Streptavidin magnetic beads

A series of pull-down assays was performed using ID5 fragments (F1, F2 and F3) solubilized in PBS. His-tagged IQGAP1-F was biotinylated (EZ-Link Sulfo-NHS-Biotinylation kit, Thermo Scientific) and immobilized for one hour on Streptavidin magnetic beads (GE Healthcare). The loaded beads were blocked for 15 min with PBS containing 0.1% Tween and 5% milk powder before washing them twice with PBS. Then, 100 µl beads were incubated for 1 h with 300 µl of either purified His-tagged ID5_F1, ID5_F2 or ID5_F3 (concentration 30 µM). Before eluting the proteins, the beads were washed three times with PBS. Then, the first elution step was performed using 100 µl elution buffer (50 mM sodium phosphate, 100 mM NaCl, 2% SDS, 2 M Urea). The beads were incubated in elution buffer for 5 min at 98 °C. The supernatant was removed and loaded on an SDS-PAGE after addition of loading dye. For the second elution step, the beads were incubated in 30 µl 1x SDS loading dye for 5 min at 98 °C. The supernatant was directly loaded on a gel. The gels were further analyzed by Western blot using anti-His antibodies.

The total RNA was transcribed using an AmpliScribe T7-Flash Biotin-RNA Transcription Kit (Epicenter, China). RNA was purified by RNeasy Plus Mini Kit transcription and DNase I (QIAGEN, Germany), and labeled with biotin by Biotin RNA Labeling Mix (Ambio Life, USA). The labeled RNA was then added with the RNA structure buffer (10 mM Tris [pH 7.0], 0.1M KCl, and 10 mM MgCl₂) and left to react at 90°C for 2 min. RNA was then incubated on ice for 20 min and left at room temperature for 20 min. Afterward, RNA and protein products were mixed together and left to incubate at room temperature for 1 h. This was followed by the addition of Streptavidin magnetic beads (GE Healthcare) at room temperature for 1 h and a rinse with ddH₂O in preparation for the next experiment.

Snap‐frozen roots of Fo‐inoculated seedlings were ground in liquid nitrogen and 100 mg was dissolved in 1.2 ml lysis buffer (50 mM Tris‐HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 10% glycerol, 10 mM DTT, 2% PVPP, 0.15% Nonidet P40 and protease inhibitor cocktail (Roche)). An aliquot of the supernatant was kept as input sample; the remaining material was incubated with 40 µl magnetic streptavidin beads (GE Healthcare, Chicago, IL, USA) and placed on a rotator for 2 h at 4°C. After washing three times, captured proteins were released by shaking the beads in 100 µl SDS loading buffer for 5–6 min at 96°C. As input, 20 µl of the lysate was loaded onto SDS gels, together with 10 µl of streptavidin‐captured fraction.