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W4238340147 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. https://doi.org/10.7554/eLife.08709.001 eLife digest Plant diseases reduce harvests of the world's most important food crops including wheat, rice, potato, and corn. These diseases are important for both global food security and local subsistence farming. To fight these diseases, crops (like all plants) have an immune system that can detect the telltale molecules produced by disease-causing microbes (also known as pathogens) and mount a defence response to protect the plant. Nucleotide-binding, leucine-rich repeat receptors (or NLRs for short) are plant proteins that survey the inside of plant cells looking for these telltale molecules. These receptors have played a central role in efforts to breed disease resistance into crop plants for decades, but little is known about how they work. Maqbool, Saitoh et al. have now used a range of biochemical, structural biology and activity-based assays to study how one NLR from rice directly interacts with a molecule from the rice blast fungus. This fungus causes the most important disease of rice (called rice blast), and the fungal molecule in question is also known as an ‘effector’ protein. A technique called X-ray crystallography was used to reveal the three-dimensional structure of the effector bound to part of the NLR called the ‘integrated HMA domain’. Biochemical techniques were then used to measure how strongly the effector (and other related effectors) interacted with this domain of the NLR. These results, combined with a close examination of the three-dimensional structure, allowed a set of changes to be made to the effector that stopped it interacting with the NLR protein domain in the laboratory. Maqbool, Saitoh et al. then performed experiments in rice plants and showed that changes to the effector that stopped it interacting with the NLR domain also stopped the effector from triggering a defence response in plants. Similar results were also obtained in experiments that used the model plant Nicotiana benthamiana. In the middle of the 20th century, an American plant pathologist called Harold Henry Flor proposed that the outcomes of interactions between plants and disease-causing microbes were based on interactions between specific biological molecules. The findings of Maqbool, Saitoh et al. provide a new structural basis for this model. A detailed picture of these molecular interactions will allow researchers to engineer tailored NLRs that detect a wider range of pathogen molecules. In the future such an approach could contribute to efforts to protect the world's most important crops from plant diseases. https://doi.org/10.7554/eLife.08709.002 Introduction Plant diseases are a continuous threat to crop production and a major constraint on achieving food security. Rice blast disease, caused by the fungal pathogen Magnaporthe oryzae, is the biggest pre-harvest biotic threat to global rice production (Pennisi, 2010; Dean et al., 2012; Liu et al., 2014). This disease can cause the loss of enough rice to feed 212–742 million people annually (Fisher et al., 2012), and result in up to 100% yield loss in infected areas (Dean et al., 2012; Liu et al., 2014). The sustainability of rice production is critical, it is a staple food crop for greater than half the world's population. Approaches to controlling blast disease have mainly been via the deployment of rice resistance (R) genes, which encode intracellular immune receptors known as NLRs (Nucleotide-binding, Leucine-rich-repeat (LRR) Receptors). NLRs are a conserved component of plants' innate immune systems and survey the host environment for perturbations caused by invading pathogens (Dangl and Jones, 2001; Chisholm et al., 2006; Jones and Dangl, 2006; van der Hoorn and Kamoun, 2008; Dodds and Rathjen, 2010). Most NLRs respond to the presence or activities of translocated pathogen effectors, proteins delivered by adapted pathogens to affect the physiology of the host to benefit the parasite (Dodds and Rathjen, 2010; Win et al., 2012; Wirthmueller et al., 2013). The recognition event often results in a robust immune response and localised cell death, which limits disease caused by biotrophic pathogens on their hosts. Most NLRs comprise a multi-domain architecture with central nucleotide-binding (NB-ARC) and C-terminal LRR regions. They also usually contain N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domains (Takken and Goverse, 2012). In at least some cases, NLRs function in pairs to deliver disease resistance, and these pairs can be tightly linked genetically (Eitas and Dangl, 2010; Cesari et al., 2014; Le Roux et al., 2015; Sarris et al., 2015). The protein:protein interactions that underlie NLR pair function are starting to be elucidated (Cesari et al., 2014; Williams et al., 2014), but many unknowns remain. Interestingly, most NLR pairs studied to date use one of the NLRs to detect the presence of specific effectors by direct binding (Kanzaki et al., 2012; Cesari et al., 2013; Williams et al., 2014; Zhai et al., 2014). One mechanism by which this can be achieved is via unconventional integrated domains in the NLRs (known as integrated decoy or sensor domains) that show evolutionary relationships to putative virulence targets (Cesari et al., 2014; Wu et al., 2015). Such domains can be integrated at different positions either before, in-between or after the standard NLR regions and are increasingly identified in NLRs of both model and crop plants (Cesari et al., 2014). How these unusual integrated domains function in the direct molecular recognition of effectors, and how this results in initiation of immune signalling, are emerging as fundamental questions in plant NLR biology. To date, ∼100 NLRs in rice have been described to confer resistance to strains of M. oryzae, and 23 of these have been cloned (Liu et al., 2014). Identification of the pathogen effectors (also known as AVRs [avirulence proteins]) that are recognised by these NLRs has lagged behind and only six have been cloned to date, AVR-Pia (Yoshida et al., 2009), AVR-Pita (Orbach et al., 2000), AVR-Pik (Yoshida et al., 2009), AVR-Pii (Yoshida et al., 2009), AVR-Piz-t (Li et al., 2009) and AVR1-CO39 (Ribot et al., 2013). AVR-Pia and AVR1-CO39 are recognised by the RGA4/RGA5 NLR pair (Okuyama et al., 2011; Cesari et al., 2013) through direct binding to a Heavy-Metal Associated domain (HMA, also known as RATX1) integrated into RGA5 after the LRR (Cesari et al., 2013). RGA4/RGA5 physically interact to prevent cell death mediated by RGA4 in the absence of AVR-Pia; the presence of the effector relieves this suppression (Cesari et al., 2014). Intriguingly, the NLR pair Pik-1/Pik-2, which recognises AVR-Pik (Figure 1) (Ashikawa et al., 2008; Yoshida et al., 2009), also binds the effector via an HMA domain but this domain is integrated between the CC and NB-ARC regions of Pik-1 (Figure 1B). The integrated HMA domains of RGA5 and Pik-1 appear to have evolved from a family of rice proteins that only contain the HMA domain (Cesari et al., 2013, 2014; Wu et al., 2015). Interestingly, the rice protein Pi21, a disease susceptibility factor, contains an HMA domain that is not part of an NLR protein (Fukuoka et al., 2009). Figure 1 Download asset Open asset Schematic representations of (A) Magnaporthe oryzae AVR-Pik effector alleles with position of polymorphic residues shown, the effector domain is shown in green with the signal peptide (SP) in grey (amino acids are denoted by their single letter codes), (B) Rice Pik resistance proteins, highlighting the position of integrated HMA domain in the classical plant NLR architecture of Pik-1 (CC = coiled coil, HMA—Heavy Metal Associated domain, NB-ARC = Nucleotide-binding Apaf-1, R-protein, CED4-shared domain, LRR = Leucine Rich Repeat domain), domain boundaries are numbered, based on the Pikp sequences. https://doi.org/10.7554/eLife.08709.003 Both AVR-Pik (Figure 1A) and the HMA region of Pik-1 exhibit nucleotide polymorphisms between pathogen isolates and rice cultivars that result