From f1635bd94cbb94ed0967f9396fe6bf6b2aaf0009 Mon Sep 17 00:00:00 2001 From: Viktoria Petrova <vipet103@hhu.de> Date: Mon, 25 Dec 2023 13:20:06 +0100 Subject: [PATCH] add protocol to study Barley --- studies/Barley/protocols/PlasmidConstruction.md | 2 ++ .../protocols/RootStainingOfRIrregularis.md | 2 ++ 2 files changed, 4 insertions(+) create mode 100644 studies/Barley/protocols/PlasmidConstruction.md create mode 100644 studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md diff --git a/studies/Barley/protocols/PlasmidConstruction.md b/studies/Barley/protocols/PlasmidConstruction.md new file mode 100644 index 0000000..2ca1622 --- /dev/null +++ b/studies/Barley/protocols/PlasmidConstruction.md @@ -0,0 +1,2 @@ +## 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.78,79 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. \ No newline at end of file diff --git a/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md b/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md new file mode 100644 index 0000000..7b96001 --- /dev/null +++ b/studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RootStainingOfRIrregularis.md @@ -0,0 +1,2 @@ +## Root Staining +Roots were stained according to a previously published protocol.83 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. \ No newline at end of file -- GitLab