Combinatorial cis-regulatory elements shape temporal effector expression in Ustilago maydis

Plant-pathogenic fungi secrete small proteins, termed effectors, to reprogramme host metabolism and suppress immune responses during infection. Although transcriptional waves of effector expression have been described in several pathosystems, the cis-regulatory elements underlying their temporal coordination remain largely unknown. Here, we investigate the temporal regulation of effector genes in the biotrophic smut fungus Ustilago maydis, a model organism for fungal plant pathogenesis. By integrating transcriptome reanalysis with comparative promoter motif enrichment across biotrophic fungi, we identify distinct promoter motifs associated with defined infection phases. In U. maydis, three candidate cis-regulatory elements correlate with early, proliferative and late infection stages, respectively. Positional enrichment relative to transcription start sites supports their regulatory relevance. Functional dissection of the native pep1, stp1, and cmu1 promoters revealed strongly context-dependent contributions of these elements and synthetic promoter assays showed that the early-phase motif acts as an activating and the proliferative motif as a repressive element. Together, our findings support a combinatorial cis-regulatory code in which activating and repressive promoter elements are integrated in a promoter-specific manner to shape temporal effector expression during U. maydis infection. These elements provide a framework for engineering synthetic fungal promoters with tunable transcriptional outputs.