in changes at the amino acid level (Yoshida et al., 2009; Kanzaki et al., 2012; Wu et al., 2014; Zhai et al., 2014). These changes are most likely associated with co-evolutionary dynamics between M. oryzae and rice, predicted to play out at the molecular level via direct protein:protein interactions (Kanzaki et al., 2012). The interaction of AVR-Pik allele AVR-PikD with the Pik-1 NLR Pikp-1 is thought to be the oldest in co-evolutionary time (Kanzaki et al., 2012). Cultivars of rice containing the Pikp allele are resistant to M. oryzae isolates expressing AVR-PikD, but are susceptible to pathogen isolates expressing other AVR-Pik alleles (Kanzaki et al., 2012). While the structural basis of function and recognition of plant pathogen effectors has advanced in recent years (Wirthmueller et al., 2013; Williams et al., 2014), only a few studies have focused on the M. oryzae/rice system. For example, the only structure known for a M. oryzae effector is that of AVR-Piz-t (Zhang et al., 2013), which adopts a six-stranded β-sandwich structure and contains a single disulphide bond. To date, there is no available structural information on domains from rice NLRs, and no structural data from any system showing how plant pathogen effectors are directly recognised at the molecular level by an NLR. To better understand the mechanisms of direct recognition of effectors by NLRs, we have investigated the interaction between the M. oryzae effector AVR-Pik and the rice NLR Pikp-1. We determined the affinity of interaction of AVR-PikD to the HMA domain of the rice NLR Pikp-1 (Pikp-HMA) in vitro, and compared the relative binding of AVR-Pik alleles AVR-PikE, AVR-PikA and AVR-PikC to this HMA. The crystal structure of AVR-PikD bound to Pikp-HMA was determined and this guided mutagenesis of the effector, targeting residues at the interface with Pikp-HMA. The binding of these mutants to the Pikp-HMA was tested in yeast and in vitro. We also used a combination of AVR-Pik alleles and AVR-PikD mutants in both the host rice and heterologous Nicotiana benthamiana systems to probe the degree to which AVR-NLR interactions mediate immunity-related readouts. Long after Harold Henry Flor proposed the gene-for-gene hypothesis of host–parasite interactions (Flor, 1955, 1971), our study establishes the structural basis of direct recognition of a pathogen effector by a plant NLR. Results The rice NLR Pikp-HMA domain selectively interacts with M. oryzae effector AVR-PikD in yeast and in vitro Previously, the full-length and CC domain (containing the HMA) of Pikp-1 have been shown to interact with AVR-PikD in yeast-2-hybrid (Y2H) assays (Kanzaki et al., 2012; Wu et al., 2014; Zhai et al., 2014). Here we show that the Pikp-HMA domain alone selectively interacts with the AVR-Pik allele AVR-PikD in yeast (Figure 2A, Figure 2—figure supplement 1, Table 1). Weak interaction was also observed with AVR-PikE (as evidenced by limited growth on the selective–LTH plate and some blue colouration in the X-gal assay), but not AVR-PikA or AVR-PikC, which is consistent with previous experiments (Kanzaki et al., 2012). Following expression, purification and verification of the proteins by intact mass spectrometry (‘Materials and methods’, Figure 2—figure supplement 2, Table 2), we showed that AVR-PikD and Pikp-HMA form a stable complex in vitro that can be purified by analytical gel filtration (Figure 2B). Using this qualitative assay, we also found that Pikp-HMA can form a complex with AVR-PikE and AVR-PikA, but not AVR-PikC (Figure 2—figure supplement 3). Figure 2 with 4 supplements see all Download asset Open asset AVR-Pik effector alleles interact with the Pikp-HMA domain with different affinities. (A) Y2H assays showing the binding of effector alleles to the Pikp-HMA using two read-outs, growth on–Leu-Trp-His+3AT (-LTH) plates and the X-gal assay. (B) Analytical Gel Filtration traces depicting the retention volume of Pikp-HMA, AVR-PikD and the complex, with SDS-PAGE gels of relevant fractions (similar results were obtained for AVR-PikE and AVR-PikA, but