Control of white-opaque phenotypic switching in Candida albicans by the Efg1p morphogenetic regulator

Abstract

Phenotypic switching in Candida albicans spontaneously generates different cellular morphologies and is manifested in strain WO-1 by the reversible switching between the white and opaque phenotypes. We present evidence that phenotypic switching is regulated by the Efg1 protein, which is known as an essential element of hyphal development (dimorphism). Firstly, EFG1 is expressed specifically in cells of the white but not the opaque phenotype. During mass conversion from the opaque to the white phenotype, the EFG1 transcript level correlates with competence of switching of opaque cells to the white form. Secondly, overexpression of EFG1 by a PCK1p-EFG1 fusion forces opaque-phase cells to switch to the white form with a high level of efficiency. Thirdly, low-level expression of EFG1 in strain CAI-8 generates a cellular phenotype similar to that of opaque cells in that cells bud as short rods, which cannot be induced to form hyphae in standard conditions; such cells (unlike authentic opaque cells) lack typical surface "pimples." Importantly, the opaque-specific OP4 transcript is induced in the opaque-like cells generated by strain CAI8 as a response to low-level expression of EFG1. The results suggest that high EFG1 expression levels induce and maintain the white cell form while low EFG1 expression levels induce and maintain the opaque cell form. It is proposed that changes in EFG1 expression determine or contribute to phenotypic switching events in C. albicans.

FIG. 1

EFG1 expression is specific for the white phase of strain WO-1. poly(A) RNA of the white (w) and opaque (o) phases of strain WO-1 grown at 25°C in YPD medium was analyzed by Northern blotting using EFG1 and ACT1 probes. The migration of rRNA is as indicated.

FIG. 2

Transcript levels during a temperature-induced shift from the opaque to the white phase. The opaque phase of strain WO-1 was pregrown at 25°C in S4D medium. At t = 0 h, cells were transferred to 42°C and incubated further at this temperature. Total RNA of cells was isolated at the indicated times and analyzed by using EFG1, WH11, or ACT1 probes. At each time point, the number of white- and opaque-phase cells was determined microscopically. In addition, a sample of the culture was plated on solid SD medium containing phloxine B, followed by incubation at 25°C and determination of white and opaque colonies after 3 days of growth. W, white-phase cells.

FIG. 3

EFG1 overexpression forces the opaque and white shift. Transformants of the opaque phase of strain Red3/6 contained plasmid pAPE(2)/ADE (PCK1p-EFG1) (A and B) or empty vector pBT-4 (C and D). Cells were pregrown at 25°C in S4D medium and then plated on SCAA medium (EFG1 expression) (B and D) or on S4D medium (no EFG1 expression) (A and C) and incubated at 25°C. Media contained phloxine B, which stains colonies of opaque cells red (which appear grey on figure) but not colonies of white-phase cells.

FIG. 4

Transcripts in transformants of strain Red3/6. Three transformants of the opaque phase of strain Red3/6 that contained plasmid pAPE(2)/ADE (PCK1p-EFG1) (1 to 3) were diluted into SCAA medium (EFG1 expression) (C) and S4D medium (no EFG1 expression) (S). Cultures were grown at 25°C to an OD₆₀₀ of 1, and total RNA of cells was prepared. Twenty micrograms of RNA was analyzed by Northern blotting using the indicated probes. Ethidium bromide-stained rRNA was used as loading control. w, white; o, opaque.

FIG. 5

Low EFG1 expression levels induce an opaque-phase-like cellular phenotype. Strain SS4 (efg1/PCK1p-EFG1) and strain CAI8 (EFG1/EFG1) containing empty control vectors (pBT-4 and pBI) as a control were grown in S4D medium, in which the PCK1 promoter is repressed (low-level EFG1 expression in strain SS4). Cells were visualized by scanning electron microscopy. Photographs represent images of 20 by 20 μm.

FIG. 6

Phase-specific transcripts in strains with low-level EFG1 expression. Strains SS4 (efg1/PCK1p-EFG1) and CAI8[pBT-4, pBI] were grown in SCAA medium (EFG1 overexpression in strain SS4) (C) or S4D (low-level EFG1 expression in strain SS4) (S). Total RNA was analyzed by Northern blotting using the indicated probes. 18S rRNA was used as a loading control for Northern gels.