Crucial Role of Antioxidant Proteins and Hydrolytic Enzymes in Pathogenicity of Penicillium expansum

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, 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 Full PubMed Scopus Google Scholar). against ROS, pathogens have defense including catalase and superoxide as as the as glutathione and and the of S. PubMed Scopus Google Scholar, P. P. W. The molecular against reactive oxygen species in Microbiol. PubMed Scopus Google Scholar). B. Oxidative stress is in cell in Saccharomyces S. PubMed Scopus Google that yeast for the catalase, superoxide and were more to the oxidative stress the that antioxidant important in oxidative stress by defense from in the site of invasion, a pathogen the host by the cell of the which is of a of proteins of the plant pathogen a in this and biochemical secretory proteins have as G. J. The and the two of in PubMed Google Scholar, J. J. of the of PubMed Google and W. of three in for the Microbiol. PubMed Scopus Google Scholar, W. a with a is with the other PubMed Scopus Google Scholar). However, have mainly on the and of on the analysis of fungal secretory proteins are of proteins from 2005; 5: PubMed Scopus Google Scholar). In proteomics analysis has to a for the of protein expression in response to various in yeast proteome analysis of the yeast response to the 2005; 5: PubMed Scopus Google Scholar, G. analysis of Saccharomyces under stress by 2003; PubMed Scopus Google Scholar, K. G. S. J. proteome analysis of the response in Saccharomyces cerevisiae..J. Biol. Chem. Full Full PubMed Scopus Google and of proteins by in using and 2004; 4: PubMed Scopus Google Scholar, S. S. S. B. on stress response of 2005; 5: PubMed Scopus Google Scholar, Proteomic analysis of proteins expressed by under oxidative 2005; 5: PubMed Scopus Google Scholar, J. M. protein expression by to various PubMed Scopus Google Scholar). the other is a of about proteomics analysis from filamentous fungi of methods and for and of PubMed Scopus Google Scholar, M. M. J. and search in the analysis of proteins from the 2005; 5: PubMed Scopus Google Scholar). P. expansum is at the protein level. In a study, we that which are widely in the the virulence of P. expansum in various fruits. are plant that plant growth and have used in industry and as a for of and However, is about using to fruit In the study, analysis of the cellular and extracellular proteome in P. expansum under condition and borate stress to the pathogenesis of this fungal pathogen. to the antioxidant proteins and hydrolytic that are differentially expressed upon exposure to borate. this is the for virulence proteins in P. expansum on the of proteomics 0.67% yeast nitrogen base without amino acids plus 2% dextrose from from from dextrose and 2′,7′-dichlorodihydrofluorescein were from TEMED, and were from from from using a The fungal pathogen P. expansum used in this The and from fruit to The were on potato dextrose for at were by to the of the culture and with a were by of to Enhancement of biocontrol of by and the 2005; PubMed Scopus Google Scholar). The of borate on the growth of P. expansum in using the of B. E. and of a with against fungi that cause decay in PubMed Scopus Google with of a of P. expansum were to of a to a of The culture with borate at in the of potassium and the of the to with The of potassium on a The at on a at and were after a three and the of P. expansum were at in of with potassium were after for and and with potassium to extracellular proteins and other The proteins were the of S. analysis of rice proteome 2004; 4: PubMed Scopus Google and and of methods and for and of PubMed Scopus Google Scholar). were by on with of 2% 2% and The cell were by for at and the for at with at a of The proteins were by for at and three with to The in of and 2% and used at of P. expansum were from potato dextrose and in of with potassium of used to the contamination of proteins from at to the culture to synthesis G. The of the plant 2004; 4: PubMed Scopus Google and the of the to with The on a at for at with of in the were by The extracellular proteins in the were for with Proteomic of and extracellular proteins from and of PubMed Scopus Google Scholar). The were by at for at The and the protein three with the proteins were in as for the protein to the of and S. to proteins in rice 2004; 4: PubMed Scopus Google with The 2% and The were in to of and of proteins and of extracellular proteins by the of and for the of of protein the of PubMed Scopus Google were to the at for for and for The and the were in and with at for with two in the using with The of the the and with in without The and The were with Coomassie Brilliant and The and of protein using The were using a with and in of protein expression in using for at from protein from were for and spots the limit of were in the to of spots one as the of The of a protein on the of that the in of protein spots and the as a of the of spots on the analysis of the using The of spots on three and the for used to the significant