Fig 1.
Fluorescence microscopy reveals chitin enrichment of penetration pegs in S. schoenii.
(A-D) Live-cell images showing chitin enrichment of calcofluor white (CW)-stained S. schoenii (elongated) and S. cerevisiae cells (ovoid). (A) Several penetration pegs (two marked by daggers; the left one next to a septum separating mother and daughter S. schoenii cells) are located inside S. cerevisiae prey cells. The S. cerevisiae cells carry a H4-GFP tag but only the one cell at the bottom (marked by an arrow) is showing nuclear fluorescence, while all others do not and are thus dead. S. cerevisiae bud scars (two positions marked by arrowheads) stain brightly with CW. (B) A predator yeast cell (center) with two penetration pegs that attacked two S. cerevisiae prey cells to the left and right of it is shown. (C) A S. cerevisiae mother cell connected with a daughter cell (the septum between both cells is marked by an asterisk) were attacked simultaneously by different predator cells; one of the four penetration pegs is marked by a dagger. (D) Two examples of S. schoenii predation pegs inside S. cerevisiae prey cells are shown indicating bulbous tips of the penetration pegs. (E) Micrographs showing co-staining of penetration pegs with CW (upper panels, images obtained with DAPI-filterset) and Wheat Germ Agglutinin (WGA CF-488A, lower panels, images acquired with the GFP filterset) of the same cells. (F) Images of cells co-stained with CW (upper panels) and concanavalin A (conA CF488A, lower panels). (E,F) Panels on the right side show predator-prey cell interactions, while panels on the left depict predator cells without prey cells. (A-F), Scale bars, 5 μm.
Fig 2.
Time-lapse fluorescence microscopy defines distinct stages of the predation cycle in S. schoenii.
(A) Schematic drawing of a mixture of elongated S. schoenii predator and ovoid S. cerevisiae prey cells at the onset of a time-lapse recording (line drawing on the left based on the DIC + GFP image at 4’). The time series consisted of brightfield images (DIC), GFP-images recording the nuclear H4-GFP and CW-images monitoring the appearance of penetration pegs and septa. Upper panels show combined DIC and GFP images, lower panels CW and GFP images. Nuclei of S. schoenii are encircled with oval lines (black solid lines in the upper images, white solid lines in the lower images). Nuclei of S. cerevisiae are marked with dashed circles. (B) Penetration peg formation is marked by a dagger (20’-24’). S. schoenii nuclei are marked in the upper panel, while S. cerevisiae nuclei are marked in the lower panel until their disappearance. The position of the tip of the S. schoenii daughter cell is marked by an arrowhead. (C) Progression of the cell cycle after predation in the predator cell. The growing daughter cell bud tip is marked by arrowheads. S. schoenii nuclei are encircled in the upper images indicating mitosis at 96’. After mitosis a septum was generated, which is marked by a ‘+`. (D) The mother cell entered a new cell cycle marked by bud emergence (arrowhead, 144’). The S. cerevisiae prey cell is large-budded, its nucleus undivided (marked by a dashed circle) and deposition of chitin at the bud neck is marked by an asterisk. Contact of the predator yeast daughter cell with the prey cell resulted in penetration peg formation (dagger), disappearance of prey cell H4-GFP and shrinking of the prey cell (daughter cell is marked by black dashed circles at 208’ and 236’). Underneath the prey cell killed in (B) a predator cell grew (D), which was not involved in the predation of this prey cell and was not the result of further growth of the penetration peg (see also S1 Movie). Scale bar, 5 μm.
Fig 3.
SsKIL1 and SsSTE12 are both required for penetration peg formation and predation in S. schoenii.
