Crucial Role of Antioxidant Proteins and Hydrolytic Enzymes in Pathogenicity of Penicillium expansum
2006; Elsevier BV; Volume: 6; Issue: 3 Linguagem: Inglês
10.1074/mcp.m600179-mcp200
ISSN1535-9484
AutoresGuozheng Qin, Shiping Tian, Zhulong Chan, Boqiang Li,
Tópico(s)Plant pathogens and resistance mechanisms
ResumoPenicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase. Penicillium expansum, a widespread filamentous fungus, is a major causative agent of fruit decay and may lead to the production of mycotoxin that causes harmful effects on human health. In this study, we compared the cellular and extracellular proteomes of P. expansum in the absence and presence of borate, which affects the virulence of the fungal pathogen. The differentially expressed proteins were identified using ESI-Q-TOF-MS/MS. Several proteins related to stress response (glutathione S-transferase, catalase, and heat shock protein 60) and basic metabolism (glyceraldehyde-3-phosphate dehydrogenase, dihydroxy-acid dehydratase, and arginase) were identified in the cellular proteome. Catalase and glutathione S-transferase, the two antioxidant enzymes, exhibited reduced levels of expression upon exposure to borate. Because catalase and glutathione S-transferase are related to oxidative stress response, we further investigated the reactive oxygen species (ROS) levels and oxidative protein carbonylation (damaged proteins) in P. expansum. Higher amounts of ROS and carbonylated proteins were observed after borate treatment, indicating that catalase and glutathione S-transferase are important in scavenging ROS and protecting cellular proteins from oxidative damage. Additionally to find secretory proteins that contribute to the virulence, we studied the extracellular proteome of P. expansum under stress condition with reduced virulence. The expression of three protein spots were repressed in the presence of borate and identified as the same hydrolytic enzyme, polygalacturonase. Penicillium expansum is a widely spread fungal pathogen that causes blue mold rot in a variety of fruits, including apples, pears, peaches, and cherries (1Marek P. Annamalai T. Venkitanarayanan K. Detection of Penicillium expansum by polymerase chain reaction..Int. J. Food Microbiol. 2003; 89: 139-144Crossref PubMed Scopus (63) Google Scholar). It causes significant economic losses to the fruit industry and is also of potential public health concern because it produces toxic secondary metabolites, including patulin, citrinin, and chaetoglobosins (2Andersen B. Smedsgaard J. Frisvad J.C. Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products..J. Agric. Food Chem. 2004; 52: 2421-2428Crossref PubMed Scopus (233) Google Scholar). Patulin is produced by various filamentous fungi with P. expansum being generally regarded as one of the main producers (3Lennox J.E. McElroy L.J. Inhibition of growth and patulin synthesis in Penicillium expansum by potassium sorbate and sodium propionate in culture..Appl. Environ. Microbiol. 1984; 48: 1031-1033Crossref PubMed Google Scholar). Because of the potential carcinogenic effects of this mycotoxin, Europe and the United States have established a maximum limit for patulin contamination in apple-based products (3Lennox J.E. McElroy L.J. Inhibition of growth and patulin synthesis in Penicillium expansum by potassium sorbate and sodium propionate in culture..Appl. Environ. Microbiol. 1984; 48: 1031-1033Crossref PubMed Google Scholar). Control of decay caused by P. expansum has become important for ensuring the quality and safety of various fruits. Currently the disease caused by P. expansum is mainly controlled by the intensive use of synthetic fungicides. However, concerns about public health and the development of fungicide resistance by pathogens have prompted the search for alternative methods (4Janisiewicz W.J. Enhancement of biocontrol of blue mold with the nutrient analog 2-deoxy-d-glucose on apples and pears..Appl. Environ. Microbiol. 