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Dan Klessig
 &emdash;  Professor

Dan Klessig
Office/Lab: 223/206-210
Graduate Fields: Plant Biology; Plant Pathology & Plant-Microbe Biology


  • How does the multifaceted plant hormone salicyle acid come disease in plants and are similar mechanisms utilized in humans? 2017

    Dempsey, D. A. and Klessig, D.F.
    BioMed Central Biology 15,  23
    Full text...
  • Plant and human MORC proteins have DNA modifying activities similar to type II topoisomerases, but require additional factor(s) for full activity. 2017

    Manohar, M., Choi, H.W., Manosalva, P., Austin, C.A., Peters, J.E., and Klessig, D.K.
    Mol Plant Microbe Inter 30,  87-100
    Full text...
  • DAMPs, MAMPs, and NAMPs in plant innate immunity. 2016

    Choi, H.W. and Klessig, D.F.
    BMC Plant Biol 16,  232
    Full text...
  • How does aspirin work in plants and humans? 2017

    Klessig, D. F.
    Frontiers for Young Minds 5,  14
    Full text...
  • Multiple targets of salicylic acid and its derivatives in plants and animals 2016

    Klessig, D.F., Tian, M., and Choi H.W.
    Front. Immunol. 7,  1-10
    Full text...
  • Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid 2016

    Choi, H.W., Manohar, M., Manosalva, P., Tian, M., Moreau, M., and Klessig, D.F.
    PLOS Pathog. 12,  e1005518
    Full text...
  • Newly identified targets of Aspirin and its primary metabolite, salicylic acid 2016

    Klessig, D.F.
    DNA Cell Biol. 35,  163-166
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  • Aspirin’s Active Metabolite Salicylic Acid Targets Human High Mobility Group Box 1 to Modulate Inflammatory Responses 2015

    Choi, H.W., Tian, M., Song, F., Venereau, E., Preti, A., Park, S.W., Hamilton, K., Swapna, G.V.T., Manohar, M., Moreau, M., Agresti, A., Gorzanelli, A., De Marchis, F., Wang, H., Antonyak, M., Micikas, R., Gentile, D.R., Cerione, R.A., Schroeder, F.C., Montelione, G.T., Bianchi, M.E., and Klessig, D.F.
    Mol. Med. 21,  526-535
    Full text...
  • Human GAPDH is a target of aspirin’s primary metabolite salicylic acid and its derivatives 2015

    Choi, H.W., Tian, M., Manohar, M., Harraz, M.M., Park, S.-W., Schroeder, F.C., Snyder, S.H., and Klessig, D.F.
    PLOS ONE 10,  e0143447
    Full text...
  • Identification of multiple salicylic acid-binding proteins using two high throughput screens. Front 2015

    Manohar, M., Tian, M., Moreau, M., Park, S-W., Choi, H.W., Fei, Z., Friso, G., Asif, M., Manosalva, P., von Dahl, C.C., Shi, K., Ma, S., Dinesh-Kumar, S.P., O'Doherty, I., Schroeder, F.C., van Wijk, K.J., and Klessig, D.F.
    Plant Sci. 5,  777
    Full text...
  • Salicylic acid inhibits the replication of Tomato Bushy Stunt Virus by directly targeting a host component in the replication complex. 2015

    Tian, M., Sasvani, Z., Gonzalez, P.A., Friso, G., Rowland, E., Liu, X-M., van Wijk, K.J., Nagy, P.D., and Klessig, D.
    Molecular Plant-Microbe Interactions
    Full text...
  • Conserved nematode signaling molecules elicit plant defenses and pathogen resistance 2015

    Manosalva, P., Manohar, M., von Reuss, S.H., Chen, S., Micikas, R.J., Koch, A., Choe, A., Kaplan, F., Xiaohong Wang, X., Kogel, K-H., Sternberg, P.W., Williamson, V. M., Schroeder, F.C., and Klessig, D.F.
    Nature Comm 6,  7795
    Full text...
  • The GHKL ATPase MORC1 modulates species-specific plant immunity in Solanaceae 2015

    Manosalva, P., Manohar, M., Kogel, K-H., Kang, H-G., and Klessig, D.F.
    Molecular Plant-Microbe Interactions 28,  927-942
    Full text...
  • The Compromised Recognition of Turnip Crinkle Virus1 Subfamily of Microrchidia ATPases Regulates Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens 2014

