FEBS Letters 581 (2007) 3217–3222

a-, the major saponin in , induces programmed cell death mediated by reactive oxygen species in the fungal pathogen Fusarium oxysporum

Shin-ichi Itoa,*, Takashi Iharaa, Hideyuki Tamuraa, Shuhei Tanakaa, Tsuyoshi Ikedab, Hiroshi Kajiharac, Chandrika Dissanayakec, Fatma F. Abdel-Motaald, Magdi A. El-Sayedd

a Department of Biological and Environmental Sciences, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753-8515, Japan b Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan c The United Graduate School of Agricultural Sciences, Tottori University, Tottori 680-8553, Japan d Department of Botany, Faculty of Science, South Valley University, Aswan, Egypt

Received 2 April 2007; revised 21 May 2007; accepted 8 June 2007

Available online 15 June 2007

Edited by Ulf-Ingo Flu¨gge

brane integrity followed by leakage of cell components and cell Abstract The tomato saponin a-tomatine has been proposed to kill sensitive cells by binding to cell membranes followed by leak- death [3,4], references therein]. However, a-tomatine has been age of cell components. However, details of the modes of action reported to inhibit the growth of some fungi lacking in of the compound on fungal cells are poorly understood. In the their cell membranes, suggesting that a-tomatine possesses an present study, mechanisms involved in a-tomatine-induced cell unknown fungicidal action [4]. death of fungi were examined using a filamentous pathogenic Fusarium oxysporum is a phytopathogen causing a vascular Fusarium oxysporum. a-Tomatine-induced cell death of wilt disease in many important crops and can be hard to be F. oxysporum (TICDF) occurred only under aerobic conditions controlled [5]. F. oxysporum is also known as a serious emerg- and was blocked by the mitochondrial F0F1-ATPase inhibitor ing pathogen of humans due to the increasing number of se- oligomycin, the caspase inhibitor D-VAD-fmk, and syn- vere cases reported and to its broad resistance to the thesis inhibitor cycloheximide. Fungal cells exposed to a-toma- available antifungal drugs [6,7]. Recently we found F. oxyspo- tine showed TUNEL-positive nuclei, depolarization of transmembrane potential of mitochondria, and reactive oxygen rum strains that are highly sensitive to a-tomatine [8]. Under- species (ROS) accumulation. These results suggest that TICDF standing the mechanisms involved in the sensitivity of the occurs through a programmed cell death process in which mito- strains may provide new insight into the fungicidal action of chondria play a pivotal role. Pharmacological studies using this compound and lead to the development of novel anti- inhibitors suggest that a-tomatine activates phosphotyrosine ki- fungal agents active against F. oxysporum. nase and monomeric G-protein signaling pathways leading to In the present study, we demonstrate that a-tomatine-in- 2+ Ca elevation and ROS burst in F. oxysporum cells. duced cell death of F. oxysporum (TICDF) occurs through a 2007 Federation of European Biochemical Societies. Pub- programmed cell death accompanying a rapid generation of lished by Elsevier B.V. All rights reserved. reactive oxygen species (ROS) in the cells, which is a novel fun- gicidal action of a-tomatine. We also demonstrate that a-tom- Keywords: Glycoalkaloid; Apoptosis; Fusarium oxysporum atine appears to activate tyrosine kinase and monomeric GTP- binding protein (G-protein) signaling pathways leading to Ca2+ elevation and ROS burst in F. oxysporum cells.

