Volume 1, Issue 6, Pages 807-817 (May 1998) Osmotin, a Plant Antifungal Protein, Subverts Signal Transduction to Enhance Fungal Cell Susceptibility  Dae-Jin Yun, José Ignacio Ibeas, Hyeseung Lee, María A Coca, Meena L Narasimhan, Yukifumi Uesono, Paul M Hasegawa, José M Pardo, Ray A Bressan  Molecular Cell  Volume 1, Issue 6, Pages 807-817 (May 1998) DOI: 10.1016/S1097-2765(00)80080-5

Figure 1 Characterization of ore Mutants (A) ore mutants are resistant to osmotin. Cells of a naturally osmotin-resistant control strain GRF167 (1), wild-type osmotin-sensitive BWG7a (2), and the ore mutants ore12, ore13, ore14, and ore4 (3–6, respectively) were grown on YPD with (left) or without (right) 4 μM osmotin. (B) Sterile ore mutants are defective in pheromone-induced growth arrest. The halo bioassay was performed by spotting 15 μl of a 3 μM (left) or 9 μM (right) solution of α factor (Sigma) on a lawn of wild-type BWG7a ([w.t.], left) or ore13 mutant (right) cells, spread on YPD. The plates were photographed after 2 days of incubation at 28°C. (C) Fertility of ore9 and ore11 mutants is recovered by complementation with STE20. Cells of wild-type BWG7a (w.t.), the Δste20 derivative DJ4, mutants ore9 and ore11, and the ore mutants transformed with pSTE20-5, a centromere plasmid containing the full-length STE20 gene (Leberer et al. 1992), were replica-plated on diploid-selective minimal media seeded with mating tester strain F15 (left) or YPD (right) and allowed to grow for 2 days at 28°C. (D) Osmotin sensitivity of ore9 and ore11 mutants is restored by complementation with STE20. The osmotin tolerance of the strains shown in (C) was measured in liquid cultures. Exponentially growing cultures were diluted to OD600 nm of 0.01 and grown for 16–20 hr with shaking at 30°C in the presence of the indicated amounts of osmotin. The final OD600 nm was determined by appropriate dilutions. The values are normalized to the OD600 nm of control cultures grown without osmotin and given as percentages. Molecular Cell 1998 1, 807-817DOI: (10.1016/S1097-2765(00)80080-5)

Figure 2 Elements of the Pheromone Response Pathway Are Required for Osmotin Sensitivity (A) Suppression of osmotin sensitivity by mutations in genes encoding proteins of the mating signal transduction pathway. Aliquots (2.5 μl) from exponentially growing cultures of BWG7a (w.t.) and the ste mutants indicated (strains DJ1, DJ2, DJ3, DJ4, DJ5, DJ6, DJ7, DJ10, and DJ11; see Table 2) at an OD600 nm of 0.04 and serial dilutions (1:10, 1:100) were spotted on YPD media supplemented with the indicated concentrations of osmotin and allowed to grow for 2 days at 28°C. (B) GPA1 does not function in osmotin sensitivity. A halo bioassay was performed on strain BWG7a transformed with either the high-copy-number vector pAD4M (left) or the same vector overexpressing GPA1 (Dohlman et al. 1996; right). 15 μL of either 5 μM α factor (upper) or 5 μM osmotin (lower) were spotted, and cells were allowed to grow for 2 days at 28°C. Molecular Cell 1998 1, 807-817DOI: (10.1016/S1097-2765(00)80080-5)

Figure 3 Osmotin-Induced Signal Flux through the STE MAPK Module (A and B) Phosphorylation of STE7 in response to osmotin. Cells of strain BWG7a, without (−) or with (+) pNC267 (Zhou et al. 1993), which contains STE7 fused in-frame with a c-Myc epitope tag and under control of the CYC1 promoter, were treated (+) with either α factor (5 μM) or osmotin ([A], 2 μM; [B], 8 μM) for the indicated time periods at 28°C. Shown are immunoblots of total protein from cell extracts (30 μg per lane) separated by SDS–PAGE, reacted with (A) Myc1-9E10 antibody and (B) STE7 antibody polyclonal antibody, and developed by the ECL method. (C) Pheromone pretreatment enhances the cytotoxic effect of osmotin. BWG7a cells were first incubated with (squares) or without (circles) 5 μM α factor for 1 hr at 28°C and then with the indicated concentrations of osmotin for 3 hr, after which they were diluted and plated on YPD media. After 2 days of incubation at 28°C, the number of viable cells were counted and are shown normalized to the value without added osmotin. Values are the average of two experiments. Molecular Cell 1998 1, 807-817DOI: (10.1016/S1097-2765(00)80080-5)

Figure 4 sst2 Mutation Affects Pheromone Response and Osmotin-Induced Cell Death (A) Δsst2 mutation increases sensitivity to osmotin. The relative sensitivity to osmotin of BC159 and a congenic Δsst2 mutant (BC180) was assayed in liquid cultures. Values shown are the mean and SE of three independent experiments and are normalized to the OD600 nm of control cultures without osmotin. (B) Overexpression of GPA1 does not affect the osmotin sensitivity of a Δsst2 mutant. Strain BC180 (Δsst2) was transformed with either the high-copy-number vector pAD4M (left) or the same vector overexpressing GPA1 (Dohlman et al. 1996; right), and the halo bioassay was performed with 15 μl of either 5 μM α factor (upper) or 5 μM osmotin (lower). Molecular Cell 1998 1, 807-817DOI: (10.1016/S1097-2765(00)80080-5)

Figure 5 Mating-Specific Morphogenesis and Transcription Are Not Detected during Osmotin-Induced Cell Death Exponentially growing BWG7a cells ([A] and [B]) or BWG7a cells transformed with pSB231, a centromere plasmid containing a FUS1-lacZ transcriptional fusion (C), were incubated with α factor (5 μM) or osmotin (2 μM, [A] and [C]; 1 μM, [B]) at 28°C for the indicated time periods. Shown are: (A) the microscopic appearance of the cells under Nomarski optics, (B) the DNA contents per cell obtained by flow cytometric analysis, and (C) the β-galactosidase activities of the cells at various time points of the treatments indicated. Molecular Cell 1998 1, 807-817DOI: (10.1016/S1097-2765(00)80080-5)

Figure 6 STE Genes Regulate Sensitivity to Osmotin through Effects on the Cell Wall Osmotin sensitivity was assayed by spotting 3 μl of 2 μM osmotin solution in 1 M sorbitol directly on a lawn of cells or spheroplasts of strain BWG7a (wild type) and its isogenic Δste7 mutant (DJ7). Cells and spheroplasts were embedded in YPD agar supplemented with 1 M sorbitol as described in Experimental Procedures. Plates were photographed after 2 days at 28°C. The size of the inhibition halo reflects the degree of sensitivity to osmotin. Molecular Cell 1998 1, 807-817DOI: (10.1016/S1097-2765(00)80080-5)

Figure 7 Model for the Signal Transduction Pathway in Osmotin-Induced Cell Death Osmotin treatment activates a MAPK cascade that shares the components STE20, STE11, STE7, KSS1, and STE12 with the pheromone response, invasive, and pseudohyphal growth pathways, and additionally, STE4, STE5, FUS3, and STE18 with the pheromone response pathway. We propose that osmotin subverts another existing MAPK cascade, with unidentified natural stimulus, by a process that may or may not require a Gα protein and a receptor. Activation of this cascade induces changes in the cell wall that facilitate osmotin access to the plasma membrane and consequently, cell death. Molecular Cell 1998 1, 807-817DOI: (10.1016/S1097-2765(00)80080-5)