Figure 1.
Setting up killer K2 toxin genome-wide screens.
A. Primary screens. The yeast knockout library was arrayed on agar plates containing either 2×105 (screen 1) or 2×106 (screen 2) K2 toxin-producing cells or 300 U (screen 3) or 600 U (screen 4) of K2 toxin preparation. Cells were grown for 2 days at 25°C. A vital blue stain was used to ease the detection of dead cells. The plates were imaged with a digital color camera. Typically, small cyan colonies are hypersensitive to K2 toxin (see one example circled in blue), while large white colonies are hyper resistant (circled in white). Note that in the screens 3 and 4, the plates were imaged in grayscale only allowing scoring for the size of the colonies that appear in dark grey. B. Secondary screens: agar diffusion assays. Candidates identified in the primary screens were seeded inside the agar layer of test plates and either overlaid with purified K2 toxin (in a “punched-well” in the agar plate, or on the surface of the plate, in screens 5 and 6, respectively) or with K2 toxin-producing cells (screen 7). In screens No. 5–7, the test plates were imaged in gray scale. The mutant cells, inoculated inside the agar, appear as a light gray background whereas the “halo”, representing the area where cell growth was inhibited by the toxin, is transparent. In screen No. 7, a colony of K2-producing cells was deposited on the surface of the plate and appears in black. The primary screens were performed each once only. The secondary screens were each repeated three times.
Figure 2.
Distribution of cellular processes and cellular components involved in K2 resistance and hypersensitivity.
A. Agar diffusion assays. Representative examples of “halo” observed for strains with different degree of sensitivity/resistance towards K2 toxin (see Figure 1B, screen 5, for details). An isogenic control (wt, strain BY4741) is provided in the center. Strains Δkre1, Δchw41, and Δmrpl3 are resistant (show a smaller “halo” than wild-type or no “halo”). Strains Δrpl14a, Δspt8, and Δhog1 are hypersensitive (show a larger “halo” than wild-type). B. Distribution of cellular pathways and cellular components, according to Gene Ontology, associated with the 205 genes associated with K2 resistance and 127 with K2 hypersensitivity. The number of genes identified in each class is indicated.
Figure 3.
Statistically enriched gene ontology terms among putative K2 effectors.
Fold enrichment (F.E.) was calculated by dividing the frequency of specific gene cluster to the total frequency for each GO term, according to the data highlighted in Table S3. Biological processes (1 to 9) and cellular components (a to h) are listed in each panel. A. Resistance. B. Hypersensitivity. C. Deconvolution of the genes involved in biological regulation shown in panel B.
Figure 4.
Gene products involved in K2 susceptibility are highly interconnected within cell.
Physico-functional networks were established with STRING (see Materials and Methods and Figure S2). Gene products are depicted as color-coded nodes, according to cellular processes, and are connected by edges. Color coding is as follows: red, mitochondrion organization & translation; deep purple, proton transport & ATP synthesis; brown, phospholipid transport; orange, ER associated protein catabolism; yellow, cell wall organization & biogenesis; light green, carbohydrate metabolism; dark green, osmosensory signaling; light purple, chromosome organization & gene expression; blue, homeostatic process. Some nodes are connected through intermediates, which were not all represented here for simplification (see Figure S2 for details). A. Mutations leading to increased resistance. B. Mutations leading to increased hypersensitivity.
Figure 5.
K2 toxin binding properties of mutants with altered K2 resistance.
K2 toxin binding assay, as described in [28]. The histograms depict the remaining K2 toxin activity following incubation of K2 toxin preparations either with wild-type reference cells or with the mutant cells to be tested. This experiment was repeated three times. Error bars indicate the standard error of the mean. The effects on remaining toxin activity were all significant (p value <0.05). The remaining toxin activity is provided as a percentage of the activity of mock-treated K2 toxin preparations (see Materials and Methods). The table is an attempt to correlate K2 toxin binding with the resistance/sensitivity phenotype and the level of β-glucan. Toxin binding (*) was calculated as a percentage of the remaining toxin activity. Incubation with wild-type cells led to 45–65% of remaining activity;++corresponds to 0–10%,+to 10–40%; − to 70–90% and – to 90–100%. K2 resistance phenotypes are taken from Tables S1 and S2. The level of β-glucan (**) in these strains was previously published [27], and is as follows. Increase (I) of β-glucan: ++, 65<I<100; +, 45<I<65; (+), 25<I<45; (++), I<25%. Decrease (D) of β-glucan: − − −, 85<D<100; − −, 65<D<85; −, 45<D<65; (−), 25<D<45; (− −), D<25%.
Figure 6.
Genes involved in K2 biology are highly specific.
The Venn diagrams depict the number of gene products known to contribute to the phenotypes of the three major killer K toxins. The number of mutants, involved in K2 resistance/hypersensitivity identified in our screens, is a combination of those exhibiting “strong” and “weak” phenotypes. The number of genes, affecting K1 and K28 biology are presented according to references [27] and [28], respectively.
Figure 7.
Model for K2 toxin entry and response of the host cell.
The K2 toxin (grey oval) initially binds to cell wall-localized β-1,6-glucan. K2 is recognized by the plasma membrane-localized receptor Kre1 prior to integrating into the cell membrane and disrupting the electrochemical ion gradient leading to the leakage of K+ ions. Disrupting the functional integrity of the plasma membrane triggers multiple stress signaling responses involving the cell wall integrity pathway (in pink), the HOG pathway (in green), and phosphoinositide signaling (in purple). Rho1 and Pkc1 efficiently relay cell wall stress from the plasma membrane into the nucleus, leading to the activation of specific transcriptional programs. Ion and pH homeostasis maintenance mechanisms are activated to prevent cell death through ion leakage. Plasma membrane ion channels and their regulators (in yellow), vacuolar H+-ATPase and other vacuolar ion homeostasis keeping proteins (in blue), mitochondrial F0F1 ATP-synthase and H+/K+-antiporter (in red) are all involved. Key: Gene products depicted in uppercase denote K toxin effectors which are specific to K2 toxin. Lowercase genes are also involved in K1 and/or K28 susceptibility. Gene products circled in red are involved in K2 resistance; those circled in green in K2 hypersensitivity. For the K2 effectors, the color-coding is as follows: Orange, proteins associated with cell wall organization/biogenesis; Purple, phosphoinositide-related; Pink, CWI cascade; Green, HOG pathway; Red, mitochondrial constituents; Blue, vacuolar constituents; Yellow, ion transporter at the PM. CW, cell wall; PM, plasma membrane; CWI, cell wall integrity pathway. The cell wall consists of an inner layer, which is composed of β-1,3-glucan (in brown) and chitin (yellow waves), and an outer layer, densely packed with mannoproteins (in orange), extensively modified by N- and O-glycosylation (not represented). Components of the inner and outer cell wall layers are connected by β-1,6-glucan (in green).