Alternaria brassicicolais an important, necrotrophic fungal pathogen that causes black spot disease on Brassicas. In order to study pathogenicity mechanisms, gene deletion mutants were generated for 21 putative regulatory genes including kinases and transcription factors subjectively selected from the annotatedA. brassicicolagenome. Except for Ste12, the deletion of the SNF1 kinase, XlnR, and CreA homologues that control cell wall-degrading enzyme production did not significantly affect virulence in contrast to other pathogenic fungi. Only deletion of XlnR but not CreA, Ste12 or SNF1 impaired the fungus' ability to utilize sole carbon sources suggestingAlternariaregulates expression of cell wall-degrading enzymes in a novel manner. In addition, two novel virulence factors encoding a transcription factor (AbPro1) and a two-component histidine kinase gene (AbNIK1) were discovered. Deletion ofAbPro1resulted in a 70% reduction in virulence and a 25% reduction in vegetative growth ratesin vitro.Deletion ofAbNIK1resulted in a near complete loss of virulence, increased sensitivity to osmotic stress, and no changes in vegetative growth ratesin vitro.Interestingly, addition of long polypeptides to spores of both Δabste12and Δabnik1during inoculations resulted in a complete restoration of pathogenicity through a yet to be defined mechanism.