Dimorphism in fungi is believed to constitute a mechanism of response to adverse conditions and represents an important attribute for the development of virulence by a number of pathogenic fungal species. We have isolated YlRAC1, a gene encoding a 192-amino-acid protein that is essential for hyphal growth in the dimorphic yeast Yarrowia lipolytica and which represents the first Rac homolog described for fungi. YlRAC1 is not an essential gene, and its deletion does not affect the ability to mate or impair actin polarization in Y. lipolytica. However, strains lacking functional YlRAC1 show alterations in cell morphology, suggesting that the function of YlRAC1 may be related to some aspect of the polarization of cell growth. Northern blot analysis showed that transcription of YlRAC1 increases steadily during the yeast-to-hypha transition, while Southern blot analysis of genomic DNA suggested the presence of several RAC family members in Y. lipolytica. Interestingly, strains lacking functional YlRAC1 are still able to grow as the pseudohyphal form and to invade agar, thus pointing to a function for YlRAC1 downstream of MHY1, a previously isolated gene encoding a C 2 H 2 -type zinc finger protein with the ability to bind putative stress response elements and whose activity is essential for both hyphal and pseudohyphal growth in Y. lipolytica.

The yeast-to-hypha morphological transition (dimorphism) is typical of many pathogenic fungi. Dimorphism has been attributed to changes in temperature and nutritional status and is believed to constitute a mechanism of response to adverse conditions. We have isolated and characterized a gene, MHY1, whose transcription is dramatically increased during the yeast-to-hypha transition inYarrowia lipolytica. Deletion of MHY1 is viable and has no effect on mating, but it does result in a complete inability of cells to undergo mycelial growth. MHY1 encodes a C2H2-type zinc finger protein, Mhy1p, which can bind putative cis-acting DNA stress response elements, suggesting that Mhy1p may act as a transcription factor. Interestingly, Mhy1p tagged with a hemagglutinin epitope was concentrated in the nuclei of actively growing cells found at the hyphal tip.

Most pathogenic fungi have the ability to alternate between a unicellular yeast form and different filamentous forms (hyphae and pseudohyphae). This attribute is generally regarded as an important virulence factor and has also attracted attention because of its implications in the study of eukaryotic cell differentiation. To identify genes that are involved in the regulation of these events, chemical mutagenesis of the dimorphic yeast Yarrowia lipolytica was performed and morphological mutants that were unable to form hyphal cells were isolated. Screening of a Y. lipolytica genomic DNA library for genes able to complement this defect led to the isolation of YlBMH1, a gene encoding a 14-3-3 protein and whose transcription levels are increased during the yeast-tohypha transition. Remarkably, overexpression of YlBMH1 was able to enhance pseudohyphae formation in a strain lacking functional YlRAC1 but caused no visible effects in ∆mhy1 and ∆bem1 cells, thus suggesting that YlBMH1 is involved in the regulation of both hyphal and pseudohyphal growth in Y. lipolytica. The identification of YlBMH2, a gene encoding a second 14-3-3 protein (YlBmh2p) that contains a 19 aa insertion absent in all other members of the 14-3-3 family, is also reported. Differently from YlBMH1, the transcription levels of YlBMH2 do not show any apparent variation during the induction of hyphal growth, and its overexpression has no effects on cells lacking functional MHY1, YlRAC1 or YlBEM1. Taken together, these observations suggest that, in spite of their high conservation, YlBmh1p and YlBmh2p have different cellular functions.

The ability to switch between a unicellular yeast form and different filamentous forms (fungal dimorphism) is an important attribute of most pathogenic fungi. Dimorphism involves a series of events that ultimately result in dramatic changes in the polarity of cell growth in response to environmental factors. We have isolated and characterized YlBEM1, a gene encoding a protein of 639 amino acids that is essential for the yeast-to-hypha transition in the yeast Yarrowia lipolytica and whose transcription is significantly increased during this event. Cells with deletions of YlBEM1 are viable but show substantial alterations in morphology, disorganization of the actin cytoskeleton, delocalization of cortical actin and chitin deposition, multinucleation, and loss of mating ability, thus pointing to a major role for YlBEM1 in the regulation of cell polarity and morphogenesis in this fungus. This role is further supported by the localization of YlBem1p, which, like cortical actin, appears to be particularly abundant at sites of growth of yeast, hyphal, and pseudohyphal cells. In addition, the potential involvement of YlBem1p in septum formation and/or cytokinesis is suggested by the concentration of a green fluorescent protein-tagged version of this protein at the mother-bud neck during the last stages of cell division. Interestingly, overexpression of MHY1, YlRAC1, or YlSEC31, three genes involved in filamentous growth of Y. lipolytica, induced hyphal growth of bem1 null mutant cells.