    Langen, G., von Einem, S., Koch, A.,Imani, J., Pai, S.B., Manohar, M., Ehlers, K., Choi, H.W., Claar, M., Schmidt, R., Mang, H-G., Bordiya, Y., Kang, H-G., Klessig, D.F., and Kogel, K-H.
    Plant Physiology 164(2),  866-878.
    Full text...
  • Salicylic acid binding of mitochondrial alpha-ketoglutarate dehydrogenase E2, which interacts with the downstream mitochondrial electron transport chain, plays a crucial role in basal defense against TMV in tomato 2014

    Liao, Y., Tian, M., Zhang, H., Li, X., Xia, X., Zhou, J., Zhou, Y., Yul, J., Shi, K., and Klessig, D.F.
    New Phytologist doi: 10.1111,  nph13137
    Full text...
  • Crystal structure of the Arabidopsis thaliana TOP2 oligopeptidase. Acta. Crystallogr 2014

    Wang, R., Rajagopalan, K., Sadre-Bazzaz, K., Moreau, M., Klessig, D.F., and Tong, L.
    F. Struct. Biol. Commun. 70,  555
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  • SOS – too many signals for systemic acquired resistance? 2012

    Dempsey, D.A., and Klessig, D.F.
    Trends Plant Sci. 17,  538-545
    Full text...
  • Structure of the Arabidopsis thaliana TOP2 oligopeptidase 2014

    Wang, R., Rajagopalan, K., Sadre-Bazzaz, K., Moreau, M., Klessig, D.F., and Tong, L.
    Acta Cryst. F70,  555–559
    Full text...
  • CRT1 is a nuclear-translocated MORC endonuclease that participates in multiple levels of plant immunity 2012

    Kang, H.G., Hyong, W.C., von Einem, S., Manosalva, P., Ehlers, K., Liu, P.P., Buxa, S.V., Moreau, M., Mang, H.G., Kachroo, P., Kogel, K.H., and Klessig, D.F.
    Nature Communications 3,  1297
    Full text...
  • Abscisic acid deficiency antagonizes high-temperature inhibition of disease resistance through enhancing nuclear accumulation of resistance proteins SNC1 and RPS4 in Arabidopsis 2012

    Mang, H.G., Qian, W.Q., Zhu, Y., Qian, J., Kang, H.G., Klessig, D.F., and Hua, J.
    Plant Cell 24,  1271-1284
    Full text...
  • Salicylic acid binds NPR3 and NPR4 to regulate NPR1-dependent defense responses 2012

    Moreau, M., Tian, M., and Klessig, D.F.
    Cell Res. 22,  1631-1633
    Full text...
  • The combined use of photoaffinity labeling and surface plasmon resonance-based technology identifies multiple salicylic acid-binding proteins 2012

    Tian, M., von Dahl, C.C., Liu, P.P., Friso, G., van Wijk, K.J., and Klessig, D.F.
    Plant J. 72,  1027-1038
    Full text...
  • Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation 2012

    Zheng, X.Y., Spivey, N.W., Zeng, W.Q., Liu, P.P., Fu, Z.Q., Klessig, D.F., He, S.Y., and Dong, X.N.
    Cell Host Microbe 11,  587-596
    Full text...
  • The extent to which methyl salicylate is required for signaling systemic acquired resistance is dependent on exposure to light after infection. 2011

    Liu, P.P., von Dahl, C.C., and Klessig, D.F.
    Plant Physiology 157,  2216-2226
    Full text...
  • Interconnection between methyl salicylate and lipid-based long-distance signaling during the development of systemic acquired resistance in Arabidopsis and tobacco 2011

    Liu, P.P., von Dahl, C.C., Park, S.W., and Klessig, D.F.
    Plant Physiology 155,  1762-1768
    Full text...
  • Salicylic acid biosynthesis and metabolism 2011

    Dempsey, D.A., Vlot, A.C., Wildermuth, M.C., and Klessig, D.F.
    In The Arabidopsis Book American Society of Plant Biologists e0156
    Full text...
  • NO synthesis and signaling in plants–where do we stand? 2010

