Daniel Klessig

Daniel Klessig
Office/Lab: 223/206-210
Phone: 607-254-4560
Office/Lab: 223/206-210
Email: dfk8@cornell.edu
Office Phone: 607-254-4560
Lab Phone: 607-254-1255
Affiliations: Adjunct Professor, Section of Plant Pathology & Plant-Microbe Biology / School of Integrative Plant Science / Cornell University
Graduate Fields: Plant Biology; Plant Pathology & Plant-Microbe Biology
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 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.

  • Plants Found to Speak Roundworm’s Language

  • Aspirin-Like Compounds Could Treat Numerous Human Diseases

    People have used aspirin to treat pain, fever and inflammation for more than a century, and the drug is also used to reduce the risk of strokes, heart attacks and some cancers. An estimated 100 billion aspirin tablets are taken worldwide each year, but how it works is still only partially understood. Dan Klessig, a […] Read more »
  • Worm Pheromones Protect Major Crops

    Protecting crops from pests and pathogens without using toxic pesticides has been a longtime goal of farmers. Researchers at Boyce Thompson Institute have found that compounds from an unlikely source – microscopic soil roundworms – could achieve this aim. As described in research published in the May 2019 issue of Journal of Phytopathology, these compounds […] Read more »
  • Ascribe Bioscience Receives SBIR Award from NSF

    On January 29, 2019, Ascribe Bioscience became the first company based on technology developed at the Boyce Thompson Institute (BTI) to receive a Small Business Innovation Research (SBIR) grant. The agbiotech startup will use the $225,000 Phase I award from the National Science Foundation (NSF) to develop a seed treatment technology that protects plants from […] Read more »
  • Klessig’s Research Featured by Parkinson’s Disease Foundation

    The Spring 2016 issue of the Parkinson's Disease Foundation newsletter featured recent work by Dan Klessig. The newsletter highlighted his recent PLOS ONE paper on salicylic acid and human GAPDH. Read more »

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.

