EXTRACELLULAR PROTEINS IN PLANT PATHOGEN INTERACTIONS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0170609
• Project Start Date: Oct 1, 2011
• Project End Date: Sep 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
NORTH CAROLINA STATE UNIV
(N/A)
RALEIGH,NC 27695

Performing Department
Horticultural Science

Non Technical Summary
Because the cell wall is a major interface between plant cells and their environment, the rapid, regulated secretion of specific proteins into this extracellular space is an important defense response. Secretion of defense proteins in both plants and animals was originally thought to be solely via an endoplasmic reticulum (ER)/Golgi-mediated pathway, with a defined signal peptide directing the protein to the ER for routing, modification and subsequent secretion via the Golgi. However, the existence of alternate secretion mechanisms was suggested whein it was reported that interleukin 1 (IL1b), a cytokine with no signal peptide, was secreted from human defense cells in response to bacterial attack. Since then, numerous Golgi-independent / leaderless eukaryotic secretion mechanisms have been reported, and the importance of these pathways, particularly in response to stress, is well established. Although non-Golgi secretion has been documented in many eukaryotes, our reports documenting the secretion of several normally cytoplasmic enzymes, including a mannitol dehydrogenase (MTD) and a cytosolic superoxide dismutase (SOD) in response to salicylic acid (SA) are among the first reports suggesting that non-classical secretion also occurs in plants. The existence of leaderless secretion in animals is well accepted, and several general mechanisms have been described. Although these mechanisms are quite diverse, all appear to first involve modification of the secreed protein in response to a stress mediated signal, enabling it to interact with the relevant secretion machinery. These proteins are then apparently transported across the membranes in a folded or native configuration. As a result, the targeting signal is an integrated feature of the protein's three-dimensional structure rather than a simple linear amino acid sequence, thus making classical deletion analysis based on primary sequence consensus problematic. We purpose to use a variety of complementary approaches to assess the mechanism(s) of secretion of potentially non-Golgi secreted proteins previously identified. 1) We will use epitope tagging to unambiguously localize these proteins. 2) Proteins of interest also will be purified by immunoprecipitation and used both to assess protein modifications associated with secretion, and to identify specific components of these pathways by identifying interacting proteins. 3) Finally, specific mechanisms suggested by these analyses would then be assessed using Arabidopsis lines with mutations in relevant pathway proteins. In addition to broadening our understanding of how plants respond to pathogens, describing a new regulated protein secretion method in plants could provide a powerful new tool for production of protein pharmaceuticals. Current methods typically involve expression of a protein of interest in large scale yeast fermentation cultures. The protein of interest must then be purified from the resulting mix. Specific, signal-mediated secretion of a protein of interest would, in contrast, provide a powerful single-step purification that could largely bypass complex and expensive methods currently used.

Animal Health Component

20%

Research Effort Categories

Basic

70%

Applied

20%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent

Knowledge Area


Subject Of Investigation


Field Of Science

Keywords

protein secretion

leaderless protein secretion

nonclassical secretion

extracellular proteins

Goals / Objectives
Regulated protein secretion is a critical component of many processes in both animals and plants. In animals, inflammatory mediators such as the cytokines are secreted in response to infection, and secreted proteins such as the fibroblast growth factors (FGFs) play key roles in cell and tissue differentiation, development, and healing. Similarly, in plants, proteins such as chitinases and β-glucanases are secreted to attack invading pathogens, while other proteins such as superoxide dismutase and peroxidases are involved in regulating induction of systemic defense responses and cell wall strengthening. Because extracellular proteins play roles in so many processes, understanding the mechanisms underlying their secretion is critical. Until relatively recently, protein transport to the extracellular space was thought to be exclusively through the ER/Golgi. Recent findings, however, show that, in both plants and animals, a surprising number of proteins secreted soon after the induction of defense responses lack a recognized secretion signal. Despite the apparent importance of these proteins, we know little about the mechanisms involved in their secretion. Characterizing pathogen-induced secretion mechanisms that are distinct from the ER/Golgi-mediated paradigm in plants is important for a number of reasons. First, although a number of stress and pathogen-induced non-Golgi secretion mechanisms have been described in animals, none have been described in plants. Because a number of key defense responses appear to involve nonclassical protein secretion, understanding the mechanisms involved is critical if we hope to improve pathogen resistance in plants. In addition to broadening our understanding of how plants respond to microbial attack, finding a new way to initiate rapid, regulated protein secretion could provide a powerful new tool for production of protein pharmaceuticals. Current commercial production typically involves expression of a protein of interest in large scale yeast cultures. The protein of interest must then be purified from the resulting complex mix of proteins. Specific, signal-mediated secretion of a protein of interest would, in contrast, constitute a powerful single-step purification that could largely bypass limitations of current separation technologies.

Project Methods
To localize and purify proteins for analyses, fusions will be made using epitope tags such as Flag or GFP. Extensive use has previously been made of FLAG-labeled MTD in our lab. Additional proteins to be assessed initially will include the Arabidopsis Cu/Zn SOD and the secreted jacalin lectin). Confocal microscopy will be used for initial assessment of the tissue/cell type secretion of these tagged. For example, untransformed and FLAG-fusion expressing transgenic Arabidopsis would be treated with either SA or sterile water. Treated and untreated tissues will be fixed and localization of fusion proteins assessed by treating fixed tissues with anti-FLAG primary antibody, followed by visualization with Alexa Fluor conjugated secondary antibodies. We will next use freeze-substitution immunoelectron microscopy to further resolve changes in localization and secretion of the Flag fusion in response to SA. Briefly, tissues from +/- SA-treated Flag- transgenic and untransformed control plants will be frozen in liquid N2 and substituted in ethanol containing glutaraldehyde, paraformaldehyde and uranyl acetate. Substituted tissues will be embedded, sectioned, blocked and incubated with anti-Flag primary followed by gold-conjugated secondary Ab. Sections will then be stained and binding of secondary Ab examined by TEM at the Center for Electron Microscopy at NCSU. In animals, there are several distinct non-Golgi protein secretion pathways. These pathways not only have distinct subcellular routing, but also have characteristic protein modifications and interactions. We will use immunoprecipitation to isolate proteins of interest, and LC-MSE to assess secretion-associated modifications. We will use coimmunoprecipitation to identify protein(s) interacting with our proteins of interest during the secretion process. Depending on the tag used, we can use FLAG, GFP or TAP immunoprecipitation. In addition, given their abundance and previous ease of identification, the Cu/Zn SOD and the secreted jacalin lectin may be co-precipitated directly using antibodies to the primary proteins themselves. Because protein modifications effecting protein-protein interactions are likely initiated by pathogen attack, we will compare interactions in + and - SA treated plants. Further, since secreted proteins likely interact with both membrane-bound and cytosolic proteins, both cytosolic and membrane fractions will be assessed. The results of these studies will be used to identify Arabidopsis mutants potentially deficient in one or more components of the hypothesized secretion pathway. The SALK collection contains a nearly complete library of tagged mutations for use in these analyses. Protein secretion will be assessed in these mutant lines as described above. To ensure that the inability of a specific mutant to secrete a protein of interest is caused by the mapped mutation, the ability of the analogous wild type sequence to restore SA-mediated secretion will be assessed.