(A) Representative images of predator-prey interactions of the indicated S. schoenii strains with S. cerevisiae, H4-GFP cells. Cells were stained with CW and CW- and GFP-fluorescence images were acquired and overlaid using ImageJ. Several penetration pegs (absent in Sskil1 and Ssste12) are marked by daggers; several prey cells showing nuclear H4-GFP fluorescence are marked by arrows. WT, wildtype; scale bar, 5 μm. (B) Diagram to show the experimental design for in situ analysis of predation. Cells of different S. schoenii strains were mixed with S. cerevisiae H4-GFP cells and spread on glass slides placed in petri dishes and covered with SD medium. Petri dishes were incubated as indicated. Then glass slides were excised from the matrix, cells were stained with CW and evaluated directly under the microscope. Events scored only included prey cells in direct contact with S. schoenii cells; dead cells penetrated by a pegs or alive unpenetrated prey cells were quantified. (C) Enumeration of predator-prey interactions of the indicated strains prepared as described in (B). WT and Sskil1/SsKIL1 strains were assayed in triplicate, the Sskil1 and Ssste12 mutant strains were assessed by 2 biological replicates and 3 technical replicates per biological replicate, Mean ± s.e.m. (D) Experimental design of a plate based predation assay. A lawn of cells of the S. schoenii tester strain was plated on SD-plates and ~600 S. cerevisiae prey cells were spread onto this lawn. S. cerevisiae CFUs were counted within the indicated square. (E) Top row shows representative images of petri plates corresponding to the squares shown in (D). S. cerevisiae CFUs that were formed against the lawn of predator cells enumerated as shown in the graph underneath the corresponding plate images. WT and Sskil1 (B054) were assayed with 6 biological replicates and 5 technical replicates. Sskil1 (G238), Sskil1/SsKIL1 and the Ssste12 mutant strains were assessed with 3 biological replicates and 5 technical replicates per biological replicate, Mean ± s.e.m.
Fig 4.
Time-lapse microscopy reveals thigmotropism that preceeds predation in S. schoenii.
(A) Selected frames of an in vivo time-lapse recording (S3 Movie) showing S. schoenii (SsKIL1 wildtype, elongated cell) predation of an S. cerevisiae prey cell (ovoid cell). Both strains are tagged by histone H4-GFP genes to monitor cell viability. The nucleus of S. schoenii is encircled with oval lines (black solid lines in the upper images, white solid lines in the lower images); the nucleus of the attacked S. cerevisiae cell is marked with a dashed circle in each frame. Penetration peg formation is marked by a dagger (72’). Note the chitin rich bud neck in S. cerevisiae (marked by an asterisk at 72’) is present before the penetration peg appears. (B) Selected micrographs of a time-lapse recording (S4 Movie) showing the interaction of a Sskil1 cell (elongated) with a S. cerevisiae prey cell (ovoid, displaying a nuclear H4-GFP). (A,B) At each time point (indicated in minutes, top left in each frame) brightfield (DIC), CW and GFP images were acquired. DIC and GFP images (upper panels) and CW and GFP images (lower panels) were stacked using ImageJ. Strains were incubated on SD medium. Scale bars, 5 μm.
Fig 5.
SsSTE12 is dispensable for mating.
(A) Experimental design of marker assisted breeding in S. schoenii. Strains were equipped with either hygromycin (HygR)or nourseothricin (clonNATR) resistance genes and plated on sporulation media. Spores were enriched by zymolyase/Triton X-100-treatment after wash-off and collection in test tubes and then plated on double selective medium to identify progeny harboring both markers. (B) representative images of selection plates of the indicated mating combinations. (C) Plot of CFUs (= the number of hybrids, “N hybrids”) identified with the indicated strain combinations. Mating was assessed using 2 biological and three technical replicates for each combination, Mean ± s.e.m.
Fig 6.
Comparative global transcriptional profiling defines the roles of SsKIL1 and SsSTE12 in the predation response of S. schoenii.
(A) Venn diagram illustrating overlapping DEGs with at least log2 ≥ 2 differential expression under predation conditions between the wild type and the Sskil1 and Ssste12 mutant strains. Two distinct gene sets were identified: the hunger response common to all strains (a) and the predation response constrained to the wild type (B), each with a set of enriched GO terms. (C,D) Consensus DNA-binding motifs, predicted using MEME, for the hunger and predation response gene sets with best matching transcription factor binding site matches (TFBS) to S. cerevisiae transcriptional regulators.