1. Introduction 2. Materials and methods

a-Tomatine, a glycoside lycotetraose is attached to the 3-OH 2.1. Strains and culture conditions group of the aglycon tomatidine, is the major saponin in toma- F. oxysporum strains (MAFF103057, 103058, 727516, and 744001) to (Lycopersicon esculentum). Tomato plants contain a-toma- were obtained from the National Institute of Agricultural Sciences, tine at 10–30 mg/kg, which is enough to kill microbes [1]. Tsukuba, Japan. Fungal strains were maintained on PDA medium (Daigo, Tokyo, Japan) at 25 C. For liquid cultures, fungal strains Indeed, a-tomatine has been shown to kill a broad range of were grown in Dextrose medium (PD medium; Daigo, Tokyo, fungi in vitro and functions as a resistance substance (phytoan- Japan) at 25 C with shaking at 100 rpm. Spore suspensions of F. oxy- ticipin) against phytopathogens in tomato [2]. The fungicidal sporum were prepared from 4-day-old liquid cultures, harvested by fil- mode of action of a-tomatine proposed is that a-tomatine tration through three layers of gauze cloth, centrifuged at 3000 · g for forms a complex with fungal membrane sterols with free 3b- 20 min and suspended in distilled water. hydroxyl groups, resulting in pore formation and loss of mem- 2.2. Chemicals a-Tomatine, A23187, 1,2-bis(2-aminophenoxy)ethane-N,N,N0,N00- tetraacetic acid (BAPTA), cycloheximide, dimethylthiourea, neomycin, *Corresponding author. Fax: +81 839335820. ryanodine, and 8-(diethylamino) octyl-3,4,5-trimethoxybenzoate E-mail address: [email protected] (S. Ito). hydrochloride (TMB-8) were purchased from Sigma–Aldrich

0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.06.010 3218 S. Ito et al. / FEBS Letters 581 (2007) 3217–3222

