1 naa

The antibiotics hygromycin B (catalog number 10843555001, Roche) and kanamycin (catalog number KA0408, Real-times) were tested for toxicity to shoot bud growth and thereby assessed for their suitability as selective agents. To determine the optimum concentration of each, multiple shoot clusters induced from stem node explants were cultured on a shoot induction medium (MS mediumplus 0.1 mg/L 6-BA, 1.0 mg/L 1-NAA) (6-BA, catalog number B3408, Sigma–Aldrich; 1-NAA, catalog number N0640, Sigma–Aldrich) supplemented with various concentrations of hygromycin B (5, 10, 20, 30, and 40 mg/L) or kanamycin (50, 100, 150, 200, and 250 mg/L). Three replicate experiments were performed with at least nine explants cultured for each treatment. The explants were transferred to fresh medium containing the same concentration of antibiotic every week to maintain the appropriate selective pressure. The percentage of surviving explants and optimal antibiotic concentration were evaluated after 3 weeks.

Unless indicated otherwise in Table 1, S. cerevisiae strains used in this work were all constructed using conventional yeast genetic methods (Sherman 2002 (link)). Unless stated otherwise, yeast cultures were grown in standard rich (YP) medium or defined minimal (SC) medium (Sherman 2002 (link)) containing 2% glucose/dextrose and were supplemented with the appropriate nutrients to permit growth of auxotrophs and/or select for plasmids. Cultures were propagated at 30°C, unless indicated otherwise. Induction of genes under the control of GAL promoters was carried out either by adding 2% galactose to strains pregrown in the appropriate SC medium with 2% raffinose and 0.2% sucrose followed by incubation over the course of 3 h or, for strains containing the Gal4 human estrogen receptor herpes simplex virus transactivator VP16 fusion protein (Gal4-ER-VP16 [GEV]) (Quintero et al. 2007 (link)), by addition of 20 µM β-estradiol (final concentration) followed by incubation over the course of 3 h. For strains containing a protein tagged with an AID, the cells were cultured in YPD or the appropriate SC medium buffered with 50 mM potassium phosphate (pH 6.2), and degradation was induced by addition of a synthetic auxin (1-NAA) (Sigma-Aldrich) for the indicated time periods.

For siRNA treatment cells were seeded in six-well plates (110,000 cells/well). Cells to be analyzed by immunocytochemistry were seeded on coverslips. Twenty-four hours after seeding cells were transfected with siRNA (20 nM final concentration) using Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher) in OptiMEM medium. Forty-eight hours after transfection cells were left untreated or treated as specified in the figure legends and either harvested for western blot analysis or fixed for immunofluorescence analysis. When cell treatment with drugs was performed the following conditions were used: bafilomycin (Santa Cruz Biotech. – 400 nM for 2 h); wortmannin (Sigma – 1 µM for 2 or 3 h, as specified in the figure legend); puromycin (ThermoFisher – 5 µg/ml for 3 h); MG132 (Boston Biochem – 10 µM for 3 h); 1-NAA (Sigma – 500 µM – 1 mM for 3 or 12 h, as specified in the figure legend); doxycycline (50 ng/ml for 12 h).