AVR-PikC did not bind [Figure 2—figure supplement 3]). (C) Binding curves derived from Surface Plasmon Resonance multi-cycle kinetics data for Pikp-HMA binding to AVR-Pik alleles, Kd values are shown (NB = No Binding). The sensorgrams of the data used to derive these curves are shown in Figure 2—figure supplement 4. https://doi.org/10.7554/eLife.08709.004 Table 1 Summary Table showing the outcomes of in vitro and in planta assays used to investigate the interactions and responses of AVR-Pik effectors with Pikp-dependent readouts https://doi.org/10.7554/eLife.08709.009 AVR-PikDAVR-PikEAVR-PikAAVR-PikCAVR-PikDHis46GluAVR-PikDIle49GluAVR-PikDArg64AlaAVR-PikDAsp66ArgAVR-PikDAla67AspAVR-PikDPro47Ala/Gly48AspInteraction with Pikp-HMA in Y2H++++−−−+++−−+++Interaction with Pikp-HMA in SPR++++++−−++−−+++Recognition in Pikp+ rice plants++++(−)(−)−+−−++++++CD response in Nicotiana benthamiana+++−−−−++−−−+++ Y2H = yeast-2-hybrid, SPR = Surface Plasmon Resonance, Pikp+ = rice cv. K60, CD = cell death. Parentheses depict results from (Kanzaki et al., 2012). Table 2 Intact masses for proteins expressed and purified in this study https://doi.org/10.7554/eLife.08709.010 ProteinVectorMolecular Mass (Da)CalculatedObservedΔPikp-HMApOPINS3C*7805.237804.97−0.26AVR-PikDpOPINS3C*10,835.3110,832.95−2.36§AVR-PikDpOPINA†10,812.3310,809.99−2.34AVR-PikDpOPINE‡11,786.3311,784.16−2.17AVR-PikEpOPINS3C*10,812.2710,809.91−2.36AVR-PikEpOPINE‡11,763.2911,760.96−2.33AVR-PikApOPINS3C*10,844.2710,841.80−2.47AVR-PikApOPINE‡11,795.2911,793.01−2.28AVR-PikCpOPINS3C*10,856.2810,853.72−2.56AVR-PikCpOPINE‡11,807.3011,804.97−2.33AVR-PikDHis46GlupOPINE‡11,778.3011,776.07−2.23AVR-PikDIle49GlupOPINE‡11,802.2811,800.04−2.24AVR-PikDArg64AlapOPINE‡11,701.2211,698.94−2.28AVR-PikDAsp66ArgpOPINE‡11,827.4311,825.31−2.12AVR-PikDAla67AsppOPINE‡11,830.3411,828.20−2.14AVR-PikDPro47Ala, Gly48AsppOPINE‡11,818.3211,816.20−2.12 * Non-native residues remaining after 3C cleavage: N-terminal Gly–Pro. † Non-native residues remaining: N-terminal Met. ‡ Non-native residues remaining after 3C cleavage: N-terminal Gly–Pro; C-terminal Lys-His-His-His-His-His-His. § The measured mass of each AVR-Pik protein should be 2.0156 Da (2 × 1.0078) less than its calculated mass due to formation of the di-sulphide bond. Next we used Surface Plasmon Resonance (SPR) to determine the binding affinity of AVR-PikD to Pikp-HMA. The purified effector, with a non-cleavable 6xHis tag at the C-terminus, was immobilised on a Ni2+-NTA chip; Pikp-HMA was used as the analyte. Using a multi-cycle kinetics approach, we found that Pikp-HMA bound to immobilised AVR-PikD with a Kd of 31 ± 2 nM (Figure 2C, Figure 2—figure supplement 4A, Table 1). SPR studies were expanded to include AVR-PikE, AVR-PikA and AVR-PikC (Figure 2C, Figure 2—figure supplement 4, Table 1). For AVR-PikE, even though this was not fully saturatable under the conditions of our assay, we obtained an apparent Kd of 367 ± 41 nM, a greater than 10-fold weaker binding compared to AVR-PikD (Figure 2C). For AVR-PikA we could determine an apparent Kd of 710 ± 111 nM (also not saturatable in the assay). We detected essentially no binding for AVR-PikC to Pikp-HMA in this assay (Figure 2C). This is consistent with the Y2H data and also correlates with the published recognition specificity in planta, although M. oryzae isolates expressing AVR-PikE were reported to not be recognised by cultivars of rice expressing Pikp (Kanzaki et al., 2012). Crystal structure of the Pikp-HMA/AVR-PikD complex Although we were able to express and purify AVR-PikD from Escherichia coli, we were unable to obtain crystals of this protein for structure determination by X-ray crystallography. However, following a co-expression strategy with 6xHis tagged Pikp-HMA and untagged AVR-PikD (see ‘Materials and methods’), we obtained crystals of this complex in multiple conditions. Optimisation of one of these conditions (Figure 3—figure supplement 1) produced crystals diffracting X-rays to 1.6 Å resolution. The structure of the Pikp-HMA/AVR-PikD complex was solved using