(St. Louis, MO, USA). Ascorbic acid, lanthanum chloride (LaCl3), was measured with a luminometer BLR-201 (Aloka, Tokyo, Japan) nifedipine, oligomycin, Ruthenium Red, and standard oxalate solution by peroxide-dependent chemiluminescence of luminol. Fungal mycelial were obtained from Wako (Osaka, Japan). a-Cyano-(3,4-dihydroxy)- mat (100 mg) was suspended in 30 ml of CA medium. a-Tomatine was cinnamonitrile (AG18) and N-[2(S)-[2(R)-Amino-3-mercaptopropyla- added to the mycelial suspension at the final concentration of 40 lM. mino]-3-methylbutyl]-Phe-Met-OH (B581) were from Calbiochem At varying time, the mycelial suspension was filtrated with a mem- (San Diego, CA, USA). 1,2-Bis(2-aminophenoxy)ethane-N,N,N0,N00- brane filter (pore size 0.45 lm), and the filtrate (1 ml) was mixed with tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) was from 3.5 ml of 50 mM potassium phosphate buffer (pH 7.8) and 500 llof Nakarai tesque (Kyoto, Japan). D-Erythro-sphingosine was from 1.2 mM luminol in 50 mM potassium phosphate buffer in a vial set Alexis (San Diego, CA, USA). Ethylene glycol-bis(2-aminoethyle- in the luminometer. The reaction was started by adding 500 llof ther)-N,N,N0,N00-tetraacetic acid (EGTA) was from Kanto Chemical 10 mM potassium ferricyanide. (Tokyo, Japan). Wartmannin was from Alomone Labs (Jerusalem, Israel). Z-VAD-fmk was from Promega (Tokyo, Japan). a-Tomatine 2.7. 2D gel electrophoresis, immunostaing, and proteomic analysis (4 mM) was dissolved in 50 mM sodium citrate buffer (pH 4.0) or in F. oxysporum cultures were filtrated with a membrane filter and dimethylsulfoxide (DMSO). A23187, B581, BAPTA, BAPTA-AM, resultant fungal mat (1 g) was resuspended in 100 ml of CA medium. neomycin, nifedipine, ryanodine, tyrphostin A23, and wartmannin a-Tomatine (40 lM) was added aseptically to the culture, and then were dissolved in DMSO (final concentration: 10 mM). Solutions of the culture was incubated with shaking. were prepared and cycloheximide (50 mM), EGTA (100 mM), LaCl3(100 mM), and analyzed according to the methods of Ito et al. [8]. For detection of TMB-8 (10 mM) were made up in water. D-Erythro-sphingosine tyrosine-phosphorylated proteins, proteins after 2D PAGE were blot- (10 mM), oligomycin (50 lM), and Ruthenium Red (10 mM) were dis- ted onto a polyvinylidene difluoride membrane (immobilom-P, Milli- solved in dichloromethane, methanol, and 50% DMSO, respectively. pore, Bedford, MA), and the membrane was stained with anti- For each treatment, control experiments were performed using the phosphotyrosine antibody (Amersham, Piscataway, NJ). The blots appropriate volume of solvent. were developed using the ECL phosphorylation detection system (Amersham) and exposed on an X-ray film. For proteomic analysis, 2.3. Cell death assay in-gel digestion with trypsin was performed according to the method F. oxysporum spores were suspended in CA medium [1% (w/v) casa- reported by Kikuchi et al. [10]. mino acids, 10 mM ammonium sulfate, and 0.05% (w/v) yeast nitrogen base, pH 5.0] at 1 · 106 spores/ml and incubated with shaking (100 rpm) at 25 C for 16 h. a-Tomatine was added to the vegetative mycelia and the culture was incubated at 25 C with shaking. After 3. Results varying times of incubation, an aliquot (50 ll) of the culture was taken and mixed with 5 ll of Evans blue dye (10 mg/ml). After 5 min of incu- F. oxysporum cells were exposed to a-tomatine in a 1 ml cul- bation, fungal cells were transferred onto glass slides and observed by ture either in a 2-ml eppendorf tube with no shaking (low-aer- light microscopy. In experiments to the determine inhibitory effects of chemicals on TICDF, chemicals, made up as stock solutions, were obic condition) or in a 15 ml tube with a sponge plug with added to the fungal culture to give the appropriate concentration either shaking at 100 rpm (aerobic condition). Fungicidal action of 30 min before, after, or simultaneously with a-tomatine exposure. a-tomatine was observed only under the aerobic