1994; 60: 2671-2676Crossref PubMed Google Scholar). This requires more knowledge about the pathogenesis at the biochemical and molecular level. During the course of invasion, a plant pathogen encounters the defense strategies of the host, including the accumulation of barrier-forming substances and the production of antimicrobial compounds that act directly to prevent pathogen invasion (5Campo S. Carrascal M. Coca M. Abián J. Segundo B.S. The defense response of germinating maize embryos against fungal infection: a proteomics approach..Proteomics. 2004; 4: 383-396Crossref PubMed Scopus (131) Google Scholar). In addition, the pathogen will also face the attack of reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; PDB, potato dextrose broth; YNB, 0.67% yeast nitrogen base without amino acids plus 2% dextrose; 2D, two-dimensional; CBB, Coomassie Brilliant Blue; DCHF-DA, 2′,7′-dichlorodihydrofluorescein diacetate; DNP, 2,4-dinitrophenylhydrazone; TEMED, N,N,N′,N′-tetramethylethylenediamine; EST, expressed sequence tag. primarily superoxide (O2) and hydrogen peroxide (H2O2), generated by the host at the site of infection (5Campo S. Carrascal M. Coca M. Abián J. Segundo B.S. The defense response of germinating maize embryos against fungal infection: a proteomics approach..Proteomics. 2004; 4: 383-396Crossref PubMed Scopus (131) Google Scholar). These ROS may cause oxidative damage to cell components including proteins, lipids, and nucleic acids (6Yan S. Tang Z. Su W. Sun W. Proteomic analysis of salt stress-responsive proteins in rice root..Proteomics. 2005; 5: 235-244Crossref PubMed Scopus (409) Google Scholar, 7Reverter-Branchat G. Cabiscol E. Tamarit J. Ros J. Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae..J. Biol. Chem. 2004; 279: 31983-31989Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). To protect cells against ROS, pathogens have developed several defense mechanisms, including enzymes (e.g. catalase and superoxide dismutase) as well as the nonenzymatic protective molecules such as glutathione and thioredoxin (8Ames B.N. Shigenaga M.K. Hagen T.M. Oxidants, antioxidants, and the degenerative diseases of aging..Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5446) Google Scholar, 9Moradas-Ferreira P. Costa V. Piper P. Mager W. The molecular defenses against reactive oxygen species in yeast..Mol. Microbiol. 1996; 19: 651-658Crossref PubMed Scopus (235) Google Scholar). Davidson et al. (10Davidson J.F. Whyte B. Bissinger P.H. Schiestl R.H. Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae..Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5116-5121Crossref PubMed Scopus (374) Google Scholar) reported that yeast mutants deleted for the genes encoding catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the oxidative stress than the isogenic wild-type yeast, demonstrating that antioxidant enzymes play an important role in oxidative stress resistance. Under protection by defense mechanisms from adverse environmental conditions in the site of invasion, a pathogen infects the host by crossing through the cell wall of the plant, which is composed of a complex matrix of polysaccharides. Extracellular proteins of the plant pathogen play a crucial role in this process. Through genetic and biochemical approaches, many secretory proteins have been characterized, such as pectate lyases (11Shevchik V.E. Condemine G. Robert-Baudouy J. Hugouvieux-Cotte-Pattat N. The exopolygalacturonate lyase PelW and the oligogalacturonate lyase Ogl, two cytoplasmic enzymes of pectin catabolism in Erwinia chrysanthemi 3937..J. Bacteriol. 1999; 181: 3912-3919Crossref PubMed Google Scholar, 12Shevchik V.E. Kester H.C. Benen J.A. Visser J. Robert-Baudouy J. Hugouvieux-Cotte-Pattat N. Characterization of the exopolygalacturonate lyase PelX of Erwinia chrysanthemi 3937..J. Bacteriol. 