Gentamicin

Agrobacteria were transformed with pGWB655-AtSFH1 nodulin and mutant versions (encoding mRFP-AtSfh1 nodulin chimera), pH7WGF2-BRI1, encoding a Bri1-GFP translational fusion used as a plasma membrane marker (kindly donated by Klaus Harter, University of Tübingen, Tübingen, Germany), and pBIN61-p19, encoding the silencing suppressor p19 (Voinnet et al., 2003 (link)). Transformants were then used for cotransfection of N. benthamiana leaves. For this, Agrobacteria transformants were grown at 28°C in Luria–Bertani (LB) medium supplemented with 100 μg/ml rifampicin (Genaxxon Bioscience, Ulm, Germany), 30 μg/ml gentamicin (Duchefa Biochemie B.V., Haarlem, The Netherlands), and appropriate antibiotic for plasmid selection to stationary phase. Subsequently, 1 ml of saturated culture was added to 4 ml of fresh medium and grown for an additional 4 h. Transformants were then sedimented at 4000 × g for 20 min at 4°C and resuspended in 10 mM 2-(N-morpholino)ethanesulfonic acid (MES; Carl Roth GmbH, Karlsruhe, Germany), 10 mM MgCl₂, and 150 μM 3′,5′-dimethoxy-4′-hydroxyacetophenone (acetosyringone; Sigma-Aldrich) and infiltrated into the abaxial air spaces of 2-wk-old tobacco plants. Leaves were imaged 5 d after infiltration using a confocal laser scanning microscope (Leica TCS SP8).