condition After 1 h incubation with a-tomatine, dead cells were determined with (Fig. 1A), suggesting that the major oxygen-consuming orga- Evans blue dye staining described above. Each experiment was per- nelle, mitochondria, would be involved in TICDF. Since all formed in triplicate. At the concentrations used in this study, the chem- icals alone had no effect on the viability of F. oxysporum cells in the four strains of F. oxysporum examined showed similar re- absence of a-tomatine. sponses against a-tomatine, we performed subsequent experi- ments using one of the strains, F. oxysporum f. sp. raphani 2.4. Electrolytes leakage assay MAFF103058. Electrolyte loss studies were performed according to the method of We examined whether a specific inhibitor of mitochondrial Steel et al. [9]. Electrical conductivity of the fungal suspension was F F -ATP synthase, oligomycin, would block the fungicidal measured using a conductivity meter (ES-12, Horiba, Tokyo, Japan). 0 1 Maximum conductivity was determined by adding chloroform at the action of a-tomatine. Oligomycin markedly inhibited TICDF, end of each experiment. suggesting that ATP is necessary for TICDF (Fig. 1B). This re- sult raised the possibility that a-tomatine may induce pro- 2.5. Analysis of programmed cell death markers grammed cell death (PCD), since ATP is required for PCD For terminal deoxynucleotidyl transferase-mediated dUTP nick end in eukaryotic cells [11]. labeling (TUNEL), F. oxysporum cells exposed to a-tomatine (40 lM) In PCD, caspases are activated and novel proteins are syn- were fixed with 3.7% formaldehyde for 30 min, washed with PBS, and the fungal suspension was transferred onto polylysine-coated slides thesized. Thus, we examined whether the broad-spectrum cas- (Matsunami, Tokyo, Japan) and air-dried to fix fungal cells on the pase inhibitor Z-VAD-fmk and the protein synthesis inhibitor slides. After removing excess PBS with a filter paper, 100 ll of PBS cycloheximide would block TICDF. Both Z-VAD-fmk and containing Kitarase (0.4 mg/ml; Yakult, Tokyo, Japan) and Novozyme cycloheximide reduced TICDF in a dose-dependent manner (1 mg/ml; Novo Nordisk, Bagsvaerd, Denmark) was added to the fungal cells on the slides and incubated in a humid chamber for 3 h (Fig. 1B). PCD is characterized by a set of distinct morpholog- for removing cell walls from the fungal cells. Cell permeabilization ical changes including DNA fragmentation and depolarization and the TUNEL reactions were carried out by using an In situ Cell of the transmembrane potential of mitochondria. DNA frag- Death Detection kit (Roche diagnostics, Tokyo, Japan) according to mentation is detected in situ by the TUNEL assay, as 30-OH the manufacturer’s instructions. TUNEL-positive cells were detected DNA ends labeled with FITC-conjugated dUTP via terminal using an epifluorescence microscope (BH2-RFK, Olympus, Tokyo, Ja- pan). Depolarization of mitochondrial membrane potential of F. oxy- deoxynucleotidyltransferase can be visualized by fluorescence sporum cells was determined by incubation of fungal cells with microscopy [12]. The TUNEL assay of F. oxysporum cells ex- MitoCapture cation dye (Calbiochem) according to the manufacturer’s posed to a-tomatine showed high percentage (>80%) of posi- instructions. tive cells (Fig. 1C). Depolarization of the transmembrane potential of mitochondria, which is early event in the PCD 2.6. Detection of ROS in fungal cells [13], is detected by a cationic dye (Mitocapture) that fluoresces Generation of ROS by F. oxysporum cells was detected by epifluo- rescence microscopy by monitoring the conversion of non-fluorescent differently in healthy and PCD cells. Healthy cells show accu- dihydrorhodamine 123 (DHR 123; Sigma–Aldrich) to fluorescent rho- mulation of Mitocapture in the mitochondria, producing a red damine 123. In time course experiments, production of ROS (H2O2) fluorescence, whereas in PCD cells the dye remains in the cyto- S. Ito et al. / FEBS Letters 581 (2007) 3217–3222 3219