1999; 181: 1652-1663Crossref PubMed Google Scholar) and polygalacturonases (13Nasser W. Shevchik V.E. Hugouvieux-Cotte-Pattat N. Analysis of three clustered polygalacturonase genes in Erwinia chrysanthemi 3937 revealed an anti-repressor function for the PecS regulator..Mol. Microbiol. 1999; 34: 641-650Crossref PubMed Scopus (40) Google Scholar, 14Hugouvieux-Cotte-Pattat N. Shevchik V.E. Nasser W. PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases..J. Bacteriol. 2002; 184: 2664-2673Crossref PubMed Scopus (19) Google Scholar). However, these studies have mainly focused on the identification, purification, and characterization of single secreted proteins. Few publications on the global analysis of fungal secretory proteins are available (15Medina M.L. Haynes P.A. Breci L. Francisco W.A. Analysis of secreted proteins from Aspergillus flavus..Proteomics. 2005; 5: 3153-3161Crossref PubMed Scopus (84) Google Scholar). In recent years, proteomics analysis has proven to be a powerful method for studying the changes of protein expression profiles in response to various stresses in yeast (16Teixeira M.C. Santos P.M. Fernandes A.R. Sá-Correia I. A proteome analysis of the yeast response to the herbicide 2,4-dichlorophenoxyacetic acid..Proteomics. 2005; 5: 1889-1901Crossref PubMed Scopus (42) Google Scholar, 17Hu Y. Wang G. Chen G.Y.J. Fu X. Yao S.Q. Proteome analysis of Saccharomyces cerevisiae under metal stress by two-dimensional differential gel electrophoresis..Electrophoresis. 2003; 24: 1458-1470Crossref PubMed Scopus (90) Google Scholar, 18Vido K. Spector D. Lagniel G. Lopez S. Toledano M.B. Labarre J. A proteome analysis of the cadmium response in Saccharomyces cerevisiae..J. Biol. Chem. 2001; 276: 8469-8474Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar) and bacterium (19Kim S.J. Jones R.C. Cha C.J. Kweon O. Edmondson R.D. Cerniglia C.E. Identification of proteins induced by polycyclic aromatic hydrocarbon in Mycobacterium vanbaalenii PYR-1 using two-dimensional polyacrylamide gel electrophoresis and de novo sequencing methods..Proteomics. 2004; 4: 3899-3908Crossref PubMed Scopus (86) Google Scholar, 20Topanurak S. Sinchaikul S. Phutrakul S. Sookkheo B. Chen S.T. Proteomics viewed on stress response of thermophilic bacterium Bacillus stearothermophilus TLS33..Proteomics. 2005; 5: 3722-3730Crossref PubMed Scopus (18) Google Scholar, 21Chuang M.H. Wu M.S. Lin J.T. Chiou S.H. Proteomic analysis of proteins expressed by Helicobacter pylori under oxidative stress..Proteomics. 2005; 5: 3895-3901Crossref PubMed Scopus (42) Google Scholar, 22Vasseur C. Labadie J. Hébraud M. Differential protein expression by Pseudomonas fragi submitted to various stresses..Electrophoresis. 1999; 20: 2204-2213Crossref PubMed Scopus (28) Google Scholar). On the other hand, there is still a lack of information about proteomics analysis from filamentous fungi (23Nandakumar M.P. Marten M.R. Comparison of lysis methods and preparation protocols for one- and two-dimensional electrophoresis of Aspergillus oryzae intracellular proteins..Electrophoresis. 2002; 23: 2216-2222Crossref PubMed Scopus (47) Google Scholar, 24Matis M. Žakelj-Mavrič M. Peter-Katalinić J. Mass spectrometry and database search in the analysis of proteins from the fungus Pleurotus ostreatus..Proteomics. 2005; 5: 67-75Crossref PubMed Scopus (13) Google Scholar). P. expansum is very poorly characterized at the protein level. In a preliminary study, we found that borates, which are distributed widely in the environment, could significantly reduce the virulence of P. expansum in various fruits. Borates are essential plant micronutrients that help plant growth and have been used extensively in industry and agriculture as a safe method for control of fungi, bacteria, and many insects. However, there is no information about using borates to control fruit decay. In the present study, comparative analysis of the cellular and extracellular proteome was performed in P. expansum under normal condition and borate stress to gain insights into the pathogenesis of this fungal pathogen. Particular attention was paid to the antioxidant proteins and hydrolytic enzymes that are differentially expressed upon exposure to borate. To our knowledge, this is the first report for identifying virulence proteins in P. expansum on the basis of proteomics analysis. 