    Moreau, M., Lindermayr, C., Durner, J., and Klessig, D.F.
    Physiol. Plant 138,  372-383
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  • The lesion-mimic mutant cpr22 shows alterations in abscisic acid signaling and abscisic acid insensitivity in a salicylic acid-dependent manner 2010

    Mosher, S., Moeder, W., Nishimura, N., Jikumaru, Y., Joo, S.H., Urquhart, W., Klessig, D.F., Kim, S.K., Nambara, E., and Yoshioka, K.
    Plant Physiology 152,  1901-1913
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  • Methyl esterase 1 (StMES1) is required for systemic acquired resistance in potato 2010

    Manosalva, P.M., Park, S.W., Forouhar, F., Tong, L., Fry , W.E., and Klessig, D.F.
    Mol. Plant Microbe Interact. , 1151-1163.,  1151-1163.
    Full text...
  • Endosome-associated CRT1 functions early in resistance gene-mediated defense signaling in Arabidopsis and tobacco 2010

    Kang, H.G., Oh, C.S., Sato, M., Katagiri, F., Glazebrook, J., Takahashi, H., Kachroo, P., Martin, G.B., and Klessig, D.F.
    Plant Cell 22,  918-936
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  • Altering expression of benzoic acid/salicylic acid carboxyl methyltransferase 1 compromises systemic acquired resistance and PAMP-triggered immunity in arabidopsis 2010

    Liu, P.P., Yang, Y., Pichersky, E., and Klessig, D.F.
    Mol. Plant Microbe Interact. 23,  82-90
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  • Systemic acquired resistance is induced by R gene-mediated responses independent of cell death. 2010

    Liu, P.P., Bhattacharjee, S., Klessig, D.F., and Moffett, P.
    Mol. Plant Pathol. 11,  155-160
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  • Cryptochrome 2 and phototropin 2 regulate resistance protein-mediated viral defense by negatively regulating an E3 ubiquitin ligase 2010

    Jeong, R.D., Chandra-Shekara, A.C., Barman, S.R., Navarre, D., Klessig, D.F., Kachroo, A., and Kachroo, P.
    P. Natl. Acad. Sci. U S A 107,  13538-13543
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  • Salicylic acid, a multifaceted hormone to combat disease 2009

    Vlot, A.C., Dempsey, D.A., and Klessig, D.F.
    Annu. Rev. Phytopathol. 47,  177-206
    Full text...
  • Use of a synthetic salicylic acid analog to investigate the roles of methyl salicylate and its esterases in plant disease resistance 2009

    Park, S.W., Liu, P.P., Forouhar, F., Vlot, A.C., Tong, L., Tietjen, K., and Klessig, D.F.
    Journal of Biological Chemistry 284,  7307-7317
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  • Enhanced defense responses in Arabidopsis induced by the cell wall protein fractions from Pythium oligandrum require SGT1, RAR1, NPR1 and JAR1 2009

    Kawamura, Y., Takenaka, S., Hase, S., Kubota, M., Ichinose, Y., Kanayama, Y., Nakaho, K., Klessig, D.F., and Takahashi, H.
    Plant Cell Physiol. 50,  924-934
    Full text...
  • Identification of likely orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana. 2008

    Vlot, A.C., Liu, P.P., Cameron, R.K., Park, S.W., Yang, Y., Kumar, D., Zhou, F.S., Padukkavidana, T., Gustafsson, C., Pichersky, E., and Klessig, D.F.
    Plant J. 56,  445-456
    Full text...
  • Systemic acquired resistance: the elusive signal(s) 2008

    Vlot, A.C., Klessig, D.F., and Park, S.W.
    Curr. Opin. Plant Biol. 11,  436-442
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  • The Structure of YqeH: An AtNOS1/AtNOA1 ortholog that couples GTP hydrolysis to molecular recognition 2008

    Sudhamsu, J., Lee, G.I., Klessig, D.F., and Crane, B.R.
    Journal of Biological Chemistry, 283,  32968-32976
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  • High level expression of a virus resistance gene, RCY1, confers extreme resistance to Cucumber mosaic virus in Arabidopsis thaliana 2008

    Sekine, K.T., Kawakami, S., Hase, S., Kubota, M., Ichinose, Y., Shah, J., Kang, H.G., Klessig, D.F., and Takahashi, H.
    Mol. Plant Microbe Interact. 21,  1398-1407
    Full text...
  • AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase 2008

