assays/BarleyPhenotyping/protocols/BarleyMaterialAndGrowthConditions.md

 ## Barley plant material 
 
-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. 42 From here on, we use ‘‘control’’ to name this non-mutagenized cultivar that carries normal copies of GBP1 and GBP2. 
+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. From here on, we use ‘‘control’’ to name this non-mutagenized cultivar that carries normal copies of *GBP1* and *GBP2*. 
 
-## Barley growth conditions 
+## Barley growth conditions (6 days old) 
 
-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). 82 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 mmol$m2$s1 ).
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+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 six days in a growth chamber under long-day conditions (day/night cycle of 16/8 h, 22 C/18 C, light intensity of 108 mmol m-2 s-1). Root and shoot fresh weight as well as length of the barley plants were measured. 
+
+## Barley growth conditions (28 days old) 
+
+Seeds were germinated on wet filter paper at room temperature in the dark under sterile conditions for three days before transfer to pots (15 cm) containing heat-treated soil mixture (Cologne Agricultural Soil:vermiculite; 1:1). Seedlings were grown in the greenhouse for 28 days until harvest. Root and shoot fresh weight as well as length of the barley plants were measured. 
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assays/HeterologousProteinProductionAndPurification/protocols/PlasmidConstructionforGBP1Expression.md

+## 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 50- and 30-UTRs of RNA-2 from the cowpea mosaic virus as translational enhancers, and C-terminal hemagglutinin (HA) and StrepII tags. 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, Du¨ ren, 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 X 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, Du¨ ren, 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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