Identification of factors for SA sensing in Ustilago maydis by NGS

A next generation sequencing approach was performed to identify genes involved in SA sensing in the biotrophic fungus Ustilago maydis. It was part of a forward genetic screen based on UV mutagenesis of axenic U. maydis culture. Preceding next generation sequencing, U. maydis SG200Psrg1mCherry-3xHA, harboring an ectopic integration of mCherry-3xHA under control of the SA-responsive srg1 promoter, was subjected to UV mutagenesis. Subsequently, the obtained mutants were screened for loss of mCherry fluorescence indicating their inability to sense SA and to activate the srg1 promoter. Seven mutants with loss of fluorescence were identified. A cosmid complementation assay narrowed down the region important for SA sensing to the end of chromosome 20 with UMAG_05966 as essential factor for SA perception and/or signalling. To support these findings, the identified UV mutants displaying the SA sensing phenotype (UV1-7) as well as the progenitor strain were sequenced by Illumina paired end 125 bp sequencing.