    Moreau, M., Lee, G.I., Wang, Y., Crane, B.R., and Klessig, D.F.
    Journal of Biological Chemistry 283,  32957-32967
    Full text...
  • Plant resistance to viruses: Natural resistance associated with dominant genes 2008

    Moffett, P., and Klessig, D.F.
    In Encyclopedia of Virology (Mahy, B.W.J. and van Regenmortel, M. eds) Oxford 0: Elsevier
    Full text...
  • Methyl salicylate is a critical mobile signal for plant systemic acquired resistance 2007

    Park, S.-W., Kaiyomo, E., Kumar, D., Mosher, S.L., and Klessig, D.F.
    Science 318,  113-116
    Full text...
  • The search for the salicylic acid receptor led to discovery of the SAR signal receptor. 2008

    Kumar, D., and Klessig, D.F.
    Plant Signal Behav. 3,  691-692
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  • CRT1, an Arabidopsis ATPase that interacts with diverse resistance proteins and modulates disease resistance to turnip crinkle virus 2008

    Kang, H.G., Kuhl, J.C., Kachroo, P., and Klessig, D.F.
    Cell Host & Microbe 3,  48-57
    Full text...
  • HRT-mediated hypersensitive response and resistance to Turnip crinkle virus in Arabidopsis does not require the function of TIP, the presumed guardee protein 2008

    Jeong, R.D., Chandra-Shekara, A.C., Kachroo, A., Klessig, D.F., and Kachroo, P.
    Mol. Plant Microbe Interact. 21,  1316-1324
    Full text...
  • The involvement of the Arabidopsis CRT1 ATPase family in disease resistance protein-mediated signaling 2008

    Kang, H.G., and Klessig, D.F.
    Plant Signal Behav. 3,  689-690
    Full text...
  • Inactive methyl indole-3-acetic acid ester can be hydrolyzed and activated by several esterases belonging to the AtMES esterase family of Arabidopsis 2008

    Yang, Y., Xu, R., Ma, C.J., Vlot, A.C., Klessig, D.F., and Pichersky, E.
    Plant Physiology 147,  1034-1045
    Full text...
  • The Arabidopsis gain-of-function mutant ssi4 requires RAR1 and SGT1b differentially for defense activation and morphological alterations. 2008

    Zhou, F.S., Mosher, S., Tian, M.Y., Sassi, G., Parker, J., and Klessig, D.F.
    Mol. Plant Microbe Interact. 21,  40-49
    Full text...
  • Validation of RNAi Silencing Specificity Using Synthetic Genes: Salicylic Acid-binding Protein 2 is Required for Innate Immunity in Plants 2006

    Kumar, D., Gustafsson, C., and Klessig, D.F.
    Plant J. 45,  863-868
    Full text...
  • The Chimeric Arabidopsis CYCLIC NUCLEOTIDE-GATED ION CHANNEL11/12 Activates Multiple Pathogen Resistance Responses 2006

    Yoshioka, K., Moeder, W., Kang, H.-G., Kachroo, P., Masmoudi, K., Berkowitz, G., and Klessig, D.F.
    Plant Cell 18,  747-763.
    Full text...
  • Crystal structure and biochemical studies identify tobacco SABP2 as a methylsalicylate esterase and further implicate it in plant innate immunity 2005

    Forouhar, F., Yang, Y., Kumar, D., Chen, Y., Fridman, E., Park, S.W., Chiang, Y., Acton, T.B., Montelione, G.T., Pichersky, E., Klessig, D.F., and Tong, L.
    P. Natl. Acad. Sci. U S A 102,  1773-1778
    Full text...
  • Silencing of the Mitogen-activated Protein Kinase MPK6 Compromises Disease Resistance in Arabidopsis 2004

    Menke, F.L.H., van Pelt, J.A., Pieterse, C.M.J., and Klessig, D.F.
    Plant Cell 16,  897-907
    Full text...
  • Interconnection between methyl salicylate and lipid-based long-distance signaling during systemic acquired resistance in Arabidopsis and tobacco 2001

    Liu, P.-P., von Dahl, C. C., Park, S.-W., and Klessig, D. F.
    Plant Physiol. 155,  1762-1768
    Full text...
  • Double-Stranded RNA-Binding Protein 4 Is Required for Resistance Signaling against Viral and Bacterial Pathogens 2013

    Zhu, S.F., Jeong, R.D., Lim, G.H., Yu, K.S., Wang, C.X., Chandra-Shekara, A.C., Navarre, D., Klessig, D.F., Kachroo, A., and Kachroo, P.
    Cell Reports 4,  1168-1184
    Full text...

