For bacterial growth conditions, strains, Nod factor preparations, plant growth and inoculations, and microscopy techniques, see the article by D'Haeze et al. (1998) and references therein.
Final concentrations of inhibitors used are the following: 7 pM L-a-(2-aminoethoxyvinyl)-glycine (AVG), 0.1 mM (aminooxy) acetic acid (AOA), 10 mM a-aminoisobutyric acid (AIB), 0.5 mM C0CI2, 1 pM Ag2S04, 0.05% (v/v) 2,5-norbornadiene, 1 pM diphenyleneiodonium chloride (DPI), 200 pM diethyldithio carbamic acid (DDC), 1 mM ascorbic acid, 1 mM LaCl3, and 10"3% (m/v) ruthenium red. All inhibitors delivered as powders were dissolved in water and filter sterilized, except DPI that was dissolved in dimethylsulfoxide (DMSO). In the latter case, the stock concentration was such that no more than 10 pi of DMSO solution was added per tube. No influence of this DMSO concentration was observed on plant growth or nodulation. Inhibitors were purchased from Sigma-Aldrich, except 2,5-norbornadiene (Avocado, La Tour du Pin, France). Roots were treated with inhibitors two days before Nod factors or the wild-type strain were added. For DDC, ascorbic acid, and ruthenium red, solutions with inhibitors were refreshed 4, 4, and 2 times, respectively, prior to observations. The effect of inhibitors on Nod factor-induced root hair formation and ORS571 -induced nodulation was performed in at least four independent experiments using at least four plants.
Tubes were filled with Norris medium, leaving an air space of 1 cm to avoid leakage and contaminations. For scoring the effect of inhibitors on nodulation, the part of the root present in the upper airspace was not considered since it was not in contact with the medium and since some nodules were observed at the most upper part of the primary root. For the assay in which Ag+ ions were added at different time points compared to addition of the wild-type strain, the solutions with inhibitor and wild-type strain were refreshed each time nodules were counted. The same was done when this experiment was carried out using AO A. The two latter assays were each done twice, independently, using 4 plants per time point at which Ag+ or AOA was added.
Hydrogen peroxide and ethylene were applied in a final concentration of 1 mM and 21 pl/tube, respectively. When roots were treated with ethylene, a sticky substance was used to tighten the tube as well as possible from the air, in order to keep the ethylene inside the tube. These experiments were done at least three times, independently using 16 plants per treatment. For the assay in which ethylene was added at different time points compared to addition of ORS571, a second dose of ethylene was applied, five days after the first one.
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