Progress 10/01/11 to 09/30/16

Outputs
Target Audience:Progress related to this effort was presented at scientific meetings, including regional and national meetings of the American Society of Horticultural Science, American Society of Plant Biology and at the annual Meetings of the American Society of Mass Spectrometry, as well as part of the departmental outreach program (Hort Science Summer Institute).Target audiences included growers and breeders of a wide variety of horticultural and agricultural crops, basic research oriented plant scientists and HS students participating in the HS Summer Institute. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training in cutting edge cell biology and Mass Spectrometry to the graduate student(s) involved in the project. The research during this period has been conducted by two graduate students working under the direction of senior research personnel in the labs including PI's and postdoctoral researchers. This research provides students with the opportunity to learn and help develop the next-generation of mass spectrometry proteomic applications. How have the results been disseminated to communities of interest?Results have been disseminated at local, regional and national meetings in the form of papers, posters and presentations as indicated in this report. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Although protein secretion and vacuolar targeting were long believed to be mediated solely by ER/Golgi pathways, Golgi-independent trafficking and secretion have now been described in both animals and plants. Specific mechanisms involved, however, have yet to be completely elucidated. We used Arabidopsis thaliana cells expressing celery mannitol dehydrogenase (MTD), a protein lacking a recognized leader sequence, to begin identifying components of the protein complex involved in its pathogen-induced secretion. Using in vivo crosslinking to preserve protein interactions triggered by the endogenous pathogen response signal salicylic acid (SA), we "captured" MTD-interacting complexes using co-immunopurification, and identified constituent proteins using quantitative liquid chromatography-tandem mass spectrometry (LC/MS/MS). After eliminating false positives, 25 and 46 high-quality, unique interactors remained in three out of three experimental repetitions while 74, and 131 interactors were present in 2 out of 3 repetitions at 10 and 20 min post-treatment, respectively. These interactors were not only found in MTD-containing crosslinked complexes, but also displayed significant qualitative changes in response to SA. Finally, while many of these proteins had been previously associated with protein trafficking via vesicular mechanisms, a number were either proteins of unknown function or proteins with reported roles in other processes that might also function in unconventional secretory pathways.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Bhattarai K, Louws FJ, Williamson JD, Panthee DR (2016) Diversity analysis of tomato genotypes based on morphological traits with commercial breeding significance for fresh market production in eastern USA. Austr. J. Crop Sci. 10:1098-1103
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Patel TK, Williamson JD (2016) Mannitol in plants, fungi, and plant-fungal interactions. Trends Plant Sci. 21(6):486-97. doi: 10.1016/j.tplants.2016.01.006. Epub 2016 Feb 3.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: 38) LaHovary C, Danehower DA, Ma G, Reberg-Horton C, Williamson JD, Baerson SR, Burton JD (2016) Phytotoxicity and Benzoxazinone Concentration in Field Grown Cereal Rye (Secale cereale). Int J Agron vol. 2016, Article ID 6463826
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Patel TK, Krasnyanski SF, Allen GC, Louws FJ, Panthee DR, Williamson JD (2015) 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 Prog. 16: 115-117.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Patel TK, Krasnyanski SF, Allen GC, Louws FJ, Panthee DR, Williamson JD (2015) Tomato plants overexpressing a celery mannitol dehydrogenase (MTD) have decreased susceptibility to Botrytis cinerea. Am J Plant Sci. 6:1116-1125.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Williamson JD, A Desai, SF Krasnyanski, F Ding, W-w Guo, T-T Nguyen, HA Olson, JM Dole, GC Allen (2013) Overexpression of mannitol dehydrogenase in zonal geranium confers increased resistance to the mannitol secreting fungal pathogen Botrytis cinerea. Plant Cell Tiss Org. Cult. 115:367-375
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Bhattarai K, Louws FJ, Williamson JD, Panthee DR (2016) The differential response of tomato to Xanthomonas-specific Pathogen Associated Molecular Patterns and correlation with Bacterial Spot (Xanthomonas perforans) resistance. HortRes. 3, Article: 16035.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Progress related to this effort was presented at scientific meetings, including regional and national meetings of the American Society of Horticultural Science and at the 63rd Meeting of the American Society of Mass Spectrometry, as well as part of thedepartmental outreach program (Hort Science Summer Institute).Target audiences includedgrowers and breeders of a wide variety of horticultural and agricultural crops,basic research oriented plant scientists and HS students participating in the HS Summer Institute. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided training in cutting edge cell biology and Mass Spectrometry to the graduate student(s) involved in the project. The research during this period has been conducted by two graduate students working under the direction of senior research personnel in the labs including PI's and postdoctoral researchers. This research provides students with the opportunity to learn and help develop the next-generation of mass spectrometry proteomic applications. How have the results been disseminated to communities of interest?Results have ben disseminated at local, regional and national meetings in the form of posters and presentations as indicated in this report. What do you plan to do during the next reporting period to accomplish the goals?During the next period we will continue to validate the composition og the MTD-"interactome" by repeating and comparing the results of the reported interacting protein identifications. If additin we plan to begin efforts to identify protein mogifications associated with the initiation of the MTD-interactome formation. Our current analyses not only identified several intriguing potential SA-triggered, MTD-associated interactors, but also validate our proposed approach for interactor isolation and identification. These observations also led us to modify our proposed approach for identifying secretion-associated post translational modifications (PTMs). If, as hypothesized, modification of MTD or one or more interactors triggers formation of the "interactome", the MTD in these completes should be primarily in the modified form. Thus, our ability to isolate the interactome should also facilitate isolating (or enriching for) the modified form of MTD (or its interactors). Additionally, since secretion undoubtedly involves formation of a membrane-associated complex, we also conducted preliminary immunoblot analysis of cellular fractions. These analyses confirmed that a 40 kD MTD-crossreacting protein appeared in crude membrane fractions at the same time that MTD is disappearing from the cytosol. Thus, while completing analyses of cytosolic MTD interactors, we are also initiating analysis of MTD-interacting proteins in membrane/microsomal fractions.

Impacts
What was accomplished under these goals? Initial analysis using FLAG IP and quantitative LC/MS/MS showed a significant decrease in total cytosolic MTD-FLAG (50% relative to cytosolic marker proteins) within 15-20 minutes of SA treatment. Using our newly developed approach combining in vivo formaldehyde protein crosslinking and co-IP coupled with a gel-shift assay, we observed a concomitant shift of MTDcross- reacting material into higher molecular weight regions of the gel. As these high MW complexes most likely represent the MTD "interactome", this entire gel region was sliced into ca. 2 mm (ca. 5 kDa) pieces, and the included proteins in-gel trypsinized and analyzed by LC/MS/MS. To date, retarded complexes from 3 independent SA treatments (+SA) have been analyzed and compared to an untreated (-SA) control. MTD interactors were identified that (1) were shifted into high MW complexes (relative to their MW and that of MTD) in response to SA, (2) were present in at least two of three replicates from SA-treated cultures, and (3) were absent from the untreated (-SA) control, as well as absent from untransformed wildtype controls. Using in vivo formaldehyde crosslinking to preserve protein interactions triggered by the endogenous pathogen response signal salicylic acid (SA), we "captured" the MTD-interacting complex using co-immunoprecipitation, and quantitative LC-MS/MS to identify proteins potentially involved in MTD secretion. After eliminating false positives, approximately 30 candidate protein remained that were not only crosslinked to MTD, but also displayed significant increases/decreases in response to SA 20 minutes post-treatment. In addition tothese 20 minute proteins (e.g. chaperonins, AAA-ATPases and their regulators, as well as proteases) which have been implicated in various trafficking pathways, including exocytosis and autophagy, we have identifies another distinct group ofproteins that interact with MTD at 10 minutes post treatment. These proteins include protein modifying enzymes such as kinases as well as interactors hypothesized to be part of a preformed complex involved in anchoring MTD (e.g. actin). In addition to initial interactors identified at

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Ho TC, Blackburn RK, Williamson JD, Goshe MB (2015) Pathogen-triggered protein-protein interactions mediating nonclassical secretion of mannitol dehydrogenase in plants. ASMS Conference on Mass Spectrometry. Poster # MP496. J. Am. Soc. Mass Spec. 26, Supplement 1, p 76. Publication Date: First online: 18 April 2015 Page Number(s): 76
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Christophe LaHovary, David Danehower, Guoying Ma, Chris Reberg-Horton, John Williamson, Scott Baerson and J. Burton (2016) Phytotoxicity and Benzoxazinone Concentration in Field Grown Cereal Rye (Secale cereale). Int J Agron accepted for publication.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Patel TK, Krasnyanski SF, Allen GC, Louws FJ, Panthee DR, Williamson JD (2015) Tomato plants overexpressing a celery mannitol dehydrogenase (MTD) have decreased susceptibility to Botrytis cinerea. Am J Plant Sci. 6:1116-1125.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Patel TK, Krasnyanski SF, Allen GC, Louws FJ, Panthee DR, Williamson JD (2015) 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 Prog. 16: 115-117.
• Type: Journal Articles Status: Submitted Year Published: 2016 Citation: Patel TK, Williamson JD (2015) Mannitol in plants, fungi, and plant-fungal interactions. Trends Plant Sci. Submitted in 2015; Accepted for publication 1/5/2016.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Progress related to this effort was presented at several scientific meetings, and as part of the American Floral Endowment web-based reporting and out reach program. Target audiences include growers and breeders of a wide variety of horticultural and agricultural crops as well as more basic research oriented plant scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project has provided training in cutting edge cell biology and Mass Spectrometry to the graduate student(s) involved in the project. The research during this period has been conducted by two graduate students working under the direction of senior research personnel in the labs including PI's and postdoctoral researchers. This research provides students with the opportunity to learn and help develop the next-generation of mass spectrometry proteomic applications. How have the results been disseminated to communities of interest? Results have ben disseminated at local, regional and national meetingsin the form of posters and presentations as indicated in this report What do you plan to do during the next reporting period to accomplish the goals? During the next period we will be validating the composition og the MTD-"interactome" by repeating and comparing the results of the reported interacting protein identifications. If possible we plan to begin efforts to identifyprotein mogifications associated with the initiation of the MTD-interactome formation. Our current analyses not only identified several intriguing potential SA-triggered, MTD-associated interactors, but also validate our proposed approach for interactor isolation and identification. These observations also led us to modify our proposed approach for identifying secretion-associated post translational modifications (PTMs). If, as hypothesized, modification of MTD or one or more interactors triggers formation of the "interactome", the MTD in these completes should be primarily in the modified form. Thus, our ability to isolate the interactome should also facilitate isolating (or enriching for) the modified form of MTD (or its interactors). Additionally, since secretion undoubtedly involves formation of a membrane-associated complex, we also conducted preliminary immunoblot analysis of cellular fractions. These analyses confirmed that a 40 kD MTD-cross-reacting protein appeared in crude membrane fractions at the same time that MTD is disappearing from the cytosol. Thus, while completing analyses of cytosolic MTD interactors, we are also initiating analysis of MTD-interacting proteins in membrane/microsomal fractions.