0.67% yeast nitrogen base without amino acids plus 2% dextrose (YNB) was from Difco. Urea was purchased from ICN (Aurora, OH). Carrier ampholyte was from Amersham Biosciences. Potato dextrose broth (PDB), thiourea, DTT, CHAPS, SDS, and 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA) were purchased from Sigma. PMSF, acrylamide, bisacrylamide, TEMED, ammonium persulfate, β-mercaptoethanol, and Nonidet P-40 were from Amresco (Solon, OH). Two-dimensional SDS-PAGE standard was from Bio-Rad. Sequencing grade trypsin was from Promega (Madison, WI). Water was prepared using a Milli-Q system (Millipore, Bedford, MA). The fungal pathogen P. expansum Link (CGMCC3.3703) was used in this study. The fungus was inoculated and reisolated from apple fruit to maintain pathogenicity. The isolates were routinely grown on potato dextrose agar plates for 14 days at 23 °C. Spores were collected by adding sterile distilled water containing 0.05% (v/v) Tween 80 to the surface of the culture and gently scrubbing with a sterile spatula. Spore suspensions were obtained by filtering through four layers of sterile cheesecloth to remove any hyphal fragments (25Qin G.Z. Tian S.P. Enhancement of biocontrol activity of Cryptococcus laurentii by silicon and the possible mechanisms involved..Phytopathology. 2005; 95: 69-75Crossref PubMed Scopus (128) Google Scholar). The inhibitory effect of borate on the growth of P. expansum was assayed in PDB medium using the method of López-García et al. (26López-García B. González-Candelas L. Pérez-Payá E. Marcos J.F. Identification and characterization of a hexapeptide with activity against phytopathogenic fungi that cause postharvest decay in fruits..Mol. Plant-Microbe Interact. 2000; 13: 837-846Crossref PubMed Scopus (77) Google Scholar) with minor modification. Briefly aliquots of a spore suspension of P. expansum were added to wells of a 24-well microtitration plate containing PDB medium to obtain a final concentration of 5 × 105 spores/ml. The culture medium was supplemented with borate at 0 or 0.1% (w/v) in the form of potassium tetraborate, and the pH value of the medium was adjusted to 5.8 with HCl. The concentration of potassium tetraborate was chosen based on a preliminary study (data not shown). The microtitration plate was incubated at 25 °C on a rotary shaker at 100 rpm. Spore germination and germ tube elongation were assayed microscopically after a 12–17-h incubation period. Each treatment was replicated three times, and the experiment was repeated twice. Spores of P. expansum (5 × 105 spores/ml) were cultured at 25 °C in 250-ml conical flasks containing 50 ml of PDB medium supplemented with 0 or 0.1% potassium tetraborate. Mycelia were collected after incubation for 48, 60, and 72 h and washed thoroughly with 10 mm potassium phosphate buffer, pH 7.0, to remove bound extracellular proteins and other contaminants. The intercellular proteins were extracted following the method of Kim et al. (27Kim S.T. Yu S. Kim S.G. Kim H.J. Kang S.Y. Hwang D.H. Jang Y.S. Kang K.Y. Proteome analysis of rice blast fungus (Magnaporthe grisea) proteome during appressorium formation..Proteomics. 2004; 4: 3579-3587Crossref PubMed Scopus (62) Google Scholar) and Nandakumar and Marten (23Nandakumar M.P. Marten M.R. Comparison of lysis methods and preparation protocols for one- and two-dimensional electrophoresis of Aspergillus oryzae intracellular proteins..Electrophoresis. 