathogen effectors.	Mar 2021
I03-Macromolecular Crystallography	Protein engineering expands the effector recognition profile of a rice NLR immune receptor Juan Carlos De La Concepcion , Marina Franceschetti , Dan Maclean , Ryohei Terauchi , Sophien Kamoun , Mark J. Banfield Elife 8 DOI: 10.7554/eLife.47713 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Plant nucleotide binding, leucine-rich repeat (NLR) receptors detect pathogen effectors and initiate an immune response. Since their discovery, NLRs have been the focus of protein engineering to improve disease resistance. However, this approach has proven challenging, in part due to their narrow response specificity. Previously, we revealed the structural basis of pathogen recognition by the integrated heavy metal associated (HMA) domain of the rice NLR Pikp (Maqbool et al., 2015). Here, we used structure-guided engineering to expand the response profile of Pikp to variants of the rice blast pathogen effector AVR-Pik. A mutation located within an effector-binding interface of the integrated Pikp–HMA domain increased the binding affinity for AVR-Pik variants in vitro and in vivo. This translates to an expanded cell-death response to AVR-Pik variants previously unrecognized by Pikp in planta. The structures of the engineered Pikp–HMA in complex with AVR-Pik variants revealed the mechanism of expanded recognition. These results provide a proof-of-concept that protein engineering can improve the utility of plant NLR receptors where direct interaction between effectors and NLRs is established, particularly where this interaction occurs via integrated domains.	Sep 2019
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen Juan Carlos De La Concepcion , Marina Franceschetti , Abbas Maqbool , Hiromasa Saitoh , Ryohei Terauchi , Sophien Kamoun , Mark J. Banfield Nature Plants 354 DOI: 10.1038/s41477-018-0194-x Diamond Proposal Number(s): [9475, 13467] Show Abstract ▼ Abstract: Accelerated adaptive evolution is a hallmark of plant–pathogen interactions. Plant intracellular immune receptors (NLRs) often occur as allelic series with differential pathogen specificities. The determinants of this specificity remain largely unknown. Here, we unravelled the biophysical and structural basis of expanded specificity in the allelic rice NLR Pik, which responds to the effector AVR-Pik from the rice blast pathogen Magnaporthe oryzae. Rice plants expressing the Pikm allele resist infection by blast strains expressing any of three AVR-Pik effector variants, whereas those expressing Pikp only respond to one. Unlike Pikp, the integrated heavy metal-associated (HMA) domain of Pikm binds with high affinity to each of the three recognized effector variants, and variation at binding interfaces between effectors and Pikp-HMA or Pikm-HMA domains encodes specificity. By understanding how co-evolution has shaped the response profile of an allelic NLR, we highlight how natural selection drove the emergence of new receptor specificities. This work has implications for the engineering of NLRs with improved utility in agriculture.	Jul 2018

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