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John D. Williamson - One of the best experts on this subject based on the ideXlab platform.
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Tomato Plants Overexpressing a Celery Mannitol Dehydrogenase (MTD) Have Decreased Susceptibility to Botrytis cinerea
American Journal of Plant Sciences, 2015Co-Authors: Takshay K. Patel, Sergei F. Krasnyanski, George C. Allen, Frank J. Louws, Dilip R. Panthee, John D. WilliamsonAbstract:The oxidative burst is a critical early event in plant-pathogen interactions that leads to a localized, programmed cell death (PCD) called the hypersensitive response (HR). The HR and associated PCD retard infection by biotrophic pathogens, but can, in fact, enhance infection by necrotrophic pa- thogens like Botrytis cinerea. In addition to signaling the induction of the HR, reactive oxygen spe- cies (ROS) produced during the oxidative burst are antimicrobial. We hypothesize that pathogens such as B. cinerea survive the antimicrobial effects of ROS, at least partially by secreting the anti- oxidant Mannitol during infection. This is supported by the previous observation that overexpres- sion of the catabolic enzyme Mannitol Dehydrogenase (MTD) can decrease a plants susceptibility to Mannitol-secreting pathogens like B. cinerea. To extend the above hypothesis, and test the gen- eral utility of this approach in an important horticultural crop, we overexpressed celery MTD in tomato (Solanum lycopersicum cv. "Moneymaker"). In these studies, we observed a significant in- crease (up to 90%) in resistance to B. cinerea in transgenic tomatoes expressing high amounts of MTD.
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Progeny of Selfed Plants from Tomato Breeding Line ‘NC1 Grape’ Overexpressing Mannitol Dehydrogenase (MTD) Have Increased Resistance to the Early Blight Fungus, Alternaria solani
Plant Health Progress, 2015Co-Authors: Takshay K. Patel, Sergei F. Krasnyanski, George C. Allen, Frank J. Louws, Dilip R. Panthee, John D. WilliamsonAbstract:Reactive oxygen species (ROS) made by plants in response to pathogen infection not only initiate local and systemic defenses, they are also antimicrobial. A number of fungi are hypothesized to secrete the antioxidant Mannitol to protect against this antimicrobial ROS during infection. This hypothesis is supported by reports that overexpression of the Mannitol catabolic enzyme Mannitol Dehydrogenase (MTD) in plants increases resistance to Mannitol-secreting pathogens like Botrytis cinerea and Alternaria alternata. To extend this hypothesis and test the general utility of this approach, we overexpressed celery MTD in a tomato breeding line (NC1 Grape) currently used in our program. Although we reported earlier that MTD overexpression provides resistance to Botrytis gray mold in a greenhouse tomato, this is the first report of overexpression in an elite breeding variety providing heritable, whole-plant resistance to A. solani (tomato early blight). In this study, progeny from a high-MTD-expressing line had infection rates <65% those of nontransformed plants, and transformants outgrew infection by 7 days post-inoculation. Finally, our results suggest that screening for higher innate MTD expression in plants, rather than screening solely for the presence of the Mtd gene, might be a more effective way to identify parental lines for use in conventional breeding of early blight resistance. Accepted for publication 7 August 2015. Published 14 August 2015.
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Overexpression of Mannitol Dehydrogenase in zonal geranium confers increased resistance to the Mannitol secreting fungal pathogen Botrytis cinerea
Plant Cell Tissue and Organ Culture (PCTOC), 2013Co-Authors: John D. Williamson, Aparna Desai, Sergei F. Krasnyanski, Wei-wen Guo, Thanh-tuyen Nguyen, Heather A. Olson, John M Dole, Fei Ding, George C. AllenAbstract:The sugar alcohol Mannitol is a carbohydrate with well-documented roles in both metabolism and osmoprotection in plants and fungi. In addition, however, Mannitol is an antioxidant, and current research suggests that pathogenic fungi can secrete Mannitol into the plant’s extracellular spaces during infection to suppress reactive oxygen-mediated host defenses. In response to pathogen attack, plants have been shown to secrete the normally symplastic enzyme, Mannitol Dehydrogenase (MTD). Given that MTD converts Mannitol to the sugar mannose, extracellular MTD may be an important defense against Mannitol-secreting fungal pathogens. Previous work demonstrated that overexpression of MTD in tobacco did, in fact, provide increased resistance to the Mannitol-secreting fungal pathogen Alternaria alternata. In the present work we demonstrate that the fungal pathogen Botrytis cinerea also can secrete Mannitol, and that overexpression of MTD in zonal geranium (Pelargonium × hortorum) in turn provides increased resistance to B. cinerea. These results are not only an important validation of previous work, but support the idea that MTD-overexpression might be used to engineer a broad variety of plants for resistance to Mannitol-secreting fungal pathogens like B. cinerea for which specific resistance is lacking.