2002; 23: 2216-2222Crossref PubMed Scopus (47) Google Scholar). Mycelia were lysed by sonification (with cooling on ice) with 1 ml of lysis buffer containing 0.5 m Tris-HCl, pH 8.3, 2% (v/v) Nonidet P-40, 20 mm MgCl2, 2% (v/v) β-mercaptoethanol, and 1 mm PMSF. The cell debris were removed by centrifugation (16,000 × g for 20 min at 4 °C), and the supernatant was precipitated for 30 min at 4 °C with ice-cold TCA at a final concentration of 10% (w/v). The proteins were collected by centrifugation (16,000 × g for 45 min at 4 °C) and washed three times with cold acetone to remove remaining TCA. The precipitate was finally solubilized in 300 μl of thiourea/urea lysis buffer (2 m thiourea, 7 m urea, 4% (w/v) CHAPS, 1% (w/v) dithiothreitol, and 2% (v/v) carrier ampholytes, pH 3–10) and either used immediately or stored at −80 °C until use. Spores of P. expansum were collected from potato dextrose agar plate and cultured in 250-ml conical flasks containing 50 ml of YNB medium supplemented with 0 or 0.1% potassium tetraborate. YNB instead of PDB was used to avoid the contamination of proteins from PDB. Galacturonate at 0.2% was added to the culture medium to induce pectinase synthesis (28Kazemi-Pour N. Condemine G. Hugouvieux-Cotte-Pattat N. The secretome of the plant pathogenic bacterium Erwinia chrysanthemi..Proteomics. 2004; 4: 3177-3186Crossref PubMed Scopus (145) Google Scholar), and the pH of the medium was adjusted to 5.8 with NaOH. The fungus was grown on a rotary shaker at 200 rpm for 7 days at 25 °C with an initial concentration of 1 × 106 spores/ml. After incubation, hyphae in the suspension were removed by filtration through 0.2-μm-pore size membrane. The extracellular proteins in the cell-free filtrate were precipitated for 30 min with ice-cold 20% (w/v) TCA (29Nouwens A.S. Willcox M.D.P. Walsh B.J. Cordwell S.J. Proteomic comparison of membrane and extracellular proteins from invasive (PAO1) and cytotoxic (6206) strains of Pseudomonas aeruginosa..Proteomics. 2002; 2: 1325-1346Crossref PubMed Scopus (82) Google Scholar). The precipitates were harvested by centrifugation at 16,000 × g for 45 min at 4 °C. The supernatant was discarded, and the protein pellet was washed three times with −20 °C acetone. After air drying, the proteins were solubilized in 200 μl lysis buffer as described for the intracellular protein fraction. Two-dimensional (2D) gel electrophoresis was carried out according to the method of Abbasi and Komatsu (30Abbasi F.M. Komatsu S. A proteomic approach to analyze salt-responsive proteins in rice leaf sheath..Proteomics. 2004; 4: 2072-2081Crossref PubMed Scopus (232) Google Scholar) with minor modification. The IEF gel mixture contained 8 m urea, 3.3% (w/v) acrylamide, 0.19% (w/v) bisacrylamide, 2% (v/v) Nonidet P-40, 5% (v/v) carrier ampholytes (pH 3.5–10.0:pH 5.0–8.0 = 1:1), 0.015% (w/v) ammonium persulfate, and 0.1% (v/v) TEMED. The gels were polymerized in glass tubes (Daiichi Pure Chemicals, Tokyo, Japan) to obtain gels of 13.5-cm length and 3-mm diameter. Approximately 500 μg of intracellular proteins and 400 μg of extracellular proteins determined by the method of Bradford (31Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding..Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) were applied to the gel. IEF was performed at 200 V for 30 min, 400 V for 15 h, and then 800 V for 1 h. The cathode buffer was 20 mm NaOH, and the anode buffer was 20 mm H3PO4. Following IEF, gels were incubated in equilibration buffer containing 10% (v/v) glycerol, 2.5% (w/v) SDS, 125 mm Tris-HCl (pH 6.8), and 5% (v/v) β-mercaptoethanol with gentle agitation at room temperature for 15 min with two changes. SDS-PAGE in the second dimension was conducted using 15% polyacrylamide gels with 5% stacking gels. The gel strip of the first dimension was placed onto the stacking gel and sealed with 0.5% agarose melted in equilibration buffer without β-mercaptoethanol. The running buffer contained 25 mm Tris (pH 8.3), 195 mm