molecular replacement to position a Pikp-HMA dimer (see below) in the asymmetric unit, followed by automated rebuilding with the sequence of both proteins supplied. This was sufficient to produce an initial model containing both Pikp-HMA and AVR-PikD that could be used to complete structure determination (see ‘Materials and methods’, X-ray Data Collection and Refinement statistics are given in Table 3). Table 3 X-ray data collection and refinement statistics https://doi.org/10.7554/eLife.08709.011 Pikp-HMAPikp-HMA/AVR-PikDNativeIodideData collection Wavelength (Å)1.202.000.90 Space groupP6522P6522P41212Cell dimensions a, b, c (Å)54.65, 54.65, 235.2254.73, 54.73, 235.80118.41, 118.41, 35.81 α, β, γ, (°)90.00, 90.00, 120.0090.00, 90.00, 120.0090.00, 90.00, 90.00 Resolution (Å)*47.33–2.10 (2.15–2.10)117.90–2.80 (2.87–2.80)39.47–1.60 (1.64–1.60) Rmerge (%)8.4 (117.6)8.7 (45.8)4.7 (65.1) I/σI32.3 (4.6)34.7 (7.3)32.3 (4.7)Completeness (%) Overall100 (99.9)99.9 (98.9)100 (100) Anomalous99.9 (99.4)Redundancy Overall45 (46.8)32.8 (24.4)17.7 (17.4) Anomalous19.4 (13.3) CC(1/2) (%)100 (94.0)100 (98.0)100 (92.8)Refinement and model Resolution (Å)47.33–2.10 (2.15–2.10)39.47–1.60 (1.64–1.60) Reflections12356 (861)32549 (2379) Rwork/Rfree (%)20.2/22.9 (20.6/19.6)17.8/20.5 (19.6/24.7)No. atoms Protein10631762 Water44138B-factors (Å2) Protein29.9623.38 Water57.3134.07R.m.s deviations Bond lengths (Å)0.0130.016 Bond angles (°)1.571.79Ramachandran plot (%)† Favoured97.198.7 Allowed2.91.3 Outliers00 MolProbity Score1.48 (98th percentile)1.21 (98th percentile) * The highest resolution shell is shown in parentheses. † As calculated by MolProbity. The structure of the Pikp-HMA/AVR-PikD complex reveals an intimate interface formed between these proteins that buries 18.7% of the effector's solvent accessible surface area (1031.0 Å2, Figure 3A,B). The majority of the interaction is formed with a monomer of Pikp-HMA, with 87.5% of the effector's buried surface area (902.2 Å2) and nine residues contributing hydrogen bond and/or salt bridge interactions. This suggests that the AVR-PikD/Pikp-HMA monomer interaction most likely represents the biologically significant interface. No hydrogen bonds or salt bridge interactions are formed with the second monomer of the Pikp-HMA dimer. Further, due to steric clash that would occur, it is not possible for an AVR-PikD/Pikp-HMA heterotetramer (2:2 complex) to assemble. All interface analysis was performed using PDBePISA (Krissinel and Henrick, 2007). Figure 3 with 4 supplements see all Download asset Open asset Structure of the AVR-PikD/Pikp-HMA complex. (A) Schematic representation of the AVR-PikD/Pikp-HMA(monomer), highlighting interfacing residues. The effector is shown in green cartoon, with side chains as sticks and green carbon atoms (no surface). The Pikp-HMA is shown in blue cartoon, with side chains as sticks and blue carbon atoms; the molecular surface of this protein is also depicted. Effector residues selected for mutation are labelled, as are important interface residues of Pikp-HMA discussed in the text. Hydrogen bonds/salt-bridges are shown as dashed lines and the di-sulphide bond as yellow bars. (B) Buried surface areas of AVR-PikD (left, purple) and Pikp-HMA (right, brown) separated and shown from the perspective of the partner molecule. Cartoon and amino acid side chains shown are as for panel (A). (C) Comparison of the Pikp-HMA (monomer, blue) with yeast Ccc2A (wheat) showing the conservation of the HMA fold. The copper ion bound to Ccc2a is shown as a red sphere. (D) Comparison of AVR-PikD (green) and AVR-Piz-t (pink) structures showing the conservation of the β-sandwich structure, and the N-terminal extension of AVR-PikD. https://doi.org/10.7554/eLife.08709.012 Structure of Pikp-HMA in the Pikp-HMA/AVR-PikD complex Each of the Pikp-HMA monomers adopts the HMA-domain fold (Pfam: PF00403), comprising a four-stranded antiparallel β-sheet and two α-helices packed in an α/β sandwich. The closest structural homologue of Pikp-HMA (defined by PDBeFold [Krissinel and Henrick, 2004]) is