Intern Projects

Over the past decade we have hosted 10 undergraduate research interns. All have worked closely with senior postdoctoral fellows or research associates, with most of them studying various aspects of SA-mediated defense signaling in plants. In addition, two helped characterize CRT1/MORC1.

Click the links to return to the Intern FacultyInternship Program, or Apply for an Internship pages on the BTI website.



    • Technology Area: Biotic Stress – Disease
    • Title: Salicylic acid induced map kinase and its use for enhanced disease resistance in plants
    • US Patent/Application(s): 5,977,442
    • Publication: Plant Cell 1997PNAS 1998
    • Technology Area: Biotic Stress – Disease
    • Title: Salicylic Acid Binding Protein (SABP2)
    • US Patent/Application(s): 7,169,966
    • Publication: PNAS 2003
    • Technology Area: Enabling Technology
    • Title: Methods for determining specificity of RNA silencing and for genetic analysis of the silenced gene or protein
    • US Patent/Application(s): 7,592,504
    • Publication: Plant J 2006
    • Technology Area: Biotic Stress – Disease
    • Title: Methods and compositions for improving salicylic acid-independent systemic acquired disease resistance in plants
    • US Patent/Application(s): 6,495,737
    • Publication: Plant J 1998
    • Technology Area: Biotic Stress – Disease
    • Title: Method of using a pathogen-activatable map kinase to enhance disease resistance in plants
    • US Patent/Application(s): 6,765,128
    • Publication: PNAS 1998
    • Technology Area: Biotic Stress – Disease
    • Title: High-affinity salicylic acid-binding protein and methods of use
    • US Patent/Application(s): 6,136,552
    • Publication: Plant Physiol 1997
    • Technology Area: Biotic Stress – Disease
    • Title: Genes Associated with enhanced disease resistance in plants
    • US Patent/Application(s): 5,939,601
    • Publication: PNAS 1996
    • Technology Area: Biotic Stress – Disease
    • Title: Compositions and methods for the generation of disease-resistant crops
    • US Patent/Application(s): PCT/US2012/043976
    • Publication: None
    • Technology Area: Biotic Stress – Disease
    • Title: Assays to identify inducers of plant defense resistance
    • US Patent/Application(s): 5,989,846
    • Publication: Science 1993

Research Utilization

Plant Disease Resistance

The central objective of research in the Klessig lab is to understand plant defense responses to pathogens. To this end the lab has identified a number of critical defense components, several of which are related to the central role that salicylic acid (SA, the active ingredient of aspirin) plays in activating plant immune responses. Three are SA-binding proteins (SABPs): catalase and ascorbate peroxidase, the two major enzymes for detoxifying hydrogen peroxide, and SABP2, a methyl esterase that converts biologically inactive methyl salicylate (MeSA) into active SA. SABP2 plays a key role in systemic acquired resistance in tissue distal to the sites of infection, where it converts the long-distance mobile signal MeSA to SA. This conversion results in the induction and/or priming of plant defense responses, resulting in a heightened defense response.

There are striking similarities between plant and animal defense mechanisms. The Klessig lab has developed novel, effective and sensitive approaches to identify SABPs, revealing many more targets of SA, with potential applications in both plant and animal disease management. Human SABPs have recently been identified that play key roles in important diseases, including heart disease, arthritis, certain cancers, and neurodegenerative diseases such as Alzheimer’s or Parkinson’s. The identification of these SABPs provides insight into how aspirin, one of the oldest drugs known to humanity, may carry out its remarkable and myriad therapeutic effects and provides an avenue to identify novel natural or synthetic derivatives of SA/aspirin with improved therapeutic properties.

