I24-Microfocus Macromolecular Crystallography
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Open Access
Abstract: Many of the world's most important food crops such as rice, barley and maize accumulate silicon (Si) to high levels, resulting in better plant growth and crop yields. The first step in Si accumulation is the uptake of silicic acid by the roots, a process mediated by the structurally uncharacterised NIP subfamily of aquaporins, also named metalloid porins. Here, we present the X-ray crystal structure of the archetypal NIP family member from Oryza sativa (OsNIP2;1). The OsNIP2;1 channel is closed in the crystal structure by the cytoplasmic loop D, which is known to regulate channel opening in classical plant aquaporins. The structure further reveals a novel, five-residue extracellular selectivity filter with a large diameter. Unbiased molecular dynamics simulations show a rapid opening of the channel and visualise how silicic acid interacts with the selectivity filter prior to transmembrane diffusion. Our results will enable detailed structure–function studies of metalloid porins, including the basis of their substrate selectivity.
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Oct 2021
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I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[14493]
Open Access
Abstract: SUGARWINs are PR-4 proteins associated with sugarcane defense against phytopathogens. Their expression is induced in response to damage by Diatraea saccharalis larvae. These proteins play an important role in plant defense, in particular against fungal pathogens, such as Colletothricum falcatum (Went) and Fusarium verticillioides. The pathogenesis-related protein-4 (PR-4) family is a group of proteins equipped with a BARWIN domain, which may be associated with a chitin-binding domain also known as the hevein-like domain. Several PR-4 proteins exhibit both chitinase and RNase activity, with the latter being associated with the presence of two histidine residues H11 and H113 (BARWIN) [H44 and H146, SUGARWINs] in the BARWIN-like domain. In sugarcane, similar to other PR-4 proteins, SUGARWIN1 exhibits ribonuclease, chitosanase and chitinase activities, whereas SUGARWIN2 only exhibits chitosanase activity. In order to decipher the structural determinants involved in this diverse range of enzyme specificities, we determined the 3-D structure of SUGARWIN2, at 1.55Å by X-ray diffraction. This is the first structure of a PR-4 protein where the first histidine has been replaced by asparagine and was subsequently used to build a homology model for SUGARWIN1. Molecular dynamics simulations of both proteins revealed the presence of a flexible loop only in SUGARWIN1 and we postulate that this, together with the presence of the catalytic histidine at position 42, renders it competent as a ribonuclease. The more electropositive surface potential of SUGARWIN1 would also be expected to favor complex formation with RNA. A phylogenetic analysis of PR-4 proteins obtained from 106 Embryophyta genomes showed that both catalytic histidines are widespread among them with few replacements in these amino acid positions during the gene family evolutionary history. We observe that the H11 replacement by N11 is also present in two other sugarcane PR-4 proteins: SUGARWIN3 and SUGARWIN4. We propose that RNase activity was present in the first Embryophyta PR-4 proteins but was recently lost in members of this family during the course of evolution.
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Sep 2021
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I04-Macromolecular Crystallography
I24-Microfocus Macromolecular Crystallography
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Diamond Proposal Number(s):
[23316, 17844]
Abstract: Sugars are essential sources of energy and carbon and also function as key signalling molecules in plants. Sugar transport proteins (STP) are proton-coupled symporters responsible for uptake of glucose from the apoplast into plant cells. They are integral to organ development in symplastically isolated tissues such as seed, pollen and fruit. Additionally, STPs play a vital role in plant responses to stressors such as dehydration and prevalent fungal infections like rust and mildew. Here we present a structure of Arabidopsis thaliana STP10 in the inward-open conformation at 2.6 Å resolution and a structure of the outward-occluded conformation at improved 1.8 Å resolution, both with glucose and protons bound. The two structures describe key states in the STP transport cycle. Together with molecular dynamics simulations that establish protonation states and biochemical analysis, they pinpoint structural elements, conserved in all STPs, that clarify the basis of proton-to-glucose coupling. These results advance our understanding of monosaccharide uptake, which is essential for plant organ development, and set the stage for bioengineering strategies in crops.
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Sep 2021
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[22992]
Abstract: Soil structure is one of the most important environmental factors affecting root architectural development and consequently plant yield. Understanding how plant roots respond to soils with variable soil structure is important as it enables soil management practices that promote optimal root growth. Many contemporary, non-invasive experiments investigating how plant root architecture responds to soil structural variations have often focused on compaction, often neglecting the role of soil aggregate size in determining root configuration. To better understand this, in this study, we used non-invasive neutron and X-Ray imaging to investigate how variable aggregate size affects the early root architectural establishment in wheat plants. Sandy loam soil derived macro-aggregates of two distinct sizes (0.25−0.5 and 2−4 mm) were used to infer the suitability of each aggregate size for use in wheat seedbeds. We also grew wheat seedlings in partitioned containers with the two different aggregate size classes filled side by side to establish whether there would be preferential growth of roots in either aggregate size class. Our results showed significantly increased root growth in the smaller 0.25−0.5 mm aggregates as compared to the larger 2−4 mm aggregates. This was mainly as a result of enhanced lateral root growth when the wheat plants were grown in the finer aggregates. On the other hand, coarser aggregates induced significantly increased seminal root axes which partially offset the differences in total root length between the two aggregate sizes. Plants growing in partitioned containers similarly indicated preferential root growth in smaller aggregate with an even more pronounced difference in root growth in the smaller aggregates. As inferred from our results, seedbeds dominated by smaller macro-aggregates (finer soil tilth) may be optimal to enhance wheat seedling root growth in sandy loam soils.