Fig. 1. Fungicidal action of a-tomatine. (A) Requirement of aerobic condition for a-tomatine-induced cell death of F. oxysporum. F. oxysporum cells were exposed to 40 lM(, n)or0lM(, h)a-tomatine under either the low-aerobic condition (n, h) or the aerobic condition (, ). At the times indicated, cell death was evaluated by Evans blue staining. (B) Inhibition of a-tomatine-induced cell death of F. oxysporum by oligomycin, Z-VAD- fmk, and cycloheximide. Each inhibitor was added to F. oxysporum culture 30 min before the addition of a-tomatine. Cell death was evaluated by Evans blue staining after 1 h exposure to a-tomatine. Data are means ± S.E.M. *P < 0.01 vs. tomatine; **P < 0.05 vs. tomatine (Student’s t-test). (C) TUNEL assay of F. oxysporum cells exposed to a-tomatine. F. oxysporum cells were exposed to 0 lM (control), 40 lM a-tomatine (tomatine) for 40 min followed by TUNEL assays. Oligomycin (500 nM) was added to F. oxysporum culture 30 min before a-tomatine exposure (tomatine + oli- gomycin). Fugal cells were also stained with DAPI to visualize nuclei. (D) Depolarization of mitochondrial membrane potential of F. oxysporum cells treated with a-tomatine.

plasm due to the change in mitochondrial membrane potential lytes were leaked out from the fungal cells [9]. We examine for and emits a green fluorescence. As shown in Fig. 1D, F. oxy- leakage of electrolytes from F. oxysporum cells exposed to a- sporum cells exposed to a-tomatine showed obvious green fluo- tomatine. Only about 7% of total electrolytes were leaked rescence. These results suggest that TICDF occurs through a out from the cells even after 60 min exposure to a-tomatine de- PCD process. spite most of cells had been killed by the compound at that An accumulation of ROS appears to be a central phenome- time, suggesting that leakage of electrolytes is not responsible non in most instances of PCD [14]. ROS generation of fungal for cell death of F. oxysporum caused by a-tomatine (data not cells was visualized under a fluorescence microscope by shown). Thus, a hypothesis that a-tomatine disrupts cell mem- DHR123, which is oxidized to a fluorescent derivative by branes followed by leakage of electrolytes resulting in cell intracellular ROS and deposit in the cells. Fungal cells exposed death is unlikely in TICDF. to a-tomatine showed elevated DHR123 staining (Fig. 2A). a-Tomatine may induce leakage and/or secretion of proteins We also performed the time course experiments of ROS gener- from F. oxysporum cells. To address the question, we analyzed ation in fungal cells exposed to a-tomatine. Rapid generation extracellular proteins in culture fluids of F. oxysporum exposed of ROS () with a peak at around 30 min to a-tomatine by 2D PAGE. Several proteins were detected after the exposure to a-tomatine was revealed (Fig. 2B). Pre- only from the culture fluids of F. oxysporum exposed to a-tom- treatment with ROS scavengers, dimethylthiourea and ascor- atine (Fig. 3A, spots 1, 2, 3, 5, and 6). Although these protein bic acid blocked TICDF in a dose-dependent manner spots were subjected to peptide mass fingereprinting analysis, (Fig. 2C). no protein was found to have a high match score in databases. It has been reported that a-tomatine-sensitive fungi showed Interestingly, some proteins detected in TICDF were tyrosine- leakage of electrolytes; more than 70% of total cellular electro- phospholyrated, suggesting that tyrosine kinase may be acti- 3220 S. Ito et al. / FEBS Letters 581 (2007) 3217–3222