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data independent liquid chromatography mass spectrometry lc mse detection and quantification of the secreted apium graveolens pathogen defense protein Mannitol Dehydrogenase
Rapid Communications in Mass Spectrometry, 2010Co-Authors: Kevin Blackburn, Fang-yi Cheng, John D. Williamson, Michael B. GosheAbstract:Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, Mannitol Dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol Mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete Mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted Mannitol. In the current study, LC/MSE was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MSE can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis. Copyright © 2010 John Wiley & Sons, Ltd.
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Data‐independent liquid chromatography/mass spectrometry (LC/MSE) detection and quantification of the secreted Apium graveolens pathogen defense protein Mannitol Dehydrogenase
Rapid communications in mass spectrometry : RCM, 2010Co-Authors: Kevin Blackburn, Fang-yi Cheng, John D. Williamson, Michael B. GosheAbstract:Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, Mannitol Dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol Mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete Mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted Mannitol. In the current study, LC/MSE was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MSE can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis. Copyright © 2010 John Wiley & Sons, Ltd.
D. Mason Pharr - One of the best experts on this subject based on the ideXlab platform.
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Salicylic acid stimulates secretion of the normally symplastic enzyme Mannitol Dehydrogenase: a possible defense against Mannitol-secreting fungal pathogens
Planta, 2009Co-Authors: Fang-yi Cheng, E. Zamski, D. Mason Pharr, John D. WilliamsonAbstract:The sugar alcohol Mannitol is an important carbohydrate with well-documented roles in both metabolism and osmoprotection in many plants and fungi. In addition to these traditionally recognized roles, Mannitol is reported to be an antioxidant and as such may play a role in host–pathogen interactions. Current research suggests that pathogenic fungi can secrete Mannitol into the apoplast to suppress reactive oxygen-mediated host defenses. Immunoelectron microscopy, immunoblot, and biochemical data reported here show that the normally symplastic plant enzyme, Mannitol Dehydrogenase (MTD), is secreted into the apoplast after treatment with the endogenous inducer of plant defense responses salicylic acid (SA). In contrast, a cytoplasmic marker protein, hexokinase, remained cytoplasmic after SA-treatment. Secreted MTD retained activity after export to the apoplast. Given that MTD converts Mannitol to the sugar mannose, MTD secretion may be an important component of plant defense against Mannitol-secreting fungal pathogens such as Alternaria . After SA treatment, MTD was not detected in the Golgi apparatus, and its SA-induced secretion was resistant to brefeldin A, an inhibitor of Golgi-mediated protein transport. Together with the absence of a known extracellular targeting sequence on the MTD protein, these data suggest that a plant’s response to pathogen challenge may include secretion of selected defensive proteins by as yet uncharacterized, non-Golgi mechanisms.
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Constitutive expression of a celery Mannitol Dehydrogenase in tobacco enhances resistance to the Mannitol‐secreting fungal pathogen Alternaria alternata
Plant Journal, 2002Co-Authors: Dianne B. Jennings, Margaret E. Daub, D. Mason Pharr, John D. WilliamsonAbstract:Summary Our previous observation that host plant extracts induce production and secretion of Mannitol in the tobacco pathogen Alternaria alternata suggested that, like their animal counterparts, plant pathogenic fungi might produce the reactive oxygen quencher Mannitol as a means of suppressing reactive oxygen- mediated plant defenses. The concurrent discovery that pathogen attack induced Mannitol Dehydrogenase (MTD) expression in the non-Mannitol-containing host tobacco suggested that plants, unlike animals, might be able to counter this fungal suppressive mechanism by catabolizing Mannitol of fungal origin. To test this hypothesis, transgenic tobacco plants constitutively expressing a celery Mtd cDNA were pro- duced and evaluated for potential changes in resistance to both Mannitol- and non-Mannitol-secreting pathogens. Constitutive expression of the MTD transgene was found to confer significantly enhanced resistance to A. alternata, but not to the non-Mannitol-secreting fungal pathogen Cercospora nicotianae. These results are consistent with the hypothesis that MTD plays a role in resistance to Mannitol-secreting fungal plant pathogens.
