Merge branch 'ceplas-dm-viktoria' into 'main'

Ceplas dm viktoria

See merge request !2

assays/AssessmentOfPlantResistanceToPseudomonasSyringae/protocols/AssessmentOfPlantResistanceToPseudomonasSyringaeProtocol.md

+## Assessment of plant resistance to *Pseudomonas syringae*
+
+To assess bacterial growth in naive 5-week-old plants, overnight log phase cultures of *Pseudomonas syringae* pathovar *maculicola* were washed three times with 10 mM MgCl2 and diluted to a final optical density at 600nm (OD600)=0.001 before infiltrating the resulting bacterial suspensions from the abaxial side into three fully grown leaves using a 1 ml syringe without a needle. The infiltration was performed between 10.00 h and 11.00 h, as previously described (DOI: 10.1016/j.cell.2018.02.049). Approximately 60 h later, the bacterial growth was quantified by measuring the bacterial bioluminescence in leaf discs (10 mm in diameter) of infiltrated leaves (one disc per leaf, three discs per plant) using a Sirius FB12 luminometer (Berthold Detection Systems). For each independent experiment, at least 18 replicate leaves from 6–7 plants per genotype were measured before performing a statistical analysis of the resulting values.
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assays/Co-cultivationWithBurkholderiaGlumae/protocols/Co-cultivationWithBurkholderiaGlumaeProtocol.md

+## Co-cultivation with *Burkholderia glumae*
+
+Plants were grown in the 12-well plates as described above for 14 d. For inoculation, overnight bacterial cultures were washed twice with sterile 10 mM MgCl2 and the final OD600 was measured. The *B. glumae* PG1 was diluted stepwise to OD600=0.0005 in 10 mM MgCl2. An 8 µl aliquot of these suspensions was used for inoculation into each well and 8 µl of 10 mM MgCl2 was used as mock treatment. Samples for camalexin (shoots) and DNA (roots) were harvested after 3 d of inoculation.
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assays/DeterminationOfBacterialTiter/protocols/DeterminationOfBacterialTiterProtocol.md

+## Determination of bacterial titer
+
+Bacterial titer in the roots was determined using the qPCR method as described in Koprivova et al. (2023). Genomic DNA was extracted by a buffer containing 0.025 M EDTA, 0.2 M Tris pH 8.0, 0.25 M NaCl, and 0.5% SDS. After 10 min incubation at 65 °C and centrifugation, the supernatant was precipitated with isopropanol, washed with 70% ethanol, and resuspended in 100 µl of sterile water. For the qPCR, 13 ng of corresponding DNA samples were used with *Arabidopsis*- (At primer AT4G26410) and *B. glumae* PG1-specific primer (Burk1 for NR042931). The qPCR conditions were the same as for expression analysis. The qPCRs were performed in duplicate for each of the four independent samples. The qPCR results were related to the bacterial titer as established in Koprivova et al. (2023).
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assays/MetaboliteAnalysis/protocols/AnionsAnalysis.md

+## Anions
+
+For the measurement of phosphate, nitrate, and sulfate anions, 1 ml of sterile H2O was added to ~20 mg of homogenized shoot material, shaken for 1 h at 4 °C and heated to 95 °C for 15 min. The samples were centrifuged at maximum speed for 15 min at 4 °C, and 200 μl of the supernatants were transferred to an ion chromatography vial. Standard curves were generated using 0.5, 1, and 2 mM KH2PO4, KNO3, and K2SO4. The inorganic anions were measured with the Dionex ICS-1100 chromatography system and separated using a Dionex IonPac AS22 RFIC 43 250 mm analytic column (Thermo Scientific). The running buffer was made up of 4.5 mM NaCO3 and 1.4 mM NaHCO3 as described in Dietzen et al. (2020).
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