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The yeast Yarrowia lipolytica undergoes a morphological transition from yeast-to-hyphal growth in response to environmental conditions. A forward genetic screen was used to identify mutants that reliably remain in the yeast phase, which were then assessed by whole-genome sequencing. All the smooth mutants identified, so named because of their colony morphology, exhibit independent loss of DNA at a repetitive locus made up of interspersed ribosomal DNA and short 10- to 40-mer telomere-like repeats. The loss of repetitive DNA is associated with downregulation of genes with stress response elements (5'-CCCCT-3') and upregulation of genes with cell cycle box (5'-ACGCG-3') motifs in their promoter region. The stress response element is bound by the transcription factor Msn2p in Saccharomyces cerevisiae We confirmed that the Y. lipolytica msn2 (Yl msn2 ) ortholog is required for hyphal growth and found that overexpression of Yl msn2 enables hyphal growth in smooth strains. The cell cycle box is bound by the Mbp1p/Swi6p complex in S. cerevisiae to regulate G 1 -to-S phase progression. We found that overexpression of either the Yl mbp1 or Yl swi6 homologs decreased hyphal growth and that deletion of either Yl mbp1 or Yl swi6 promotes hyphal growth in smooth strains. A second forward genetic screen for reversion to hyphal growth was performed with the smooth-33 mutant to identify additional genetic factors regulating hyphal growth in Y. lipolytica Thirteen of the mutants sequenced from this screen had coding mutations in five kinases, including the histidine kinases Yl chk1 and Yl nik1 and kinases of the high-osmolarity glycerol response (HOG) mitogen-activated protein (MAP) kinase cascade Yl ssk2 , Yl pbs2 , and Yl hog1 Together, these results demonstrate that Y. lipolytica transitions to hyphal growth in response to stress through multiple signaling pathways. IMPORTANCE Many yeasts undergo a morphological transition from yeast-to-hyphal growth in response to environmental conditions. We used forward and reverse genetic techniques to identify genes regulating this transition in Yarrowia lipolytica We confirmed that the transcription factor Yl msn2 is required for the transition to hyphal growth and found that signaling by the histidine kinases Yl chk1 and Yl nik1 as well as the MAP kinases of the HOG pathway (Yl ssk2 , Yl pbs2 , and Yl hog1 ) regulates the transition to hyphal growth. These results suggest that Y. lipolytica transitions to hyphal growth in response to stress through multiple kinase pathways. Intriguingly, we found that a repetitive portion of the genome containing telomere-like and rDNA repeats may be involved in the transition to hyphal growth, suggesting a link between this region and the general stress response., (Copyright © 2018 Pomraning et al.)

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane-associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.