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Instruments / Technical Area	Publication Details	Published
I03-Macromolecular Crystallography I04-Macromolecular Crystallography	A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum Adam R. Bentham , Yohann Petit-Houdenot , Joe Win , Izumi Chuma , Ryohei Terauchi , Mark J. Banfield , Sophien Kamoun , Thorsten Langner Plos Pathogens 17 DOI: 10.1371/journal.ppat.1009957 Diamond Proposal Number(s): [18565, 13467] Open Access Show Abstract ▼ Abstract: Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps.	Nov 2021
I03-Macromolecular Crystallography	Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain Aleksandra Bialas , Thorsten Langner , Adeline Harant , Mauricio P. Contreras , Clare E. M. Stevenson , David M. Lawson , Jan Sklenar , Ronny Kellner , Matthew J Moscou , Ryohei Terauchi , Mark J. Banfield , Sophien Kamoun Elife 10 DOI: 10.7554/eLife.66961 Diamond Proposal Number(s): [18565] Open Access Show Abstract ▼ Abstract: A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal-associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.	Jul 2021
I03-Macromolecular Crystallography	Multiple variants of the fungal effector AVR-Pik bind the HMA domain of the rice protein OsHIPP19, providing a foundation to engineer plant defence Josephine H. R. Maidment , Marina Franceschetti , Abbas Maqbool , Hiromasa Saitoh , Chatchawan Jantasuriyarat , Sophien Kamoun , Ryohei Terauchi , Mark J. Banfield Journal Of Biological Chemistry 7 DOI: 10.1016/j.jbc.2021.100371 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Microbial plant pathogens secrete effector proteins which manipulate the host to promote infection. Effectors can be recognised by plant intracellular nucleotide-binding leucine-rich repeat (NLR) receptors, initiating an immune response. The AVR-Pik effector from the rice blast fungus Magnaporthe oryzae is recognised by a pair of rice NLR receptors, Pik-1 and Pik-2. Pik-1 contains a non-canonical integrated heavy metal-associated (HMA) domain, which directly binds AVR-Pik to activate plant defences. The host targets of AVR-Pik are also HMA domain-containing proteins, namely heavy metal-associated isoprenylated plant proteins (HIPPs) and heavy metal-associated plant proteins (HPPs). Here, we demonstrate that one of these targets interacts with a wider set of AVR-Pik variants compared to the Pik-1 HMA domains. We define the biochemical and structural basis of the interaction between AVR-Pik and OsHIPP19, and compare the interaction to that formed with the HMA domain of Pik-1. Using analytical gel filtration and surface plasmon resonance, we show that multiple AVR-Pik variants, including the stealthy variants AVR-PikC and AVR-PikF which do not interact with any characterised Pik-1 alleles, bind to OsHIPP19 with nanomolar affinity. The crystal structure of OsHIPP19 in complex with AVR-PikF reveals differences at the interface that underpin high-affinity binding of OsHIPP19-HMA to a wider set of AVR-Pik variants than achieved by the integrated HMA domain of Pik-1. Our results provide a foundation for engineering the HMA domain of Pik-1 to extend binding to currently unrecognised AVR-Pik variants and expand disease resistance in rice to divergent pathogen strains.	Feb 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	Cross-reactivity of a rice NLR immune receptor to distinct effectors from the rice blast pathogen Magnaporthe oryzae provides partial disease resistance Freya A. Varden , Hiromasa Saitoh , Kae Yoshino , Marina Franceschetti , Sophien Kamoun , Ryohei Terauchi , Mark J. Banfield Journal Of Biological Chemistry DOI: 10.1074/jbc.RA119.007730 Diamond Proposal Number(s): [13467] Open Access Show Abstract ▼ Abstract: Unconventional integrated domains in plant intracellular immune receptors of the nucleotide-binding leucine-rich repeat (NLRs) type can directly bind translocated effector proteins from pathogens and thereby initiate an immune response. The rice (Oryza sativa) immune receptor pairs Pik-1/Pik-2 and RGA5/RGA4 both use integrated heavy metal–associated (HMA) domains to bind the effectors AVR-Pik and AVR-Pia, respectively, from the rice blast fungal pathogen Magnaporthe oryzae. These effectors both belong to the MAX effector family and share a core structural fold, despite being divergent in sequence. How integrated domains in NLRs maintain specificity of effector recognition, even of structurally similar effectors, has implications for understanding plant immune receptor evolution and function. Here, using plant cell death and