In a separate project, the lab has identified a novel ATPase named CRT1 (Compromised for Recognition of Turnip Crinkle Virus), that plays important roles in regulating disease resistance. It is closely related to the mammalian MORC1 gene (Microchidia), which appears to play a role in epigenetic modification of DNA. Silencing or inactivating CRT1 in plants affects four distinct levels of immunity: resistance (R) gene-mediated resistance, basal resistance, non-host resistance, and systemic acquired resistance in Arabidopsis, tobacco, tomato, potato, soybean, and barley. Moreover, CRT1 is involved in immunity against a broad spectrum of pathogens including virus, bacteria, fungi, and oomycetes. Overexpression or suppression of these defense components has a significant impact on disease susceptibility, suggesting biotech approaches to modulating plant disease susceptibility.

In a collaboration with Frank Schroeder’s lab at BTI, the Klessig lab has been studying the effects of ascarosides, small molecules synthesized by nematodes, on plant responses to disease and pathogenic nematodes. Plant pathogenic nematodes are important agricultural pests, and the possibility of enhancing nematode and disease resistance via treatment with small molecules has significant practical potential. Preliminary results in Arabidopsis, tobacco, tomato, potato, and barley are very encouraging.

Collaboration and Consulting Opportunities

  • Engineering plant disease resistance: effector-triggered immunity and basal immunity
  • Plant immunity
    • Role(s) of salicylic acid, signaling in systemic acquired resistance
    • Engineering broad spectrum disease resistance with CRT1- a player in 4 levels of immunity
  • Role of salicylic acid binding proteins in human disease

Collaborations and Consulting

    • Technology Area: Biotic Stress – Disease
    • Research Area: Roles of salicylic acid in immunity / Characterization of multiple signaling components in immunity
    • Opportunities:
      • Roles of salicylic acid in immunity
      • Signaling in systemic acquired resistance
      • Use of defense signaling components to engineer disease resistance
      • Specificity of RNA silencing
      • New highly potent derivatives of salicylic acid for treatment of prevalent, devastating human diseases, including cancer, arthritis, and Alzheimer’s

In the News

Research Overview

Our research is focused on understanding, at the biochemical, molecular and cellular levels, how plants protect themselves against microbial pathogens. The major goal is to determine the mechanisms of action of salicylic acid (SA) in activation and regulation of the plant’s immune responses. We also are now employing the technology developed and knowledge gained from our work on SA and plant immunity to identify the targets of aspirin (acetyl SA) and its major metabolite SA in humans. The molecular/biochemical function of CRT1/MORC1 in multiple levels of plant immunity is also being deciphered. In addition, in collaboration with Frank Schroeder the induction of plant immune responses by nematode ascarosides is being investigated.

A. Systemic acquired resistance (SAR) in Arabidopsis, tobacco, and potato

Methyl salicylate (MeSA)

Methyl salicylate (MeSA) – mediated induction of systemic acquired resistance

Systemic acquired resistance (SAR) is a state of heightened defense to a broad spectrum of pathogens that is activated throughout a plant following local infection. Development of SAR requires translocation of one or more mobile signals from the site of infection through the vascular system to distal (systemic) tissues. Between 2007 and 2011 we reported the identification of the first long-distance mobile signal, methyl salicylate, in several plant species in a series of five papers. In 2011 we published the inter-relationship between methyl salicylate and lipid signal(s). More recently several other mobile signals have been reported in addition to methyl salicylate and a DIR1/GLY1-dependent lipid signal. These include the dicarboxylic acid azelaic acid, the abietane diterpenoid dehydroabietinal, jasmonic acid, and the amino acid-derivative pipecolic acid. Our 2012 mini-review (Dempsey and Klessig) entitled “SOS – too many signals for systemic acquired resistance?” attempts to make sense of these newly discovered mobile signals.

B. Identification and characterization of new SA-binding proteins


In addition to disease resistance, SA affects many other plant processes, including flowering, seed germination, adventitious root initiation, and thermogenesis. In order to identify new SABPs through which SA exerts its many effects, we developed during the past several years two high throughput screens to identify candidate SABPS (cSABPs). The first utilizes SA analogs 4-azido SA (4AzSA) or 3-aminoethyl SA (3AESA), in conjunction with either a photoaffinity labeling technique or surface plasmon resonance (SPR)-based technology, to identify and evaluate cSABPs from Arabidopsis. The photoaffinity labeling and SPR-based approaches appear to be more sensitive than the traditional approach for identifying plant SA-binding activity using size exclusion chromatography with radiolabeled SA, as these proteins exhibited little to no SA-binding activity in such an assay. These novel approaches therefore complement conventional techniques and has help dissect the SA signaling network in plants. Our first results were reported in 2012 (Tian et al., Plant J 72:1027, 2012).