Impacts
What was accomplished under these goals? Initial analysis using FLAG IP and quantitative LC/MS/MS showed a significant decrease in total cytosolic MTD-FLAG (50% relative to cytosolic marker proteins) within 15-20 minutes of SA treatment. Using our newly developed approach combining in vivo formaldehyde protein crosslinking and co-IP coupled with a gel-shift assay, we observed a concomitant shift of MTD-cross-reacting material into higher molecular weight regions of the gel. As these high MW complexes most likely represent the MTD "interactome", this entire gel region was sliced into ca. 2 mm (ca. 5 kDa) pieces, and the included proteins in-gel trypsinized and analyzed by LC/MS/MS. To date, retarded complexes from 3 independent SA treatments (+SA) have been analyzed and compared to an untreated (-SA) control. MTD interactors were identified that (1) were shifted into high MW complexes (relative to their MW and that of MTD) in response to SA, (2) were present in at least two of three replicates from SA-treated cultures, and (3) were absent from the untreated (-SA) control. A number of intriguing candidates appeared in our resulting MTD-"interactome" that previously have been implicated in exocytosis. These included, among others, myosin, Sec3A and B, various chaperonin/HSP's, ATPases and actin. The tail-anchored, endoplasmic reticulum (ER) interacting myosins are linked to functions ranging from ER streaming to organelle and exocytotic vesicle transport in plants. Other interactors that have also been implicated in exosome trafficking include sec3s, various HSP's/ chaperonins, AAA-ATPases and actin.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Takshay Patel T, JD Williamson, SF Krasnyanski, DR Panthee, GC Allen, A Desai. (2014) Overexpression of Celery Mannitol Dehydrogenase (MTD) in Tomato Increases Resistance to the Mannitol Secreting Fungal Pathogen Botrytis cinera. HortScience 49 (9) (Supplement) S22.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Bhattarai K, Louws FJ, Williamson JD, Panthee DR. (2014) Screening of tomato (Solanum lycopersicum L.) lines for bacterial spot (Xanthomonas spp.) resistance. HortScience 49 (9) (Supplement) S244.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Patel T SF Krasnyanski, DR Panthee, GC Allen, JD Williamson (2014) Mannitol Dehydrogenase (MTD) over-expression in tomato increases resistance to Botrytis cinerea. ASPB meeting Portland abstract P29040-A.
• Type: Theses/Dissertations Status: Published Year Published: 2014 Citation: Patel T (2014) Mannitol Dehydrogenase (MTD) over-expression in tomato increases resistance to Botrytis cinerea. NC State University. MS Thesis
• Type: Other Status: Published Year Published: 2014 Citation: Williamson JD, Allen GC, Dole JM (2014) Engineering Fungal Resistance in Zonal Geranium using a Gene for Mannitol Dehydrogenase. AFE Special Research Report # 136: Plant Breeding & Genetic Engineering.
• Type: Other Status: Published Year Published: 2014 Citation: Williamson JD, GC Allen, JM Dole (2014) Engineering Fungal Resistance in Bedding Plants using a Gene for Mannitol Dehydrogenase; Part II. AFE Special Research Report # 308: Plant Breeding & Genetic Engineering.
• Type: Websites Status: Published Year Published: 2014 Citation: Williamson JD, Allen GC, Dole JM (2014) Engineering Fungal Resistance in Zonal Geranium using a Gene for Mannitol Dehydrogenase. AFE Special Research Report # 136: Plant Breeding & Genetic Engineering. Williamson JD, GC Allen, JM Dole (2014) Engineering Fungal Resistance in Bedding Plants using a Gene for Mannitol Dehydrogenase; Part II. AFE Special Research Report # 308: Plant Breeding & Genetic Engineering. Williamson JD, JM Dole, GC Allen, SF Krasnyanski (2014) New Genetic Engineering Research Aims to Increase Postharvest Life of Cut Flowers AFE News Release. http://endowment.org/new-afe-funded-research-aims-increase-postharvest-life-cut-flowers

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Target audiences include growers and breeders of a wide variety of horticultural and agricultural crops as well as more basic research oriented plant scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The research during this period has been conducted by three graduate students working under the direction of senior research personnel in the labs including PI's and postdoctoral researchers. This research provides students with the opportunity to learn and help develop the next-generation of mass spectrometry proteomic applications, but it also has the potential to generate powerful new tools for bioengineering of all types. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We will next use chemical crosslinking and Flag-coimmunoprecipitation to identify protein(s) interacting with our proteins of interest during the secretion process. Because protein modifications effecting protein-protein interactions are likely initiated by pathogen attack, we will compare interactions in + and - SA treated plants. Further, since secreted proteins likely interact with both membrane-bound and cytosolic proteins, both cytosolic and membrane fractions will be assessed. The combined results of the above protein-protein interaction and localization studies should give us a reasonable idea of the mechanism involved in MTD secretion. These analyses will be used to identify Arabidopsis mutants potentially deficient in one or more components of the hypothesized secretion pathway.

