E for brightfield and DTSSP Crosslinker Purity & Documentation fluorescence (Lumar; Zeiss, Jena, Germany) four days after plating. Pictures were acquired using the SPOT PursuitXS Monochrome camera controlled by the SPOT Basic Image Capture Computer software (SPOT Imaging, Sterling Heights, MI, USA). Colonies of cells displaying the expected phenotype have been employed for inoculation of liquid YEPSlight medium (Schirawski et al., 2005a) and tested once again for loss of fluorescence immediately after spotting in serial dilution on YNBN medium (Rabe et al., 2013) supplemented with two glucose and ten mM salicylate. For complementation analysis, SA sensing mutants had been transformed with an U. maydis cosmid library (Weinzierl, 2001). Colonies have been replica plated on YNBN plates containing two glucose, ten mM salicylate, and 200 mg ml21 Hygromycin B and screened for rescue of mCherry fluorescence by fluorescence stereomicroscopy. Rescue mutants were grown in YEPSlight supplemented with 200 mg ml21 Hygromycin B to retain the autonomously replicating cosmids. Cosmids were reisolated by employing the genomic DNA isolation protocol in line with Hoffman and Winston (1987) and amplified in E. coli. The complementing U. maydis fragment, which can be inserted within the cosmid, was sequenced with primers 50 pScos seq and 30 pScos seq. Sequenced reads have been mapped for the U. maydis genome by using CLC Key Workbench (Qiagen, Hilden, Germany) to narrow down regions that harbour genes for SA sensing.Experimental proceduresPlasmids, strains and culture conditionsPlasmids had been generated in accordance with common molecular cloning procedures described in Sambrook et al. (1989). Primers, plasmids, and cloning techniques employed in thisC V 2016 The Authors. Molecular Microbiology Published by John Wiley Sons Ltd., Molecular Microbiology, 102, 290Salicylic acid sensing by U. maydisU. maydis development assay on minimal mediaGrowth assays on salicylate and tryptophan minimal medium were performed as described in Rabe et al. (2013). In brief, U. maydis strains had been grown in YEPSlight till OD600nm 5 0.8 was reached. Cells had been washed 3 occasions with H20dd and resuspended in H20dd to OD600nm 5 1. Adjusted cultures have been spotted in serial dilutions on YNBN minimal medium, pH 7.0, supplemented with either 10 mM sodium salicylate or 10 mM Ltryptophan.Quantitative true time PCR and microarray analysesQuantitative genuine time PCR was performed as described in Rabe et al. (2013). In short, RNA from axenic culture or from infected plant material was extracted applying the TRIzol approach in accordance with the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA), DNA was removed by DNase I remedy (DNAfree Kit; Thermo Fisher Scientific, Waltham, MA, USA) and RNA was reverse transcribed working with the RevertAid Very first Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative Acid corrosion Inhibitors Reagents actual time PCR measurements were performed with a Roche R LightCyclerV 96 program (Roche Diagnostics, Rotkreuz, Switzerland) as outlined by manufacturer’s instructions. Relative expression values had been calculated with the 22DDCt strategy (Livak and Schmittgen, 2001). Graphical outputs and statistical analyses were performed making use of GraphPad Prism (v6.0; GraphPad Application, La Jolla, CA, USA). For international transcriptional profiling, maize plants (Early Golden Bantam) had been grown inside a plant development chamber and infected with SG200 or SG200Drss1 as described previously (Doehlemann et al., 2008). Samples had been collected in 3 independently carried out experiments by sampling 12 plants per.