The second high throughput screen utilizes a protein microarray (PMA) to identify proteins that bind SA analogs. The initial screen, which used a 5,000 PMA (developed by S. Popescu, S. Dinesh-Kumar and M. Snyder) in conjunction with photoaffinity crosslinking to 4AzSA, yielded several dozen cSABPs, most of these were false positives. After further optimization we screened a new 10,000 PMA. Bioinformatic analysis indicated that the results were much more reproducible from microarray to microarray than in the previous screen. Using a stringent cutoff of P < 0.01, 41 cSABPs were identified. A subset of these, together with a subset from the 4AzSA crosslinking – immuno-selection screen were further characterized. The results were reported in Manohar et al., 2015, which revealed the identity of nine new SA-binding proteins (SABPs) and summarized the results from our two new, powerful screens for SABPs, which led to the discovery of a total of 23 new SABPs.


     Several members of the Arabidopsis GAPDH family, including two chloroplast-localized and two cytosolic isoforms, were identified as SABPs. Since cytosolic GAPDH is an important host factor involved in Tomato Bushy Stunt Virus (TBSV) replication, the effects of SA on its replication were evaluated in collaboration with Peter Nagy using three different replication system. SA inhibited TBSV replication by disrupting the binding of cytosolic GAPDH to the negative (-) RNA strand of TBSV. Thus, this study reveals a novel mechanism through which SA mediates resistance by targeting host factors used for virus replication (Tian et al., 2015).


Our results indicate that, as in humans, Arabidopsis HMGB3 functions as a Damage-Associated Molecular Pattern (DAMP), and that SA modulates this function. We discovered that extracellular HMGB3 induced a series of innate immune responses, including i) MAPK activation, ii) enhanced defense-related gene expression, iii) callose deposition, and iv) enhanced resistance to Botrytis cinerea, that are activated via a pathway that is depended on the receptor-like kinases BAK1 and BKK1. Further supporting its role as a DAMP, HMGB3 was released into the apoplast following B. cinerea infection. In addition, silencing multiple HMGB genes in transgenic plants reduced resistance to B. cinerea. HMGB3 exhibited authentic SA-binding activity in vitro, and its ability to activate MAPKs, induce callose deposition, and enhance resistance was inhibited by SA in vivo. These findings are consistent with our recent discovery that SA binds human HMGB1 (HsHMGB1), thereby inhibiting its pro-inflammatory activities (see below – Choi et al., 2015). Sequence alignment revealed that SA-binding sites in HsHMGB1 are conserved in the HMG box domain of Arabidopsis HMGB proteins. An SA-binding site mutant of HMGB3 retained its DAMP activity, but this activity was no longer inhibited by SA, consistent with its reduced ability to bind SA. Together these results provide cross-kingdom evidence that HMGB proteins function as DAMPs and that SA is their conserved inhibitor.

How does aspirin work in people?C. Human SAPBs

During the past three years, more of our focus has been on identifying and characterizing novel targets of SA, the active ingredient of aspirin that mediates aspirin’s multiple pharmacological effects, such as reduction in fever, pain, and inflammation, as well as the risk of stroke, heart attack, and cancer. We have discovered that SA binds to human High Mobility Group Box 1 (HMGB1) and Glyceraldehyde 3-Phosphate Dehydrogenase (GAPDH) and alters their activities.


We demonstrated that HMGB1 is a novel SA-binding protein using affinity chromatography, 4AzSA crosslinking, SPR, and NMR. SA-binding sites on HMGB1 were identified in the HMG-box domains by NMR and confirmed by mutational analysis. Extracellular HMGB1 is a DAMP, with multiple redox states. SA suppresses both the chemo-attractant activity of fully reduced HMGB1 and the increased expression of pro-inflammatory cytokine genes and COX-2 gene induced by disulfide HMGB1. Natural and synthetic SA derivatives with stronger binding affinity for HMGB1 than SA and greater potency for inhibition of HMGB1 were identified, providing proof-of-concept that new SA-based molecules with high efficacy against inflammation are attainable. An HMGB1 protein mutated in one of the SA-binding sites identified by NMR chemical shift perturbation studies retained chemo-attractant activity, but lost binding of and inhibition by SA and its derivatives, thereby firmly establishing that SA binding to HMGB1 directly suppresses its pro-inflammatory activities. Identification of HMGB1 as a pharmacological target of SA/aspirin provides new insights into the mechanisms of action of one of the world’s longest and most used natural and synthetic drugs (Choi et al., 2015). We are trying to extend this work to animal model systems for various diseases including arthritis, cancer, and Alzheimer’s disease with collaborators. In addition, HMGB1 and GAPDH are being used to identify new, more potent SA derivatives – either synthetic or from medicinal plants. For more information, see the interview in MedicalResearch.com.