Internship Program | Projects & FacultyApply for an Internship

Arabidopsis SMALL DEFENSE-ASSOCIATED PROTEIN 1 Modulates Pathogen Defense and Tolerance to Oxidative Stress
Dutta, A., Choudhary, P., Gupta-Bouder, P., Chatterjee, S., Liu, P.P., Klessig, Daniel F., Raina, R.
Front Plant Sci..
Plant metabolism of nematode pheromones mediates plant-nematode interactions
Manohar, M., Tenjo-Castano, F., Chen, S., Zhang, Y.K., Kumari, A., Williamson, V.M., Wang, X., Kless…
Nat Commun..
A genome-wide screen for human salicylic acid (SA)-binding proteins reveals targets through which SA may influence development of various diseases
Choi, H.W., Wang, L., Powell, A.F., Strickler, S.R., Wang, D., Dempsey, D.A., Schroeder, Frank C., K…
Scientific Reports.
Nematode ascaroside enhances resistance in a broad spectrum of plant–pathogen systems
Klessig, Daniel F., Manohar, M., Baby, S., Koch, A., Danquah, W.B., Luna, E., Park, H.J., Kolkman, J…
Journal of Phytopathology.
Mimicking the Host Regulation of Salicylic Acid: A Virulence Strategy by the Clubroot Pathogen Plasmodiophora brassicae
Djavaheri, M., Ma, L., Klessig, Daniel F., Mithöfer, A., Gropp, G., Borhan, H.
Molecular Plant-Microbe Interactions.
Systemic Acquired Resistance and Salicylic Acid: Past, Present and Future
Klessig, Daniel F., Choi, H. W., Dempsey, D. A.
Molecular Plant-Microbe Interactions.
Plant and Human MORC Proteins Have DNA-Modifying Activities Similar to Type II Topoisomerases, but Require One or More Additional Factors for Full Activity
Manohar, M., Choi, H. W., Manosalva, P., Austin, C. A., Peters, Joe E., Klessig, Daniel F.
Molecular Plant-Microbe Interactions.
MORC Proteins: Novel Players in Plant and Animal Health
Koch, A., Kang, H. G., Steinbrenner, J., Dempsey, D. A., Klessig, Daniel F., Kogel, K. H.
Frontiers in Plant Science.
How does the multifaceted plant hormone salicylic acid combat disease in plants and are similar mechanisms utilized in humans?
Dempsey, D. A., Klessig, Daniel F.
BMC Biology.
Multiple Targets of Salicylic Acid and Its Derivatives in Plants and Animals
Klessig, Daniel F., Tian, M., Choi, H. W.
Frontiers in Immunology.
Newly Identified Targets of Aspirin and Its Primary Metabolite, Salicylic Acid
Klessig, Daniel F.
DNA and Cell Biology.
DAMPs, MAMPs, and NAMPs in plant innate immunity
Choi, H. W., Klessig, Daniel F.
BMC Plant Biology.
Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid
Choi, H. W., Manohar, M., Manosalva, P., Tian, M., Moreau, M., Klessig, Daniel F.
PLoS Pathogens.
Pathogen Infection and MORC Proteins Affect Chromatin Accessibility of Transposable Elements and Expression of Their Proximal Genes in Arabidopsis
Bordiya, Y., Zheng, Y., Nam, J. C., Bonnard, A. C., Choi, H. W., Lee, B. K., Kim, J., Klessig, Danie…
Molecular Plant-Microbe Interactions.
The GHKL ATPase MORC1 Modulates Species-Specific Plant Immunity in Solanaceae
Manosalva, P., Manohar, M., Kogel, K. H., Kang, H. G., Klessig, Daniel F.
Molecular Plant-Microbe Interactions.
Salicylic acid binding of mitochondrial alpha-ketoglutarate dehydrogenase E2 affects mitochondrial oxidative phosphorylation and electron transport chain components and plays a role in basal defense against tobacco mosaic virus in tomato
Liao, Y., Tian, M., Zhang, H., Li, X., Wang, Y., Xia, X., Zhou, J., Zhou, Y., Yu, J., Shi, K., Kless…
New Phytologist.
Aspirin’s Active Metabolite Salicylic Acid Targets High Mobility Group Box 1 to Modulate Inflammatory Responses
Choi, H. W., Tian, M., Song, F., Venereau, E., Preti, A., Park, S. W., Hamilton, K., Swapna, G. VT, …
Molecular medicine (Cambridge, Mass.).
Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex
Tian, M., Sasvari, Z., Gonzalez, P. A., Friso, Giulia, Rowland, E., Liu, X. M., Van Wijk, Klaas, Nag…
Molecular Plant-Microbe Interactions.
Conserved nematode signalling molecules elicit plant defenses and pathogen resistance
Manosalva, P., Manohar, M., Von Reuss, S. H., Chen, S., Koch, A., Kaplan, F., Choe, A., Micikas, R. …
Nature Communications.
Human GAPDH Is a Target of Aspirin’s Primary Metabolite Salicylic Acid and Its Derivatives
Choi, H. W., Tian, M., Manohar, M., Harraz, M. M., Park, S. W., Schroeder, Frank, Snyder, S. H., Kle…

High-affinity salicylic acid-binding protein and methods of use
Dan Klessig
US Patent: 6,136,552
Genes Associated with enhanced disease resistance in plants
Dan Klessig
US Patent: 5,939,601
Methods for determining specificity of RNA silencing and for genetic analysis of the silenced gene or protein
Dan Klessig
US Patent: 7,592,504
Methods and compositions for improving salicylic acid-independent systemic acquired disease resistance in plants
Dan Klessig
US Patent: 6,495,737
Method of using a pathogen-activatable map kinase to enhance disease resistance in plants
Dan Klessig
US Patent: 6,765,128
Salicylic Acid Binding Protein (SABP2)
Dan Klessig
US Patent: 7,169,966
Salicylic acid induced map kinase and its use for enhanced disease resistance in plants
Dan Klessig
US Patent: 5,977,442


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