pathogenicity assays and protein–protein interaction analyses, we show that the rice NLR pair Pikp-1/Pikp-2 triggers an immune response leading to partial disease resistance towards the “mis-matched” effector AVR-Pia in planta, and that the Pikp-HMA domain binds AVR-Pia in vitro. We observed that the HMA domain from another Pik-1 allele, Pikm, cannot bind AVR-Pia, and does not trigger a plant response. The crystal structure of Pikp-HMA bound to AVR-Pia at 1.9 Å resolution revealed a binding interface different from those formed with AVR-Pik effectors, suggesting plasticity in integrated domain–effector interactions. The results of our work indicate that a single NLR immune receptor can bait multiple pathogen effectors via an integrated domain, insights that may enable engineering plant immune receptors with extended disease resistance profiles.	Jul 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
B21-High Throughput SAXS I02-Macromolecular Crystallography I04-1-Macromolecular Crystallography (fixed wavelength)	Structural basis of host Autophagy-related protein 8 (ATG8) binding by the Irish potato famine pathogen effector protein PexRD54 Abbas Maqbool , Richard Hughes , Yasin F Dagdas , Nicholas Tregidgo , Erin Zess , Khaoula Belhaj , Adam Round , Tolga O Bozkurt , Sophien Kamoun , Mark Banfield Journal Of Biological Chemistry DOI: 10.1074/jbc.M116.744995 Diamond Proposal Number(s): [7641] Show Abstract ▼ Abstract: Filamentous plant pathogens deliver effector proteins to host cells to promote infection. The Phytophthora infestans RXLR-type effector PexRD54 binds potato ATG8 via its ATG8-family interacting motif (AIM) and perturbs host selective autophagy. However, the structural basis of this interaction remains unknown. Here we define the crystal structure of PexRD54, which comprises a modular architecture including five tandem repeat domains, with the AIM sequence presented at the disordered C-terminus. To determine the interface between PexRD54 and ATG8, we solved the crystal structure of potato ATG8CL in complex with a peptide comprising the effector’s AIM sequence, and established a model of the full-length PexRD54/ATG8CL complex using small angle X-ray scattering. Structure-informed deletion of the PexRD54 tandem domains reveals retention of ATG8CL binding in vitro and in planta. This study offers new insights into structure/function relationships of oomycete RXLR effectors and how these proteins engage with host cell targets to promote disease.	Jul 2016
I04-Macromolecular Crystallography	Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor Abbas Maqbool , H Saitoh , M Franceschetti , Cem Stevenson , A Uemura , H Kanzaki , S Kamoun , R Terauchi , M. J Banfield Elife 4 DOI: 10.7554/eLife.08709 PMID: 26304198 Diamond Proposal Number(s): [7641, 9475] Open Access Show Abstract ▼ Abstract: Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.	Aug 2015
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Oomycetes, effectors, and all that jazz Tolga O. Bozkurt , Sebastian Schornack , Mark J. Banfield , Sophien Kamoun Current Opinion In Plant Biology 15 (4), 483 - 492 DOI: 10.1016/j.pbi.2012.03.008 Diamond Proposal Number(s): [7641] Show Abstract ▼ Abstract: Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete–plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology.	Aug 2012
I02-Macromolecular Crystallography I04-Macromolecular Crystallography	Sequence Divergent RXLR Effectors Share a Structural Fold Conserved across Plant Pathogenic Oomycete Species Joe Win , Ksenia V. Krasileva , Sophien Kamoun , Ken Shirasu , Brian J. Staskawicz , Mark J. Banfield PLoS Pathogens 8 (1) DOI: 10.1371/journal.ppat.1002400 PMID: 22253591 Diamond Proposal Number(s): [7641] Open Access Show Abstract ▼ Abstract: The availability of genome sequences for some of the most devastating eukaryotic plant pathogens has led a revolution in our understanding of how these parasites cause disease, and how their hosts respond to invasion. One of the most significant discoveries from the genome sequences of plant pathogenic oomycetes is the plethora of putative translocated effector proteins these organisms encode. Many effector genes display signatures of rapid evolution and tend to reside in dynamic regions of the pathogen genomes. Once inside the host, effector proteins modulate cellular processes, mainly suppressing plant immunity. Effectors can also be recognized directly or indirectly by the plant immune system through the action of disease resistance proteins	Jan 2012

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