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Commit 24d8f5c9 authored by Viktoria Petrova's avatar Viktoria Petrova
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add mermaid graph

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......@@ -4,3 +4,154 @@ Wanke, A., van Boerdonk, S., Mahdi, L. K., Wawra, S., Neidert, M., Chandrasekar,
## Copyright
Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.
## ARC structure
``` mermaid
%%{
init: {
'theme': 'base',
'themeVariables': {
'background': '#fff',
'lineColor': '#2d3e50',
'primaryTextColor': '#2d3e50'
}
}
}%%
flowchart TB
subgraph Legend
l1(Sample)
l2>Process]
l3(Material)
l4(Data)
end
subgraph studies\NicotianaBenthamiana
direction TB
s1(wild type seeds)---p1>PlantMaterialAndGrowthConditions.md]-->s2(N. benthamiana plants)
end
subgraph studies\HordeumVulgare
direction TB
s3(barley seeds)---p2>PlantMaterialAndGrowthConditions.md]-->s4(barley plants)
end
subgraph studies\CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays
direction TB
p3>CarbohydrateSubstratesForImmunityAndEnzymaticDigestionAssays.md]-->s5(carbohydrate substrates)
end
subgraph assays\HeterologousProteinProductionAndPurification
direction TB
a1(codon-optimized GBP1 sequence)---p4>PlasmidConstruction.md]-->a2(pXCScpmv-GBP1-HAStrep)
a3(catalitically inactive GBP1 sequence)---p4-->a4(pXCScpmv-GBP1_E500A-HAStrep)
a2----p6>"`isolation
sequencing
_Agrobacterium tumefaciens_ transformation`"]
a4---p5>"`gel electrophoresis
purification
_E.coli_ transformation`"]---p6
p6-->a5(Agrobacterium_GBP1)
p6-->a6(Agrobacterium_GBP1_E500A)
a5 & a6---p7>infiltration]
s2----p7
p7---p8>HeterologousProteinProductionAndPurification.md]-->a7(purified proteins)
end
subgraph assays\EnzymaticCarbohydrateDigestionAndTLC
direction TB
a7 & s5---p9>EnzymaticCarbohydrateDigestion.md]-->a8(digestion products)
a8---p10>TLC.md]-->d1("`gr3b_lrg.jpg
gr4a_lrg.jpg
gr4b_lrg.jpg
gr4c_lrg.jpg
FigS4A.jpg
FigS4B.jpg
FigS5A.png`")
end
subgraph assays\MALDI-TOFAnalysis
direction TB
a8---p11>MALDI-TOFAnalysis.md]-->d2("`gr3c_large.jpg
FigS4C.jpg`")
end
subgraph assays\CRISPRCas9-basedMutagenesis
direction TB
s4---p12>CRISPRCas9-basedMutagenesis.md]-->a9(barley mutants)-->d15(gr1b_lrg.jpg)
end
subgraph assays\OxidativeBurstAssay
direction TB
s2 & s4 & a9---p13>PreparationOfThePlantMaterial.md]-->a10(plant material)
a10---p14>ROSBurstAssay.md]-->d3("`gr5_lrg.jpg
FigS1A.jpg
FigS1B.jpg
FigS5B.jpg
FigS5C.jpg`")
end
subgraph assays\FungalMaterialGrowthConditionsAndBarleyColonizationAssays
direction TB
s4 & a9---p15>RhizophagusIrregularis.md]-->a11(R. irregularis colonized plants)-->d11(FigS3B.jpg)
s4 & a9---p16>BlumeriaHordei.md]-->a12(B. hordei colonized plants)-->d13(FigS3D.jpg)
s4 & a9---p17>SerendipitaIndicaSerendipitaVermiferaAndBipolarisSorokiniana.md]-->a13(S. indica colonized plants) & a14(S. vermifera colonized plants) & a15(B. sorokiniana colonized plants)
a15-->d12(FigS3C.jpg)
d14(gr6b_lrg.jpg)
end
subgraph assays\qRT-PCR
