Snf1 protein kinase and the repressors Nrg1 and Nrg2 regulate FLO11, haploid invasive growth, and diploid pseudohyphal differentiation

Abstract

The Snf1 protein kinase of Saccharomyces cerevisiae is important for many cellular responses to glucose limitation, including haploid invasive growth. We show here that Snf1 regulates transcription of FLO11, which encodes a cell surface glycoprotein required for invasive growth. We further show that Nrg1 and Nrg2, two repressor proteins that interact with Snf1, function as negative regulators of invasive growth and as repressors of FLO11. We also examined the role of Snf1, Nrg1, and Nrg2 in two other Flo11-dependent processes. Mutations affected the initiation of biofilm formation, which is glucose sensitive, but also affected diploid pseudohyphal differentiation, thereby unexpectedly implicating Snf1 in a response to nitrogen limitation. Deletion of the NRG1 and NRG2 genes suppressed the defects of a snf1 mutant in all of these processes. These findings suggest a model in which the Snf1 kinase positively regulates Flo11-dependent developmental events by antagonizing Nrg-mediated repression of the FLO11 gene.

FIG. 1.

Snf1 kinase regulates FLO11 expression. (A) Wild-type (WT) and snf1 mutant strains (MCY4460 and MCY4471) were grown to mid-log phase at 25°C in YEP-2% glucose (glucose repressed, R) and then shifted to YEP-0.05% glucose for the indicated times. Total RNAs were prepared and fractionated on a 0.8% agarose-formaldehyde gel, and the FLO11 mRNA was detected by Northern blot analysis. Prior to membrane transfer, the gel was stained with ethidium bromide to visualize the rRNA, which served as a loading control. (B) Deletion of REG1 increases invasion and FLO11 expression. Cells were assayed for invasive growth as described in Materials and Methods. After 3 to 4 days of incubation at 26°C, plates were photographed, washed, and photographed again; at this temperature, wild-type cells require 5 to 6 days for substantial invasion. Strains were also grown in YEP-2% glucose to mid-log phase, and FLO11 mRNA levels were assessed by Northern blot analysis. Visualization of the rRNA confirmed uniform sample loading (data not shown).

FIG. 2.

Nrg1 and Nrg2 affect agar invasion and FLO11 expression. (A) Strains with the indicated genotypes were assayed for invasive growth. Plates were photographed before wild-type cells had invaded the agar to any significant extent so that the increased invasiveness caused by the double nrg1Δ nrg2Δ mutation would be apparent. The same strains were grown to mid-log phase in YEP-2% glucose and subjected to Northern blot analysis of FLO11 mRNA and, as a control, ACT1 mRNA. (B) Wild-type and flo11 strains were transformed with plasmids expressing GAD-Nrg1, GAD-Nrg2, or GAD from the ADH1 promoter (pV40, pV39, or pACTII, respectively) (37). After growth on SC-2% glucose plates lacking leucine (for plasmid selection), cells were resuspended in sterile 10 mM Tris-HCl (pH 7.5)-1 mM EDTA and spotted onto plates for invasive growth assays. We were also able to detect invasive growth on selective SC-2% glucose plates, with similar results (data not shown).

FIG. 3.

The Snf1-Nrg pathway affects adherence to plastic surfaces. Cells were assayed for adherence to polystyrene (29) as described in Materials and Methods. All strains tested were MATa. (A) Cells were resuspended in SC with 2% or 0.1% glucose, transferred to the wells of a microtiter plate, and incubated for the indicated times. (B) Cells were resuspended in SC-2% glucose, and incubation was for 6 h. Duplicate samples are shown. (C) Cells were grown in SC-2% glucose to an OD₆₀₀ of 2 and resuspended in SC-0.1% glucose. Incubation was for 2.5 h. Duplicate samples are shown. We also monitored the growth of samples of the same cultures for 2.5 h after resuspension; differences in growth rate did not correlate with differences in adherence (not shown). The nrg1 nrg2 cells also adhered better than wild-type cells when resuspended in SC-2% glucose, and when assayed together, nrg1 nrg2 and reg1 cells adhered similarly (data not shown).

FIG. 4.

Snf1, Nrg1, and Nrg2 affect diploid pseudohyphal growth. Diploid cells were streaked on solid low ammonia (SLAD) medium (8) and incubated at 30°C for 5 days. Colonies were viewed using a Nikon Eclipse E800 fluorescent microscope. Images were taken with an Orca100 (Hamamatsu) camera using Open Lab (Improvision) software and processed using Adobe Photoshop 5.5 software. Diploid strains were MCY4472, MCY4473, MCY4474, and MCY4475, which were transformed with pLCLG-Staf, a centromeric plasmid with URA3 and LEU2 (12) to confer prototrophy.

FIG. 5.

Model for regulation of FLO11 gene expression by the Snf1-Nrg pathway. (A) In haploid cells, the Snf1 kinase is activated in response to glucose limitation and relieves Nrg-mediated repression of FLO11. Expression of FLO11 is critical for invasive growth and biofilm formation. It is also possible that Snf1 affects FLO11 by other Nrg-independent mechanisms. (B) In diploid cells, the Snf1 kinase is activated under conditions of nitrogen limitation that lead to pseudohyphal differentiation. The simple model is that Snf1 responds to a low nitrogen signal, but there is no evidence to exclude other possibilities (see text).