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Analysis of celery (Apium graveolens) Mannitol Dehydrogenase (Mtd) promoter regulation in Arabidopsis suggests roles for MTD in key environmental and metabolic responses
Plant Molecular Biology, 2001Co-Authors: E. Zamski, Wei-wen Guo, D. Mason Pharr, Yuri T. Yamamoto, John D. WilliamsonAbstract:Of the growing list of promising genes for plant improvement, some of the most versatile appear to be those involved in sugar alcohol metabolism. Mannitol, one of the best characterized sugar alcohols, is a significant photosynthetic product in many higher plants. The roles of Mannitol as both a metabolite and an osmoprotectant in celery ( Apium graveolens ) are well documented. However, there is growing evidence that `metabolites' can also have key roles in other environmental and developmental responses in plants. For instance, in addition to its other properties, Mannitol is an antioxidant and may have significant roles in plant-pathogen interactions. The Mannitol catabolic enzyme Mannitol Dehydrogenase (MTD) is a prime modulator of Mannitol accumulation in plants. Because the complex regulation of MTD is central to the balanced integration of Mannitol metabolism in celery, its study is crucial in clarifying the physiological role(s) of Mannitol metabolism in environmental and metabolic responses. In this study we used transformed Arabidopsis to analyze the multiple environmental and metabolic responses of the Mtd promoter. Our data show that all previously described changes in Mtd RNA accumulation in celery cells mirrored changes in Mtd transcription in Arabidopsis . These include up-regulation by salicylic acid, hexokinase-mediated sugar down-regulation, and down-regulation by salt, osmotic stress and ABA. In contrast, the massive up-regulation of Mtd expression in the vascular tissues of salt-stressed Arabidopsis roots suggests a possible role for MTD in Mannitol translocation and unloading and its interrelation with sugar metabolism.
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Roles for Mannitol and Mannitol Dehydrogenase in active oxygen-mediated plant defense
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Dianne B. Jennings, D. Mason Pharr, Marilyn Ehrenshaft, John D. WilliamsonAbstract:Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize Mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use Mannitol to suppress ROS-mediated plant defenses. Here we show induction of Mannitol production and secretion in the phytopathogenic fungus Alternaria alternata in the presence of host-plant extracts. Conversely, we show that the catabolic enzyme Mannitol Dehydrogenase is induced in a non-Mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing Mannitol of fungal origin.
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Characterization of NAD-dependent Mannitol Dehydrogenase from celery as affected by ions, chelators, reducing agents and metabolites
Plant Science, 1998Co-Authors: J.m.h. Stoop, John D. Williamson, Mark A. Conkling, John Mackay, D. Mason PharrAbstract:Abstract NAD-dependent Mannitol Dehydrogenase (MTD) from celery (Apium graveolens L. var. dulce (Mill.) Pers.) provides the initial step by which Mannitol is committed to central metabolism and plays a critical role in regulating Mannitol concentration in the plant. The pH optimum for Mannitol oxidation occurs at pH 9.5 whereas the optimum for mannose reduction occurs at pH 6.5. Michaelis–Menten kinetics were exhibited for Mannitol and NAD with Km values of 64 and 0.14 mM, respectively at pH 9.5. The Km for mannose and NADH were 745 mM and 1.27 μM, respectively at pH 6.5. The high Km for mannose is consistent with a reaction in situ favoring Mannitol oxidation rather than mannose reduction. The observed down-regulation of MTD in salt stressed celery is not due to a direct inhibition by NaCl or macronutrients. Inhibition by the chelator 1,10-phenanthroline suggests that zinc is required for MTD activity. Reducing agents DTT, DTE and β-mercaptoethanol inactivated MTD reversibly. At pH 7.0, ADP and to a lesser extend AMP and ATP were competitive inhibitors, with respect to NAD, having apparent Ki’s of 0.24, 0.64 and 1.10 mM, respectively.
David M. Pharr - One of the best experts on this subject based on the ideXlab platform.
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Subcellular localization of celery Mannitol Dehydrogenase. A cytosolic metabolic enzyme in nuclei.
Plant physiology, 1997Co-Authors: Y. T. Yamamoto, E. Zamski, John D. Williamson, Mark A. Conkling, David M. PharrAbstract:Mannitol Dehydrogenase (MTD) is the first enzyme in Mannitol catabolism in celery (Apium graveolens L. var dulce [Mill] Pers. cv Florida 638). Mannitol is an important photoassimilate, as well as providing plants with resistance to salt and osmotic stress. Previous work has shown that expression of the celery Mtd gene is regulated by many factors, such as hexose sugars, salt and osmotic stress, and salicylic acid. Furthermore, MTD is present in cells of sink organs, phloem cells, and Mannitol-grown suspension cultures. Immunogold localization and biochemical analyses presented here demonstrate that celery MTD is localized in the cytosol and nuclei. Although the cellular density of MTD varies among different cell types, densities of nuclear and cytosolic MTD in a given cell are approximately equal. Biochemical analyses of nuclear extracts from Mannitol-grown cultured cells confirmed that the nuclear-localized MTD is enzymatically active. The function(s) of nuclear-localized MTD is unknown.