direction TB
a12 & a13 & a14 & a15---p18>qRT-PCR.md]-->d4("`gr1c_lrg.jpg
gr2_lrg.jpg
gr6d_lrg.jpg`")
end
subgraph assays\StainingForConfocalMicroscopy
direction TB
a13---p19>HordeumVulgare.md]-->d5("`gr6a_lrg.jpg
FigS6A.jpg
FigS6B.jpg
FigS6C.jpg`")
a11---p20>RootStainingOfRIrregularis.md]-->d6(FigS3A.jpg)
end
subgraph assays\ProteinPull-down
direction TB
a16(laminarin)---p21>BiotinylationOfLaminarin.md]-->a17(biotinylated laminarin)
s4 & a17---p22>ProteinPull-downWithBiotinylatedLaminarin.md]-->a18(pull-down proteins)
a18---p23>SDS-PAGE separation]-->d7(FigS1C.jpg)
end
subgraph assays\MS-MSAnalysisOfThePull-downProteins
direction TB
a18---p24>MS-MSAnalysisOfThePull-downProteins.md]-->d8("`gr1a_lrg.jpg
mmc2.xlsx`")
end
subgraph assays\MultipleSequenceAlignment
d9(gr4a_lrg.jpg)
end
subgraph assays\QuantificationAndStatisticalAnalysis
pr25>QuantificationAndStatisticalAnalysis.md]-->d10("`gr6c_lrg.jpg
mmc3.xlsx`")
end
%% Defining node styles
classDef S fill:#b4ce82, stroke:#333;
classDef M fill:#ffc000;
classDef D fill:#c21f3a,color:white;
classDef P stroke-width:0px;
%% Assigning styles to nodes
class l1,a7,a8,a18 S;
class l3,s1,s2,s3,s4,s5,a1,a2,a3,a4,a5,a6,a9,a10,a11,a12,a13,a14,a15,a16,a17 M;
class d1,d2,d3,d4,d5,d6,d7,d8,d9,d10,d11,d12,d13,d14,d15,l4 D;
class l2,p1,p2,p3,p4,p5,p6,p7,p8,p9,p10,p11,p12,p13,p14,p15,p16,p17,p18,p19,p20,p21,p22,p23,p24,p25 P;
%% Box style
style Studies fill:#fff, stroke-width:2px, stroke:#333;
style Assays fill:#fff, stroke-width:2px, stroke:#333;
\ No newline at end of file
## Enzymatic carbohydrate digestion
Carbohydrate digestion assays were performed using either purified barley GBP (heterologously expressed in *N. benthamiana*) or barley BGLUII (Chandrasekar et al., 2022) (available from Megazyme, E-LAMHV). Preparations of the fungal CW and EPS matrix were incubated overnight in sterile Milli-Q water at 65 °C prior to enzymatic digestion. Substrate and enzyme concentrations, buffer compositions, digestion temperature and time are described in the figure captions. Digestion was stopped by denaturing the enzymes at 95 °C for 10 min and the digestion products were stored at -20 °C prior to use.
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## Thin layer chromatography (TLC)
An aliquot of each sample was subjected to TLC using a silica gel 60 F254 aluminum TLC plate (Merck Millipore, Burlington, USA), using a running buffer containing ethyl acetate/acetic acid/methanol/formic acid/water at a ratio of 8:4:1:1:1 (v/v). D-glucose, laminaribiose β-1-3-(Glc)2, laminaritriose β-1-3-(Glc)3, gentiobiose β-1-6-(Glc)2, and laminaripentaose β-1-3-(Glc)5 at a concentration of 1.5 mg·mL−1 were used as standards (Megazyme, Bray, Ireland). To visualize the glucan fragments, the TLC plate was sprayed with glucan developer solution (45 mg N-naphthol, 4.8 mL H2SO4, 37.2 mL ethanol and 3 mL water) and baked at 95 °C until the glucan bands became visible (approximately 4-5 min).
