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- PDB-9fyc: The barley MLA13-AVRA13 heterodimer -

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Basic information

Entry
Database: PDB / ID: 9fyc
TitleThe barley MLA13-AVRA13 heterodimer
Components
  • CC-NBS-LRR resistance protein MLA13
  • CSEP0372 putative effector protein
KeywordsANTIFUNGAL PROTEIN / Complex / Apoptosis / Immune receptor / Mildew
Function / homology
Function and homology information


defense response to other organism / ADP binding
Similarity search - Function
Virus X resistance protein-like, coiled-coil domain / Rx, N-terminal / Rx N-terminal domain / Disease resistance protein Winged helix domain / Disease resistance protein, plants / Apoptotic protease-activating factors, helical domain / NB-ARC / NB-ARC domain / : / Leucine-rich repeat region ...Virus X resistance protein-like, coiled-coil domain / Rx, N-terminal / Rx N-terminal domain / Disease resistance protein Winged helix domain / Disease resistance protein, plants / Apoptotic protease-activating factors, helical domain / NB-ARC / NB-ARC domain / : / Leucine-rich repeat region / Leucine-rich repeat domain superfamily / Winged helix-like DNA-binding domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
CSEP0372 putative effector protein / CC-NBS-LRR resistance protein MLA13
Similarity search - Component
Biological speciesHordeum vulgare (barley)
Blumeria graminis (grass mildew)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsBehrmann, E. / Schulze-Lefert, P. / Flores-Ibarra, A. / Lawson, A.W.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)INST 216/949-1 FUGG Germany
German Research Foundation (DFG)INST 216/512/1 FUGG Germany
CitationJournal: EMBO J / Year: 2025
Title: The barley MLA13-AVR heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors.
Authors: Aaron W Lawson / Andrea Flores-Ibarra / Yu Cao / Chunpeng An / Ulla Neumann / Monika Gunkel / Isabel M L Saur / Jijie Chai / Elmar Behrmann / Paul Schulze-Lefert /
Abstract: Co-evolution between cereals and pathogenic grass powdery mildew fungi is exemplified by sequence diversification of an allelic series of barley resistance genes encoding Mildew Locus A (MLA) ...Co-evolution between cereals and pathogenic grass powdery mildew fungi is exemplified by sequence diversification of an allelic series of barley resistance genes encoding Mildew Locus A (MLA) nucleotide-binding leucine-rich repeat (NLR) immunoreceptors with an N-terminal coiled-coil domain (CNLs). Each immunoreceptor recognises a matching, strain-specific powdery mildew effector encoded by an avirulence gene (AVR). We present here the cryo-EM structure of barley MLA13 in complex with its cognate effector AVR-1. The effector adopts an RNase-like fold when bound to MLA13 in planta, similar to crystal structures of other RNase-like AVR effectors unbound to receptors. AVR-1 interacts via its basal loops with MLA13 C-terminal leucine-rich repeats (LRRs) and the central winged helix domain (WHD). Co-expression of structure-guided MLA13 and AVR-1 substitution variants show that the receptor-effector interface plays an essential role in mediating immunity-associated plant cell death. Furthermore, by combining structural information from the MLA13-AVR-1 heterocomplex with sequence alignments of other MLA receptors, we engineered a single amino acid substitution in MLA7 that enables expanded effector detection of AVR-1 and the virulent variant AVR-V2. In contrast to the pentameric conformation of previously reported effector-activated CNL resistosomes, MLA13 was purified and resolved as a stable heterodimer from an in planta expression system. Our study suggests a common structural principle for RNase-like effector binding to MLAs and highlights the utility of structure-guided engineering of plant immune receptors for broadening their pathogen effector recognition capabilities.
History
DepositionJul 3, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 12, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 26, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2Jun 11, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CC-NBS-LRR resistance protein MLA13
B: CSEP0372 putative effector protein


Theoretical massNumber of molelcules
Total (without water)121,8272
Polymers121,8272
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein CC-NBS-LRR resistance protein MLA13


Mass: 108095.391 Da / Num. of mol.: 1 / Mutation: K98E, K100E
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hordeum vulgare (barley) / Production host: Nicotiana benthamiana (plant) / References: UniProt: Q8GSK4
#2: Protein CSEP0372 putative effector protein


Mass: 13731.284 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Blumeria graminis (grass mildew) / Gene: BGHDH14_bghG002861000001001 / Production host: Nicotiana benthamiana (plant) / References: UniProt: N1JFM8
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: MLA13-AVRA13 heterodimer complex / Type: COMPLEX / Entity ID: #2, #1 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.109 MDaYES
210.02 MDaYES
Source (natural)Organism: Hordeum vulgare (barley)
Source (recombinant)Organism: Nicotiana benthamiana (plant)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMreduced glutathioneC10H17N3O6S1
250 mMTris-HClNH2C(CH2OH)3HCl1
3150 mMsodium chlorideNaCl1
42 mMDTTC4H10O2S21
50.1 %polysorbate 20C58H114O261
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportGrid material: GRAPHENE OXIDE / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 3

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Processing

EM softwareName: PHENIX / Version: 1.21_5207 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48191 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 106.24 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028270
ELECTRON MICROSCOPYf_angle_d0.488911169
ELECTRON MICROSCOPYf_chiral_restr0.04211262
ELECTRON MICROSCOPYf_plane_restr0.00341443
ELECTRON MICROSCOPYf_dihedral_angle_d4.51461118

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