add references to protocols

studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/RhizophagusIrregularis.md

 ## Rhizophagus irregularis
-Barley control and mutant seeds were sterilized and germinated as described previously.75 Germinated seedlings were transferred to 9 × 9 cm pots containing autoclaved 1:1 silica sand:vermiculite mixture inoculated with 700 mg *R. irregularis* spore inoculum (10,000 spores·g-1 moist diatomaceous earth powder [50% water]) (Symplanta, Darmstadt, Germany). Approximately 500 mg of the inoculum was evenly mixed into the lower two-third substrate layer, further 200 mg were evenly sprinkled into a hole in the upper third of the substrate layer, into which the seedlings were transplanted. The seedlings were grown in the greenhouse and watered weekly with 30 mL of deionized water and tap water in a 1:1 ratio. Roots were harvested at 28 dpi and stored in 50% EtOH at 4 °C until staining.
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+Barley control and mutant seeds were sterilized and germinated as described previously (Mahdi et al., 2022). Germinated seedlings were transferred to 9 × 9 cm pots containing autoclaved 1:1 silica sand:vermiculite mixture inoculated with 700 mg *R. irregularis* spore inoculum (10,000 spores·g-1 moist diatomaceous earth powder [50% water]) (Symplanta, Darmstadt, Germany). Approximately 500 mg of the inoculum was evenly mixed into the lower two-third substrate layer, further 200 mg were evenly sprinkled into a hole in the upper third of the substrate layer, into which the seedlings were transplanted. The seedlings were grown in the greenhouse and watered weekly with 30 mL of deionized water and tap water in a 1:1 ratio. Roots were harvested at 28 dpi and stored in 50% EtOH at 4 °C until staining.
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studies/FungalMaterialGrowthConditionsAndBarleyColonizationAssays/protocols/SerendipitaIndicaSerendipitaVermiferaAndBipolarisSorokiniana.md

 ## Serendipita indica, Serendipita vermifera and Bipolaris sorokiniana
-The growth conditions for barley, *S. indica* (DSM11827, Si), *S. vermifera* (MAFF305830, Sv) and *B. sorokiniana* (ND90Pr, Bs) and the preparation of fungal suspensions for plant inoculation have been described previously.41,75 Four-day-old barley seedlings were transferred to sterile jars on 1/10 PNM (pH 5.7) and inoculated with 3 mL of either sterile water as control, *Si* chlamydospores (500,000 spores·mL-1), *Sv* mycelium (1 g per 50 mL), or *Bs* conidia (5,000 spores·mL-1). Plants were grown on a day/night cycle of 16/8 h at 22/18 °C and 60% humidity under a light intensity of 108 μmol·m−2·s−1. Plant roots were harvested six days post inoculation (dpi), washed thoroughly to remove extraradical fungal hyphae, and frozen in liquid nitrogen. Four barley plants were pooled per biological replicate. 
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+The growth conditions for barley, *S. indica* (DSM11827, Si), *S. vermifera* (MAFF305830, Sv) and *B. sorokiniana* (ND90Pr, Bs) and the preparation of fungal suspensions for plant inoculation have been described previously (Sarkar et al., 2019; Mahdi et al., 2022). Four-day-old barley seedlings were transferred to sterile jars on 1/10 PNM (pH 5.7) and inoculated with 3 mL of either sterile water as control, *Si* chlamydospores (500,000 spores·mL-1), *Sv* mycelium (1 g per 50 mL), or *Bs* conidia (5,000 spores·mL-1). Plants were grown on a day/night cycle of 16/8 h at 22/18 °C and 60% humidity under a light intensity of 108 μmol·m−2·s−1. Plant roots were harvested six days post inoculation (dpi), washed thoroughly to remove extraradical fungal hyphae, and frozen in liquid nitrogen. Four barley plants were pooled per biological replicate. 
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studies/HordeumVulgare/protocols/CRISPRCas9-basedMutagenesis.md

 ## CRISPR/Cas9-based mutagenesis of GBP homologues in barley
-The CRISPOR web tool80 (version 4.97) was used to design two single guide RNAs for each *GBP1* and *GBP2*:
+The CRISPOR web tool (Concordet et al., 2018) (version 4.97) was used to design two single guide RNAs for each *GBP1* and *GBP2*:
 GBP1_gRNA1: 5’-CCCGGCACGCTTCTTCGCGCCGG-3’
 GBP1_gRNA2: 5’-TGGCGCCTTCGGATGAACAGCGG-3’
 GBP2_gRNA1: 5’-TAAGATCCGTCGAGGCAGTATGG-3’
 GBP2_gRNA2: 5’-GTACAGCCGTTGCTACCCGACGG-3’
-Golden Gate cloning was used to load each guide sequence from complementary oligonucleotides into shuttle vectors pMGE625, pMGE626, pMGE628, pMGE629. The four guide expression cassettes were then assembled into binary vector pMGE599 to create pMP202. Vectors and cloning protocols have been previously described87 and were kindly provided by Johannes Stuttmann. Stable transformation of pMP202 in cv. Golden Promise Fast was performed as previously described.88
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+Golden Gate cloning was used to load each guide sequence from complementary oligonucleotides into shuttle vectors pMGE625, pMGE626, pMGE628, pMGE629. The four guide expression cassettes were then assembled into binary vector pMGE599 to create pMP202. Vectors and cloning protocols have been previously described (Kumar et al., 2018) and were kindly provided by Johannes Stuttmann. Stable transformation of pMP202 in cv. Golden Promise Fast was performed as previously described (Amanda et al., 2022).
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studies/NicotianaBenthamiana/protocols/PlasmidConstruction.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 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.
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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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