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assays/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/image.png

52.5 KiB

## Heterologous protein production and purification from N. benthamiana
*A. tumefaciens* GV3101::pMP90RK strains carrying the binary vectors for protein production (antibiotic selection: 30 μg·mL-1 Rifampicin, 25 μg·mL-1 Kanamycin, 50 μg·mL-1 Carbenicillin) and *A. tumefaciens* GV3101 strains carrying the binary vector for viral p19 silencing inhibitor expression (antibiotic selection: 30 μg·mL-1 Rifampicin, 30 μg·mL-1 Gentamicin, 100 μg·mL-1 Carbenicillin) were grown in selection LB liquid medium at 28 °C, 180 rpm for three days. The cultures were centrifuged (3,500 g for 15 min), resuspended in infiltration buffer (10 mM MES pH 5.5, 10 mM MgCl2, 200 μM acetosyringone) to an OD600 of 1 and incubated for 1 h in the dark at 28 °C, 180 rpm. Each of the two *A. tumefaciens* strains carrying the GBP1 production constructs was mixed with the *A. tumefaciens* strain carrying the p19-expressing construct in a 1:1 ratio. The bacterial suspensions were infiltrated into the four youngest, fully developed leaves of four-week-old *N. benthamiana* plants with a needleless syringe. Five days after infiltration, the leaves were detached from the plant and ground in liquid nitrogen. Protein purification was carried out according to Werner and coworkers (Werner et al., 2008) with minor modifications: The ground plant material (up to the 5 mL mark of 15-mL tube) was thoroughly resuspended in 5 mL of ice-cold extraction buffer (100 mM Tris pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 10 mM DTT, 100 μg·mL-1 Avidin) and centrifuged at 10,000 g, 4 °C for 10 min. The supernatant was filtered through a PD-10 desalting column (Sigma-Aldrich, Taufkirchen, Germany), transferred to a new tube, and supplemented with 75 μL·mL-1 Strep-Tactin Macroprep (50% slurry) (IBA Lifesciences GmbH, Göttingen, Germany). Samples were incubated in a rotary wheel at 4 °C for 1 h, followed by centrifugation for 30 s at 700 g. The supernatant was discarded, and the beads were washed three times with 2 mL of washing buffer (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.005% Triton X-100, 2 mM DTT). Proteins were eluted from the beads by adding 100 μL of elution buffer (wash buffer containing 10 mM biotin) and incubating at 800 rpm for 5 min at 25 °C. The samples were centrifuged at 700 g for 20 s and the elution was repeated two more times. The elution fractions were pooled and dialyzed overnight against cold Milli-Q water (dialysis tubing with 6-8 kDa cut-off). Proteins were stored on ice at 4 °C for further use. The success of protein purification was analyzed by SDS PAGE and Western Blotting.
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*A. tumefaciens* GV3101::pMP90RK strains carrying the binary vectors for protein production (antibiotic selection: 30 μg·mL-1 Rifampicin, 25 μg·mL-1 Kanamycin, 50 μg·mL-1 Carbenicillin) and *A. tumefaciens* GV3101 strains carrying the binary vector for viral p19 silencing inhibitor expression (antibiotic selection: 30 μg·mL-1 Rifampicin, 30 μg·mL-1 Gentamicin, 100 μg·mL-1 Carbenicillin) were grown in selection LB liquid medium at 28 °C, 180 rpm for three days. The cultures were centrifuged (3,500 g for 15 min), resuspended in infiltration buffer (10 mM MES pH 5.5, 10 mM MgCl2, 200 μM acetosyringone) to an OD600 of 1 and incubated for 1 h in the dark at 28 °C, 180 rpm.