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Aug 2021
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[24489]
Abstract: At the morphological and anatomical level the ionome, or the elemental composition of an organism, is an understudied area of plant biology. In particular, the ionomic responses of plant-pathogen interactions are scarcely described, and there are no studies on immune reactions. In this study we explored two X-ray fluorescence based ionome visualisation methods (benchtop and synchrotron-based micro XRF (µXRF), as well as the quantitative Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES) method, to investigate the changes that occur in the ionome of compatible and incompatible plant-pathogen interactions. We utilised the agronomically important and comprehensively studied interaction between potato, Solanum tuberosum, and the late blight oomycete pathogen, Phytophthora infestans, as an example. We used one late blight susceptible potato cultivar and two resistant transgenic plant lines (only differing from the susceptible cultivar in one or three resistance genes) both in control and P. infestans inoculated conditions. In the lesions from the compatible interaction we observed rearrangements of several elements. This included, a decrease of the mobile macro nutrient potassium (K), and an increase of iron (Fe), and manganese (Mn) in the lesion versus the tissue outside the lesion. Interestingly, we observed distinctly different distribution patterns of accumulation at the site of inoculation in the resistant lines for calcium (Ca), Magnesium, Mn and silicon (Si) compared to the susceptible cultivar. The results reveal different ionomes in diseased plants compared to resistant plants. Our results demonstrate a technical advance and pave the way for deeper studies of the plant-pathogen ionome in the future.
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Aug 2021
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[21741]
Open Access
Abstract: Non-vesicular lipid transfer at ER and plasma membrane (PM) contact sites (CS) is crucial for the maintenance of membrane lipid homeostasis. Extended synaptotagmins (E-Syts) play a central role in this process as they act as molecular tethers of ER and PM and as lipid transfer proteins between these organelles. E-Syts are proteins constitutively anchored to the ER through an N-terminal hydrophobic segment and bind the PM via a variable number of C-terminal C2 domains. Synaptotagmins (SYTs) are the plant orthologous of E-Syts and regulate the ER–PM communica- tion in response to abiotic stress. Combining different structural and biochemical techniques, we demonstrate that the binding of SYT1 to lipids occurs through a Ca2+-dependent lipid-binding site and by a site for phosphorylated forms of phosphatidylinositol, thus integrating two different molecular signals in response to stress. In addition, we show that SYT1 displays three highly flexible hinge points that provide conformational freedom to facilitate lipid extraction, protein loading, and subsequent transfer between PM and ER.
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Aug 2021
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I04-Macromolecular Crystallography
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Federico
Sabbadin
,
Saioa
Urresti
,
Bernard
Henrissat
,
Anna O.
Avrova
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Lydia R. J.
Welsh
,
Peter J.
Lindley
,
Michael
Csukai
,
Julie N.
Squires
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Paul H.
Walton
,
Gideon J.
Davies
,
Neil C.
Bruce
,
Stephen C.
Whisson
,
Simon J.
Mcqueen-Mason
Diamond Proposal Number(s):
[9948]
Abstract: The oomycete Phytophthora infestans is a damaging crop pathogen and a model organism to study plant-pathogen interactions. We report the discovery of a family of copper-dependent lytic polysaccharide monooxygenases (LPMOs) in plant pathogenic oomycetes and its role in plant infection by P. infestans. We show that LPMO-encoding genes are up-regulated early during infection and that the secreted enzymes oxidatively cleave the backbone of pectin, a charged polysaccharide in the plant cell wall. The crystal structure of the most abundant of these LPMOs sheds light on its ability to recognize and degrade pectin, and silencing the encoding gene in P. infestans inhibits infection of potato, indicating a role in host penetration. The identification of LPMOs as virulence factors in pathogenic oomycetes opens up opportunities in crop protection and food security.
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Aug 2021
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I03-Macromolecular Crystallography
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Aleksandra
Bialas
,
Thorsten
Langner
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Adeline
Harant
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Mauricio P.
Contreras
,
Clare E. M.
Stevenson
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David M.
Lawson
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Jan
Sklenar
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Ronny
Kellner
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Matthew J
Moscou
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Ryohei
Terauchi
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Mark J.
Banfield
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Sophien
Kamoun
Diamond Proposal Number(s):
[18565]
Open Access
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.
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Jul 2021
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B18-Core EXAFS
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Diamond Proposal Number(s):
[15231]
Open Access
Abstract: Cynara cardunculus L. is a multipurpose crop, characterized by high production of biomass suitable for energy purposes and green chemistry. Taking advantage of its already demonstrated ability to grow in polluted environments that characterize many world marginal lands, the aim of this work was to investigate the response of different cardoon genotypes to exposure to cadmium (Cd) and arsenic (As) pollution, in order to use this crop for rehabilitation of contaminated sites and its biomass for energy production. In this study, seeds of two wild cardoon accessions harvested in rural and industrial Sicilian areas and of a selected line of domestic cardoon were used, and the grown plants were spiked with As and Cd, alone or in combination, at two different concentrations (500 and 2000 μM) and monitored for 45 days. The growth parameters showed that all the plants survived until the end of experiment, with growth stimulation in the presence of low concentrations of As and Cd, relative to metal-free controls. Biomass production was mostly allocated in the roots in As treatment and in the shoots in Cd treatment. Cd EXAFS analysis showed that tolerance to high concentrations of both metals was likely linked to complexation of Cd with oxygen-containing ligands, possibly organic acids, in both root and leaf biomass with differences in behaviour among genotypes. Under As+Cd contamination, the ability of the plants to translocate As to aboveground system increased also showing that, for both metal(loid)s, there were significant differences between genotypes studied. Moreover, the results showed that Cynara cardunculus var. sylvestris collected in an industrial area is the genotype that, among those studied, had the best phytoextraction capability for each metal(loid).
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Jun 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[13467]
Open Access
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.
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Mar 2021
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