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Sugar Repression of Mannitol Dehydrogenase Activity in Celery Cells
Plant physiology, 1997Co-Authors: Rogerio Prata, John D. Williamson, Mark A. Conkling, David M. PharrAbstract:We present evidence that the activity of the Mannitol-catabolizing enzyme Mannitol Dehydrogenase (MTD) is repressed by sugars in cultured celery (Apium graveolens L.) cells. Furthermore, this sugar repression appears to be mediated by hexokinases (HKs) in a manner comparable to the reported sugar repression of photosynthetic genes. Glucose (Glc)-grown cell cultures expressed little MTD activity during active growth, but underwent a marked increase in MTD activity, protein, and RNA upon Glc starvation. Replenishment of Glc in the medium resulted in decreased MTD activity, protein, and RNA within 12 h. Addition of mannoheptulose, a competitive inhibitor of HK, derepressed MTD activity in Glc-grown cultures. In contrast, the addition of the sugar analog 2-deoxyglucose, which is phosphorylated by HK but not further metabolized, repressed MTD activity in Mannitol-grown cultures. Collectively, these data suggest that HK and sugar phosphorylation are involved in signaling MTD repression. In vivo repression of MTD activity by galactose (Gal), which is not a substrate of HK, appeared to be an exception to this hypothesis. Further analyses, however, showed that the products of Gal catabolism, Glc and fructose, rather than Gal itself, were correlated with MTD repression.
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Immunolocalization of Mannitol Dehydrogenase in celery plants and cells.
Plant physiology, 1996Co-Authors: E. Zamski, John D. Williamson, Mark A. Conkling, Y. T. Yamamoto, David M. PharrAbstract:Immunolocalization of Mannitol Dehydrogenase (MTD) in celery (Apium graveolens L.) suspension cells and plants showed that MTD is a cytoplasmic enzyme. MTD was found in the meristems of celery root apices, in young expanding leaves, in the vascular cambium, and in the phloem, including sieve-element/companion cell complexes, parenchyma, and in the exuding phloem sap of cut petioles. Suspension cells that were grown in medium with Mannitol as the sole carbon source showed a high anti-MTD cross-reaction in the cytoplasm, whereas cells that were grown in sucrose-containing medium showed little or no cross-reaction. Gel-blot analysis of proteins from vascular and nonvascular tissues of mature celery petioles showed a strong anti-MTD sera cross-reactive band, corresponding to the 40-kD molecular mass of MTD in vascular extracts, but no cross-reactive bands in nonvascular extracts. The distribution pattern of MTD within celery plants and in cell cultures that were grown on different carbon sources is consistent w ith the hypothesis that the Mtd gene may be regulated by sugar repression. Additionally, a developmental component may regulate the distribution of MTD within celery plants.
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Substrate stereospecificity of the NAD-dependent Mannitol Dehydrogenase from celery
Phytochemistry, 1996Co-Authors: J.m.h. Stoop, William Scott Chilton, David M. PharrAbstract:Abstract The NAD-dependent Mannitol Dehydrogenase (MTD) of celery catalyses the interconversion of d -Mannitol and d -mannose. This 1-oxidoreductase is uniquely different from all NAD-dependent polyol Dehydrogenases described to date, which are 2-oxidoreductases. The stereospecificity of Mannitol Dehydrogenase was tested in the oxidative direction in the presence of polyol and NAD cofactor and in the reductive direction in the presence of aldose and NADH. The enzyme would be expected to show the same stereospecificity in either direction. The stereospecificity in the reductive direction was tested by attempted reduction of all eight d - and l -pentoses and 15 of the 16 d - and l -hexoses. Stereospecificity in the oxidative direction was tested with the four pentitols and four of the hexitols. Mannitol Dehydrogenase showed a marked preference for aldopentose and aldohexose substrates with the same absolute configuration at C-2 as that of d -mannose. Reduction of l -dose by Mannitol Dehydrogenase was the only exception to the stated stereochemical preference among 23 aldoses and eight alditols tested. The sugar d -threose that occurs rarely in nature is a competitive inhibitor ( K i = 18 mM) of Mannitol oxidation. The physiologically important hexitols, galactitol and glucitol, are oxidized by MTD to aldoses that are not metabolized by higher plants.