Impacts
What was accomplished under these goals? My research program has focused on the previously unrecognized role of sugar alcohol metabolism in plant pathogen interactions. This research has had a number of important consequences: 1) By demonstrating that fungal pathogens use the sugar alcohol mannitol as a defense compound against active oxygen, it validates the importance of active oxygen mediated signaling in plant defenses. This work has now been independently verified by several other labs, and has stimulated a whole new area of research. 2) We previously demonstrated that the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in plants is a pathogen defense protein, and assigned a metabolic function to a class of previously uncharacterized proteins induced upon pathogen attack. We have demonstratedthat MTD can be used to genetically engineer specific crops for increased resistance to fungal pathogens. As fungal plant pathogens are responsible for a large number of economically devastating diseases, this has important economic consequences. During this reporting period we have completed analyses and final verification that MTD overexpression in zonal geranium results in increased resistance to Botrytis, the fungal pathogen causing flower and leaf blight in many important floriculture crops. These results have been published in the refereed journal PCTOC. We have further initiated analysis of protein modification and protein-protein interactions in response to pathogens. These cellular responses are key components of the cells regulatedsecretion of protective proteins such as MTD. We have currently established Arabidopsis cell cultures overexpressing a protein epitope tagged MTD and begun initial mass spec analysis of potential protein modifications in response to these treatments.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Williamson JD, A Desai, SF Krasnyanski, F Ding, W-w Guo, T-T Nguyen, HA Olson, JM Dole, GC Allen (2013)Overexpression of mannitol dehydrogenase in zonal geranium confers increased resistance to the mannitol secreting fungal pathogen Botrytis cinerea. Plant Cell Tiss Org. Cult. 115:367-375.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Carlson AS, GC Allen, JM Dole B Sosinski, JD Williamson (2013) Global Gene Expression Changes in Response to Bent Neck and Petal Blueing in Cut Roses Freedom' and Forever Young'. HortScience ASHS Student Oral Presentation. Abstract 15280.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: My research program has focused on the previously unrecognized role of sugar alcohol metabolism in plant pathogen interactions. This research has had a number of important consequences: 1) By demonstrating that fungal pathogens use the sugar alcohol mannitol as a defense compound against active oxygen, it validates the importance of active oxygen mediated signaling in plant defenses. This work has now been independently verified by several other labs, and has stimulated a whole new area of research. 2) We previously demonstrated that the mannitol catabolic enzyme mannitol dehydrogenase (MTD) in plants is a pathogen defense protein, and assigned a metabolic function to a class of previously uncharacterized proteins induced upon pathogen attack. 3) Application of these basic research findings to real world problems is a key aspect of my work. We have demonstrated, for instance, that MTD can be used to genetically engineer specific crops for increased resistance to fungal pathogens. As fungal plant pathogens are responsible for a large number of economically devastating diseases, this has important economic consequences. Most recently a project funded by the American Floral Endowment to engineer fungal resistance in bedding plants, specifically petunia and zonal geranium, using the MTD. During this reporting period we have completed analyses demonstrating that MTD overexpression in zonal geranium results in increased resistance to Botrytis, the fungal pathogen causing flower and leaf blight in many important floriculture crops. These results have been disseminated through publication on the American Floral Endowment web site as well as being republished on Plante and Cite, the website of the French centre for landscape and urban horticulture. PARTICIPANTS: Collaborators this period include Dr. George C Allen, Horticultural Science, Research Associate Professor, Partners II 1201 Box 7550, NCSU Campus, Raleigh, NC 27695. Phone [phone], Email; [e-mail]. Dr John M Dole. Horticultural Science, Professor. Kilgore Hall 120, Box 7609, NCSU Campus, Raleigh NC. Phone: [phone], Email; [e-mail]. Dr. Michael B Goshe Biochemistry Associate Professor Polk Hall 128, Box 7622 NCSU Campus Raleigh, NC 27695 Phone: [phone] EMail: [e-mail] TARGET AUDIENCES: Target audiences include growers and breeders of a wide variety of horticultural and agricultural crops as well as more basic research oriented plant scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Transformation of zonal geranium will have significant Projected Impact to the Floriculture Industry 1. New products for the industry would include lower maintenance potted plants, as well as a proven process for creation of additional resistant varieties. 2. Benefits accruing to the grower would include reduced production expenses due to decreased pest management costs and decreased fungal disease losses. 3. Advantages for floriculture consumers would be healthier plants with lower maintenance requirements in the home, and thus lower environmental impact. This work has drawn significant international recognition, with various aspects of the project being featured in news and academic publications. During this reporting period our work was feature in a 2012 review in Trends in Plant Science (Ding et al, 2012, 17:606-614).

Publications

• Williamson, J.D., G.C. Allen, and J.M. Dole. 2012 Engineering fungal resistance in bedding plants using a gene for mannitol dehydrogenase. Special Research Report # 307 to the American Floral Endowment.

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Regulated protein secretion is a critical cellular response to pathogen attack in both plants and animals. Secretion of newly synthesized defense proteins is largely via the well-studied ER/Golgi pathway. However, in animal systems, unconventional, non-Golgi secretion of stress and pathogen response proteins has also been well documented. More recently, several labs, including ours, have accumulated sufficient data to suggest that non-Golgi secretion also occurs in plants. Analyses of pathogen induced protein secretion in plants have identified a number of secreted proteins that lack a signal peptide for export by the conventional ER/Golgi pathway. These include normally cytoplasmic proteins that also have essential roles in extracellular defenses (e.g. MTD and Cu/Zn SOD), as well as proteins whose secretion via the Golgi would disrupt normal cellular processes (e.g. jacalin lectins). We hypothesize that the rapid, leaderless secretion of these normally cytosolic plant proteins, like leaderless secretion in mammalian systems, requires modification of the secreted protein itself or modification of interacting secretory pathway components. We propose to characterize potential modifications of these proteins in response to salicylic acid (SA), an endogenous inducer of pathogen defense responses, and identify proteins interacting with our target (bait) protein(s) immediately after exposure of cells to SA (at intervals within the first hour). Changes in the overall pattern of protein phosphorylation have been undertaken. Using ERLIC/IMAC combined with LC/MS/MS and LC/MSE in stable isotope-labeled cultured cells we have analyzed changes in the phosphoproteome during cell defense responses. Initial results were presented at the annual meeting or the American Society of Mass Spectrometry, and were used in the submission of a preproposal to the National Science Foundation entitled "Role of Protein Modifications and Protein-Protein Interactions in the Regulated Secretion of Leaderless Proteins in Plants". PARTICIPANTS: Principal investigator: John D. Williamson, Horticultural Science, Associate Professor, 250 Kilgore Hall, Box 7609, NCSU Campus, Raleigh NC, 27695,Phone [phone], email: [e-mail] Collaborator: Michael B. Goshe Biochemistry Associate Professor, Polk Hall 128, Box 7622 NCSU Campus Raleigh, NC 27695 Phone: [phone] EMail: [e-mail] Student Researcher: Ko-yi Cheng, Department of Biochemistry, Box 7622 NCSU Campus, Raleigh, NC 27695; Phone; 919-515-7195; email:[e-mail] TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We had previously used label-free, data-independent mass spectrometry to identify proteome changes in the Arabidopsis secretome in response to treatment with the pathogen defense elicitor salicylic acid (SA). Although this type of analysis was very good for protein identification, label-free methods such as ERLIC/IMAC/LC/MS/MS appear to be less suited to the analysis of pathogen stimulated changes in protein phosphorylation. The variance that was associated with the multi-step fractionation involved in this approach led us to try an isotope-labeling strategy to facilitate quantitative comparison of treated and untreated cells. Arabidopsis suspension cells were cultured in either a 14N- or 15N-enriched medium, then treated with either a mock inducer (e.g. hydroxybenzoic acid, HBA) or the endogenous inducer SA, and finally combined for simultaneous phosphoproteome analysis. First, we assessed isotope incorporation and found that a 15 day replacement with 15N-medium produced an isotope incorporation of ca. 95%, while a 30-day culture increased this value to ca. 98%. When we examined the 38-day culture, 15N-incorporation reached a. 99%, a value well within experimental error for the 15N sources used for our in-house prepared medium (potassium nitrate, 15N, 99% and ammonium sulfate, 15N, 99%). A subset of cells was then grown in 15N-enriched medium and treated with 1 mM SA (+SA) for 2 h while the other subset of cells grown in the 14N-medium were mock-treated (-SA). An inverse labeling was also performed by growing +SA cells in 14N-medium and -SA cells in 15N medium. Cells from each subset was harvested, and the +SA /15N-labeled cells were then combined with the -SA/14N-labeled cells, while the +SA/14N-labeled cells were combined with -SA/15N-labeled cells. Initial results using this new ERLIC/IMAC strategy using a PolyLC polyWAX column and iron-chelated NTA-agarose resin has produced promising results, revealing significant numbers of phosphopeptides. Additional work is being conducted to verify phosphopeptide identification and to determine the relative quantification of specific phosphorylation sites to unveil specific cellular plant defense mechanisms upon pathogen challenge.

Publications

• Ding, F., S. Krasnyanski, A. Dasai, E. Silverman, T-t. Nguyen , W-w. Guo, J. Dole, G. Allen, and J. Williamson. 2011. Engineering fungal resistance in bedding plants using a gene for mannitol dehydrogenase. In Annual Reports to the American Floral Endowment. (August 2010-Sept 2011).
• Chien, K-y., J.D. Williamson, and M.B. Goshe. 2011. Quantitative phosphoproteomic analysis of cell defenses using ERLIC/IMAC combined with LC/MS/MS and LC/MSe in stable isotope-labeled cultured cells ASMS Abstract 2739.
• Williamson, J. and J. Dole. 2011. Creating disease resistant bedding plants. Greenhouse Product News 21(12):26-27.