glycine, and 0.1% (w/v) SDS. The gels were stained with colloidal Coomassie Brilliant Blue (CBB) R-250 solution containing 50% (v/v) methanol, 15% (v/v) acetic acid, and 0.1% (w/v) CBB R-250. The pI and Mr of each protein was calibrated using 2D electrophoresis markers. The colloidal CBB-stained gels were scanned using a flatbed scanner (Amersham Biosciences) with 300 dpi resolution and saved in TIF format. Comparison of protein expression in 2D gel images was performed using Image Master 2D Elite software (Amersham Biosciences). To account for experimental variation, at least triplicate gels, resulting from protein extracts obtained from independent experiments, were analyzed for each treatment. Spot detection was carried out automatically, and those spots showing faint intensity near the detection limit of colloidal CBB were not included in the comparisons. Prior to automatic matching of spots between gel images, one gel was selected as the reference gel of each treatment. The amount of a protein spot was calculated based on the volume of that spot. To reflect the quantitative variations in intensity of protein spots between control and treated samples, the spot volume was normalized as a percentage of the total volume of all spots on the corresponding gel. Statistical analysis of the data was performed using SPSS software (SPSS Inc., Chicago, IL). The normalized intensity of spots on three replicate 2D gels was averaged, and the standard deviation was calculated for each treatment. A two-tailed nonpaired Student's t test was used to determine whether the relative change was statistically significant between control and borate-treated samples. Only spots that changed significantly in averaged normalized spot volume were excised for protein identification. In-gel digestion was performed as described by Shen et al. (32Shen S. Jing Y. Kuang T. Proteomics approach to identify wound-response related proteins from rice leaf sheath..Proteomics. 2003; 3: 527-535Crossref PubMed Scopus (127) Google Scholar). Coomassie Blue-stained protein spots were manually excised from the gels and cut in about 1-mm2 pieces. Gel slices were destained with 50 mm NH4HCO3 in 50% (v/v) methanol for 1 h at 40 °C. The step was repeated until the color disappeared. Gel particles were then mixed with 10 mm DTT in 100 mm NH4HCO3 for 1 h at 60 °C to reduce the proteins. The gels were dried in a vacuum centrifuge for 30 min prior to incubating with 40 mm iodoacetamide in 100 mm NH4HCO3 for 30 min at ambient temperature in the dark to alkylate the proteins. The gel pieces were washed several times with water and completely dried in a vacuum centrifuge. Enzymatic digestion was carried out by adding gel pieces into the digestion buffer containing 100 mm NH4HCO3 and 5 ng/μl trypsin. The reaction mixture was kept at 37 °C for 16 h. Digested peptides were extracted by three changes of 0.1% TFA in 50% acetonitrile. The collected solutions were concentrated to 10 μl and then desalted with ZipTipC18 (Millipore). Peptides were eluted from the column in 2 μl of 0.1% TFA in 50% acetonitrile. ESI-MS/MS was performed for the purified tryptic digests using a quadrupole time-of-flight mass spectrometer (Q-TOF-2; Micromass, Altrincham, UK) equipped with a z-spray source (29Nouwens A.S. Willcox M.D.P. Walsh B.J. Cordwell S.J. Proteomic comparison of membrane and extracellular proteins from invasive (PAO1) and cytotoxic (6206) strains of Pseudomonas aeruginosa..Proteomics. 