In addition to its central role in glycolysis, GAPDH is a major participant in disease. GAPDH is a prime suspect in several neurodegenerative disorders, including Alzheimer’s, Parkinson’s, and Huntington’s diseases. In collaboration with Sol Snyder at Johns Hopkins, we found that SA and its more potent derivatives suppress nuclear translocation of GAPDH, induced by oxidative stress-like conditions, and the resulting cell death, just like the anti-Parkinson’s disease drug deprenyl (Choi et al., 2015). For additional information, see the interview in MedicalResearch.com.

D. CRT1/MORC1 characterization

Over the past several years we have characterized CRT1/MORC1 (Microrchidia) family, a subset of the GHKL ATPase superfamily, and discovered that it interacts with multiple immune receptors (R proteins and PAMP Recognition Receptors) and functions in multiple layers of plant immunity in Arabidopsis. CRT1/MORC1 localizes to endosomal-like vesicles but a small subpopulation translocates to the nucleus upon activation of immune responses (Kang et al., 2008, 2010, 2012). While the CRT1/MORC1 family positively modulates resistance in Arabidopsis and potato, in barley (Langen et al., 2014) and tomato this family negatively affects resistance, since its silencing results in enhanced resistance. To understand this species-specific effect of altering expression of CRT1/MORC1 on immunity, we took advantage of the differential effects in closely related tomato and potato. Using domain swapping and site-directed mutagenesis we determined that this species specificity is due to differences in the proteins themselves rather than the cellular environment in which these proteins function. This species specificity is determined by just four amino acid differences in the C-t region of these 650 amino acid proteins. We found that this C-t region also is required for i) protein dimerization and ii) interaction with 14 other proteins, iii) is phosphorylated, and iv) displays signaling activity (Manosalva et al., 2015). We suspect the species-specific effect is controlled by differential interaction of the C-t of the potato vs. tomato MORC1 with other proteins , which is being assessed using proteomics. We recently discovered that both plant and human MORCs have DNA modifying activities similar to type II topoisomerases, but are unique in requiring additional factor(s) for full activity (Manohar et al. 2017). These findings provide important insight into their functions in gene silencing (plants and animals) and cancer (human).

E. Modulation of plant immunity by nematode ascarosides

This is our newest area of research and one that holds considerable commercial promise. It is done in collaboration with Frank Schroeder. We discovered that plant parasitic nematodes produce small molecules called ascarosides, an evolutionarily conserved family of nematode pheromones, and that plants respond to these nematode-specific molecular patterns by activating systemic defenses against a broad spectrum of pathogens. Picomolar to micromolar concentrations of ascr#18, the most abundant ascaroside in plant parasitic nematodes, induced hallmark defense responses, including the expression of genes associated with Microbe-Associated Molecular Pattern (MAMP)-triggered immunity and activation of MAPKs. Ascr#18 induced both SA- and JA-mediated defense signaling pathways, enhanced resistance to virulent viral and bacterial pathogens, and reduced cyst nematode infection in Arabidopsis. Furthermore, we found that ascr#18 perception via roots or leaves increases resistance in tomato, potato, and barley to foliar bacterial, oomycete, or fungal pathogens. Our results indicate that monocots and dicots recognize ascarosides as a conserved molecular signature of nematodes that triggers conserved plant defense signaling pathways, similar to perception of MAMPs (Manosalva et al., 2015). Currently we are attempting to identify the plant receptor for ascr#18, uncover the mechanism of ascr#18 priming of plant defenses, and with a large group of collaborators, assess ascr#18’s ability to enhance resistance to a broad spectrum of pathogens in all the major crop species, including corn, rice, soybean, and wheat.