A rac homolog is required for induction of hyphal growth in the dimorphic yeast Yarrowia lipolytica

Abstract

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.

FIG. 1

Colony morphology of various Y. lipolytica strains. (A) Wild-type strain E122; (B) original mutant strain CHY1220; (C) RAC1 disruptant strain CHY1220-A30; (D) MHY1 disruptant strain mhy1KO9; (E) strain CHY1220 transformed with plasmid pRAC1; (F) strain CHY1220-A30 transformed with plasmid pRAC1; (G) strain mhy1KO9 transformed with plasmid pRAC1; (H) strain CHY1220 transformed with plasmid pMHY1; (I) strain CHY1220-A30 transformed with plasmid pMHY1; (J) wild-type strain 22301-3; (K) RAC1 disruptant strain CHY1220-B36; (L) RAC1/RAC1 diploid strain E122//22301-3; (M and N) RAC1/rac1 diploid strains 22301-3//CHY1220-A30 and E122//CHY1220-B36, respectively; (O) rac1/rac1 diploid strain CHY1220-A30//CHY1220-B36. Colonies were photographed after 3 days of incubation at 28°C on YNA agar plates. Magnification, ×100.

FIG. 2

Nucleotide sequence of the YlRAC1 gene and deduced amino acid sequence of YlRac1p. The transcriptional start site of the YlRAC1 gene is indicated by an arrow. Putative STREs are indicated. Consensus sequences for intron splicing are underlined. Putative transcription termination signals are doubly underlined.

FIG. 3

Amino acid sequence alignment of Rac1p of Y. lipolytica (YlRac1p) and Rac proteins from Homo sapiens (HsRac1, HsRac2, and HsRac3), Mus musculus (MmRac1 and MmRac2), Drosophila melanogaster (DmRac1 and DmRac2), Caenorhabditis elegans (CeRac1 and CeRac2), Canis familiaris (CfRac1), and Xenopus laevis (XlRac). GenBank accession numbers are M29870 (HsRac1), CAB45265 (HsRac2), AAC51667 (HsRac3), CAA40545 (MmRac1), Q05144 (MmRac2), AAA62870 (DmRac1), P48554 (DmRac2), AAA28141 (CeRac1), AAB40386 (CeRac2), P15154 (CfRac1), and AAD50299 (XlRac).

FIG. 4

Nucleotide sequence of the YlCDC42 gene and deduced amino acid sequence of YlCdc42p. Consensus sequences for intron splicing are underlined.

FIG. 5

Amino acid sequence alignment of Cdc42p of Y. lipolytica (YlCdc42p) and its homologs in S. cerevisiae (ScCdc42p) (28), C. albicans (CaCdc42p) (46), S. pombe (SpCdc42p) (44), Caenorhabditis elegans (CeCdc42p) (15), Mus musculus (MmCdc42p) (43), and Homo sapiens (HsCdc42p) (48).

FIG. 6

Integrative disruption of the YlRAC1 gene. (A) Diagram illustrating the replacement of a 1.0-kbp ApaI-NdeI fragment of YlRAC1 by a 1.6-kbp ApaI-NdeI fragment containing the Y. lipolytica URA3 gene. (B) Southern blot analysis of SpeI-HpaI-digested genomic DNA, and PCR analysis of total genomic DNA, from wild-type strain E122 and strain CHY1220-A30, confirming the correct replacement of the YlRAC1 gene with the URA3-containing linear molecule in strain CHY1220-A30. Primers KO1 and KO2 (Table 2) are indicated by black arrows in panel A.

FIG. 7

Disruption of YlRAC1 affects cell morphology and impairs hyphal growth, but not pseudohyphal growth, in Y. lipolytica. Strains were grown in YEPD. Top panels, exponential growth phase (optical density at 600 nm [OD₆₀₀] = 1). Middle panels, late exponential growth phase (OD₆₀₀ = 4). Bottom panels and inset, stationary phase (OD₆₀₀ = 10). WT, wild-type strain E122. Δrac1, strain CHY1220-A30. Bars, 5 μm.

FIG. 8

Invasive filamentous growth by different Y. lipolytica strains. Following 5 days of incubation at 28°C on minimal agar medium containing glucose or acetate as the sole carbon source, plates were washed with running water to remove cells from the agar surface. Pictures were taken before and after washes. WT, wild-type strain E122. Δrac1, strain CHY1220-A30. Δmhy1, strain mhy1KO9.

FIG. 9

Actin localization during different stages of development of wild-type and Δrac1 cells. Actin was detected by staining of cells with Oregon Green 488 phalloidin followed by fluorescence microscopy. (A to G) Wild-type strain E122. (H and I) Δrac1 strain CHY1220-A30. (A and H) Yeast-like cells; (B, G, and I) pseudohyphal growth; (C) early germ tube formation; (D) late germ tube formation; (E and F) hyphal growth. Bars, 5 μm.

FIG. 10

YlRAC1 mRNA levels are increased during the dimorphic transition. Total RNA was isolated from E122 cells incubated at 28°C in YNBGlcNAc (induction of filamentous growth) or YNBGlc (control culture, yeast-like cells) for the times indicated and subjected to Northern blot analysis. Ten micrograms of RNA from each time point was separated on a formaldehyde agarose gel and transferred to nitrocellulose. Blots were hybridized with a probe specific for the YlRAC1 gene (1.0-kbp ApaI-NdeI fragment [see Fig. 6]). Equal loading of RNA was ensured by ethidium bromide staining (data not shown).

FIG. 11

Southern blot analysis of E122 genomic DNA. Ten micrograms of DNA per lane was digested with the indicated restriction enzymes, separated by electrophoresis, transferred to nitrocellulose, and probed with a 240-bp SacII-NdeI-labeled fragment from YlRAC1 (boxed), as described in Materials and Methods.