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: These results have been disseminated through, symposia, poster presentations at the annual meeting of the American Society of Plant Biologists, invited talks at the university and regional level and through publication in the journals listed below. Specifically: Poster presentation at the International Plant Biology Meeting (2010) Invited speaker for the NCSU, Biochemistry, Departmental Seminar (2010) Invited speaker at the NC Biotech Plant Mol Biol Retreat (2010) Invited review appearing in the journal Plant Signaling (2010). PARTICIPANTS: During this reporting period, we used a new mass spec technique called MSe, developed by Dr. Goshe's group in biochemistry, to validate MTD secretion by celery cells in response to pathogen attack. In addition, the plant transformation expein petunia an geranium were a result of collaboration with Dr. George Allen and Prof. John Dole in the Department of Horticultural Science. Collaborators: Dr. Michael B Goshe Biochemistry Associate Professor Polk Hall 128, Box 7622 NCSU Campus Raleigh, NC 27695 Phone: [phone] email: michael [e-mail] Dr George Allen, Horticultural Science, Research Assoc Professor, Partners II 1201, Box 7550, NCSU Campus, Raleigh, NC 2769 Phone: [phone]. EMail: [e-mail] Dr. John M. Dole, Horticultural Science, Professor, Kilgore Hall 158, Box 7609, NCSU Campus, Raleigh, NC 27695, Phone: [phone], email: [e-mail] Box nd Prof. Eli Zamski, Department of Agricultural Botany, The Hebrew University of Jerusalem. email: [e-mail]. PARTICIPANTS: PARTICIPANTS: Collaborators: Dr. Michael B Goshe Biochemistry Assistant Professor Polk Hall 128, Box 7622 NCSU Campus Raleigh, NC 27695 Phone: [phone] email: michael [e-mail] TARGET AUDIENCES: Finding a new method to initiate rapid, regulated protein secretion can be a powerful tool for producing foreign proteins using plants and plant cell cultures (bioprocessing). Thus the technology derived from the protein secretion research in our lab would be of considerable utility to both the pharmaceutical as well as the AgBiotech industries. In addition, the plant transformation research has the potential to not only produce plants that require less care, but also provide a proven process to create additional resistant varieties. Growers would have reduced production expenses due to lower pest-management costs and decreased shrink caused by plant mortality and aesthetic damage due to fungal disease. For the consumer, over-expression of this naturally occurring plant enzyme could result in healthier plants with lower maintenance requirements in the home and landscape, and thus lower environmental impact. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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 are well documented. However, there is evidence that "metabolites" can also have key roles in environmental and developmental responses in plants. For instance, in addition to its other properties, mannitol is an antioxidant that might 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 appear to counter this fungal mechanism by making the enzyme mannitol dehydrogenase (MTD) to catabolize mannitol of fungal origin. We had previously shown that constitutive expression of the MTD transgene in fact confers resistance to the mannitol secreting fungus Alternaria. To extendedthis work we are assessing whether or not this protective mechanism will also work for other plant-pathogen pairs. We have transformed both petunia and geranium to overexpress MTD, and are testing if these plants now have increased resistance to other significant fungal pathogens, such as Botrytis and/or Cladosporium. In separate work, we are continuing to assess the secretion of defense-related proteins in response to pathogen attack. In addition to our observation that MTD, a normally cytoplasmic enzyme, is specifically secreted after treatment with an endogenous inducer of plant defense responses salicylic acid (SA), more global work suggests that a significant number of other proteins are secreted by unconventional mechanisms in response to pathogen attack. This included studies wherein LCMSE was used to analyze proteins secreted by celery following treatment with SA. Induced secretion of MTD was found to be >18 fold greater than in untreated samples. This report has the first direct protein sequence data supporting the hypothesis that MTD is secreted in response to pathogen attack and validates the use of LCMSE in investigating secretion of defense-related proteins. Impact:Our plant transformation research has the potential to not only produce plants that require less care, but also provide a proven process to create additional resistant varieties. Growers would thus have reduced production expenses due to lower pest-management costs and decreased shrink caused by plant mortality and aesthetic damage due to fungal disease. For the consumer, this could result in healthier plants with lower maintenance requirements in the home and landscape, and thus lower environmental impact. In addition, if successful, our research will delineate a new method to initiate rapid, regulated protein secretion. This would provide a powerful new tool for producing foreign proteins using plants and plant cell cultures (bioprocessing). Thus the technology derived from the protein secretion research in our lab would be of considerable utility to both the pharmaceutical as well as the AgBiotech industries.

Publications

• Blackburn, R.K., Cheng, F.-y., Williamson, J.D., and Goshe, M.B. (2010) Data-independent liquid chromatography/mass spectrometry (LC/MSE) detection and quantification of the secreted Apium graveolens pathogen defense protein mannitol dehydrogenase. Rapid Commun. Mass Spectrom. 24:1009-1016.
• Cheng, F.-y. and Williamson, J.D. (2010) Is there leaderless protein secretion in plants Plant Signaling and Behavior 5(2):129-131.
• Ding, F., Krasnyanski, S., Silverman, E., Guo, W.-W., Dole, J., Allen G., and Williamson, J. (2010) Engineering fungal resistance in bedding plants using a gene for mannitol dehydrogenase; Part II. In Annual Reports to the American Floral Endowment.
• Ding, F., Krasnyanski, S., Guo, W.-W., Nguyen, T.-t., Dole, J.M., Allen, G., and Williamson, J.D. (2010) Expression of a celery mannitol dehydrogenase in petunia confers resistance to the mannitol-secreting fungal pathogen Botrytis cinerea. Poster Abstract P12053 ASPB Montreal.

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: Plants secrete a wide variety of defense-related proteins in response to pathogen attack. One of these, mannitol dehydrogenase (MTD), is a normally cytoplasmic enzyme whose primary role is regulation of intracellular levels of the sugar alcohol mannitol in plants. Results published during this period, however, showed that this normally cytoplasmic enzyme is specifically secreted after treatment with an endogenous inducer of plant defense responses salicylic acid (SA). Secreted MTD retained activity after export from the cell. Given that MTD catabolizes mannitol, MTD secretion forms an important component of plant defense against mannitol secreting fungal pathogens. After SA treatment, MTD was not detected in the Golgi apparatus and its SA-induced secretion was resistant to an inhibitor of Golgi-mediated protein transport. Together with the lack of a known extracellular targeting sequence on the MTD protein, this suggests plant responses to pathogens include secretion of defensive proteins by non-Golgi mechanisms. In a second study, LCMSE was used to analyze proteins secreted by celery following treatment with SA. Levels of MTD secreted by SA-treated celery cell cultures were found to be induced >18 fold over samples from those not exposed to SA. This level of induction corroborates observations in our previous biochemical study. This report is the first direct sequence data supporting the hypothesis that MTD is secreted in response to simulated pathogen attack and validates the use of LCMSE in investigating secretion of novel defense-related proteins in plants. These results have been disseminated through, symposia, poster presentations at the annual ASMS Conference on Mass Spectrometry, the annual meeting of the American Society of Plant Biologists, and through publication in the journals Planta and Rapid Communications in Mass Spectrometry. PARTICIPANTS: During this reporting period, we used a new mass spec technique called MSe, developed by Dr. Goshe's group in biochemistry, to validate MTD secretion by celery cells in response to pathogen attack. In addition, the electron microscopy forming the core of the MTD secretion study in tobacco was the result of collaboration with Dr. Eli Zamski at the Hebrew University. PARTICIPANTS: Collaborators: Dr. Michael B Goshe Biochemistry Assistant Professor Polk Hall 128, Box 7622 NCSU Campus Raleigh, NC 27695 Phone: [phone] email: [e-mail] and Prof. Eli Zamski, Department of Agricultural Botany, The Hebrew University of Jerusalem. email: [e-mail]. PARTICIPANTS: PARTICIPANTS: Collaborators: Dr. Michael B Goshe Biochemistry Assistant Professor Polk Hall 128, Box 7622 NCSU Campus Raleigh, NC 27695 Phone: [phone] email: [e-mail] and Prof. Eli Zamski, Department of Agricultural Botany, The Hebrew University of Jerusalem. email: [e-mail]. TARGET AUDIENCES: TARGET AUDIENCES: Finding a new method to initiate rapid, regulated protein secretion can be a powerful tool for producing foreign proteins using plants and plant cell cultures (bioprocessing). This technology would be of considerable utility to both the pharmaceutical as well as the AgBiotech industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The elucidation of a pathogen-induced secretion mechanism that is distinct from the classical Golgi mediated paradigm would be of enormous significance. 1) First, this is important in terms of basic cell biology. Although a number of stress and pathogen-induced nonclassical (non-Golgi mediated) secretion mechanisms have been described in animals, none have been described in plants. 2) Second, and most important here, is that the rapid, regulated secretion of proteins in response to pathogen attack is a critical component of the plants defense response. Much of the work to date has focused on the classical Golgi-mediated secretion anti microbial enzymes (e.g. beta-glucanases), enzymes involved in cell wall strengthening, and etc. The inference that a separate Golgi-independent pathway mediates pathogen-induced secretion of enzymes regulating the oxidant/ antioxidant status of the extracellular space is of great importance. 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 commandeering an existing/endogenous mechanism provides a superb tool for engineering new defenses. 3) Finally, a means of secreting proteins "on demand" without impacting Golgi-mediated protein trafficking has exciting 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