Fig. 2. a-Tomatine-induced cell death of F. oxysporum is associated Fig. 3. a-Tomatine-induced cell death of F. oxysporum is associated with ROS generation. (A) Microscopy of F. oxysporum cells exposed to with signal transduction in the cells. (A) 2D-PAGE analysis of a-tomatine. F. oxysporum cells were exposed to 0 lM (-tomatine) or extracellular proteins of F. oxysporum cultures treated with a-toma- 40 lM a-tomatine (+tomatine) for 40 min in the presence of dihydro- tine. Extracellular proteins were prepared from the fungal cultures rhodamine 123 (5 lg/ml). DIC, differential interference contrast after 1 h exposure to 0 lM (control) or 40 lM a-tomatine (Tomatine) microscopy. (B) Time course of ROS (H O ) generation in F. and analyzed by 2D-PAGE. Gels were stained with Coomassie 2 2 brilliant blue (upper right). Tyrosine-phosphorylated proteins were oxysporum cells exposed to a-tomatine. Generation of H2O2 was monitored by luminol-induced chemiluminescence in the supernatant detected with anti-phosphotyrosine antibody (lower panels). (B) Dose- of F. oxysporum culture exposed to 0 lM()or40lM() a-tomatine. dependent inhibition of a-tomatine-induced cell death by pharmaco- (C) Dose-dependent inhibition of a-tomatine-induced cell death by logical inhibitors. Each inhibitor was added to F. oxysporum culture ROS scavengers. Dimethylthiourea (DMTU) or ascorbic acid was 30 min before exposure to a-tomatine. Cell death was evaluated by added to F. oxysporum culture 30 min before the addition of a- Evans blue staining after 1 h exposure to a-tomatine. Data are * ** tomatine. Cell death was evaluated by Evans blue staining after 1 h means ± S.E.M. (n = 3). P < 0.01 vs. tomatine; P < 0.05 vs. toma- exposure to a-tomatine. Data are means ± S.E.M (n = 3). *P < 0.01 vs. tine (Student’s t-test). (C) Effects of pharmacological inhibitors added tomatine; **P < 0.05 vs. tomatine (Student’s t-test). at different times. Each inhibitor was added to the fungal culture either 30 min before, after, or simultaneously with a-tomatine exposure. Cell death was evaluated by Evans blue staining after 1 h exposure to a- tomatine. Data are means ± S.E.M. (n = 3). *P < 0.01 vs. tomatine; vated in TICDF. Indeed, TICDF was blocked by the tyrosine **P < 0.05 vs. tomatine (Student’s t-test). kinase inhibitor (AG18) (Fig. 3B). TICDF was blocked also by the farnesyltransferase inhibitor I (B581), the L-type calcium ion channel inhibitor (nifedipine), the intracellular calcium (oxalate) in a dose-dependent manner (Fig. 3B). The extracel- modulators (ryanodine and TMB-8), the intracellular calcium lular calcium chelators (BAPTA and EGTA) and the calcium chelator (BAPTA-AM), and the calcium precipitating agent channel blocker (LaCl3) also blocked TICDF (data not S. Ito et al. / FEBS Letters 581 (2007) 3217–3222 3221 shown). Chemicals including the calcium ionophore A23187, the mechanisms that trigger elevation of intracellular Ca2+ re- the protein kinase C inhibitor D-erythro-sphingosine, the main unclear. protein kinase C activator dibutyryl-cAMP, the PI3 kinase The finding that AG18, B581, and oligomycin blocked inhibitor wortmannin, and the mitochondrial calcium uni- TICDF only when they were added to the fungal culture prior porter inhibitor ruthenium red did not block TICDF. to or simultaneously with a-tomatine suggests that tyrosine To determine the order of target molecules of the inhibitors, kinase, farnesyltransferase, and mitochondrial F0F1-ATP syn- each inhibitor was added to F. oxysporum cultures either thase act upstream in a-tomatine signaling pathway(s) re- 30 min before, after, or simultaneously with a-tomatine expo- quired for TICDF. Among the targets, tyrosine kinase sure. Nifedipine, ryanodine, TMB-8, oxalate, and Z-VAD-fmk appears to act at a relatively proximal position in the signaling effectively blocked the cell death even at 30 min after a-toma- pathway(s), because AG18 was effective only when it was tine exposure. In contrast, AG18, B581, and oligomycin added to the fungal culture prior to a-tomatine. It has been blocked the cell death when they were added to the fungal cul- known that tyrosine kinase activation initiates the signaling ture before or simultaneously with a-tomatine exposure. Par- pathway early on to direct the program leading to cell death ticularly, AG18 blocked the cell death only when added to in cultured cells of plant [29] and human [30]. In yeast, a pos- the culture before a-tomatine exposure (Fig. 3C). sible crosstalk between tyrosine phosphorylation and Ras We examine the effects of the inhibitors on ROS generation pathways is suggested [31]. As shown in the present study, in F. oxysporum cells exposed to a-tomatine. When AG18, TICDF was blocked not only by the tyrosine kinase inhibitor B581, and oligomycin blocked TICDF, no DHR123-stained (AG18) but also by the farnesyltransferase inhibitor (B581), cell was observed. In contrast, when