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move PlasmidConstruction protocol

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<div class="inline-table__head no-label"><span class="inline-table__label no-caption">Tabled
1</span><span class="inline-table__title"></span></div>
<table width="100%" cellpadding="0" cellspacing="0" border="0" class="scrollable bordered" tabindex="0">
<thead>
<tr>
<th class="rowsep">REAGENT or RESOURCE</th>
<th class="rowsep">SOURCE</th>
<th class="rowsep">IDENTIFIER</th>
</tr>
</thead>
<tbody>
<tr>
<td class="rowsep" colspan="3"><strong>Bacterial and virus strains</strong></td>
</tr>
<tr>
<td><em>Escherichia coli</em> MachI</td>
<td>University of Cologne, Germany (Prof. Dr. Ute Hoecker)</td>
<td>N/A</td>
</tr>
<tr>
<td><em>Agrobacterium tumefaciens</em> GV3101::pMP90RK</td>
<td>this paper</td>
<td>N/A</td>
</tr>
<tr>
<td class="rowsep"><em>Agrobacterium tumefaciens</em> GV3101::pBIN61-p19</td>
<td class="rowsep">this paper</td>
<td class="rowsep">N/A</td>
</tr>
<tr>
<td class="rowsep" colspan="3"><strong>Chemicals, peptides, and recombinant proteins</strong></td>
</tr>
<tr>
<td>Horseradish peroxidase</td>
<td>Sigma-Aldrich</td>
<td>P6782; CAS: 9003-99-0</td>
</tr>
<tr>
<td>L-012</td>
<td>Wako Chemicals</td>
<td>120-04891; CAS: 143556-24-5</td>
</tr>
<tr>
<td>Rifampicin</td>
<td>Roth</td>
<td>4163.2; CAS: 13292-46-1</td>
</tr>
<tr>
<td>Kanamycin</td>
<td>Duchefa</td>
<td>K0126.0010; CAS: 25389-94-0</td>
</tr>
<tr>
<td>Carbenicillin</td>
<td>Roth</td>
<td>6344.1; CAS: 4800-94-6</td>
</tr>
<tr>
<td>Gentamicin</td>
<td>Roth</td>
<td>0233.2; CAS: 1405-41-0</td>
</tr>
<tr>
<td>Sodium hypochlorite</td>
<td>Roth</td>
<td>9062.4; CAS: 7681-52-9</td>
</tr>
<tr>
<td>T4 polynucleotide kinase</td>
<td>New England BioLabs</td>
<td>CAT#M0201S</td>
</tr>
<tr>
<td>T4 DNA ligase</td>
<td>New England BioLabs</td>
<td>CAT#M0202M</td>
</tr>
<tr>
<td>DpnI</td>
<td>New England BioLabs</td>
<td>CAT#R0176L</td>
</tr>
<tr>
<td>T4 DNA ligase buffer</td>
<td>New England BioLabs</td>
<td>CAT#B0202S</td>
</tr>
<tr>
<td>N-naphthol</td>
<td>Sigma Aldrich</td>
<td>N1000-10G; CAS: 90-15-3</td>
</tr>
<tr>
<td>D-glucose</td>
<td>Roth</td>
<td>6887.2; CAS: 77938-63-7</td>
</tr>
<tr>
<td>Laminaribiose β-1-3-(Glc)<sub>2</sub></td>
<td>Megazyme</td>
<td>O-LAM2; CAS: 34980-39-7</td>
</tr>
<tr>
<td>Laminaritriose β-1-3-(Glc)<sub>3</sub></td>
<td>Megazyme</td>
<td>O-LAM3; CAS: 3256-04-0</td>
</tr>
<tr>
<td>Laminaripentaose β-1-3-(Glc)<sub>5</sub></td>
<td>Megazyme</td>
<td>O-LAM5; CAS: 23743-55-7</td>
</tr>
<tr>
<td>Gentiobiose β-1-6-(Glc)<sub>2</sub></td>
<td>Megazyme</td>
<td>O-GENT; CAS: 554-91-6</td>
</tr>
<tr>
<td><em>endo</em>-1,3-β-D-glucanase (barley BGLUII)</td>
<td>Megazyme</td>
<td>E-LAMHV; CAS: 9025-37-0</td>
</tr>
<tr>
<td>Chitohexaose</td>
<td>Megazyme</td>
<td>O-CHI6; CAS: 38854-46-5</td>
</tr>
<tr>
<td>Cellohexaose</td>
<td>Megazyme</td>
<td>O-CHE; CAS: 2478-35-5</td>
</tr>
<tr>
<td>Xyloglucan oligomer (XXXG)</td>
