A. GFP visualization reveals that Eap1 and Pga10 localize primarily to the cell surface, and Csh1 and Pbr1 localize primarily in the cytosol upon induction by α-pheromone. The complemented strains EAP1_WPREΔ-EAP1/eap1, PGA10_WPREΔ-PGA10/pga10, CSH1_WPREΔ-CSH1/csh1 and PBR1_WPREΔ-PBR1/pbr1, which were tagged at the carboxy terminus with GFP, were examined. B. Northern analysis of mRNA levels of the parental control, deletion mutants and complemented strains of the four genes in the absence (−) and presence (+) of α-pheromone. C. Northern analysis of pheromone-induced expression of CSH1 and PBR1 in deletion mutants missing both the high consensus (strong) WPRE and the low consensus (lc) (weak) WPRE, lcWPRE, in the absence (−) and presence (+) of α-pheromone (α-ph). D. Western analysis of pheromone-induced expression of CSH1 and PBR1 in deletion mutants, as in panel C, using anti-GFP antibody.

A. Quantitation of shmoo formation of control and mutant strains in response to 4 hr treatment with 3×10⁻⁶ M α-pheromone (chemically synthesized 13-mer). At least 1,000 cells, the sum of four independent experiments, were analyzed and the mean±standard deviation of the percent shmoo formation presented. N.S., not significant. B. Quantitation of fusion between control and mutant opaque cells, with opaque α/α cells of the mating partner WO-1. At least 2,000 cells of each strain, the sum of four independent experiments, were analyzed and the mean±standard deviation of the percent presented. C. Examples of shmoo formation. D. Examples of mating fusants with α/α strain WO-1. −, absence of α-pheromone; +, presence of α-pheromone. Scale bars in C and D represent 4 µm.

A. Biofilm thickness measured in µm in the absence (−Op) and presence (+Op) of 10% opaque cells, for the complemented control strains, homozygous deletion strains and WPRE deletion strains of the four test genes. The opaque cells were half a/a and α/α. For each strain and condition, three individual biofilms were analyzed through three random regions, providing nine measurements. P values are provided for the measurements in the absence (−) and presence (+) of 10% opaque cells (Op). In supplemental Table S7, the p values are presented for comparisons of the complemented control and the two deletion mutants. B. Comparisons of biofilm compositions of the parent strain P37005, the complemented control strains, the homozygous mutants and the WPRE deletion mutants of the four test genes, in the absence (−) or presence (+) of opaque (Op) cells. Maximum matrix staining is representative as ++++ and minimum as +. The presence or absence of a white cell basal layer (Wh cell bas. layer) is denoted as + or −, respectively. Maximum and minimum hyphal density (Hyph. des) is represented as ++++ and +, respectively. Hyphal orientation (Hyph. orient.) was either vertical (vert.) and intertwined (int.), or horizontal (hor.). C, D. Scanning confocal microscopic images of the basal layer and hyphal region of biofilms of the EAP1 complemented control and WPRE deletion mutant of EAP1. E. β-glucan measurements of biofilm supernatants F. Examples of the pixel intensity scans used to measure thickness, for the complemented control and WPRE deletion mutant of EAP1, respectively.

A. Northern analysis demonstrating that 100 µg/ml of doxycycline (Dox) induces PBR1 transcription similarly in the absence (−) and presence (+) of α-pheromone (α-ph). B. Examples of white cells adhering to the bottom of wells for the control strain P37005-tetPBR1 and CSH1 WPRE deletion mutant CSH1_WPREΔ/csh1-tetPBR1 in the absence (−) or presence (+) of doxycycline. C. Quantitation of adherence to well bottoms. D. Northern analysis demonstrating treatment with pheromone of the mutant cek1/cek1 cek2/cek2 does not cause an increase in expression of the four test genes necessary for a full adhesion response to α-pheromone. E. Demonstration that misexpression of PBR1 in the mutant cek1/cek1 cek2/cek2, in which the three genes, EAP1, PGA10, CSH1 and the native PBR1 gene are not up-regulated, results in an increase in adhesion that is 33% that of control cells. This increase is therefore independent of α-pheromone treatment in the cek1/cek1 cek2/cek2 mutant.

Each of these genes contained one or more putative white-specific pheromone response elements (WPRE) in their promoters. A. Northern analysis of the expression of twelve genes in white and opaque a/a cells in the absence (−) and presence (+) of α-pheromone (α-ph) that were identified in a screen of 103 genes as strongly up-regulated. B. The sequence considered to represent the putative white-specific pheromone response element (WPRE) in the promoters of the 12 selected white-specific genes in panel A, using a high stringency E value threshold of ≤0.001 in the Multiple for Motif Elicitation (MEME) software. The consensus sequence for WPRE is given at the bottom of the panel. C. The sequence considered the putative opaque-specific pheromone response element (OPRE) in the promoters of six genes selectively up-regulated by pheromone in opaque cells, using a threshold of 0.001 in the MEME program. The consensus sequence for OPRE is presented at the bottom of the panel. D. Genes up-regulated by α-pheromone in both opaque and white cells contain both OPRE and WPRE. The positions of the OPRE and WPRE sequence with the highest homology to the consensus sequence is given relative to the start codon in panels B, C and D.

A. GFP visualization reveals that Cph1 localizes to the putative nucleus. The strain CPH1_OPREΔ-CPH1/cph1, which possesses a C-terminal GFP tag, was used for analysis. B. Northern analysis of the RNA levels of the parental control, deletion mutants and complemented strains for CPH1 in the absence (−) and presence (+) of α-pheromone (α-ph). C. Northern analysis of the RNA levels of the parental control, deletion mutants and complemented strains for MFA1 in the absence (−) and presence (+) of α-pheromone (α-ph).

A. Quantitation of cells adhering to the well bottom in the absence (−) and presence (+) of α-pheromone (α-ph). The mean±standard deviation (error bars) of three dishes is presented. B. Examples of the bottom of wells after washing of control and mutant strains.

A. Expression of the four genes in EAP1 mutants in the absence (−) or presence (+) of α-pheromone. B. Expression of the four genes in PBR1 mutants in the absence (−) or presence (+) of α-pheromone. To demonstrate levels of loading, 18S rRNA levels are shown for opaque and white.

This model also includes genes that are up-regulated in both the opaque and white responses, and therefore contain both an OPRE and a WPRE in their promoters. The two pathways share the same components from receptor through the MAP kinase cascade [25]. However, there are two differences, circled in red. First, an extra region of the first intracellular loop (ICI) is essential for the white, but not the opaque response [26]. Second, the transcription factor targeted by the pathway is CPH1 in the opaque response and a still unidentified factor in the white response [25].