• Cheng, F.-y., Locke, E., and Williamson, J.D. 2009. Polyols in plants and pathogens; an integration of transport and function. Curr. Topics Plant Biol. 9:101-114.
• Cheng, F.-y., Zamski, E., Guo, W.-W., Pharr, D.M., and Williamson, J.D. 2009. Salicylic acid stimulates secretion of the normally symplastic enzyme mannitol dehydrogenase (MTD): a possible defense against mannitol secreting fungal pathogens. Planta 230:1093-1103.
• Cheng, F.-y. and Williamson, J.D. 2010. Is there leaderless protein secretion in plants Plant Signaling and Behavior 5(2): 1-3. published online 2009: landesbioscience.com/journals/psb/article/ 10304.
• Blackburn, K., Cheng, F.-y., Williamson, J.D., and Goshe, M.B. 2009. Detection and Quantification of a Novel Plant Pathogen Defense Protein Mannitol Dehydgrogenase (MTD) from LC/MSE Datasets. ASMS Conference on Mass Spectrometry. Abstract:648.
• Cheng, F.-y., Zamski, E., Blackburn, K., Guo, W.-W., Pharr, D.M., Goshe, M.B., and Williamson, J.D. 2009. Salicylic acid stimulates secretion of the typically cytosolic metabolic enzyme mannitol dehydrogenase. Plant Physiol (ASPB meeting Abs P48024:).
• Ding, F., Krasnyanski, S., Guo, W.-W., Nguyen, T.-t., Dole, J., Allen G., and Williamson, J. 2009. Engineering fungal resistance in bedding plants using a gene for mannitol dehydrogenase. In Annual Reports to the American Floral Endowment.
• Locke, E.L., Dole, J.M., and Williamson, J.D. 2009. Production environment light and temperature affects postharvest vaselife of cut L.A. Lilium 'Dazzle' and Helianthus 'Sunbright'. HortScience 44: 1013. ASHS Student Oral Presentation. Abstract 1682.

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: In this study, absolute quantification by LC/MSE was used to perform a comprehensive, quantitative analysis of salicylic acid (SA)-induced changes in the proteome of the plant extracellular space (the secretome). In plants, pathogen attack initiates a complex signaling cascade that ultimately leads to the synthesis of the compound SA. SA in turn induces the expression and/or secretion of a large number of proteins collectively called pathogenesis response (PR) proteins that, acting together, lead to acquisition of pathogen resistance in the plant (Systemic Acquired Resistance). Given SA's proximal role as an endogenous inducer of pathogen defense responses in plants, direct SA treatment provides a convenient means of assessing pathogen-induced responses. Arabidopsis suspension cultures were thus treated with increasing doses of SA for various periods of time. After treatment, proteins secreted into the culture medium were collected and temporal and dosedependent changes in the secretome were characterized both qualitatively and quantitatively using LC/MSE. The results of these studies have been disseminated through symposia presentations at the annual ASMS Conference on Mass Spectrometry and the MIT Proteomics Symposium 2007, and formed the basis of a paper accepted for publication in the Journal of Proteomic Research special issue on temporal and spatial genomics. PARTICIPANTS: During this reporting period, we used a new mass spec technique called MSe, developed by Dr. Goshe's group in biochemistry, to show that a number of additional proteins that lack Golgi secretion signals are rapidly secreted in response to pathogen attack. PARTICIPANTS: Collaborator: Dr. Michael B Goshe Biochemistry Assistant Professor Polk Hall 128, Box 7622 NCSU Campus Raleigh, NC 27695 Phone: [phone] EMail: michael [e-mail] TARGET AUDIENCES: Finding a new method to initiate rapid, regulated protein secretion can be a powerful tool for producing foreign proteins using plants and plant cell cultures (bioprocessing). This technology would be of considerable utility to both the pharmaceutical as well as the AgBiotech industries. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Changes in Knowledge Absolute quantification by LC/MSE was used to perform a comprehensive, quantitative analysis of salicylic acid (SA)-induced changes in the proteome of the plant extracellular space (the secretome). Absolute quantities of individual proteins from samples treated with SA and from uninduced, HBA-treated controls at 1, 2, 6, and 18 h were compared to assess induced secretion. A total of 74 proteins were identified in the secretome with 63 of these having a >2-fold increase in secretion in response to SA at one or more time points. Previous proteomic studies using standard 2D gel-based proteomic approaches identified 18 SA-responsive and 8 chitosan/elicitor-responsive proteins in the Arabidopsis secretome. This demonstrated that a major advantage of a label-free LC/MS approach such as LC/MSE is that it does not require pairwise comparisons between samples, thus, accommodating more complex experiments. In turn, the factorial study reported here not only enabled us to assess time and dose effects of SA on protein secretion, but also led to identification of a larger number of SA-responsive proteins. Plant defense mechanisms are an interconnected set of transient as well as sustained processes that initiate immediately after pathogen attack and last for periods ranging from minutes to hours to days. Each of these responses in turn requires the expression and function of a potentially wide range of proteins, hormones or secondary metabolites. Our longitudinal study across 18 h post SA treatment generated a picture of the distribution of proteins with significantly altered secretion over four points (induced), compared with time-matched uninduced controls. These results suggest that most induced protein secretion takes place within the first 2 h after SA treatment, with the number of proteins with induced secretion decreasing after 6 h. Extracellular proteins are classically characterized by the presence of an amino-terminal signal peptide in the protein sequence. This peptide sequence constitutes a signal that directs proteins to the ER/Golgi secretory pathway. It has been traditionally believed that this N-terminal leader peptide is strictly required for targeting specific protein secretion in plants. However, when the program SignalP 3.0 was employed to predict the presence of a signal peptide in the 74 observed secreted proteins, there were 37 proteins without a recognizable signal peptide. Among the proteins showing maximal secretion within the first hour after SA treatment, 65% lacked a signal peptide. Among those showing SA-induced maxima at 2 h, 50% lacked a signal peptide. In contrast, for proteins secreted at later time-points, only 35% lacked a signal peptide. Therefore, many of the proteins that were most rapidly secreted in response to SA might be secreted by leaderless, so-called nonclassical, Golgi-independent mechanisms.

Publications

• Blackburn, K., Cheng, F.-Y., Williamson, J.D., and Goshe, M.B. 2007. Applications of MSE in plant proteomics and protein characterization. Massachusetts Institute of Technology Department of Chemical Engineering Label-Free Quantification and Identification Proteomics Symposium. http://web.mit.edu/cheme/proteomics/index.html
• Cheng, F.-Y., Blackburn, K., Williamson, J.D., and Goshe, M.B. 2008. Global Analysis of Pathogen-Induced Plant Protein Secretion Responses Using Label-Free Quantification. ASMS Conference on Mass Spectrometry. Abstract: 1979.
• Cheng, F.-Y., Blackburn, R.K., Lin, Y.-M., Goshe, M., and Williamson, J.D. 2008. Absolute protein quantification by LC/MSE for global analysis of pathogen-induced plant protein secretion responses. J. Proteome Res. (in press). Accessible Online http://pubs.acs.org/doi/full/10.1021/pr800649s November 17

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: The sugar alcohol mannitol is a major metabolite in many important crops that, in addition to its roles as an osmoprotectant, is a potent antioxidant that has a potential role in pathogen resistance . Like animals, plants use various forms of active oxygen in defense, both as signals and as cytotoxins. Fungal pathogens may suppress reactive oxygen-mediated plant defenses, in part, by secreting mannitol. The mannitol catabolic enzyme mannitol dehydrogenase (MTD) in plants appears, in turn, to counteract this suppression by catabolizing the fungal mannitol. Our discovery of pathogen-induced expression of MTD in non-mannitol-producing plants, together with the finding that transgenic expression of MTD gives plants increased resistance to the mannitol-secreting fungal pathogen Alternaria alternata, suggests that MTD is a pathogen resistance protein with potential for providing increased fungal resistance in plants. Despite this, we know little about the precise mechanisms involved. This information is vital if we hope to manipulate this interaction for production of better crops. We have previously verified that pathogen induced secretion of MTD (a necessary component of the plant's defense response) is resistant to the golgi inhibitor Brefeldin-A. This indicates that secretion of the normally cytosolic enzyme MTD is by a novel non-golgi mediated mechanism, and supports previous immunolocalization studies showing the absence of MTD in the golgi during secretion. During this reporting period, we used a new mass spec technique developed by Dr. Goshe's group in biochemistry, to show that a number of additional proteins that lack Golgi secretion signals are rapidly secreted in response to pathogen attack. PARTICIPANTS: Collaborator: Dr. Michael B Goshe Biochemistry Assistant Professor Polk Hall 128, Box 7622 NCSU Campus Raleigh, NC 27695 Phone: [phone] EMail: [e-mail]