Each of the two *A. tumefaciens* strains carrying the GBP1 production constructs was mixed with the *A. tumefaciens* strain carrying the p19-expressing construct in a 1:1 ratio. The bacterial suspensions were infiltrated into the four youngest, fully developed leaves of four-week-old *N. benthamiana* plants with a needleless syringe.
Five days after infiltration, the leaves were detached from the plant and ground in liquid nitrogen. Protein purification was carried out according to Werner and coworkers (Werner et al., 2008) with minor modifications: The ground plant material (up to the 5 mL mark of 15-mL tube) was thoroughly resuspended in 5 mL of ice-cold extraction buffer (100 mM Tris pH 8.0, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 10 mM DTT, 100 μg·mL-1 Avidin) and centrifuged at 10,000 g, 4 °C for 10 min. The supernatant was filtered through a PD-10 desalting column (Sigma-Aldrich, Taufkirchen, Germany), transferred to a new tube, and supplemented with 75 μL·mL-1 Strep-Tactin Macroprep (50% slurry) (IBA Lifesciences GmbH, Göttingen, Germany). Samples were incubated in a rotary wheel at 4 °C for 1 h, followed by centrifugation for 30 s at 700 g. The supernatant was discarded, and the beads were washed three times with 2 mL of washing buffer (50 mM Tris pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.005% Triton X-100, 2 mM DTT). Proteins were eluted from the beads by adding 100 μL of elution buffer (wash buffer containing 10 mM biotin) and incubating at 800 rpm for 5 min at 25 °C. The samples were centrifuged at 700 g for 20 s and the elution was repeated two more times. The elution fractions were pooled and dialyzed overnight against cold Milli-Q water (dialysis tubing with 6-8 kDa cut-off). Proteins were stored on ice at 4 °C for further use.
The success of protein purification was analyzed by SDS PAGE and Western Blotting.
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## Plasmid construction for the heterologous expression of barley GBP1 in *N. benthamiana*
For *in planta* protein production in *N. benthamiana*, we used the binary vector pXCScpmv-HAStrep characterized by a 35S promoter cassette, modified 5′- and 3′-UTRs of RNA-2 from the cowpea mosaic virus as translational enhancers, and C-terminal hemagglutinin (HA) and StrepII tags (Witte et al., 2004; Myrach et al., 2017). The codon-optimized GBP1 coding sequence was amplified with the primer pair ClaI_GBP1_F (5’-gacggtatcgataaaATGCCGCCACATGGTAGACG-3’) and GBP1_noSTOP_XmaI_R (5’-ataactcccgggATGGCCATATTGACGATACCAACAGC-3’) and directionally cloned into the ClaI and XmaI sites of the binary vector to produce pXCScpmv-GBP1-HAStrep. To generate a catalytically inactive version of GBP1, the first glutamate residue of the catalytic center (E500) was exchanged to an alanine residue via site-directed mutagenesis PCR with the primer pair GBP1_E500A_F (5’-CAGGCATCAACATCAGAAGCAGTG-3’) and GBP1_E500A_R (5’-GTTCCTACCATCTCCAAACTCAGTC-3’). The linearized, mutated plasmid was purified after gel electrophoresis using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). The isolated DNA fragment was treated with a self-made KLD mixture (1,000 units·mL-1 T4 polynucleotide kinase, 40,000 T4 DNA ligase units·mL-1 ligase 2,000 units·mL-1 DpnI, 1 × T4 DNA ligase buffer; all enzymes were purchased from New England BioLabs, Ipswich, USA) for 1 h at room temperature before transformation into *Escherichia coli* MachI cells. Plasmids were isolated using the NucleoSpin Plasmid Kit (Machery-Nagel, Düren, Germany) and sequenced to confirm the introduced mutation. Both plasmids (pXCScpmv-GBP1-HAStrep and pXCScpmv-GBP1_E500A-HAStrep) were introduced into *Agrobacterium tumefaciens* GV3101::pMP90RK strains for transient transformation of *N. benthamiana* leaf tissue.