the HMA domain of yeast protein Ccc2A (Banci et al., 2001), overlaying with an r.m.s.d. of 1.58 Å over 72 residues (Figure 3C). Typically, HMA domains bind heavy metals, or lighter cations such as Cu1+ or Zn2+, via two conserved Cys residues and are involved in metal transport or detoxification pathways (Bull and Cox, 1994). Interestingly, these Cys residues are not conserved in Pik-1 HMA domains, including Pikp-1. Hence, the Pikp-HMA structure does not contain a metal ion, and the loop between β1 and α1, which usually contains the metal-chelating Cys residues, is disordered. Further, this loop is positioned away from the interface with the effector (Figure 3A, Figure 3—figure supplement 2) and does not contribute to complex formation. We were also able to obtain the crystal structure of Pikp-HMA in the absence of AVR-PikD (see ‘Materials and methods’, Figure 3—figure supplements 1, 2A). The structure of the Pikp-HMA dimer in isolation is essentially identical to that found in the complex (r.m.s.d. 0.67 Å over 69 residues, for the monomer bound to AVR-PikD), with the exceptions of a minor shift in the loop spanning residues Val222—Lys228 and the N-terminal four residues (Figure 3—figure supplement 2C). Structure of AVR-PikD in the Pikp-HMA/AVR-PikD complex AVR-PikD adopts a six-stranded β-sandwich structure, stabilised by a di-sulphide bond between Cys54 and Cys70. The effector contains an N-terminal extension, comprising residues Arg31 to Pro52, prior to the start of this fold (Figure 3A,D). The extension is anchored to the β-sandwich at each end via a salt–bridge interaction involving the side chains of Asp45 and Arg110 and hydrogen bonds between both the main chain carbonyl of Arg39 and Glu38Oε1 and Arg64Nη1. Database searches using PDBeFold reveals that a close structural homologue of AVR-PikD is AVR-Piz-t (Zhang et al., 2013), another M. oryzae effector protein, despite there being essentially no sequence identity between these proteins (r.m.s.d. = 2.33 Å over 58 aligned residues, Figure 3D). This suggests that sequence divergent translocated effectors of M. oryzae may share a conserved structural scaffold, despite very different sequences, which has striking parallels to RXLR-type effectors of plant pathogenic oomycetes (Boutemy et al., 2011; Win et al., 2012). Further, structural homology is also observed to ToxB, a protein toxin from Pyrenophora tritici-repentis, the causative agent of tan spot in wheat (Nyarko et al., 2014). In each case, the identified structural homology only extends to the β-sandwich fold and the N-terminal extension of AVR-PikD appears to be unique. This raises the interesting possibility that candidate effectors from distant fungi could be identified by structure-guided sequence similarity searches. Binding interfaces in the Pikp-HMA/AVR-PikD complex Three primary sites of interaction are apparent between Pikp-HMA and AVR-PikD. The first is dominated by main-chain hydrogen bonding between the C-terminal β-stand of Pikp-HMA and β3 of AVR-PikD, which results in formation of a continuous antiparallel β-sheet comprising the four β-strands of Pikp-HMA and β3–5 of AVR-PikD. The second involves the side chain of Pikp-HMAAsp224, which forms a salt–bridge interaction with the side chain of AVR-PikDArg64, and is also held in place by a hydrogen bond of its main chain NH group to the side chain of AVR-PikDAsp66 (Figure 3A). The third interaction site centres on AVR-PikDHis46, although has contributions from residues Asn42—Ile49. This region forms part of the N-terminal extension and includes the polymorphic AVR-Pik residues 46, 47 and 48 (His46, Pro47 and Gly48 in AVR-PikD, Figures 1A and 3A). AVR-PikDHis46 is bound in a pocket on Pikp-HMA via hydrogen bonds/salt bridge interactions between AVR-PikDHis46:Nδ1/Pikp-HMASer218:Oγ and AVR-PikDHis46:Nε2/Pikp-HMAGlu230:Oε1; also, Pikp-HMAVal232 packs on top of the AVR-PikDHis46 ring and contributes hydrophobic/van der Waals interactions (Figure 3—figure supplement 3). Finally, it is worth noting that there is an extensive network of