Qubit fluorometric quantitation system

The DNA isolation method was performed on 50 mg of urediniospores as previously described in Pernaci et al. (2014) (link) and Persoons et al. (2014) (link). Quality and quantity of recovered high-molecular weight DNA was assessed by electrophoresis on agarose gel, by spectrophotometry (Nanodrop, Saint-Remy-lès-Chevreuse, France) and with the QuBit fluorometric quantitation system (Life Technologies, Villebon-sur-Yvette, France).

Known binding sites for CsrA were sought based upon data described by Kulkarni et al., (10 (link)). RNA transcripts corresponding to the promoter regions of acrAB (including wild-type and those harboring site directed mutations), and ramA were synthesized in vitro using the AmpliScribe™ T7-Flash™ Transcription Kit (Cambio Ltd, UK) and purified by ammonium acetate precipitation. Concentrations of transcripts were determined with the Qubit™ fluorometric quantitation system (Life Technologies, UK). The electrophoretic mobility shift assay was done using an electrophoretic mobility shift assay (EMSA) kit (E33075; Thermo Fisher, UK) according to the manufacturer's instructions. Briefly, 0.5 nM target RNA was incubated with CsrA-HisX6 protein for 20 min at room temperature in a 15 μl reaction mixture containing 1 × binding buffer (750 mM KCl, 0.5 mM dithiothreitol, 0.5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris–HCl, pH 7.4). Following addition of EMSA gel-loading solution, mixtures were separated by electrophoresis on a non-denaturing 8% (wt/vol) polyacrylamide gel in 0.5 × TBE buffer (22 mM Tris-HCl, 22 mM boric acid, 0.5 mM EDTA, pH 8.0). Gels were stained with 1 × SYBR Green EMSA nucleic acid stain. DNA was then visualized using a G:BOX F3 gel documentation system (Syngene, UK).