and spots that in were for protein as by S. T. to related proteins from rice 2003; PubMed Scopus Google Scholar). Coomassie protein spots were from the and in about were with in for at The the were with in for at to the The were in a for to with in for at in the to the The were with and in a by the and The at for were by three of in The were to and with were from the in of in for the using a with a Proteomic of and extracellular proteins from and of PubMed Scopus Google Scholar). the the using the of the of B. The were by with The with a on on the and of the the from to were the in and were for and were from were using to The generated were to on the public and protein against the for protein with a to as the enzyme, and one for of of and of for the and of for the were of on the 2005; 4: Full Full PubMed Scopus Google Scholar). of and and and the to a to from were as is the that the observed the and the sequence is a This that the is the one with the were significant protein a of three observed that were for the proteins were identified proteins that were identified with to three from proteins in other were to sequence analysis using and the generated were used for using as by S. P. W. the proteomes of with by and Chem. PubMed Scopus Google with the search and the a on that are on the of and the of S. P. W. the proteomes of with by and Chem. PubMed Scopus Google Scholar, B. J. S. The and the of protein by sequence 2004; Full Full PubMed Scopus Google Scholar). The used to the ROS levels in P. expansum to the methods in the fungal pathogen S. 2005; PubMed Scopus Google Scholar, and the in by Microbiol. 2004; PubMed Scopus Google Scholar). of P. expansum were in with potassium as and after and of The were with potassium and for in the same in with potassium were under a using a proteins from P. expansum were using the and of the Detection E. T. and of protein in the of Biol. Chem. 2004; 279: Full Full PubMed Scopus Google Scholar, accumulation cellular stress and cellular proteins from damage by oxygen Biol. Chem. Full Full PubMed Scopus Google Scholar). of of proteins to of were to by with of for at The by the of of were by and to using The proteins were with and the used to the proteins in the to The of including the of the and sequence are in generated from the are as The of for proteins that were identified species with to three are as The of borate on the growth of P. expansum is in The culture with borate and to to the of on the of borate. It observed that borate at to and of P. expansum, and the by the of the to after for at under borate growth observed in culture without borate at the same of in the treatment, and were observed in culture with borate. protein expression of P. expansum under condition and borate stress were using and The of borate and the of were to that a for protein of proteins expression and at expansum in potato dextrose with and potassium for and at were from of the pathogen with by as under protein on and with Coomassie Brilliant The of protein to in protein spots were on of and after spots and spots with and of proteins expression repressed under borate stress were in this study, and we on differentially expressed protein spots with to the of sequence for this protein used which has to the to proteins from with the sequence S. by of proteins from PubMed Scopus Google to were for in search the one with the to further proteins were identified with the the molecular and for the differentially expressed proteins as as their levels of expression in the and The in is as with which the of from three the differentially expressed proteins, three are related to stress response, including glutathione S-transferase catalase and heat shock protein we that the expression levels of glutathione S-transferase and catalase were reduced were to borate. of the that proteins were also differentially expressed the of exposure to borate The of proteins in protecting from oxidative damage were by further analysis of ROS levels and protein carbonylation in to borate in the proteins, three were in basic including dihydroxy-acid and The other proteins were protein and protein and the protein by with to three and The that of proteins and identified by proteins that were identified by were against K. analysis of the of to use of of and expressed sequence 2003; PubMed Scopus Google that with analysis used to proteins from for which the is the with the of in the identified with this of proteins expression under borate stress using are significant protein of with to spots in and the in expressed as from three protein from of molecular and on amino sequence of the identified molecular and from the the species of the from for the of amino sequence for the identified with borate. in a are significant protein of with to spots in and the in expressed as from three protein from of molecular and on amino sequence of the identified molecular and from the the species of the from for the of amino sequence for the identified with borate. analysis of the extracellular proteome to the