| Summary: | <i>Candida lusitaniae</i> is an emerging opportunistic pathogenic yeast capable of shifting from yeast to pseudohyphae form, and it is one of the few <i>Candida</i> species with the ability to reproduce sexually. In this study, we showed that a <i>dpp3</i>Δ mutant, inactivated for a putative pyrophosphatase, is impaired in cell separation, pseudohyphal growth and mating. The defective phenotypes were not restored after the reconstruction of a wild-type <i>DPP3</i> locus, reinforcing the hypothesis of the presence of an additional mutation that we suspected in our previous study. Genetic crosses and genome sequencing identified an additional mutation in <i>MED15</i>, encoding a subunit of the mediator complex that functions as a general transcriptional co-activator in Eukaryotes. We confirmed that inactivation of <i>MED15</i> was responsible for the defective phenotypes by rescuing the <i>dpp3</i>Δ mutant with a wild-type copy of <i>MED15</i> and constructing a <i>med15</i>Δ knockout mutant that mimics the phenotypes of <i>dpp3</i>Δ in vitro. Proteomic analyses revealed the biological processes under the control of Med15 and involved in hyphal growth, cell separation and mating. This is the first description of the functions of <i>MED15</i> in the regulation of hyphal growth, cell separation and mating, and the pathways involved in <i>C. lusitaniae</i>.
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