2002; 2: 1325-1346Crossref PubMed Scopus (82) Google Scholar). Before loading the digested peptide samples, the instrument was externally calibrated using the fragmentation spectrum of the doubly charged 1571.68-Da (785.84 m/z) ion of fibrinopeptide B. The peptides were loaded by nanoelectrospray with gold-coated borosilicate glass capillaries (Micromass). The applied spray voltage was 800 V with a sample cone working on 30 V. Dependent on the mass and charge state of the peptides, the collision energy was varied from 14 to 40 V. Peptide precursor ions were acquired over the m/z range 400–1900 Da in TOF-MS mode. Multiply charged (2+ and 3+) ions rising above predefined threshold intensity were automatically selected for MS/MS analysis, and product ion spectra were collected from m/z 50–2000. Tandem MS data were processed using MaxEnt 3.0 (Micromass) to create peak lists. The generated peak lists were uploaded to Mascot MS/MS Ions Search program (Mascot version 2.0) on the Matrix Science public web site, and protein identification was performed against the National Center for Biotechnology Information non-redundant (NCBInr) protein databases (version, April 14, 2006; 3,570,920 sequences) or EST database (version, April 29, 2006; 140,695,050 sequences) with a taxonomy restriction to "Other Fungi." Trypsin was specified as the proteolytic enzyme, and one missed cleavages was allowed. Variable modifications selected for searching included carbamidomethylation of cysteine, oxidation of methionine, and N-terminal pyroglutamine. A peptide tolerance of ±2.0 Da for the precursor ions and an MS/MS tolerance of ±1.2 Da for the fragment ions were set (33Taylor N.L. Heazlewood J.L. Day D.A. Millar A.H. Differential impact of environmental stresses on the pea mitochondrial proteome..Mol. Cell. Proteomics. 2005; 4: 1122-1133Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Peptide charges of +2 and +3 and monoisotopic mass was chosen, and the instrument type was set to ESI-QUAD-TOF. Mascot uses a probability-based "Mowse score" to evaluate data obtained from tandem mass spectra. Mowse scores were reported as −10 × log10(p) where p is the probability that the observed match between the experimental data and the database sequence is a random event. This means that the best match is the one with the highest score. Mowse scores greater than 44 were considered significant (p < 0.05). To confirm protein identification, a minimum of three observed peptides that were selected for MS/MS was required if the proteins were identified across species. For proteins that were identified with MS/MS spectra matched to less than three peptides from proteins in other species, MS/MS spectra were subjected to de novo sequence analysis using PEAKS Version 4.0 software (Bioinformatics Solutions Inc., Waterloo, Ontario, Canada), and the generated peptide sequences were used for homology searching using available on-line tools (MS BLAST, European Molecular Biology Laboratory) as described by Shevchenko et al. (34Shevchenko A. Sunyaev S. Loboda A. Shevchenko A. Bork P. Ens W. Standing K.G. Charting the proteomes of organisms with unsequenced genomes by MALDI-Quadrupole Time-of-Flight mass spectrometry and BLAST homology searching..Anal. 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The oxidant-sensitive probe DCHF-DA was used to assess the intracellular ROS levels in P. expansum according to the methods described previously (36Chen C. Dickman M.B. Proline suppresses apoptosis in the fungal pathogen Colletotrichum trifolii..Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3459-3464Crossref PubMed Scopus (370) Google Scholar, 37Chen C. Dickman M.B. Dominant active Rac and dominant negative Rac revert the dominant active Ras phenotype in Colletotrichum trifolii by distinct signalling pathways..Mol. Microbiol. 2004; 51: 1493-1507Crossref PubMed Scopus (72) Google Scholar). Spores of P. expansum were cultured in PDB medium supplemented with 0 or 0.1% potassium tetraborate as described above and collected after 2, 4, and 6 h of incubation. The spores were washed with 10 mm potassium phosphate buffer (pH 7.0) and incubated for 1 h in the same buffer containing 10 μm DCHF-DA (dissolved in dimethyl sulfoxide). After washing twice with potassium phosphate buffer, spores were
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