nifedipine, ryanodine, which specifically blocks Ras activation [32]. Thus, a possible TMB-8, oxalate, or Z-VAD-fmk blocked TICDF, most fungal crosstalk between tyrosine phosphorylation and Ras pathway cells were weakly stained by DHR123, suggesting that a in TICDF is deduced. Recent studies [24,33] showing that certain level of ROS was generated in the cells (data not ROS generation was via RAS-dependent pathway in PCD of shown). filamentous fungi support this notion. Based on our findings, we hypothesize that a-tomatin induces PCD in F. oxysporum via activation of tyrosine kinase and G-protein signaling path- 4. Discussion way(s) which lead to intracellular Ca2+ elevation and ROS burst. This is, to our knowledge, the first report describing PCD of fungi including F. oxysporum in response to the major tomato saponin a-tomatine, though the hallmarks of PCD or apopto- References sis have been reported in several fungal species in response to [1] Roddick, J.G. (1974) The steroidal glycoalkaloid a-tomatine. different stimuli other than saponin [15–25]. Phytochemistry 13, 9–25. Fungicidal mode of action of a-tomatine on sensitive cells [2] Sandrock, R.W. and VanEtten, H.D. (1998) Fungal sensitivity to has been assumed that the compound interacts with membrane and enzymatic degradation of the phytoanticipin a-tomatine. associated resulting in the formation of specific Phytopathology 88, 137–143. [3] Morrissey, J.P. and Osbourn, A.E. (1999) Fungal resistance to complexes which causes disruption of the membrane [3]. How- plant antibiotics as a mechanism of pathogenesis. Microbiol. Mol. ever, in the present study fungicidal action of a-tomatine was Biol. Rev. 63, 708–724. significantly blocked when ROS production was inhibited, sug- [4] Friedman, M. (2002) Tomato glycoalkaloids: role in the plant and gesting that the ROS is essential for TICDF. Importance of in the diet. J. Agric. Food Chem. 50, 5751–5780. ROS generation in PCD has been known in other fungi [5] Nelson, P.E., Toussoun, T.A. and Cook, R.J. (1981) Fusarium: Diseases, Biology, and Taxonomy, The Pennsylvania State [15,16,19,21,24,26,27]. Although we were not able to address University Press, University Park. the origin of the ROS in TICDF, mitochondria appear deeply [6] Ortoneda, M., Guarro, J., Madrid, M.P., Caracuel, Z., Roncero, involved since the ROS generation was completely abolished M.I.G., Mayayo, E. and Di Pietro, A. (2004) Fusarium oxysporum by the mitochondrial F F -ATP synthase inhibitor oligomycin. as a multihost model for the genetic dissection of fungal virulence 0 1 in plants and mammals. Infect. Immun. 72, 1760–1766. It is notable that TICDF was blocked by inhibitors includ- [7] Odds, F.C., Van Gerven, F., Espinel-Ingroff, A., Bartlett, M.S., ing nifedipine, ryanodine, TMB-8, oxalate, and Z-VAD-fmk Ghannoum, M.A., Lancaster, M.V., Pfaller, M.A., Rex, J.H., even after the cells had been incubated with a-tomatine for Rinaldi, M.G. and Walsh, T.J. (1998) Evaluation of possible 30 min. At this time period, most of F. oxysporum cells ap- correlations between antifungal susceptibilities of filamentous peared to accumulate a certain level of ROS. These findings fungi in vitro and antifungal treatment outcomes in animal infection models. Antimicrob. Agents Chemother. 42, 282– suggest that there are two steps in ROS generation in TICDF: 288. generation of low-level ROS in the first step and burst of ROS [8] Ito, S., Kawaguchi, T., Nagata, A., Tamura, H., Matsushita, H., in the second step which occurs around at 30 min after the Takahara, H., Tanaka, S. and Ikeda, T. (2004) Distribution of the exposure of cells to a-tomatine. These results also imply that FoToml gene encoding tomatinase in different formae speciales of Fusarium oxysporum and identification of a novel tomatinase F. oxysporum cells generating low-level ROS are not irrevers- from F. oxysporum f. sp. radicis-lycopersici,the causal agent of ibly committed into cell death process. Thus, it is likely that Fusarium crown and root rot of tomato. J. Gen. Plant Pathol. 70, ROS burst is essential for TICDF. Furthermore, elevation of 195–201. intracellular Ca2+ appears critical for ROS burst because the [9] Steel, C.C. and Drysdale, R.B. (1988) Electrolyte leakage from calcium ion channel inhibitor (nifedipine), the intracellular cal- plant and fungal tissues and disruption of membranes by a-tomatine. Phytochemistry 27, 1025–1030. cium modulators (ryanodine and TMB-8), and the calcium [10] Kikuchi, M., Hatano, N., Yokota, S., Shimozawa, N., Imanaka, precipitating agent (oxalate) can block the ROS burst. T. and Taniguchi, H. (2004) Proteomic analysis of rat liver Although elevation of intracellular Ca2+ has been known as peroxisome: presence of peroxisome-specific isozyme of Lon a pivotal event in initiating PCD in mammalian cells [28], protease. J. Biol. Chem. 279, 421–428. 3222 S. Ito et al. / FEBS Letters 581 (2007) 3217–3222