secretory protein of P. expansum and and in of protein were in compared with borate in three the contamination of proteins from the culture without amino acids to culture the The to to proteins for by Several methods for proteins from culture of P. expansum were In as in a and the were to we used to the secretory proteins in the to of extracellular proteins expression and as in The of protein to in extracellular protein spots on the have in the from to and a molecular and protein spots on after spots and spots with and analysis that proteins were differentially expressed under borate we of protein spots that of differentially expressed proteins and the were for in search protein proteins were identified with significant one with the It is that three protein spots were identified as the same in the in molecular and and indicating that of have expression repressed under borate In addition, proteins were identified as in basic including and we identified a cell synthesis protein and proteins with and proteins, and were identified species with to three analysis the of that were identified by were against and the that were for the protein spots and The of proteins of differentially expressed extracellular proteins under borate stress using to spots in and the in expressed as from three protein from of molecular and on amino sequence of the identified molecular and from the the species of the from for the of amino sequence for the identified with are significant are as of of with in a to spots in and the in expressed as from three protein from of molecular and on amino sequence of the identified molecular and from the the species of the from for the of amino sequence for the identified with borate. are significant are as of of with further the of catalase and glutathione S-transferase in scavenging ROS in we used a DCHF-DA, to ROS levels P. expansum to borate has used to ROS in yeast under oxidative stress in the fungal pathogen S. 2005; PubMed Scopus Google Scholar, and the in by Microbiol. 2004; PubMed Scopus Google Scholar). In study, of P. expansum were in the absence and presence of borate for at The were with DCHF-DA, and observed under a of the with DCHF-DA, that were (ROS) produced at that However, with the of ROS were in of P. expansum. with more under borate stress were with the DCHF-DA, indicating that of produced in to borate. The levels of ROS in are generated by metabolism 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 Full PubMed Scopus Google Scholar). that the levels of ROS in of P. expansum with borate are to the of expression of antioxidant enzymes, as catalase and glutathione S-transferase, borate Because the expression of two oxidative proteins, catalase and glutathione S-transferase, were repressed in the proteomics of the oxidative damage of cellular proteins in P. expansum by with carbonylation is a widely used of oxidative protein and has used as a for the of carbonylated proteins E. T. and of protein in the of Biol. Chem. 2004; 279: Full Full PubMed Scopus Google Scholar, accumulation cellular stress and cellular proteins from damage by oxygen Biol. Chem. Full Full PubMed Scopus Google Scholar, S. of in PubMed Scopus Google Scholar, M. J. T. protein in is by the of the J. PubMed Scopus Google Scholar). the of cellular proteins with the it is that protein carbonylation in and The levels of protein are caused by ROS generated in as E. E. P. E. Ros J. Oxidative stress specific protein damage in Saccharomyces cerevisiae..J. Biol. Chem. Full Full PubMed Google Scholar). protein were because are to oxidative damage. The levels of protein carbonylation the were to borate. These proteins in of molecular from to the of the exposure to borate the amounts of in cell proteins of were we that P. expansum were under of oxidative damage to the of expression of catalase and glutathione S-transferase by borate In this study, we proteins to virulence of P. expansum on the of analysis of cellular and extracellular proteomes under condition and borate differentially expressed proteins were identified in the cellular proteome identified proteins, the the reduced expression of two proteins, catalase and glutathione S-transferase which are in stress the that the of and has as one of the major in T. peroxide of plant catalase by S. PubMed Scopus Google Scholar). It is from to and has a of to to and in and S. J. S. B. of with superoxide PubMed Scopus Google Scholar, W.J. G. of extracellular by catalase the of the of a stress Biol. Chem. Full Full PubMed Scopus Google Scholar). glutathione S-transferase, the that compounds as by glutathione to toxic is important in protecting from oxidative stress for glutathione in the oxidative stress response in Biol. Chem. Full Full PubMed Scopus Google Scholar, M. B. glutathione with in the of and other products of J. PubMed Scopus Google Scholar, B. of in the by glutathione J. PubMed Scopus Google Scholar). These two antioxidant may act to against the ROS produced under oxidative In the study, the expression of two antioxidant defense proteins