I03-Macromolecular Crystallography
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Mauricio P.
Contreras
,
Hsuan
Pai
,
Muniyandi
Selvaraj
,
Amirali
Toghani
,
David M.
Lawson
,
Yasin
Tumtas
,
Cian
Duggan
,
Enoch Lok Him
Yuen
,
Clare E. M.
Stevenson
,
Adeline
Harant
,
Abbas
Maqbool
,
Chih-Hang
Wu
,
Tolga O.
Bozkurt
,
Sophien
Kamoun
,
Lida
Derevnina
Diamond Proposal Number(s):
[18565]
Open Access
Abstract: Parasites counteract host immunity by suppressing helper nucleotide binding and leucine-rich repeat (NLR) proteins that function as central nodes in immune receptor networks. Understanding the mechanisms of immunosuppression can lead to strategies for bioengineering disease resistance. Here, we show that a cyst nematode virulence effector binds and inhibits oligomerization of the helper NLR protein NRC2 by physically preventing intramolecular rearrangements required for activation. An amino acid polymorphism at the binding interface between NRC2 and the inhibitor is sufficient for this helper NLR to evade immune suppression, thereby restoring the activity of multiple disease resistance genes. This points to a potential strategy for resurrecting disease resistance in crop genomes.
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May 2023
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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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
Diamond Proposal Number(s):
[13467, 18565]
Open Access
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.
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May 2023
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I13-2-Diamond Manchester Imaging
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Diamond Proposal Number(s):
[13848, 16052, 17632, 20385]
Open Access
Abstract: Local coexistence of bees has been explained by flower resource partitioning, but coexisting bumblebee species often have strongly overlapping diets. We investigated if light microhabitat niche separation, underpinned by visual traits, could serve as an alternative mechanism underlying local coexistence of bumblebee species. To this end, we focused on a homogeneous flower resource—bilberry—in a heterogeneous light environment—hemi-boreal forests. We found that bumblebee communities segregated along a gradient of light intensity. The community-weighted mean of the eye parameter—a metric measuring the compromise between light sensitivity and visual resolution—decreased with light intensity, showing a higher investment in light sensitivity of communities observed in darker conditions. This pattern was consistent at the species level. In general, species with higher eye parameter (larger investment in light sensitivity) foraged in dimmer light than those with a lower eye parameter (higher investment in visual resolution). Moreover, species realized niche optimum was linearly related to their eye parameter. These results suggest microhabitat niche partitioning to be a potential mechanism underpinning bumblebee species coexistence. This study highlights the importance of considering sensory traits when studying pollinator habitat use and their ability to cope with changing environments.
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Apr 2023
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[15475, 15215, 12700]
Open Access
Abstract: The agricultural soils of West Limerick, Ireland, contain very localised, extremely high natural Se concentrations that reach levels that are very toxic to grazing livestock. The Carboniferous shales that formed in anoxic deep-water marine environments are the source of the selenium, which, along with the other redox-sensitive elements of molybdenum, uranium, arsenic and vanadium, were mobilised and reprecipitated in post-glacial anoxic marshes. The result has been a history of selenosis and molybdenosis in livestock in this important dairy province. Soils collected at 10–20 cm from five different agricultural sites were analysed, and all yielded concentrations greatly in excess of the safe Se limits of 3–10 mg kg−1; the highest value recorded was 1265.8 mg kg−1 Se. The highest recorded value for Mo in these soils was 1627.5 mg kg−1, and for U, 658.8 mg kg−1. There was a positive correlation between Se, Mo U and organic matter in the soils. Analysis of non-accumulator pasture grasses (Lolium perenne (perennial ryegrass), Festuca arundinacea (tall fescue), Dactylis glomerata (cocksfoot) and Phleum pretense (timothy grass)) revealed the shoot/leaf to contain up to 78.05 mg kg−1 Se while Trifolium repens (white clover) leaves contained 296.15 mg kg−1 Se. An in situ growing experiment using the Se accumulator species Brassica oleracea revealed 971.2 mg kg−1 Se in the leaves of premier kale, which also contained 1000.4 mg kg−1 Mo. Translocation factors (TFs) were generally higher for Mo than Se across all plant species. Combined X-ray absorption near edge spectroscopy (XANES) with micro-X-ray fluorescence (μ-XRF) showed the Se was present in the soil predominantly as the reduced immobile phase, elemental Se (Se0), but also as bioavailable organoselenium species, mainly selenomethionine (SeMet). SeMet was also the main species identified within both the Se non-accumulator and Se accumulator plants. The Se soil–plant system in West Limerick is dominated by SeMet, and uptake into the cattle pasture results in selenosis in the grazing dairy herds. The hyperaccumulating Brassica oleracea species could be used to extract both the Se and Mo to reduce the toxicity of the blighted fields.