<td>Megazyme</td>
<td>O-X3G4; CAS: 121591-98-8</td>
</tr>
<tr>
<td>Laminarin from <em>Eisenia bicyclis</em></td>
<td>Biosynth</td>
<td>YL02421; CAS: 9008-22-4</td>
</tr>
<tr>
<td>Laminarin from <em>Laminaria digitata</em></td>
<td>Sigma Aldrich</td>
<td>L9634-1G; CAS: 9008-22-4</td>
</tr>
<tr>
<td>Elf18</td>
<td>The Sainsbury Laboratory, United Kingdom (Prof. Dr. Cyril Zipfel)</td>
<td>N/A</td>
</tr>
<tr>
<td>EZ-Link Hydrazide-Biotin</td>
<td>Thermo Scientific</td>
<td>CAT#21339</td>
</tr>
<tr>
<td>Sodium cyanoborohydride</td>
<td>Sigma Aldrich</td>
<td>42077-10G; CAS: 25895-60-7</td>
</tr>
<tr>
<td>Aniline blue</td>
<td>Sigma Aldrich</td>
<td>415049-25G; CAS: 666787-07-8</td>
</tr>
<tr>
<td>Concanavalin A (ConA) - Alexa Fluor 633</td>
<td>Invitrogen, Thermo Fisher Scientific</td>
<td>CAT#C21402</td>
</tr>
<tr>
<td>Wheat Germ Agglutinin (WGA) - Alexa Fluor 488</td>
<td>Invitrogen, Thermo Fisher Scientific</td>
<td>CAT#W11261</td>
</tr>
<tr>
<td>Avidin</td>
<td>Sigma Aldrich</td>
<td>A9275-5MG; CAS: 1405-69-2</td>
</tr>
<tr>
<td>Acetosyringone</td>
<td>Roth</td>
<td>6003.2; CAS: 2478-38-8</td>
</tr>
<tr>
<td>NeutrAvidin agarose resin</td>
<td>Thermo Fisher Scientific</td>
<td>CAT#29200</td>
</tr>
<tr>
<td>Blue ink</td>
<td>Pelikan</td>
<td>301010</td>
</tr>
<tr>
<td>Gelrite</td>
<td>Duchefa</td>
<td>G1101; CAS: 71010-52-1</td>
</tr>
<tr>
<td class="rowsep">Strep-Tactin Macroprep</td>
<td class="rowsep">IBA Lifesciences GmbH</td>
<td class="rowsep">2-1505-010</td>
</tr>
<tr>
<td class="rowsep" colspan="3"><strong>Critical commercial assays</strong></td>
</tr>
<tr>
<td>NucleoSpin Gel and PCR Clean-up Kit</td>
<td>Macherey-Nagel</td>
<td>740609.50</td>
</tr>
<tr>
<td class="rowsep">NucleoSpin Plasmid Kit</td>
<td class="rowsep">Macherey-Nagel</td>
<td class="rowsep">740588.50</td>
</tr>
<tr>
<td class="rowsep" colspan="3"><strong>Deposited data</strong></td>
</tr>
<tr>
<td class="rowsep">Proteomic data</td>
<td class="rowsep">ProteomeXchange Consortium via PRIDE partner repository (Perez-Riverol et&nbsp;al.<sup>73</sup>)</td>
<td class="rowsep">PXD046129</td>
</tr>
<tr>
<td class="rowsep" colspan="3"><strong>Experimental models: Organisms/strains</strong></td>
</tr>
<tr>
<td><em>Serendipita indica</em>: DSM11827</td>
<td>German Collection of Microorganisms and Cell Cultures (DSMZ)</td>
<td>NCBI Taxonomy ID: 1109443</td>
</tr>
<tr>
<td><em>Serendipita vermifera</em>: MAFF305830</td>
<td>NARO Research Center of Genetic Resources, Japan</td>
<td>NCBI Taxonomy ID: 933852</td>
</tr>
<tr>
<td><em>Bipolaris sorokiniana</em>: ND90Pr</td>
<td>Max Planck Institute for Terrestrial Microbiology, Marburg, Germany (Shadab Nizam)</td>
<td>NCBI Taxonomy ID: 665912</td>
</tr>
<tr>
<td><em>Blumeria hordei</em>: K1</td>
<td>Hinze et&nbsp;al.<sup>74</sup></td>
<td>NCBI Taxonomy ID: 1283759</td>
</tr>
<tr>
<td><em>Rhizophagus irregularis</em>: DAOM197198</td>
<td>Symplanta</td>
<td>00101SP; https://www.symplanta.com/productcatalog.html</td>