to repressed the to borate stress and that the of the P. expansum to ROS may under borate the ROS production in P. expansum to borate using the more were with the indicating that more ROS were produced in accumulation of ROS 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 Full PubMed Scopus Google Scholar). oxidative to proteins P. expansum under borate we further studied the of protein carbonylation using The of protein in compared with more proteins were in to borate. that borate causes oxidative damage to P. expansum by of the expression of the antioxidant catalase and glutathione These proteins may for the virulence of the fungal pathogen. proteomics has widely to the protein expression in under stress proteins are to in response to stress proteome analysis of the yeast response to the 2005; 5: PubMed Scopus Google Scholar, S. S. S. B. on stress response of 2005; 5: PubMed Scopus Google Scholar). However, the that the expression of two antioxidant defense proteins, catalase and glutathione S-transferase, were repressed in P. expansum after exposure to borate stress and This may by the in exposure of to in the G. analysis of Saccharomyces under stress by 2003; PubMed Scopus Google Scholar). The of antioxidant in the are to for a in of P. expansum were with borate for This is consistent with the of Proteomic analysis of proteins expressed by under oxidative 2005; 5: PubMed Scopus Google observed that the expression levels of antioxidant were in the after under oxidative we also that the of the catalase in proteomics analysis more at The expression of catalase repressed after exposure to borate for However, this after and of in borate, that catalase important in protecting P. expansum from protein oxidative damage at the of borate the other antioxidant proteins as glutathione S-transferase and superoxide may to against the ROS for glutathione in the oxidative stress response in Biol. Chem. Full Full PubMed Scopus Google Scholar, M. protein in yeast and superoxide Biol. Chem. 2004; 279: Full Full PubMed Scopus Google Scholar). the of catalase and glutathione S-transferase were the expression of in P. expansum under borate is a molecular with a variety of as protein and the of protein B. The and Full Full PubMed Scopus Google Scholar). of to other that damage cell proteins the expression of accumulation cellular stress and cellular proteins from damage by oxygen Biol. Chem. Full Full PubMed Scopus Google Scholar). In the study, we that is in the of proteins, which were produced upon exposure to borate It is that as the to borate to in the of toxic proteins, cell upon stress with the repressed of catalase and glutathione S-transferase, further the of cellular defense against In have on the molecular with which pathogens their of secretory proteins, hydrolytic enzymes, is to the pathogenesis of proteomics has as a for virulence J.C. of of PubMed Scopus Google Scholar, of proteomics to virulence that are for Microbiol. 2004; PubMed Scopus Google Scholar). the of secretory proteins of P. expansum under stress we the extracellular proteome of this pathogen. In the protein spots that were to differentially we identified a hydrolytic enzyme, polygalacturonase. protein spots with a in and were identified as the same This may as a of protein of proteins by in using and 2004; 4: PubMed Scopus Google Scholar). In the of pathogen and as a and virulence because the of virulence M. for the of a specific in the invasion and spread of in Environ. Microbiol. PubMed Google Scholar, W. J. The is for virulence of PubMed Google Scholar). the of to of the compounds that the plant in and the expression of reduced P. expansum to borate. proteins with as and were identified in the culture of P. expansum The occurrence of proteins, which are generally regarded as being of cellular to the of contamination by cell other T. K. analysis of the extracellular proteins from 2005; 5: PubMed Scopus Google Scholar). However, in other the proteins were also with the cell the J. K. that a protein with to PubMed Google Scholar, J.C. P. T. M. of major proteins of PubMed Scopus Google Scholar, a protein on the of Biol. Chem. Full Full PubMed Scopus Google Scholar). J.C. P. T. M. of major proteins of PubMed Scopus Google that of the major proteins as dehydrogenase, and in the pathogen proteins, is of In the of a protein on the of Biol. Chem. Full Full PubMed Scopus Google it to a virulence that to the infection of the Additionally two proteins and which significant to proteins with in the protein to may potential virulence and their to In we have a proteome analysis to find two antioxidant enzymes, catalase and glutathione S-transferase, in P. expansum the identified These two proteins may for the virulence of this fungal pathogen because the of expression of proteins in a of ROS and of carbonylated proteins in Additionally identified in the extracellular and expression repressed in the presence of borate. These may for the of and potential for for in the proteomics and for in the analysis of also for and of the