[11] Leist, M., Single, B., Castoldi, A.F., Ku¨hnle, S. and Nicotera, P. [23] Amin, S., Mousabi, A. and Robson, G.D. (2004) Oxidative and (1997) Intracellular adenosine triphosphate (ATP) concentration: amphotericin B-mediated cell death in the opportunistic pathogen a switch in the decision between apoptosis and necrosis. J. Exp. Aspergillus fumigatus is associated with an apoptosis-like pheno- Med. 185, 1481–1486. type. Microbiology 150, 1937–1945. [12] Gavrieli, Y., Sherman, Y. and Ben-Sasson, S.A. (1992) Identifi- [24] Chen, C. and Dickman, M.B. (2005) Proline suppresses apoptosis cation of programmed cell death in situ via specific labelling of in the fungal pathogen Colletotrichum trifolii. Proc. Natl. Acad. nuclear DNA fragmentation. J. Cell Biol. 119, 493–501. Sci. USA 102, 3459–3464. [13] Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, [25] Roze, L.V. and Linz, J.E. (1998) Lovastatin triggers an apoptosis- A., Hirsch, T., Susin, S.A., Petit, P.X., Mignotte, B. and Kroemer, like cell death process in the fungus Mucor racemosus. Fungal G. (1995) Sequential reduction of mitochondrial transmembrane Genet. Biol. 25, 119–133. potential and generation of reactive oxygen species in early [26] Kobayashi, D., Kondo, K., Uehara, N., Otokozawa, S., Tsuji, N., programmed cell death. J. Exp. Med. 182, 367–377. Yagihashi, A. and Watanabe, N. (2002) Endogenous reactive [14] Robson, G.D. (2006) Programmed cell death in the aspergilli and oxygen species is an important mediator of miconazole antifungal other filamentous fungi. Med. Mycol. 44, S109–S114. effect. Antimicrob. Agents Chemother. 46, 3113–3117. [15] Fro¨hloch, K-U. and Madeo, F. (2000) Apoptosis in yeast – a [27] Leiter, E´ ., Szappanos, H., Oberparleiter, C., Kaiserer, L., monocellular organism exhibits altruistic behaviour. FEBS Lett. Csernoch, L., Pusztahelyi, T., Emri, T., Po´csi, I., Salvenmoser, 473, 6–9. W. and Marx, F. (2005) Antifungal protein PAF severely affects [16] Klassen, R. and Meinhardt, F. (2005) Induction of DNA damage the integrity of the plasma membrane of Aspegillus nidulans and and apoptosis in Saccharomyces cerevisiae by a yeast killer . induces an apoptosis-like phonotype. Antimicrob. Agents Che- Cell Microbiol. 7, 393–401. mother. 49, 2445–2453. [17] Silva, R.D., Sotoca, R., Johansson, B., Ludovico, P., Sansonetty, [28] Mattson, M.P. and Chan, S.L. (2003) Calcium orchestrates F., Silva, M.T., Peinado, J.M. and Corte-Real, M. (2005) apoptosis. Nat. Cell Biol. 5, 1041–1043. Hyperosmotic stress induces metacaspase- and mitochondria- [29] Otani, H., Erdos, M. and Leonard, W.J. (1993) Tyrosine kinase(s) dependent apoptosis in Saccharomyces cerevisiae. Mol. Microbiol. regulate apoptosis and bcl-2 expression in a growth factor- 58, 824–834. dependent cell line. J. Biol. Chem. 268, 22733–22736. [18] Balzan, R., Sapienza, K., Galea, D.R., Vassallo, N., Frey, H. and [30] Anderson, P. (1997) Kinase cascades regulating entry into Bannister, W.H. (2004) Aspirin commits yeast cells to apoptosis apoptosis. Microbiol. Mol. Biol. Rev. 61, 33–46. depending on carbon source. Microbiology 150, 109–115. [31] Magherini, F., Busti, S., Gamberi, T., Sacco, E., Raugei, G., [19] Narasimhan, M.L., Damsz, B., Coca, M.A., Ibeas, J.I., Pardo, Manao, G., Ramponi, G., Modesti, A. and Vanoni, M. (2006) In J.M., Hasegawa, P.M. and Bressan, R.A. (2001) A plant defense Saccharomyces cereevisiae an unbalanced level of tyrosine phos- response effecter induces microbial apoptosis. Mol. Cell 8, 921– phorylation down-regulates the Ras/PKA pathway. Int. J. 930. Biochem. Cell Biol. 38, 444–460. [20] Phillips, A.J., Sudbery, I. and Ramsdale, M. (2003) Apoptosis [32] Cox, A.D., Garcia, A.M., Westwick, J.K., Kowalczyk, J.J., induced by environmental stresses and amphotericin B in Candida Lewis, M.D., Brenner, D.A. and Der, C.J. (1994) The CAAX albicans. Proc. Natl. Acad. Sci. USA 25, 14327–14332. peptidomimetic compound B581 specifically blocks farnesy- [21] Semighini, C.P., Hornby, J.M., Dumitru, R., Nickerson, K.W. lated, but not geranylgeranylated or myristylated, oncogenic and Harris, S.D. (2006) Farnesol-induced apoptosis in Aspergillus ras signaling and transformation. J. Biol. Chem. 269, 19203– nidulans reveals a possible mechanism for antagonistic interaction 19206. between fungi. Mol. Microbiol. 59, 753–764. [33] Phillips, A.J., Crowe, J.D. and Ramsdale, M. (2006) Ras pathway [22] Cheng, J., Park, T.-S., Chio, L.-C., Fischl, A.S. and Ye, X.S. signaling accelerates programmed cell death in the pathogenic (2003) Induction of apoptosis by sphingoid long-chain bases in fungus Candida albicans. Proc. Natl. Acad. Sci. USA 103, 726– Aspergillus nidulans. Mol. Cell. Biol. 23, 163–177. 731.