Isolation and characterization of YlBEM1, a gene required for cell polarization and differentiation in the dimorphic yeast Yarrowia lipolytica

Abstract

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 YlBemlp, 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.

FIG. 1.

Colony morphology of Y. lipolytica strains. (A) Filamentous phenotype of a colony of wild-type E122 cells. (B) Smooth phenotype of a colony of CHY33169 cells. (C) Smooth phenotype of a colony of bem1KO157 cells obtained by deletion of the YlBEM1 gene. (D) Enhanced filamentous phenotype of a colony of bem1KO157 cells transformed with plasmid pBEM1. Colonies were photographed at a magnification of ×100 after 3 days of incubation at 28°C on YNA-agar plates.

FIG. 2.

Nucleotide sequence of the YlBEM1 gene and deduced amino acid sequence of YlBem1p. The transcriptional start site of the YlBEM1 gene is indicated. The putative TATA box and consensus sequences for intron splicing are underlined.

FIG. 3.

Amino acid sequence alignment of Bem1p of Y. lipolytica (YlBem1p) and its homologs from S. pombe (SpScd2p) and S. cerevisiae (ScBem1p). Amino acid sequences were aligned by use of the ClustalW program (EMBL, Heidelberg, Germany). Solid background, identical residues in at least two of the proteins; shaded background, similar residues in at least two of the proteins. The following amino acids are similar to each other: G, A, and S; V, I, L, and M; I, L, M, F, Y, and W; K, R, and H; D, E, Q, and N; and S, T, Q, and N. Percentages of identity between YlBem1p and SpScd2p and between YlBem1p and ScBem1p are given in parentheses at the end of the sequence. GenBank accession numbers are AAA50557 (SpScd2p) and CAA45320 (ScBem1p).

FIG. 4.

YlBEM1 mRNA levels are increased during dimorphic transition. Total RNA was isolated from E122 cells incubated at 28°C in YNBGlcNAc (induction of hyphal growth) or YNBGlc (control culture, growth as the yeast form) for the times indicated and subjected to semiquantitative RT-PCR analysis. The 600- and 400-bp RT-PCR products were resolved by electrophoresis on 2% agarose gels and visualized by staining with ethidium bromide. YlBEM1 mRNA expression was normalized to that of YlHIS1 mRNA. Bars in micrographs, 5 μm.

FIG. 5.

Localization of GFP-tagged YlBem1p in yeast (A and B), pseudohyphal (C), and hyphal (D) bem1KO157 cells carrying plasmid pBEM1GFP. Arrows indicate sites at which the YlBem1p-GFP chimera concentrates. Bars, 5 μm.

FIG. 6.

Deletion of the YlBEM1 gene. (A) Diagram illustrating the targeted gene replacement strategy for the YlBEM1 gene. Arrows indicate the open reading frames and directionality of the YlBEM1 and YlURA3 genes. (B) Southern blot analysis of BamHI-digested genomic DNA from the Y. lipolytica wild-type strain E122 and mutant strain bem1KO157, confirming replacement of the YlBEM1 gene segment by the YlURA3-containing linear molecule.

FIG. 7.

Deletion of the YlBEM1 gene affects cell morphology and impairs hyphal and pseudohyphal growth of Y. lipolytica in YEPD liquid medium. Top panels, exponential-growth phase (optical density at 600 nm [OD₆₀₀] = 1). Bottom panels, stationary phase (OD₆₀₀ = 10). WT, wild-type strain E122. bem1Δ, strain bem1KO157. Bars, 5 μm.

FIG. 8.

Y. lipolytica bem1 null mutants exhibit binucleation. Shown are bright-field images of cells and DAPI staining of cell nuclei from the wild-type strain E122 (WT) and the mutant strain bem1KO157 (bem1Δ), grown in YEPD liquid medium for 12 h at 28°C. Arrows indicate binucleate cells. Bars, 5 μm.

FIG. 9.

Distribution and organization of actin at different developmental stages of wild-type and bem1Δ cells. Actin was stained with Oregon Green 488 phalloidin and detected by fluorescence microscopy. (A to F) Yeast cells (A to C), pseudohyphal cells (D and E), and hyphal cells (F) of the wild-type strain E122. (G to I) Yeast-like cells of the bem1Δ strain bem1KO157. Arrows indicate actin-rich zones in E122 and bem1KO157 cells. Bars, 5 μm.

FIG. 10.

Deletion of the YlBEM1 gene affects the budding pattern and chitin deposition in Y. lipolytica. Chitin was stained with Fluorescent Brightener 28 and detected by fluorescence microscopy. WT, wild-type strain E122; bem1Δ, strain bem1KO157.

FIG. 11.

Colony (upper panels) and cell (bottom panels) morphology of Y. lipolytica strains transformed with autonomously replicating plasmids carrying the MHY1, YlRAC1, YlSEC31, and YlBEM1 genes. (A) Wild-type strain E122; (B) mutant strain bem1KO157; (C through E) strain bem1KO157 transformed with plasmid pMHY1 (C), pRAC1 (D), or pSEC31 (E); (F) mutant strain rac1KO30; (G) strain rac1KO30 transformed with plasmid pBEM1; (H) mutant strain mhy1KO9; (I) strain mhy1KO9 transformed with plasmid pBEM1. Colonies and cells were photographed after 3 days of incubation at 28°C on YNA-agar plates. Colony magnification, ×100. Bars, 5 μm.