Tagging morphogenetic genes by insertional mutagenesis in the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is distantly related to Saccharomyces cerevis iae, can be genetically modified, and can grow in both haploid and diploid states in either yeast, pseudomycelial, or mycelial forms, depending on env ironmental conditions. Previous results have indicated that the STE and RIM pathways, which mediate cellular switching in other dimorphic yeasts, are not required for Y. lipolytica morphogenesis, To identify the pathways invo lved in morphogenesis, we mutagenized a wild-type strain of Y. lipolytica w ith a Tn3 derivative. U e isolated eight tagged mutants, entirely defective in hyphal formation, from a total of 40,000 mutants and identified seven g enes homologous to S. cerevisiae CDC25, RAS2, BUD6, KEX2, GP17, SNFS, and P PH21. We analyzed their abilities to invade agar and to form pseudomycelium or hyphae under inducing conditions and their sensitivity to temperature a nd to Calcofluor white. Chitin staining was used to detect defects in their cell walls. Our results indicate that a functional Ras-cyclic AMP pathway is required for the formation of hyphae in Y. lipolytica and that perturbat ions in the processing of extracellular. possibly parietal, proteins result in morphogenetic defects.