buried solvent-mediated contacts between Pikp-HMA and AVR-PikD. Structure-based mutations in AVR-PikD perturb binding to Pikp-HMA in yeast and in vitro Based on the Pikp-HMA/AVR-PikD structure, we designed four mutations in AVR-PikD predicted to perturb complex formation through generating steric clashes/introducing charged residues, or removing a salt–bridge interaction (His46Glu, Ile49Glu, Asp66Arg and Arg64Ala), and two mutants to mimic other AVR-Pik alleles, but retain His46 (Ala67Asp [based on AVR-PikC], Pro47Ala/Gly48Asp [based on AVR-PikA]), Figure 3—figure supplement 4A. First, we screened these mutants for interaction with Pikp-HMA in the Y2H assay. We found that AVR-PikDHis46Glu, AVR-PikDArg64Ala and AVR-PikDAsp66Arg prevent the interaction (as observed on the -LTH selective growth plate and in the X-gal assay (Figure 4A, Figure 4—figure supplement 1, Table 1). However, AVR-PikDIle49Glu maintains an interaction and AVR-PikDAla67Asp and AVR-PikDPro47Ala/Gly48Asp showed intermediate binding (weak interaction on -LTH selective growth plate and in the X-gal assay [Figure 4A]). Figure 4 with 2 supplements see all Download asset Open asset Structure-based mutagenesis at the Pikp-HMA/AVR-PikD interface perturbs protein interactions in yeast and in vitro. (A) Y2H assays showing the binding of AVR-PikD mutants to Pikp-HMA using two read-outs, growth on–Leu-Trp-His+3AT (-LTH) plates and the X-gal assay. (B) Binding curves derived from Surface Plasmon Resonance single-cycle kinetics data for Pikp-HMA binding to AVR-PikD and AVR-PikD mutants, Kd values are shown where determined (ND = Not Determined, NB = No Binding). The sensorgrams of the data used to derive these curves are shown in Figure 4—figure supplement 2B. https://doi.org/10.7554/eLife.08709.017 Next, we expressed and purified each of these AVR-PikD mutants (as for wild-type and with C-terminal non-cleavable 6xHis tag) and confirmed their identity by intact mass spectrometry (Figure 4—figure supplement 2A, Table 2). We then used SPR to determine the binding affinities between these mutants and the Pikp-HMA using a single-cycle kinetics approach ([Karlsson et al., 2006] Figure 4B, Figure 4—figure supplement 2B, Table 1), having confirmed a similar affinity of Pikp-HMA for AVR-PikD (Kd = 29 ± 3.5 nM) using this approach. Consistent with the Y2H results, we could not measure any meaningful interaction of AVR-PikDHis46Glu, AVR-PikDArg64Ala and AVR-PikDAsp66Arg with Pikp-HMA (Figure 4B). For AVR-PikDIle49Glu and AVR-PikDPro47Ala/Gly48Asp we were able to determine Kds of interaction of 99 ± 18 nM and 83 ± 16 nM respectively (Figure 4B, Figure 4—figure supplement 2B). AVR-PikDAla67Asp showed a weaker response but we were unable to obtain a reliable Kd at the concentrations of Pikp-HMA used. AVR-PikDIle49Glu, AVR-PikDAla67Asp and AVR-PikDPro47Ala/Gly48Asp all interacted in the Y2H assay, with the latter two showing qualitatively weaker binding. Structure-based mutations in AVR-PikD prevent response in rice when delivered by M. oryzae To test the effects of the AVR-PikD mutations on pathogen virulence on rice plants expressing the Pikp gene, we transformed M. oryzae isolate Sasa2 with constructs encoding each of the six mutants above, with expression driven by the native AVR-PikD promoter. AVR-PikE was included in these experiments as it represents a naturally occurring point mutant at the important position 46 (His46Asn). Each of the transformed M. oryzae lines were spot inoculated (Kanzaki et al., 2002) onto leaf blades of rice cultivars Nipponbare (Pik−, lacks known Pik alleles) and K60 (which contains Pikp). The Nipponbare cultivar was susceptible to all of the M. oryzae lines, including Sasa2 wild type and empty vector control, as shown by the development of l" @default.
W4238340147 created "2022-05-12" @default.
W4238340147 date "2015-06-21" @default.
W4238340147 modified "2023-10-16" @default.
W4238340147 title "Decision letter: Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor" @default.
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