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For *in planta* protein production in *N. benthamiana*, we used the binary vector pXCScpmv-HAStrep characterized by a 35S promoter cassette, modified 5′- and 3′-UTRs of RNA-2 from the cowpea mosaic virus as translational enhancers, and C-terminal hemagglutinin (HA) and StrepII tags (Witte et al., 2004; Myrach et al., 2017). The codon-optimized GBP1 coding sequence was amplified with the primer pair ClaI_GBP1_F (5’-gacggtatcgataaaATGCCGCCACATGGTAGACG-3’) and GBP1_noSTOP_XmaI_R (5’-ataactcccgggATGGCCATATTGACGATACCAACAGC-3’) and directionally cloned into the ClaI and XmaI sites of the binary vector to produce pXCScpmv-GBP1-HAStrep.
To generate a catalytically inactive version of GBP1, the first glutamate residue of the catalytic center (E500) was exchanged to an alanine residue via site-directed mutagenesis PCR with the primer pair GBP1_E500A_F (5’-CAGGCATCAACATCAGAAGCAGTG-3’) and GBP1_E500A_R (5’-GTTCCTACCATCTCCAAACTCAGTC-3’).
The linearized, mutated plasmid was purified after gel electrophoresis using the NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany). The isolated DNA fragment was treated with a self-made KLD mixture (1,000 units·mL-1 T4 polynucleotide kinase, 40,000 T4 DNA ligase units·mL-1 ligase 2,000 units·mL-1 DpnI, 1 × T4 DNA ligase buffer; all enzymes were purchased from New England BioLabs, Ipswich, USA) for 1 h at room temperature before transformation into *Escherichia coli* MachI cells.
Plasmids were isolated using the NucleoSpin Plasmid Kit (Machery-Nagel, Düren, Germany) and sequenced to confirm the introduced mutation. Both plasmids (pXCScpmv-GBP1-HAStrep and pXCScpmv-GBP1_E500A-HAStrep) were introduced into *Agrobacterium tumefaciens* GV3101::pMP90RK strains for transient transformation of *N. benthamiana* leaf tissue.
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## MALDI-TOF analysis
The digested products of GBP1 were analyzed using Oligosaccharide Mass Profiling as previously described (Günl et al., 2011). Briefly, 2 μL of the samples were spotted onto 2 μL of crystalized dihydroxy benzoic acid matrix (10 mg·mL−1) and analyzed by MALDI-TOF mass spectrometry (Bruker rapifleX instrument, Bremen). Mass spectra were recorded in linear positive reflectron mode with an accelerating voltage of 20,000 V. The spectra of the samples were analyzed using the flexanalysis software 4.0 (Bruker Daltonics, Billerica, USA).
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## Tandem mass spectrometric (MS-MS) analysis of the pull-down proteins
LC-MS/MS analysis was performed using an Orbitrap Fusion tribrid mass spectrometer (Thermo Scientific) and a nanoflow-UHPLC system (Dionex UltiMate3000, Thermo Scientific). Peptides were trapped to a reverse phase trap column (Acclaim PepMap, C18 5 μm, 100 μm x 2 cm, Thermo Scientific) connected to an analytical column (Acclaim PepMap 100, C18 3 μm, 75 μm x 50 cm, Thermo Scientific). Peptides were eluted in a gradient of 3-40% acetonitrile in 0.1% formic acid (solvent B) over 86 min followed by gradient of 40-80% B over 6 min at a flow rate of 200 nL·min at 40 °C. The mass spectrometer was operated in positive ion mode with nano-electrospray ion source with ID 0.02mm fused silica emitter (New Objective). Voltage +2200 V was applied via platinum wire held in PEEK T-shaped coupling union with transfer capillary temperature set to 275 ºC. The Orbitrap, MS scan resolution of 120,000 at 400 m/z, range 300 to 1800 m/z was used, and automatic gain control (AGC) was set at 2e5 and maximum injection time to 50 ms. In the linear ion trap, MS/MS