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Sequence analysis of a Mannitol Dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: John D. Williamson, J.m.h. Stoop, Mark A. Conkling, M. O. Massel, David M. PharrAbstract:Abstract Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms Mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of Mannitol as an osmoprotectant. A Mannitol catabolic enzyme that oxidizes Mannitol to mannose (Mannitol Dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue Mannitol concentration are inversely related. MTD provides the initial step by which translocated Mannitol is committed to central metabolism and, by regulating Mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on Mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize Mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling Mannitol pool size reflect the importance of Mannitol metabolism in plant responses to divergent types of environmental stress.
Michael B. Goshe - One of the best experts on this subject based on the ideXlab platform.
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data independent liquid chromatography mass spectrometry lc mse detection and quantification of the secreted apium graveolens pathogen defense protein Mannitol Dehydrogenase
Rapid Communications in Mass Spectrometry, 2010Co-Authors: Kevin Blackburn, Fang-yi Cheng, John D. Williamson, Michael B. GosheAbstract:Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, Mannitol Dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol Mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete Mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted Mannitol. In the current study, LC/MSE was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MSE can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis. Copyright © 2010 John Wiley & Sons, Ltd.
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Data‐independent liquid chromatography/mass spectrometry (LC/MSE) detection and quantification of the secreted Apium graveolens pathogen defense protein Mannitol Dehydrogenase
Rapid communications in mass spectrometry : RCM, 2010Co-Authors: Kevin Blackburn, Fang-yi Cheng, John D. Williamson, Michael B. GosheAbstract:Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, Mannitol Dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol Mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete Mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted Mannitol. In the current study, LC/MSE was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MSE can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis. Copyright © 2010 John Wiley & Sons, Ltd.
Fang-yi Cheng - One of the best experts on this subject based on the ideXlab platform.
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data independent liquid chromatography mass spectrometry lc mse detection and quantification of the secreted apium graveolens pathogen defense protein Mannitol Dehydrogenase
Rapid Communications in Mass Spectrometry, 2010Co-Authors: Kevin Blackburn, Fang-yi Cheng, John D. Williamson, Michael B. GosheAbstract:Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, Mannitol Dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol Mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete Mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted Mannitol. In the current study, LC/MSE was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MSE can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis. Copyright © 2010 John Wiley & Sons, Ltd.
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Data‐independent liquid chromatography/mass spectrometry (LC/MSE) detection and quantification of the secreted Apium graveolens pathogen defense protein Mannitol Dehydrogenase
Rapid communications in mass spectrometry : RCM, 2010Co-Authors: Kevin Blackburn, Fang-yi Cheng, John D. Williamson, Michael B. GosheAbstract:Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, Mannitol Dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol Mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete Mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted Mannitol. In the current study, LC/MSE was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MSE can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis. Copyright © 2010 John Wiley & Sons, Ltd.
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Salicylic acid stimulates secretion of the normally symplastic enzyme Mannitol Dehydrogenase: a possible defense against Mannitol-secreting fungal pathogens
Planta, 2009Co-Authors: Fang-yi Cheng, E. Zamski, D. Mason Pharr, John D. WilliamsonAbstract:The sugar alcohol Mannitol is an important carbohydrate with well-documented roles in both metabolism and osmoprotection in many plants and fungi. In addition to these traditionally recognized roles, Mannitol is reported to be an antioxidant and as such may play a role in host–pathogen interactions. Current research suggests that pathogenic fungi can secrete Mannitol into the apoplast to suppress reactive oxygen-mediated host defenses. Immunoelectron microscopy, immunoblot, and biochemical data reported here show that the normally symplastic plant enzyme, Mannitol Dehydrogenase (MTD), is secreted into the apoplast after treatment with the endogenous inducer of plant defense responses salicylic acid (SA). In contrast, a cytoplasmic marker protein, hexokinase, remained cytoplasmic after SA-treatment. Secreted MTD retained activity after export to the apoplast. Given that MTD converts Mannitol to the sugar mannose, MTD secretion may be an important component of plant defense against Mannitol-secreting fungal pathogens such as Alternaria . After SA treatment, MTD was not detected in the Golgi apparatus, and its SA-induced secretion was resistant to brefeldin A, an inhibitor of Golgi-mediated protein transport. Together with the absence of a known extracellular targeting sequence on the MTD protein, these data suggest that a plant’s response to pathogen challenge may include secretion of selected defensive proteins by as yet uncharacterized, non-Golgi mechanisms.