Cis-regulatory elements orchestrate phase-specific effector gene expression in Ustilago maydis

Plant pathogenic fungi secrete small proteins, termed effectors, to reprogram host metabolism and suppress immune responses during infection. Although transcriptional waves of effector expression have been described in several pathosystems, the cis-regulatory elements encoding infection-stage specificity 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 promoter mutagenesis demonstrates that the early-phase motif GTGGG significantly contributes to effector gene expression in planta and is sufficient to drive stage-restricted gene expression in synthetic minimal promoters. Collectively, our findings demonstrate that temporal deployment of the effector repertoire is at least partially encoded at the promoter level. The identified cis-regulatory elements provide a framework for dissecting transcriptional control during biotrophic infection and offer tools for infection-stage-specific gene expression in synthetic biology applications.