Impacts
The rapid, regulated secretion of specific proteins is a critical component of plant defense responses. Most work to date has focused on secretion of proteins that are cotranslationally inserted into the Golgi secretion pathway. In contrast, a pathogen-induced mechanism for secretion of normally cytosolic proteins is of major consequence for two reasons. First, although a number of stress and pathogen-induced non-Golgi secretion mechanisms have been described in animals, none have been previously described in plants. In addition, finding a new way to initiate rapid, regulated protein secretion provides a powerful tool for producing foreign proteins using plants and plant cell cultures (bioprocessing).

Publications

• Leatherwood, W.R., Pharr, D.M., Dean, L.O., and Williamson, J.D. 2007. Carbohydrate Content and Root Growth in Seeds Germinated Under Salt Stress: Implications for Seed Conditioning. J. Am Soc Hort Sci. 132: 876-882.

Progress 10/01/05 to 09/30/06

Outputs
The sugar alcohol mannitol is a major metabolite in many important crops that, in addition to its roles as an osmoprotectant, is a potent antioxidant that has a potential role in pathogen resistance . Like animals, plants use various forms of active oxygen in defense, both as signals and as cytotoxins. Fungal pathogens may suppress reactive oxygen-mediated plant defenses, in part, by secreting mannitol. The mannitol catabolic enzyme mannitol dehydrogenase (MTD) in plants appears, in turn, to counteract this suppression by catabolizing the fungal mannitol. Our discovery of pathogen-induced expression of MTD in non-mannitol-producing plants, together with the finding that transgenic expression of MTD gives plants increased resistance to the mannitol-secreting fungal pathogen Alternaria alternata, suggests that MTD is a pathogen resistance protein with potential for providing increased fungal resistance in plants. Despite this, we know little about the precise mechanisms involved. This information is vital if we hope to manipulate this interaction for production of better crops. During this reporting period we have verified that pathogen induced secretion of MTD (a necessary component of the plant's defense response) is resistant to the golgi inhibitor Brefeldin-A. This indicates that secretion of the normally cytosolic enzyme MTD is by a novel non-golgi mediated mechanism, and verifies previous immunolocalization studies showing the absence of MTD in the golgi during secrtion.

Impacts
Impact The rapid, regulated secretion of specific proteins is a critical component of plant defense responses. Most work to date has focused on secretion of proteins that are cotranslationally inserted into the Golgi secretion pathway. In contrast, a pathogen-induced mechanism for secretion of normally cytosolic proteins is of major consequence for two reasons. First, although a number of stress and pathogen-induced non-Golgi secretion mechanisms have been described in animals, none have been previously described in plants. In addition, finding a new way to initiate rapid, regulated protein secretion provides a powerful tool for producing foreign proteins using plants and plant cell cultures (bioprocessing).

Publications

• Cheng, F., Zamski, E., Pharr, D.M. and Williamson, J.D. 2006. Pathogen-induced secretion of the normally cytoplasmic enzyme mannitol dehydrogenase (MTD) in plants. http://abstracts.aspb.org/pb2006/public/P20034.

Progress 10/01/04 to 09/30/05

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 mannitol produced by plant pathogenic fungi is necessary to suppress 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. More recently it has been shown that Alternaria mutants that do not make mannitol are much less pathogenic. For MTD to be an effective defense, it must be co-localized with pathogen-produced mannitol. However, fungal mannitol is localized in the intercellular space or apoplast, while MTD in uninfected plants is cytosolic. Using anti-MTD antibodies produced in this lab, we previously showed that an anti-MTD crossreacting protein is localized in the apoplast of pathogen challenged plants. The identity of this secreted protein was confirmed using cells expressing MTD-FLAG epitope-tagged proteins. Blotting techniques showed the protein secreted from induced tobacco cells cross-reacts with both anti-MTD and anti-FLAG antibodies. 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

• Reberg-Horton, SC, JD Burton, D Danehower, G Ma, D Monks, P Murphy, NN Ranells, JD Williamson, NG Creamer (2005) Effect of time on the alle1ochemical content of ten cultivars of rye (Secale cereale L.). J. Chem. Ecol. 31:179-194.

Progress 10/01/03 to 09/30/04

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
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

• No publications reported this period

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.

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 arguably 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 either engineering or breeding fungal resistance in plants, the identification of a new and effective single gene resistance is of great potential agronomic impact.

Publications

• 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.
• 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.

Progress 10/01/00 to 09/30/01

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. 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 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.

Progress 01/01/00 to 12/31/00

Outputs
We have now tested a number of previously created MTD expressing F2 transgenic tobacco lines for altered disease resistance against both mannitol- and non-mannitol-producing plant pathogens. Constitutive expression of the mannitol-catabolizing enzyme mannitol dehydrogenase (MTD) from celery in the non-mannitol producing plant tobacco conferred enhanced tolerance to the mannitol secreting plant pathogen, Alternaria alternata. Enhanced resistance did not correlate with increased expression of PR1a, a protein indicator of systemic acquired resistance (SAR). This confirmed that increased resistance was due to the presence of MTD rather than a coincident induction of the SAR response. In contrast, constitutive MTD expression did not enhance tolerance to the non-mannitol secreting fungal plant pathogen, Cercospora nicotianae, or to the non-mannitol secreting bacterial pathogen, Pseudomonas syringae. These results are consistent with our hypothesis that MTD's role in plant resistance to mannitol secreting fungal pathogens is to catabolize mannitol of fungal origin. To date, in addition to finding MTD in the mannitol-containing plants celery, parsley and snap-dragon, the non-mannitol plants tobacco, tomato and Arabidopsis also have a pathogen-induced MTD. Based on these results we have received a three year USDA competitive grant to investigate the precise mechanisms of resistance and to define the contribution of this gene to pathogen resistance in plants.

Impacts
These results suggest the existence of a widespread, previously unknown mechanism for the plant to counteract fungal suppression of reactive-oxygen-mediated defenses. In short, mannitol dehydrogenase appears to represent a new class of pathogen resistance gene, with exciting potential for introducing increased fungal resistance in plants.

Publications

• Yamamoto, Y.T., Prata, R.T.N., Williamson, J.D. and Pharr, D.M. 2000. Formation of a hexokinase complex is associated with changes in energy demand in celery tissues and cells. Physiol. Plant. 110:28-37.

Progress 01/01/99 to 12/31/99

Outputs
Previously produced MTD expressing transgenic plants were selfed and F1 transgenics homozygous for Mtd selected. TheseF2 plants were screened for MTD expression. High level expressers were tested for altered disease resistance. In analyzing transgenics we found that, although tobacco does not make mannitol, these plants had a pathogen inducible MTD gene. In addition, our analyses show that induction is at the level of RNA accumulation, and that tobacco MTD is similar enough to celery MTD that it immunotitrates with celery MTD sera. 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. We have shown 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 several non-mannitol producing plants 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. To date, in addition to finding MTD in the mannitol-containing plants celery, parsley and snap-dragon, the non-mannitol plants tobacco, tomato and Arabidopsis have been found to contain pathogen-induced MTD. Furthermore, constitutive ectopic expression of an active celery MTD in tobacco was shown to confer significantly enhanced resistance to the mannitol secreting fungus Alternaria alternata, but not to non-mannitol producing pathogens such as Cercospora nicotianae and Pseudomonas syringeae pv tabaci.

Impacts
Mannitol dehydrogenase appears to represent a new class of pathogen resistance gene, with potential for introducing increased fungal resistance in plants. Based on these results we have submitted a proposal to USDA to investigate the precise mechanisms of resistance and to define the contribution of this gene to pathogen resistance in plants.

