Role of RIM101 for Sporulation at Alkaline pH in Ashbya gossypii

Abstract

Microorganisms need to sense and adapt to fluctuations in the environmental pH. In fungal species, this response is mediated by the conserved pacC/RIM101 pathway. In Aspergillus nidulans, PacC activates alkaline-expressed genes and represses acid-controlled genes in response to alkaline pH and has important functions in regulating growth and conidia formation. In Saccharomyces cerevisiae, the PacC homolog Rim101 is required for adaptation to extracellular pH and to regulate transcription of IME1, the Initiator of MEiosis. S. cerevisiae rim101 mutants are defective in sporulation. In Ashbya gossypii, a filamentous fungus belonging to the family of Saccharomycetaceae, little is known about the role of pH in regulating growth and sporulation. Here, we deleted the AgRIM101 homolog (AFR190C). Our analyses show that Rim101 is important for growth and essential for sporulation at alkaline pH in A. gossypii. Acidic liquid sporulation media were alkalinized by sporulating strains, while the high pH of alkaline media (starting pH = 8.6) was reduced to a pH ~ 7.5 by these strains. However, Agrim101 mutants were unable to sporulate in alkaline media and failed to reduce the initial high pH, while they were capable of sporulation in acidic liquid media in which they increased the pH like the wild type.

Keywords: ascus; filamentous fungus; functional analysis; germination; meiosis; signal transduction.

Figure 1

Induction of sporulation in response to alkaline pH in S. cerevisiae. When a non-fermentable carbon source is metabolized by respiration the external pH increases. The alkaline pH induces proteolytic activation of Rim101 by Rim13/Rim20. Rim101 then activates IME1 transcription by repressing transcription of the repressor Smp1 [14].

Figure 2

Alignment of the zinc finger regions Rim101/PacC proteins of S. cerevisiae, A. gossypii, and Aspergillus nidulans. The conserved cysteine and histidine residues of the three C2H2 zinc-chelating residues are marked with asterisks [4].

Figure 3

Radial growth of the Ashbya parental (WT) strain and the Agrim101 mutant (AWL63) on full medium plates after one week of growth at the indicated temperatures.

Figure 4

Sporulation ability of the A. gossypii wild-type strain and rim101 mutant at different pH. (A) Spores were isolated from the central part of colonies grown for 10 days on a full medium plate. (B,C) Sporulation of mycelia on solid media buffered with Tris-HCl to different pH values. (B) Spore-forming units were determined by dilution plating of spore suspensions derived from the sporulation zones. (C) Sporulation zones of mycelia were marked with white-dotted circles.

Figure 5

Liquid sporulation assays at acidic and alkaline pH. The indicated strains were pre-grown in AFM and then transferred to CSM-sporulation media (acidic = unbuffered with a starting pH of 5.3; alkaline = Tris-buffered to a pH of 8.6 at room temperature). After one week of incubation at 28 °C, samples were analyzed by microscopy. Representative images are shown. Clumps of spores (indicated by black arrows in the WT) were abundant in sporulating strains but completely absent in homokaryotic rim101 mutants showing only hyphal filaments (indicated by white arrows). Scale bars are 10 µm.