spectra were triggered with a data dependent acquisition method using ‘top speed’ and ‘most intense ion’ settings. The selected precursor ions were fragmented sequentially in both the ion trap using CID and in the HCD cell. Dynamic exclusion was set to 15 s. Charge state allowed between 2+ and 7+ charge states to be selected for MS/MS fragmentation. Peak lists in the format of Mascot generic files (mgf files) were prepared from raw data using MSConvert package (ProteoWizard). Peak lists were searched on Mascot server v.2.3 (Matrix Science) against a *Hordeum vulgare* Morex v1.0 database (IBSC_v2, IPK Gatersleben) and an in-house contaminants database. Tryptic peptides with up to two possible mis-cleavages and charge states +2, +3, +4, were allowed in the search. The following modifications were included in the search: oxidized methionine as variable modification and carbamidomethylated cysteine as static modification. Data were searched with a monoisotopic precursor and fragment ions mass tolerance 10 ppm and 0.6 Da, respectively. Mascot results were combined in Scaffold v. 4 (Proteome Software) and exported in Excel (Microsoft Office, Table S1).
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## Preparation of plant material - **Barley**
For immunity assays in barley, the roots and shoots of seven-day-old seedlings were separated. The root tissue between 2 cm below the seed and 1 cm above the tip was cut into 5 mm pieces. Each assay was carried out with randomly selected root pieces from 16 barley seedlings. Four root pieces were transferred to each well of a 96-well microtiter plate containing 150 μL of sterile Milli-Q water. Barley shoot assays were performed on 3-mm leaf discs punched from the youngest leaves of eight individual barley seedlings.
## Preparation of plant material - *N. benthamiana*
For immunity assays in N. benthamiana, 3-mm leaf discs from the youngest, fully developed leaf of eight three-week-old plants were transferred to a 96-well plate filled with 150 μL of sterile Milli-Q water.
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## ROS burst assay
The ROS burst assay was based on previously published protocols (Chandrasekar et al., 2022; Felix et al., 1999). In brief, a 96-well plate containing water and plant material (as described above) was incubated overnight at RT to remove ROS that had resulted from mechanical damage to the tissue during preparation. The next day, the water was replaced with 100 μL of fresh Milli-Q water containing 20 μg·mL-1 horseradish peroxidase (Sigma-Aldrich, Taufkirchen, Germany) and 20 μM L-012 (Wako Chemicals, Neuss, Germany). After a short incubation period (∼15 min), 100 μL of double-concentrated elicitor solutions were added to the wells. All elicitors were dissolved in Milli-Q water without additional treatment. Measurements of elicitor-triggered apoplastic ROS production were started immediately and performed continuously with an integration time of 450 ms in a TECAN SPARK 10 M multiwell plate reader (Männedorf, Switzerland).
<img src=dataset\FigS1C.jpg width=60%>
<img src=dataset\FigS1C.jpg width=110%>
### Supplementary Figure 1 C) caption
......
## Biotinylation of laminarin
Laminarin (final concentration ∼ 60 mM) was incubated with biotin-hydrazide (120 mM) and sodium cyanoborohydride (1 M) for 2 h at 65 °C. The product was purified on a PD MidiTrap G-10 column (GE Healthcare) according to the manufacturer’s description. Success of biotinylation was validated via mass spectrometry. The immunogenic capacity of biotinylated laminarin was confirmed via ROS burst assays.
