I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
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.
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Feb 2021
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
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.
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Sep 2019
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I02-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[7641]
Open Access
Abstract: Plant NLRs are modular immune receptors that trigger rapid cell death in response to
attempted infection by pathogens. A highly conserved nucleotide-binding domain shared
with APAF-1, various R-proteins and CED-4 (NB-ARC domain) is proposed to act as a
molecular switch, cycling between ADP (repressed) and ATP (active) bound forms. Studies
of plant NLR NB-ARC domains have revealed functional similarities to mammalian homologues,
and provided insight into potential mechanisms of regulation. However, further
advances have been limited by difficulties in obtaining sufficient yields of protein suitable for
structural and biochemical techniques. From protein expression screens in Escherichia coli
and Sf9 insect cells, we defined suitable conditions to produce the NB-ARC domain from the
tomato NLR NRC1. Biophysical analyses of this domain showed it is a folded, soluble protein.
Structural studies revealed the NRC1 NB-ARC domain had co-purified with ADP, and
confirmed predicted structural similarities between plant NLR NB-ARC domains and their
mammalian homologues.
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Aug 2019
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
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.
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Jul 2019
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[1219]
Abstract: The potato blight agent Phytophthora infestans secretes a range of RXLR effectors to promote disease. Recent evidence indicates that some effectors suppress early pattern‐triggered immunity (PTI) following perception of Microbe‐Associated Molecular Patterns (MAMPs). Phytophthora infestans effector PiSFI3/Pi06087/PexRD16 was previously shown to suppress MAMP‐triggered pFRK1‐Luciferase reporter gene activity. How PiSFI3 suppresses immunity is unknown.
We employed yeast‐two‐hybrid (Y2H) assays, co‐immunoprecipitation, transcriptional silencing by RNA interference and virus‐induced gene silencing (VIGS), and X‐ray crystallography for structure‐guided mutagenesis, to investigate the function of PiSFI3 in targeting a plant U‐Box‐kinase protein (StUBK) to suppress immunity.
We discovered that PiSFI3 is active in the host nucleus and interacts in yeast and in planta with StUBK. UBK is a positive regulator of specific PTI pathways in both potato and Nicotiana benthamiana. Importantly, it contributes to early transcriptional responses that are suppressed by PiSFI3. PiSFI3 forms an unusual trans‐homodimer. Mutation to disrupt dimerization prevents nucleolar localisation of PiSFI3 and attenuates both its interaction with StUBK and its ability to enhance P. infestans leaf colonisation.
PiSFI3 is a ‘WY domain’ RXLR effector that forms a novel trans‐homodimer which is required for its ability to suppress PTI via interaction with the U‐Box‐kinase protein StUBK.
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Dec 2018
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Lennart
Wirthmueller
,
Shuta
Asai
,
Ghanasyam
Rallapalli
,
Jan
Sklenar
,
Georgina
Fabro
,
Dae Sung
Kim
,
Ruth
Lintermann
,
Pinja
Jaspers
,
Michael
Wrzaczek
,
Jaakko
Kangasjärvi
,
Daniel
Maclean
,
Frank L. H.
Menke
,
Mark J.
Banfield
,
Jonathan D. G.
Jones
Diamond Proposal Number(s):
[7641]
Open Access
Abstract: The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL‐INDUCED CELL DEATH1 (RCD1).
We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes.
We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)‐induced defense genes and alters plant growth responses to light. HaRxL106‐mediated suppression of immunity is abolished in RCD1 loss‐of‐function mutants. We report that RCD1‐type proteins are phosphorylated, and we identified Mut9‐like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1‐interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA‐induced defense marker gene expression compared with wild‐type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling.
Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.
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Aug 2018
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[9475, 13467]
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.
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Jul 2018
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I03-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[10071, 14980]
Abstract: An increasing number of surface‐associated proteins identified in Gram‐positive bacteria are characterized by intramolecular cross‐links in structurally conserved thioester, isopeptide, and ester domains (TIE proteins). Two classes of thioester domains (TEDs) have been predicted based on sequence with, to date, only representatives of class I structurally characterized. Here, we present crystal structures of three class II TEDs from Bacillus anthracis, vancomycin‐resistant Staphylococcus aureus, and vancomycin‐resistant Enterococcus faecium. These proteins are structurally distinct from class I TEDs due to a β‐sandwich domain that is inserted into the conserved TED fold to form a slipknot structure. Further, the B. anthracis TED domain is presented in the context of a full‐length sortase‐anchored protein structure (BaTIE). This provides insight into the three‐dimensional arrangement of TIE proteins, which emerge as very abundant putative adhesins of Gram‐positive bacteria.
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Jul 2018
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I02-Macromolecular Crystallography
I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[9475]
Open Access
Abstract: Plant pathogens and parasites are a major threat to global food security. Plant parasitism has arisen four times independently within the phylum Nematoda, resulting in at least one parasite of every major food crop in the world. Some species within the most economically important order (Tylenchida) secrete proteins termed effectors into their host during infection to re-programme host development and immunity. The precise detail of how nematodes evolve new effectors is not clear. Here we reconstruct the evolutionary history of a novel effector gene family. We show that during the evolution of plant parasitism in the Tylenchida, the housekeeping glutathione synthetase (GS) gene was extensively replicated. New GS paralogues acquired multiple dorsal gland promoter elements, altered spatial expression to the secretory dorsal gland, altered temporal expression to primarily parasitic stages, and gained a signal peptide for secretion. The gene products are delivered into the host plant cell during infection, giving rise to “GS-like effectors”. Remarkably, by solving the structure of GS-like effectors we show that during this process they have also diversified in biochemical activity, and likely represent the founding members of a novel class of GS-like enzyme. Our results demonstrate the re-purposing of an endogenous housekeeping gene to form a family of effectors with modified functions. We anticipate that our discovery will be a blueprint to understand the evolution of other plant-parasitic nematode effectors, and the foundation to uncover a novel enzymatic function.
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Apr 2018
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[4975]
Open Access
Abstract: Plant pathogens employ effector proteins to manipulate their hosts. Fusarium oxysporum f. sp. lycopersici (Fol), the causal agent of tomato wilt disease, produces effector protein Avr2. Besides being a virulence factor, Avr2 triggers immunity in I-2 carrying tomato (Solanum lycopersicum). Fol strains that evade I-2 recognition carry point mutations in Avr2 (e.g. Avr2R45H), but retain full virulence. Here we investigate the virulence function of Avr2 and determine its crystal structure. Transgenic tomato and Arabidopsis expressing either wild-type ΔspAvr2 (deleted signal-peptide) or the ΔspAvr2R45H variant become hypersusceptible to fungal, and even bacterial infections, suggesting that Avr2 targets a conserved defense mechanism. Indeed, Avr2 transgenic plants are attenuated in immunity-related readouts, including flg22-induced growth inhibition, ROS production and callose deposition. The crystal structure of Avr2 reveals that the protein shares intriguing structural similarity to ToxA from the wheat pathogen Pyrenophora tritici-repentis and to TRAF proteins. The I-2 resistance-breaking Avr2V41M, Avr2R45H and Avr2R46P variants cluster on a surface-presented loop. Structure-guided mutagenesis enabled uncoupling of virulence from I-2-mediated recognition. We conclude that I-2-mediated recognition is not based on monitoring Avr2 virulence activity, which includes suppression of immune responses via an evolutionarily conserved effector target, but by recognition of a distinct epitope.
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Aug 2017
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