Functional cloning of C. albicans FLO8 in S. cerevisiae flo8 mutant. C. albicans FLO8 was cloned by complementation of S. cerevisiae flo8 mutants in invasive growth (A). C. albicans FLO8 could also complement S. cerevisiae flo8 in pseudohyphal growth (B) and biofilm formation (C). Haploid wild-type (MY1384) and flo8 (HLY850) strains carrying a vector (pRS202) or FLO8 (pCF56) were grown at 30°C on YPD for 3 d (A), YPD with 0.3% agar for 13 d (C), diploid wild-type (CG68) and flo8/flo8 (HLY852) strains carrying the pRS202, or pCF56 grown on SLAD (synthetic low ammonium dextrose medium) at 30°C for 4 d (B).

Sequence alignment and functional analysis of Flo8 LUFS domain. (A) Sequence alignment of the LUFS domain between C. albicans Flo8 and other regulatory proteins. Identical residues are shaded in black and conserved residues are shaded in gray. (B) C. albicans Flo8 LUFS domain is required for its functional complementation in S. cerevisiae flo8 mutants. Diploid wild-type (CG68) and flo8/flo8 (HLY852) strains carrying a vector (pVTU) or FLO8 expression plasmids (pVTU-CaFlo8, pVTU-CaFlo8_123–817, pVTU-CaFlo8_1–122) were grown on SLAD for 4 d. (C) Transcriptional activity of Flo8 is located mostly at the C-terminal domain. Transcriptional activity of various lexA_BD-Flo8 fragments is shown in β-galactosidase activity and growth rate on SC-Leu medium.

FLO8 is essential for hyphal development and the expression of hypha-specific genes in C. albicans. (A) C. albicans flo8/flo8 cells are unable to form hyphae (top row). Cells were induced in YPD + 10% serum for 3.5 h at 37°C (top). Colony morphology of flo8/flo8 mutants (middle and bottom rows). Strains were plated on solid serum-containing medium and solid Lee's medium, incubated at 37°C for 5 and 7 d, respectively. (B) flo8/flo8 mutants were defective in the induction of hypha-specific genes as well as ALS1. Cells were grown in YPD + 10% serum at 37°C for 3.5 h or grown in YPD at 25°C for 6 h and collected for RNA extraction and Northern analysis. Strains shown in A and B are WT (SC5314), FLO8/flo8 (CCF1, +/–), flo8/flo8 (CCF3, –/–), flo8/flo8+vector [CCF4 + pBA1, –/–(v)], flo8/flo8 + ADH1p-FLO8 [CCF4 + pBA1-CaFLO8, –/–(+)_ADH1p], flo8/flo8 + FLO8 [CCF4 + pBES116-CaFLO8, –/–(+)_FLO8p]. (C and D) Flo8 LUFS domain is required for hyphal development (C) and the induction of hypha-specific genes (D). The experiments in C and D were carried out under the same conditions as in A and B, respectively. Strains –/–(+) and –/–(ΔN) in D are flo8/flo8 + ADH1p-FLO8 [CCF4 + pBA1-CaFLO8, –/–(+)_ADH1p] and flo8/flo8 + ADH1p-flo8ΔN [CCF4 + pBA1-CaFLO8ΔN, –/–(ΔN)_ADH1p].

flo8/flo8 is avirulent in a systemic model. Survival curves for strains SC5314, CAI4 + pBES116, flo8/flo8 (CCF4 + pBES116), and flo8/flo8 + FLO8 (CCF4 + pBES116-CaFLO8) in a mouse model of systemic infection are shown. For each strain, 8 ICR male mice were injected with 5 × 10⁶ cells from tail vein. Percentage of survival is indicated in the y-axis.

Comparison of gene expression between flo8/flo8 and efg1/efg1 by DNA microarray. (A) TreeView of clustering analysis. Genes that had an intensity difference (Cy5-Cy3) greater than a basal intensity as well as a fold change greater than 3 were included in the clustering analysis. Wild-type (SC5314), flo8/flo8 (CCF3), and efg1/efg1 (HLC52) strains were grown under either yeast growth conditions (YPD or Lee's, at 25°C) or hyphalinducing conditions (YPD + 10% serum or Lee's, 37°C). Strains and growth conditions used in each hybridization are indicated at the top. Yeast growth conditions were grouped to the left and hyphal-inducing conditions to the right by the clustering program. The cluster of genes that are equally induced in YPD + serum and in Lee's medium are indicated as hypha-specific genes. Genes that are induced in hyphae but with a higher fold of induction in Lee's than in YPD serum are named Lee's hyphal genes. A group of Efg1 repressed genes, including HSP31, are labeled as HSP31 cluster. A larger view for part of the genes in these three clusters is shown on the right. For HWP1 and ALS3, two different 70-mers representing each gene are present in the C. albicans 70-mer set of 6530 ORFs (QIAGEN Operon). (B) Northern analysis of HGC1, IHD1, and HSP31. RNA used in the microarray experiments was used in the Northern analysis to confirm the expression of three differentially regulated genes identified from the microarray study. PCR products of the HGC1, IHD1, and HSP31 ORFs were used for probing.

Flo8 interacts with Efg1 in vivo. (A) Yeast two-hybrid assays. EGY48(p8op-lacZ) was cotransformed with following plasmids: 1, pSH17–4+pJG4-5; 2, pRFHM1+pJG4-5; 3, pEG202-CaFLO8+pJG4-5; 4, pEG202-EFG1+ pJG4-5; 5, pEG202-EFG1+ pJG-CaFLO8; and 6, pEG202+pJG-CaFLO8. (B) Flo8 interacts with Efg1 in yeast and hyphae. C. albicans HLY3271 (Efg1-Protein A Flo8myc) were grown at 30°C in YPD (lane 2), SSA (lane 3), and Lee's (lane 4) for yeast growth, and at 37°C in YPD + serum (lane 6), SSA (lane 7), and Lee's (lane 8) for hyphal growth. C. albicans HLY3426 (Flo8myc) grown in YPD at 30°C (lane 1) and YPD + serum at 37°C (lane 5) were used as a control. Protein lysates were subjected to immunoprecipitation with IgG beads (Sigma), and the precipitated proteins were separated by 8% SDS-PAGE and probed with peroxidase-conjugated anti-c-myc (Roche Diagnostics). The anti-c-myc antibody cross-hybridized weakly with protein A, revealing the Efg1-protein A in the IP. As input control, cell lysates were analyzed by Western blotting with the peroxidase-conjugated anti-c-myc.

EFG1 stimulated invasive growth in S. cerevisiae requires Flo8. Total and invasive growth of wild-type (MY1384), ste7 (HLY367), ste12 (HLY362), tec1 (HLY2000), and flo8 (HLY850) strains carrying a vector, EFG1 or FLO8, after 5 d of growth on SC-Ura.

flo8/flo8 and cdc35/cdc35 show increased hyphal filamentation under microaerophilic condition. Cells of wild-type (SC5314), efg1/efg1 (HLC52), flo8/flo8 (CCF3), and cdc35/cdc35 (CR216) were plated with molten YPS agar and grown for 56 h or 5 d at 25°C.