</tr>
<tr>
<td>Barley: control: <em>Hordeum vulgare</em> (cv. Golden Promise Fast)</td>
<td>Gol et&nbsp;al.<sup>42</sup></td>
<td>N/A</td>
</tr>
<tr>
<td>Barley: <em>gbp1 gbp2 #1</em>: <em>Hordeum vulgare</em> (cv. Golden Promise Fast) <em>gbp1-1 gbp2-1</em></td>
<td>this paper</td>
<td>N/A</td>
</tr>
<tr>
<td>Barley: <em>gbp1 gbp2 #2: Hordeum vulgare</em> (cv. Golden Promise Fast) <em>gbp1-2 gbp2-2/gbp2-3</em></td>
<td>this paper</td>
<td>N/A</td>
</tr>
<tr>
<td class="rowsep"><em>Nicotiana benthamiana</em></td>
<td class="rowsep">The Sainsbury Laboratory, United Kingdom (Prof. Dr. Cyril Zipfel)</td>
<td class="rowsep">N/A</td>
</tr>
<tr>
<td class="rowsep" colspan="3"><strong>Oligonucleotides</strong></td>
</tr>
<tr>
<td>Primers for cloning see Table&nbsp;S1</td>
<td>this paper</td>
<td>N/A</td>
</tr>
<tr>
<td>Primers for site-directed mutagenesis see Table&nbsp;S1</td>
<td>this paper</td>
<td>N/A</td>
</tr>
<tr>
<td>Primers for CRISPR/Cas9 gRNA see Table&nbsp;S1</td>
<td>this paper</td>
<td>N/A</td>
</tr>
<tr>
<td>Primers for <em>S.&nbsp;indica</em> colonization assay (qPCR) see Table&nbsp;S1</td>
<td>Chandrasekar et&nbsp;al.<sup>33</sup></td>
<td>N/A</td>
</tr>
<tr>
<td>Primers for <em>S.&nbsp;vermifera</em> colonization assay (qPCR) see Table&nbsp;S1</td>
<td>Mahdi et&nbsp;al.<sup>75</sup></td>
<td>N/A</td>
</tr>
<tr>
<td>Primers for <em>B.&nbsp;sorokiniana</em> colonization assay (qPCR) see Table&nbsp;S1</td>
<td>Mahdi et&nbsp;al.<sup>75</sup></td>
<td>N/A</td>
</tr>
<tr>
<td>Primers for <em>H.&nbsp;vulgare</em> colonization assay (qPCR) see Table&nbsp;S1</td>
<td>Deshmukh et&nbsp;al.<sup>76</sup></td>
<td>Accession no. M60175</td>
</tr>
<tr>
<td class="rowsep">Primers for barley <em>PR10</em> expression (qPCR) see Table&nbsp;S1</td>
<td class="rowsep">Schäfer et&nbsp;al.<sup>77</sup></td>
<td class="rowsep">N/A</td>
</tr>
<tr>
<td class="rowsep" colspan="3"><strong>Recombinant DNA</strong></td>
</tr>
<tr>
<td>pXCScpmv-HAStrep</td>
<td>Witte et&nbsp;al.<sup>78</sup>; Myrach et&nbsp;al.<sup>79</sup></td>
<td>N/A</td>
</tr>
<tr>
<td>pXCScpmv-GBP1-HAStrep</td>
<td>This paper</td>
<td>N/A</td>
</tr>
<tr>
<td>pXCScpmv-GBP1_E500A-HAStrep</td>
<td>This paper</td>
<td>N/A</td>
</tr>
<tr>
<td>pMGE625</td>
<td>Martin Luther University Halle-Wittenburg, Germany (Prof. Dr. Johannes Stuttmann)</td>
<td>N/A</td>
</tr>
<tr>
<td>pMGE626</td>
<td>Martin Luther University Halle-Wittenburg, Germany (Prof. Dr. Johannes Stuttmann)</td>
<td>N/A</td>
</tr>
<tr>
<td>pMGE628</td>
<td>Martin Luther University Halle-Wittenburg, Germany (Prof. Dr. Johannes Stuttmann)</td>
<td>N/A</td>
</tr>
<tr>
<td>pMGE629</td>
<td>Martin Luther University Halle-Wittenburg, Germany (Prof. Dr. Johannes Stuttmann)</td>
<td>N/A</td>
</tr>
<tr>
<td>pMGE599</td>
<td>Martin Luther University Halle-Wittenburg, Germany (Prof. Dr. Johannes Stuttmann)</td>
<td>N/A</td>
</tr>
<tr>
<td class="rowsep">pMP202</td>