Publications

• Feusi, M.E.S., Burton, J.D., Williamson, J.D. and Pharr, D.M. 1999. Galactosyl-sucrose metabolism and UDP-galactose pyrophosphorylase from Cucumis melo L. fruit. Physiol. Plant. 106:9-16.
• Pharr, D.M., Prata, R.T.N, Jennings, D.B., Williamson, J.D., Zamski, E., Yamamoto, Y. and Conkling, M.A. 1999. Regulation of mannitol dehydrogenase: relationship to plant growth and stress tolerance. HortSci. 34:1027-1032.

Progress 01/01/98 to 12/31/98

Outputs
We have begun purification of celery hexokinase (HK) and analysis of its expression to determine the mechanism of HK mediated regulation of MTD. Additionally, two celery genomic clones previously isolated have been completely sequenced and verified. Previously produced MTD expressing transgenic plants were selfed and F1 transgenics homozygous for Mtd selected. F1 plants were screened for MTD expression. High level expressers are currently being tested for altered disease resistance. In analyzing transgenics we found that, although tobacco does not make mannitol, these plants had a pathogen inducible MTD gene. In addition, our analyses show that induction is at the level of RNA accumulation, and that tobacco MTD is similar enough to celery MTD that it immunotitrates with celery MTD sera. 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. Further, evidence indicates that MTD is present in other non-mannitol producing plants such as corn and Arabidopsis.

Impacts
(N/A)

Publications

• Stoop, J.M.H., Williamson, J.D., Conkling, M.A., MacKay, J.H. and Pharr, D.M. 1998. Characterization of NAD-dependent mannitol dehydrogenase from celery as affected by ions, chelators, reducing agents and metabolites. Plant Sci. 131:43-51.
• Williamson, J.D., Guo, W.-W. and Pharr, D.M. 1998. Cloning and characterization of a genomic clone (Accession No. AF067082) encoding mannitol dehydrogenase, a salt, sugar and SA regulated gene from celery (Apium graveolens L.) (#PGR98-137). Plant Physiol. 118:329.
• Jennings, D.B., Eherenshaft, M., Pharr, D.M. and Williamson, J.D. 1998. Roles for mannitol and mannitol dehydrogenase in active oxygen mediated plant pathogen interaction. Proc. Natl. Acad. Sci. USA 95:15129-15133.

Progress 01/01/97 to 12/31/97

Outputs
Assess MTD expression using antibodies and cDNA. Using EM immunohistochemistry, and biochemical analyses, localization of functional MTD in the nucleus as well as phloem parenchyma was confirmed. Further we showed that expression and localization of MTD are regulated by the sugar phosphorylating enzyme hexokinase(HK). We have begun purification of HK and analysis of its expression to determine the mechanism of HK mediated regulation of MTD. Isolate and characterize Mtd genomic clones. Two celery genomic clones previously isolated have been subcloned into sequencing vectors and sequencing is well underway. Approximately 50 and 80 percent, respectively, of the two genes have been sequenced and verified. Express MTD in transgenic tobacco. Previously produced MTD expressing transgenic plants were selfed and F1 transgenics homozygous for Mtd selected. These plants were in turn selfed and seed collected for production of F2 transgenics. F2 plants will be screened for MTD expression. High level expressers will be tested for altered disease resistance. In analyzing transgenics we found that, although tobacco does not make mannitol, these plants had a pathogen inducible MTD gene. Several tobacco cultivars have since been tested and all show pathogen induction of MTD expression. In addition, our analyses show that induction is at the level of RNA accumulation, and that tobacco MTD is similar enough to celery MTD that it immunotitrates with celery MTD sera. Further, evidence indicates that MTD may be pres.

Impacts
(N/A)

Publications

• YAMAMOTO, Y.T., ZAMSKI, E., WILLIAMSON, J.D., CONKLING, M.A. and PHARR, D.M. 1997. Subcellular Localization of celery mannitol dehydrogenase. A cytosolic metabolic enzyme in nuclei. Plant Physiol.
• PRATA, R.T.N., WILLIAMSON, J.D., CONKLING, M.A. and PHARR, D.M. 1997. Sugar repression of mannitol dehydrogenase activity in celery cells. Plant Physiol. 114: 307-314.

Progress 01/01/96 to 12/30/96

Outputs
Assess MTD expression using antibodies and Mtd cDNA. Localization of MTD expression in celery was assessed by immuno-histochemisry using MTD antibodies to identify specific cell types expressing MTD. These experiments showed that MTD protein accumulates primarily in the cell division region in developing roots, in analogous regions in leaf buds and in phloem tissues. These analyses also showed that active MTD protein is present in both the cytosol and nucleus. Further biochemical and molecular analyses revealed that expression of MTD is controlled by hexokinase-mediated sugar repression. Isolate and characterize Mtd genomic clone(s). Celery genomic clones were isolated, and PCR analyses verified that at least 2 contained the full Mtd gene with 3-4 kbp of upstream sequence. Direct sequencing of PCR products further verified the identity of these clones. Express MTD in transgenic tobacco. A 35S promoter-Mtd cDNA fusion construct was transformed into tobacco. Of 50 plants screened, 3 transgenic plants expressed active MTD enzyme. These plants have been inbred and seed collected for the production of F1 transgenics. The effects of ectopic MTD expression on the response of these plants to fungal pathogens will be assessed. Untransformed plants will be used as isogenic controls. Further analyses revealed that, although it does not produce mannitol and normally does not express MTD activity, tobacco apparently does exhibit pathogen/salicylate induced MTD activity.

Impacts
(N/A)

Publications

• STOOP, J. M.H, WILLIAMSON, J.D. and PHARR, D.M. 1996. Mannitol metabolism in plants; a method for coping with stress. Trends in Plant Science 1:139-144.
• ZAMSKI, E., YAMAMOTO, Y.T., WILLIAMSON, J. D., CONKLING, M.A. and PHARR, D.M. 1996. Immu.

Progress 01/01/95 to 12/30/95

Outputs
a. Assess mannitol dehydrogenase (MTD) expression using antibodies and Mtd cDNA.Using MTD antibody, we have begun looking at the organ-specific expression of MTD using proteins isolated from various organs (roots, etc.). Arabidopsis mutants that both over and under express MTD also have been obtained from Dr. Jeff Dangl at UNC, Chapel Hill, and will be used to assess the effects of these changes in Mtd expression. The localization of MTD expression is also being assessed using immuno- histochemisry using MTD antibodies to identify specific cell types expressing MTD. These experiments have shown that MTD protein accumulates primarily in the cell expansion region in developing roots, as well as in analogous regions in leaf buds. These analyses have also shown that the intracellular localization of MTD protein is most probably cytosolic. b. Isolate and characterize Mtd genomic clone(s). Toward this end Southern genomic analysis has been initiated and the Mtd gene shown to reside on a single 20kbp NotI restriction fragment and to be present as two alleles of a single gene copy in celery. c. Express MTD in transgenic tobacco already expressing an E. coli mannitol biosynthetic gene. Toward this end, a CaMV 35S promoter-Mtd fusion construct has been created and transformed into Agrobacterium for subsequent transformation into plants. Effects on the response of these mannitol utilizing plants to pathogens will be assessed. Untransformed plants (MTD expression null) will be used as isogenic controls.

Impacts
(N/A)

Publications

• PHARR, D. M., STOOP, J. M. , FEUSI-STUDER, M. E., WILLIAMSON, J. D., MASSEL, M. O. and CONKLING, M. A. 1995. Mannitol catabolism in plant sink tissues. In Curr. Topics in Plant Physiol., Vol. 13 (Madore and Lucas, eds) Amer. Society. of
• STOOP, J. M. , WILLIAMSON, J. D., CONKLING, M. A. and PHARR, D. M. 1995. Purification of NAD-dependent mannitol dehydrogenase from celery suspension cultures. Plant Physiol. 108:1219-1225.
• WILLIAMSON, J. D., STOOP, J. M. , MASSEL, M. O., CONKLING. M. A. and PHARR, D. M. 1995. Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the PR protein ELI3. Proc. Natl. Acad. Sci., USA 92:7148-7152.
• PHARR, D. M., STOOP, J. M. , WILLIAMSON, J. D., FEUSI-STUDER, M. E., MASSEL, M. O. and CONKLING, M. A. 1995. The dual role of mannitol as osmoprotectant and photoassimilate in celery. HortSci. 30:1182-1188.