Gamborg s vitamins

Arabidopsis seedlings were grown for 11 days on 0.5x MS salts with Gamborg’s vitamins (M0404; Sigma) media solidified with 0.8% agar then transferred to six well plates (six seedlings per well) containing 3 ml of liquid medium containing 0.5x MS salts with 30 µM DEX (for +DEX treatment) or 0.5xMS salt (–DEX). Seedlings were gently shaken in 12 h light, 12 h dark cycle at day/night temperature regime of 22°C/18° for. After 24 h, flg22 peptide was added to a final concentration of 1 µM. Shaking was maintained for 20 minutes then seedlings were harvested and snap frozen in liquid nitrogen. Frozen tissue was homogenized in 100 µl of extraction buffer (100 mM HEPES, pH 7.5, 5 mM EDTA, 5 mM EGTA, 2 mM dithiothreitol, 10 mM Na₃VO₄, 10 mM NaF, 50 mM ß-glycerolphosphate, 1 mM phenylmethylsulfonyl fluoride, and 10% glycerol, 1% (w/v) polyvinylpolypyrrolidone). After centrifugation at 13,000 rpm for 30 min at 4°C, supernatants were reserved as clarified extract. Protein concentrations of clarified extracts were determined using a Bradford assay (BIO-RAD, Hercules, CA, USA) and twenty µg of total protein was separated by SDS-PAGE and immunoblotted.

Arabidopsis thaliana Columbia-0 (Col-0) seeds were obtained from Lehle Seeds (Round Rock, TX, USA). UNG (AT3G18630) T-DNA insertion hemizygous lines were obtained from the Arabidopsis Biological Resource Center, line number CS308282. Hemizygous T-DNA lines were self-crossed to obtain homozygous lines (Genotyping primers: wild-type 5′-TGTCAAAGTCCTGCAATTCTTCTCACA-3′ and 5′-TCGTGCCATATCTTGCAGACCACA-3′, and UNG 5′-ATAATAACGCTGCGGACATCTACATTTT-3′ and 5′-ACTTGGAGAAGGTAAAGCAATTCA-3′). All plants were grown in walk-in growth chambers under a 16:8 light:dark schedule at 22 °C. Plants grown on agar were surface sterilized and grown on 1× Murashige and Skoog Basal Medium (MSA) with Gamborg’s vitamins (Sigma, St. Louis, MO, USA) with 5 μg/mL Nystatin Dihydrate to prevent fungal contamination.

Catharanthus roseus, Vinca Little Bright Eye¹, was used for this work. Seeds were surface sterilized and then germinated on B5 medium (Sigma, St. Louis, MO, USA) supplemented with Gamborg’s vitamins (Sigma, St. Louis, MO, USA). Seeds were germinated in the dark at 26°C for 2 weeks. The seedlings were then transferred to a 16-h-light/8-h-dark cycle with a light intensity of approximately 44 μmol m^-2 s^-1 for 4 weeks before inoculation with Agrobacterium tumefaciens.

Seeds were harvested from mature plants and surface-sterilized with 12.5 g/L sodium hypochlorite and 0.05% (v/v) Triton X-100 for 5 min. Seeds were washed 4 times in sterile water and then plated onto media consisting of 0.5 × Murashige and Skoog Basal Salts, 3% (w/v) sucrose, pH 5.7 (KOH), 0.8% (w/v) plant agar and 1 × Gamborg’s vitamins (Sigma-Aldrich) without selection. Whole seedlings were harvested 10 days after germination. DNA was extracted from whole seedlings using the method described by Edwards et al. [37 (link)]. Edits in the T1 progeny were determined using amplicon deep sequencing on a MiSeq (Illumina) as described above.