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## Protein pull-down with biotinylated laminarin
Barley leaves from 2-week-old plants were treated for 15 min with biotinylated laminarin, followed by vacuum infiltration for 2 min. Untreated laminarin and a biotinylated version of the bacterial elongation factor Tu peptide (elf18) were used as controls. The tissue was frozen in liquid nitrogen and ground to fine powder. Then, 10 mg·mL-1 of extraction buffer (10 mM MES, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% IGEPAL, proteinase inhibitor cocktail) were added to the powder. To avoid pH-dependent binding effects to the NeutrAvidin beads, two buffer conditions (pH 5.6 and pH 8.0) were used for all treatments. Samples were incubated rotating at 4 °C for 60 min and centrifuged at 10,645 g, 4 °C for 15 min. Supernatant was filtered to remove pieces, mixed with 50 μL of high-capacity NeutrAvidin agarose resin (Thermo Fisher Scientific, Schwerte, Germany), and incubated (inverting) at 4 °C for 3 h. The sample was briefly centrifuged at 60 g for 1 min. After discarding the supernatant, the beads were washed four times with 10 mL of wash buffer (10 mM MES [pH 5.6 or pH 8.0], 50 mM NaCl, 10 mM MgCl2, 0.5% IGEPAL). Proteins were eluted by boiling the beads with 50-70 μL of 2x SDS loading (including reducing agent) for 5 min. Proteins were separated by SDS-PAGE (NuPAGE; Invitrogen, Waltham, United States) after staining with Coomassie brilliant blue G-250, cut out for mass spectrometric analysis and digested with trypsin.
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Barley leaves from 2-week-old plants were treated for 15 min with biotinylated laminarin, followed by vacuum infiltration for 2 min. Untreated laminarin and a biotinylated version of the bacterial elongation factor Tu peptide (elf18) were used as controls.
The tissue was frozen in liquid nitrogen and ground to fine powder. Then, 10 mg·mL-1 of extraction buffer (10 mM MES, 50 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% IGEPAL, proteinase inhibitor cocktail) were added to the powder. To avoid pH-dependent binding effects to the NeutrAvidin beads, two buffer conditions (pH 5.6 and pH 8.0) were used for all treatments. Samples were incubated rotating at 4 °C for 60 min and centrifuged at 10,645 g, 4 °C for 15 min. Supernatant was filtered to remove pieces, mixed with 50 μL of high-capacity NeutrAvidin agarose resin (Thermo Fisher Scientific, Schwerte, Germany), and incubated (inverting) at 4 °C for 3 h. The sample was briefly centrifuged at 60 g for 1 min. After discarding the supernatant, the beads were washed four times with 10 mL of wash buffer (10 mM MES [pH 5.6 or pH 8.0], 50 mM NaCl, 10 mM MgCl2, 0.5% IGEPAL). Proteins were eluted by boiling the beads with 50-70 μL of 2x SDS loading (including reducing agent) for 5 min.
Proteins were separated by SDS-PAGE (NuPAGE; Invitrogen, Waltham, United States) after staining with Coomassie brilliant blue G-250, cut out for mass spectrometric analysis and digested with trypsin.
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## Hordeum vulgare
Root tissue of barley control and mutant plants colonized by *S. indica* was harvested at 6 dpi and then stained as previously described (Hilbert et al., 2019). Briefly, roots were incubated at 95 °C for 2 min in 10% KOH, washed 3 times for 30 min in deionized water and 3 times for 30 min in PBS (pH 7.4). The roots were stained for 5 min under vacuum and then washed three times with deionized water. Fungal structures were visualized using 10 μg·mL-1 fluorescently labeled wheat germ agglutinin (WGA-AF488, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) in PBS (pH 7.4) and imaging was conducted with an excitation wavelength of 488 nm and emission detection between 500-540 nm. Papillae and root cell wall appositions were stained with 10 μg·mL-1 fluorescently labeled Concanavalin A (ConA-AF633, Invitrogen, Thermo Fisher Scientific, Schwerte, Germany) in PBS (pH 7.4) and imaged by excitation at 633 nm and detection at 650-690 nm.
