Identification And Characterization Of Genes Regulating Yeast-to-hypha Transition In Yeast Yarrowia Lipolytica

Previous studies on the yeast-to-hypha transition of Yarrowia lipolytica revealed that the MAPK and the cAMP-PKA pathways play a role in yeast-to-hypha transition. To gain insight into how MAPK pathway and cAMP-PKA pathway function, we identified homologues of Saccharomyces cerevisiae Tec1p and Ste12p, YlTec1p and YlSte12p, in dimorphic yeast Yarrowia lipolytica. We characterized the role of these regulators during hyphal development by gene knockout and overexpression assay. To uncover some new regulatory pathways, we employed a zeta-mediated insertional mutagenesis method to find new regulatory genes.First, we identified a homologue of Saccharomyces cerevisiae Teclp, YlTec1p. YlTec1p contains an evolutionarily conserved TEA/ATTS DNA-binding domain. Expression of YlTECl in S. cerevisiae tec1△cells rescued invasive growth defect and activated a FLO11-lacZ reporter, indicating that YlTec1p is functionally related to Tec1p. However, YlTEC1expression failed to activate a FRE-lacZ reporter, suggesting that difference exists between these two transcription factors. YlTEC1plays a negative role in yeast-to-hypha transition in Y. lipolytica based on gene deletion and overexpression studies. We show that YlTec1p activates rather than represses gene expression in Y. lipolytica by yeast one-hybrid assay, and YlTec1p is critical for the activation of FLO11-lacZ in Y. lipolytica. In addition, YlTec1p localized to the nucleus and its nuclear localization decreased during hyphal growth. Our study also suggests that YlTEC1may not be largely regulated by the cAMP-PKA pathway. Finally, we searched for several target gene candidates of YlTec1p based on homologue of Teclp and CaTeclp regulated genes, and assayed their transcription activity in wild type and tec1△mutant, we find transcription of Y1MSB2is dependent on YlTec1p.And then we identified another downstream transcription factor YlSte12p. Gene deletion results and overexpression studies show that YlSTE12also plays a negative role in hypha formation of Yarrowia lipolytica. YlStel2p and YlTec1p act independently, and the transcription of YlTEC1and YlSTE12are independent of each other. YlStel2p also localized to the nucleus, which is consistent with its function in transcriptional regulation. YlSTE12may not be regulated by the cAMP-PKA pathway. Overexpression of YlSTE12in WT strains inhibited hyphal development, whereas in Ylkss1△strain, it enhanced hypha formation. We speculated that YlSte12p was regulated by YlKsslp via phosphorylation. There are5potential phosphorylated sites in YlSte12p, our study indicates that S108and S111are essential to the function of YlSte12p.We also characterized YIHOG1, YlPHD1, YlSOK2and YlFLO8in the regulation of hyphal development in Yarrowia lipolytica. We found that hog1△. cells show a enhancement in filamentous growth. Overexpression of YIHOG1, YlPHD1, YISOK2and YlFLO8have no impact on filamentous growth.In addition, we screened mutants which could not form hyphae or show a enhancement in hyphae formation by zeta element-mediated random insertion. We identified the disrupted genes of some of the mutants. One of the mutants hyp166could always keep in yeast form in several growth conditions, and we identified YlSFL1as the inactived gene in hyp166mutant by TAIL-PCR. Our gene deletion and overexpression studies indicate that YlSFL1plays a positive role in yeast-to-hypha transition.