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Mar 2023
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[18565]
Open Access
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.
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Oct 2022
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Krios II-Titan Krios II at Diamond
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Diamond Proposal Number(s):
[21404, 27980]
Open Access
Abstract: Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants1,2,3. The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space4,5,6,7,8,9. Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline–proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development.
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Sep 2022
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Open Access
Abstract: Since its discovery, bovine theileriosis has caused major socioeconomic losses in sub-Saharan Africa. Acaricide resistance of the intermediate host, paucity of therapeutics, and lack of sufficiently cross-protective vaccines increase the risk of parasite spread due to global warming. Here, we highlight three important areas that require investigation to develop next-generation vaccines.
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Aug 2022
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[22992]
Open Access
Abstract: Purpose: Freshwater is an increasingly scarce natural resource, essential for agricultural production. As plants consume 70% of the world’s freshwater, a reduction in their water use would greatly reduce global water scarcity. Plants with improved Water Use Efficiency (WUE) such as those with altered expression of the Epidermal Patterning Factor (EPF) family of genes regulating stomatal density, could help reduce plant water footprint. Little however, is known about how this modification in Arabidopsis thaliana. L. affects root architectural development in soil, thus we aim to improve our understanding of root growth when stomatal density is altered. Methods: We used X-Ray synchrotron and neutron imaging to measure in three dimensions, the root system architecture (RSA) of Arabidopsis thaliana. L. plants of three different genotypes, namely that of the wild type Columbia (Col 0) and two different EPF mutants, EPF2OE and epf2-1 (which show reduced and increased stomatal density, respectively). We also used the total biomass and carbon isotope discrimination (Δ) methods to determine how WUE varies in these genotypes when grown in a sandy loam soil under controlled conditions. Results: Our results confirm that the EPF2OE line had superior WUE as compared to the wild type using both the Δ and total biomass method. The epf2-1 mutant, on the other hand, had significantly reduced WUE using the Δ but not with the biomass method. In terms of root growth, the RSAs of the different genotypes had no significant difference between each other. There was also no significant difference in rhizosphere porosity around their roots as compared to bulk soil for all genotypes. Conclusion: Our results indicate that the EPF mutation altering stomatal density in Arabidopsis thaliana. L. plants did not have an adverse effect on root characteristics thus their wide adoption to reduce the global freshwater footprint is unlikely to compromise their soil foraging ability.
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Aug 2022
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[2164]
Open Access
Abstract: Protein synthesis in plant crops contributes to the balance of food and fuel on our planet that influences human metabolic activity and lifespan. Protein synthesis can be regulated with respect to changing environmental cues via deposition of chemical modifications into rRNA. Here, we present the structure of a plant ribosome from tomato and quantitative mass spectrometry analysis of its rRNAs. The study reveals fine features of the ribosomal proteins, 71 plant-specific rRNA modifications, and re-annotates 30 rRNA residues in the available sequence. On the protein level, isoAsp is found in position 137 of uS11, and previously believed to be universal zinc finger is missing in eL34, suggesting a lower effect of zinc deficiency on protein synthesis in plants. On the rRNA level, the plant ribosome differs markedly from its human counterpart with respect to the spatial distribution of modifications. Thus, it represents an additional layer of gene expression regulation highlighting the molecular signature of a plant ribosome. The results provide a reference model of a plant ribosome for structural studies and an accurate marker for molecular ecology.
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May 2022
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
Abstract: Grain phytate, a mixed metal ion salt of inositol hexakisphosphate, accounts for 60-80% of stored phosphorus in plants and is a potent antinutrient of non-ruminant animals including humans. Through neofunctionalization of purple acid phytases (PAPhy) some cereals such as wheat and rye have acquired particularly high mature grain phytase activity. As PAPhy activity supplies phosphate, liberates metal ions necessary for seedling emergence and obviates antinutrient effects of phytate, its manipulation and control are targeted crop traits. Here we show the X-ray crystal structure of the b2 isoform of wheat PAPhy induced during germination. This high resolution crystal structure suggests a model for phytate recognition which, validated by molecular dynamics simulations, implicates elements of two sequence inserts (termed PAPhy motifs) relative to a canonical metallophosphoesterase (MPE) domain in forming phytate-specific substrate specificity pockets. These motifs are well-conserved in PAPhys from monocot cereals, enzymes which are characterised by high specificity for phytate. Tested by mutagenesis, residues His229 in PAPhy motif 4 and Lys410 in the MPE domain, both conserved in PAPhys, are found to strongly influence phytase activity. These results explain the observed phytase activity of cereal PAPhys and open the way to the rational engineering of phytase activity in planta.
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Feb 2022
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