Primers used in this study.

<div><p><i>Saccharomycopsis</i> yeasts are natural organic sulfur auxotrophs due to lack of genes required for the uptake and assimilation of sulfate/sulfite. Starvation for methionine induces a shift to a predatory, mycoparasitic life strategy that is unique amongst ascomycetous yeasts. Similar to fungal plant pathogens that separated from <i>Saccharomycopsis</i> more than 400 million years ago, a specialized infection structure called penetration peg is used for prey cell invasion. Penetration pegs are highly enriched with chitin. Here we demonstrate that an ancient and conserved MAP kinase signaling pathway regulates penetration peg formation and successful predation in the predator yeast <i>S</i>. <i>schoenii</i>. Deletion of the MAP kinase gene <i>SsKIL1</i>, a homolog of the <i>Saccharomyces cerevisiae ScKSS1/ScFUS3</i> and the rice blast <i>Magnaporthe oryzae MoPMK1</i> genes, as well as deletion of the transcription factor <i>SsSTE12</i> generate non-pathogenic mutants that fail to form penetration pegs. Comparative global transcriptome analyses using RNAseq indicate loss of the <i>Ss</i>Kil1-<i>Ss</i>Ste12-dependent predation response in the mutant strains, while a methionine starvation response is still executed. Within the promoter sequences of genes upregulated during predation we identified a <i>cis</i>-regulatory element similar to the <i>Sc</i>Ste12 pheromone response element. Our results indicate that, re-routing MAP-kinase signaling by re-wiring Ste12 transcriptional control towards predation specific genes contributed to the parallel evolution of this predacious behaviour in predator yeasts. Consequently, we found that <i>SsSTE12</i> is dispensable for mating.</p></div>