Progress 11/01/00 to 10/31/04
Outputs
The sugar alcohol mannitol is a significant photosynthetic product in many plants with well-documented roles as both a metabolite and an osmoprotectant. In addition to its other properties, mannitol is an antioxidant, and as such may play a significant role in host-pathogen interactions. Current research suggests that many pathogenic fungi secrete mannitol to suppress reactive oxygen-mediated host defenses. Our work further suggests that plants counter this by making MTD to catabolize mannitol of fungal origin. For this to be an effective defense the plant's pathogen-induced MTD must be co-localized with pathogen-produced mannitol. Yet work in this and other labs indicated that, while pathogen-secreted mannitol is most likely extracellular, in healthy plants MTD is cytosolic. Here we present evidence that the normally cytosolic enzyme MTD is exported into the extracellular space in response to salicylic acid. Given the absence of any previously identified extracellular
targeting sequence in MTD, these findings imply the existence of a previously unknown pathogen-activated protein secretion mechanism in plants.
Impacts
A pathogen-induced secretion mechanism distinct from the classical Golgi-mediated paradigm is of enormous significance for a number of reasons. First, although a number of stress and pathogen-induced nonclassical secretion mechanisms have been described in animals (cf. Nickel, 2003), none have been previously described in plants. Second, not only is this important to our basic understanding of how plants respond to microbial invasion, but identifying a new way to initiate rapid, specifically regulated secretion of an introduced protein by recruiting an existing/endogenous mechanism provides a superb tool for engineering new defenses. Finally, a means of secreting proteins "on demand" without impacting Golgi- mediated protein trafficking has implications for bioprocessing. Not only should a Golgi-independent secretion mechanism have less impact on normal cellular processes, but use of an endogenous mechanism that allows tightly regulated secretion of introduced gene
products may help facilitate the acceptance of engineered plants.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Mannitol, one of the best-characterized sugar alcohols, is a significant photosynthetic product in many higher plants. The roles of mannitol as both an osmoprotectant and a metabolite in celery and parsley are well documented. However, there is growing evidence that many "metabolites", and the enzymes mediating their synthesis and catabolism, also have key roles in environmental and developmental responses in plants. For instance, in addition to its other properties, mannitol is an antioxidant and may play a significant role in plant-pathogen interactions. Our work suggests that many plant pathogenic fungi make mannitol as an antioxidant to suppress reactive oxygen-mediated plant defenses. Conversely, plants might counter this suppression by synthesizing mannitol dehydrogenase (MTD) to catabolize mannitol of fungal origin. This was corroborated by our demonstration that over-expression of MTD in transgenic plants conferred resistance to the mannitol secreting fungus
Alternaria. For MTD to be an effective defense it must be co-localized with pathogen-produced mannitol. Work in other labs suggests that mannitol is localized in the intercellular space or apoplast. Yet our own work indicated that, in uninfected plants, MTD is cytosolic. Using antibodies previously produced in this lab, Dr. Eli Zamski of the Hebrew University showed that the mannitol catabolic enzyme MTD is localized in the apoplast of pathogen challenged plants. This was confirmed using protein-blotting techniques that revealed the appearance of a 36 kD MTD antibody cross-reacting protein in the medium of pathogen induced tobacco cells. Given the absence of an apoplastic-export sequence in MTD, these findings imply the existence of a previously unknown pathogen-activated protein secretion mechanism in plants.
Impacts
Fungal pathogens probably constitute the most economically devastating group of plant pathogens. Our results are consistent with the hypothesis that MTD can play a major role in promoting resistance to mannitol-secreting fungal plant pathogens. Given the scarcity of suitable genes presently available for engineering fungal resistance in plants, the identification of a new and effective single-gene resistance mechanism is of great potential benefit to the agronomic community.
Publications
- Williamson, J.D., Jennings, D.B., Ehrenshaft, M., Guo, W.-W., and Pharr, D.M. 2002. Sugar alcohols, salt stress, and fungal resistance: Polyols-multifunctional plant protection. J. Amer. Soc. Hort. Sci. 127:467-473.
- Barb, A.W., Pharr, D.M., and Williamson, J.D. 2003. Nicotiana tabacum culture selected for growth on mannose has elevated phosphomannose isomerase activity. Plant Science 165:639-648.
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Progress 10/01/01 to 09/30/02
Outputs
Some of the most versatile genes with potential for use in plant improvement 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 an osmoprotectant and a metabolite in celery (Apium graveolens) are well documented. However, there is growing evidence that many "metabolites" also have key roles in environmental and developmental responses in plants. For instance, in addition to its other properties, mannitol is an antioxidant and may play significant roles in plant-pathogen interactions. Our previous work suggested that many plant pathogenic fungi make mannitol as an antioxidant to suppress reactive oxygen-mediated plant defenses. Conversely, plants might counter this fungal suppressive mechanism by synthesizing mannitol dehydrogenase (MTD) to catabolize mannitol of fungal origin. To test this hypothesis transgenic
plants expressing a celery MTD were 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 the mannitol secreting fungus Alternaria alternata, but not to the non-mannitol secreting fungal pathogen Cercospora nicotianae.
Impacts
Fungal pathogens constitute one of the most economically devastating groups of plant pathogens. Our results show that MTD can play a major role in promoting resistance to mannitol-secreting fungal plant pathogens. Given the scarcity of suitable genes presently available for breeding or engineering fungal resistance in plants, the identification of a new and effective single gene resistance is of great potential benefit to the agricultural community.
Publications
- Williamson, J.D. 2002. Biotechnology; past, present and future. J. Am. Soc. Hort. Sci. 127:462-466.
- Williamson, J.D., Jennings, D.B., Ehrenshaft, M., Guo, W.-W. and Pharr, D.M. 2002. Sugar alcohols, salt stress, and fungal resistance. J. Am. Soc. Hort. Sci. 127:467-473.
- Jennings, D.B., Daub, M.E., Pharr, D.M. and Williamson, J.D. 2002. Constitutive expression of a celery mannitol dehydrogenase in tobacco enhances resistance to the mannitol-secreting fungal pathogen Alternaria alternata. Plant J. 32:41-49.
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Progress 11/01/00 to 10/31/01
Outputs
Some of the most versatile genes with promise for plant improvement 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 integration of mannitol metabolism in celery, its study is crucial in clarifying the physiological roles of mannitol metabolism in environmental and metabolic responses.
In pursuit of this we used transformed Arabidopsis to analyze the multiple environmental and metabolic responses of the Mtd promoter. Our data showed that all previously described changes in Mtd RNA accumulation in celery cells mirrored changes in Mtd transcription in Arabidopsis. Most importantly here, these included 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 suggest a role for MTD in mannitol translocation and unloading and its interrelation with sugar metabolism.
Impacts
One of the limiting factors in genetic engineering in plants is the limited number of suitable promoters for expression of foreign genes. We have isolated and characterized a plant promoter that drives very high levels of specifically regulated gene expression. This should be of value in expressing high levels of specific gene products in response to a number of important environmental stressors, particularly pathogen attack.
Publications
- Zamski, E., W.-W. Guo, Y.T. Yamamoto, D.M. Pharr, and J.D. Williamson. 2001. Analysis of celery (Apium graveolens) mannitol dehydrogenase (Mtd) promoter regulation in Arabidopsis suggests roles for MTD in key environmental and metabolic responses. Plant Mol. Biol. 47:621-631.
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