<td class="rowsep">Martin Luther University Halle-Wittenburg, Germany (Prof. Dr. Johannes Stuttmann)</td>
<td class="rowsep">N/A</td>
</tr>
<tr>
<td class="rowsep" colspan="3"><strong>Software and algorithms</strong></td>
</tr>
<tr>
<td>Flexanalysis software (v. 4.0)</td>
<td>Bruker Daltonics</td>
<td>https://www.bruker.com/en.html</td>
</tr>
<tr>
<td>CRISPOR web tool (v. 4.97)</td>
<td>Concordet and Haeussler<sup>80</sup></td>
<td>http://crispor.tefor.net/</td>
</tr>
<tr>
<td>ImageJ (v. 1.53)</td>
<td>Schneider et&nbsp;al.<sup>81</sup></td>
<td>https://imagej.nih.gov/ij/</td>
</tr>
<tr>
<td>SparkControl (v. 2.1)</td>
<td>Tecan</td>
<td>https://lifesciences.tecan.com/multimode-plate-reader?p=tab--3</td>
</tr>
<tr>
<td>Scaffold (v. 4)</td>
<td>Proteome Software</td>
<td>https://www.proteomesoftware.com/products/scaffold-5</td>
</tr>
<tr>
<td>MSConvert package</td>
<td>ProteoWizard</td>
<td>https://proteowizard.sourceforge.io/download.html</td>
</tr>
<tr>
<td class="rowsep">Mascot server (v. 2.3)</td>
<td class="rowsep">Matrix Science</td>
<td class="rowsep">https://www.matrixscience.com/server.html</td>
</tr>
<tr>
<td class="rowsep" colspan="3"><strong>Other</strong></td>
</tr>
<tr>
<td>TECAN SPARK 10&nbsp;M multiwell plate reader</td>
<td>Tecan</td>
<td>N/A</td>
</tr>
<tr>
<td>PD MidiTrap G-10 column</td>
<td>Cytiva</td>
<td>28918011</td>
</tr>
<tr>
<td>TCS SP8 confocal microscope</td>
<td>Leica</td>
<td>https://www.leica-microsystems.com/</td>
</tr>
<tr>
<td>AxioStar light microscope</td>
<td>Carl Zeiss</td>
<td>N/A</td>
</tr>
<tr>
<td>Silica gel 60 F254 aluminum TLC plate</td>
<td>Merck Millipore</td>
<td>105554</td>
</tr>
<tr>
<td>MALDI-TOF mass spectrometer (rapifleX instrument)</td>
<td>Bruker</td>
<td>https://www.bruker.com/en/products-and-solutions/mass-spectrometry/maldi-tof/rapiflex.html</td>
</tr>
<tr>
<td>Reverse phase trap column (Acclaim PepMap, C18 5&nbsp;μm, 100&nbsp;μm x 2&nbsp;cm)</td>
<td>Thermo Scientific</td>
<td>N/A</td>
</tr>
<tr>
<td>Analytical column (Acclaim PepMap 100, C18 3&nbsp;μm, 75&nbsp;μm x 50&nbsp;cm)</td>
<td>Thermo Scientific</td>
<td>CAT#164570</td>
</tr>
<tr>
<td>Orbitrap Fusion Tribrid mass spectrometer</td>
<td>Thermo Scientific</td>
<td>https://www.thermofisher.com/de/de/home/industrial/mass-spectrometry/liquid-chromatography-mass-spectrometry-lc-ms/lc-ms-systems/orbitrap-lc-ms/orbitrap-tribrid-mass-spectrometers.html#fusion-tribrid</td>
</tr>
<tr>
<td>Nanoflow-UHPLC system (UltiMate3000 Dionex)</td>
<td>Thermo Scientific</td>
<td>N/A</td>
</tr>
<tr>
<td>PD-10 desalting column</td>
<td>Sigma Aldrich</td>
<td>GE17-0851-01</td>
</tr>
</tbody>
</table>
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studies/NicotianaBenthamiana/protocols/PlasmidConstruction.md → assays/HeterologousProteinProductionAndPurification/protocols/PlasmidConstruction.md
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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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