Callose was stained by aniline blue according to a protocol adapted from Mason and coworkers (Mason et al., 2020). Roots were incubated at 95 °C for 2 min in 10% KOH, washed 3 times for 30 min in deionized water and 3 times for 30 min in PBS (pH 7.4). Roots were washed for 1 h at RT under continuous shaking in 67 mM K2HPO4 (pH 12). The samples were then incubated for 1 h at RT under continuous shaking in an aniline blue solution composed of 0.01% aniline blue (w/v) in 67 mM K2HPO4 (pH 12). Roots were washed again for 1 h at RT under continuous shaking in 67 mM K2HPO4 (pH 12). Imaging was conducted with an excitation wavelength of 405 nm and emission detection from 490-510 nm.
Images were taken with a Leica TCS SP8 confocal microscope (Wetzlar, Germany). The percentage area of ConA staining was quantified using ImageJ81 in maximum intensity projections of 10-slice Z-stacks with an image depth of 10 μm. At least 24 different root regions of each genotype were analyzed.
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## Root staining of *R. irregularis*
Roots were stained according to a previously published protocol (Vierheilig et al., 1998). Briefly, roots were incubated for 15 min at 95 °C in 10% KOH, washed with 10% acetic acid and incubated for 5 min at 95 °C with a staining solution of 5% ink (Pelikan, Falkensee, Germany) in 5% acetic acid. After staining, the roots were carefully washed with tap water, then incubated in 5% acetic acid at 4 °C for at least 20 min. The ink-stained root tissue was cut into 1 cm segments with a scalpel and 30 segments of similar diameter were randomly selected from each genotype. Cross-section points were determined from 10 random cuts per root segment. Ink-stained *R. irregularis* structures such as intraradical hyphae (IRH), extraradical hyphae (ERH), arbuscules and vesicles were visualized with a light microscope (AxioStar, Carl Zeiss, Jena, Germany) at 10× magnification. Colonization with R. irregularis was scored as positive if IRH, arbuscules or vesicles were present. The roots of four biological replicates per genotype were examined.
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<img src=dataset\gr1c_lrg.jpg width=60%>
<img src=dataset\gr1c_lrg.jpg width=100%>
### Figure 1 C) caption
......
## Carbohydrate substrates for immunity and enzymatic digestion assays
All laminarioligomeres, gentiobiose, chitohexaose, cellohexaose and xyloglucan oligomer (XXXG) were purchased from Megazyme (Bray, Ireland). Laminarin from *Laminaria digitata* was purchased from Sigma-Aldrich (Taufkirchen, Germany) and laminarin from *Eisenia bicyclis* was purchased from Biosynth (Staad, Switzerland). Substrates derived from *S. indica* CW and EPS matrix were purified and prepared as previously described (Chandrasekar et al., 2022).
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## PlantMaterialAndGrowthConditions
All experiments, including the generation of CRISPR/Cas9 knock-out lines, were performed with the spring barley (*H. vulgare L.*) cv. Golden Promise Fast, an introgression line carrying the Ppd-H1 allele that confers fast flowering (Gol et al., 2021) From here on, we use “control” to name this non-mutagenized cultivar that carries normal copies of *GBP1* and *GBP2*. For ROS burst assays, barley seeds were surface sterilized with 6% sodium hypochlorite for 1 h and then washed extensively (5 × 30 mL sterile water). Seeds were germinated on wet filter paper at room temperature in the dark under sterile conditions for three days before transfer to sterile jars containing solid 1/10 plant nutrition medium (PNM), pH 5.7 and 0.4% Gelrite (Duchefa, Haarlem, the Netherlands). Seedlings were cultured for four days in a growth chamber under long-day conditions (day/night cycle of 16/8 h, 22 °C/18 °C, light intensity of 108 μmol·m−2·s−1).
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## PlantMaterialAndGrowthConditions
Seeds of *N. benthamiana* wild-type lines were sown on soil and grown for 3 weeks in the greenhouse under long-day conditions (day/night cycle of 16/8 h, 22–25 °C, light intensity of ∼140 μmol·m−2·s−1, maximal humidity of 60%).
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