Boyce Thompson Institute for Plant Research
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Dan Klessig

Dan Klessig
Dan Klessig
Professor
Office/Lab: 223/206
dfk8@cornell.edu
Office: 607-254-4560
Lab: 607-254-1255

Research Summary

Research Summary

The major focus of our research is to understand, at the biochemical, molecular and cellular levels, how plants protect themselves against microbial pathogens. The interaction of tobacco and Arabidopsis with their viral pathogens Tobacco Mosaic Virus (TMV) and Turnip Crinkle Virus (TCV), respectively, are the two principal (but not exclusive) model systems used. Our main goal is to decipher the signal transduction pathway(s) that leads to induction of plant defenses.

During the past two decades we have identified salicylic acid (SA) and nitric oxide (NO) as two important defense signals involved in the activation of defense responses to pathogens. Current studies are directed at defining components in the SA- and NO-mediated signalingpathways and determining their mechanisms of action using a combination of biochemical, pharmacological, molecular and genetic approaches. The TCV-Arabidopsis pathosystem is being utilized to help understand the early, as well as late, steps in activation of defense responses.

SA-binding proteins

One of the major projects during the past several years is the continued characterization of tobacco SA-binding proteins (SABPs) as part of our effort to decipher the SA-mediated signal transduction network. Much of our recent effort has been focused on SABP2 since it was a likely receptor for SA, given its very high affinity and specificity for SA and extremely low abundance. Silencing of SABP2 using RNAi technology showed that it is involved in both basal resistance and resistance (R) gene-mediated resistance, as well as in systemic acquired resistance (SAR; Kumar and Klessig, 2003). Biochemical and structural studies of SABP2 revealed i) its methyl salicylate (MeSA) esterase activity, ii) its 3-D structure, and iii) feedback inhibition of its esterase activity by binding of SA in its active site (Forouhar et al., 2005). To assess the specificity of RNAi-mediated silencing and to facilitate structure-function analyses of SABP2, a novel approach, termed synthetic gene complementation, was developed (Kumar et al., 2006).

Characterization of SABP2’s role in SAR revealed that its MeSA esterase activity is required in the systemic tissue to perceive/process the mobile SAR signal and this esterase activity must be inhibited by SA binding in the primary infected tissue to generate the SAR signal. These results together with quantification of MeSA in the various tissue, including phloem exudate, demonstrated that MeSA is a long-sought, phloem-mobile signal for SAR and SABP2’s function is to cleave MeSA to release active SA, which then activates/primes defenses in the systemic tissue leading to SAR (Park et al., 2007). The involvement of MeSA esterase and MeSA in SAR has been demonstrated in Arabidopsis (Vlot el al., 2008) and is currently being studied in potato. We have recently identified a large number of new putative SABPs. Their characterization is a major focus of our current and future research.

Identification of defense signaling components using genetics

Arabidopsis-TCV pathosystem:

During the 1990s and early 2000s we developed the Arabidopsis-TCV pathosystem. This led to identification of HRT, which encodes a CC (coiled coil)-NBS-LRR type R protein required for resistance to TCV, and also RRT, a yet to be cloned gene that regulates resistance to TCV. Further analyses of this pathosystem suggest that the reason why HRT, while necessary, is not sufficient for resistance is that SA levels are not sufficiently elevated after infection to enhance HRT expression and thus facilitate stable resistance. The recessive rrt allele, which together with HRT is sufficient for resistance, may correct this SA deficiency (Chandra-Shekara et al., 2004). A genetic screen has uncovered a novel ATPase that is required for resistance to TCV and other pathogens; it physically interacts with HRT and other R proteins (Kang et al., 2008). Its role in defense signaling is under investigation.

Cyclic Nucleotide-Gated Ion Channel (CNGCs) mutants:

A genetic screen of a T-DNA insertion mutant Arabidopsis library identified a mutant which constitutively expresses defense-associated PR genes (cpr22), has elevated levels of SA and displays enhanced resistance (Yoshioka et al., 2001). Further characterization of cpr22 revealed that it contains a deletion which results in loss of two CNGCs, CNGC11 and CNGC12, and formation of a chimeric CNGC11/12 with new signaling properties. This study also demonstrated that the defense signaling components NDR1, EDS1 and PAD4 mediate other resistance signaling functions in addition to SA and PR protein accumulation (Yoshioka et al., 2006).

Suppressors of SA insensitive mutants:

In the late 1990’s we isolated several Arabidopsis mutants termed suppressor of the SA insensitive (ssi1-ssi4) phenotype of npr1/sai1; NPR1/SAI1 is a key positive regulator of SA-mediated signaling. Characterization of ssi4 showed that it is a constitutively active R protein, which confers constitutive activation of SA-mediated defenses, enhanced disease resistance, elevated levels of reactive oxygen species, activation of the defense-associated MAP kinases MPK3 and MPK6, and induction of various defense genes (Shirano et al., 2002; Zhou et al., 2004). The ssi4 mutation also causes morphological alterations which differentially require SGT1b, whereas activation of defenses requires RAR1 (Zhou et al., 2008). Similarly, the ssi2 mutation leads to activation of SA-mediated defense signaling but suppression of jasmonic acid (JA)-mediated defense signaling. The mutation is in a fatty acid (stearoyl) desaturase resulting in reduced levels of oleic acid (18:1) which is responsible for both constitutive SA-mediated signaling and the resulting enhanced resistance to biotrophic pathogens like Pseudomonas syringae, and for compromised JA-mediated signaling and the resulting reduced resistance to necrotrophic pathogens such as Botrytis cinerea (P. Kachroo et al., 2001; P. Kachroo et al., 2003; A. Kachroo et al., 2003; Nandi et al., 2005).

Mitogen-activated protein kinases (MAP kinases)

In the late 1990s we purified the first plant MAP kinase (SA-induced protein kinase, SIPK) and demonstrated its involvement in protection against pathogens. In 2004, Arabidopsis’ homolog of SIPK, MPK6, was shown to be required for disease resistance (Menke et al., 2004) and in 2005, one of the first substrates of a plant MAP kinase was identified. SIPK was found to phosphorylate the defense-associated transcription factor NtWRKY1 (Menke et al., 2005).

NO and plant defense

In the late 1990s, we and the Chris Lamb/Rick Dixon group demonstrated the involvement of NO in plant immunity. Several direct targets of NO were subsequently identified, including aconitase, catalase, and ascorbate peroxidase. Further studies demonstrated that plant aconitases are bifunctional, like their mammalian counterparts. In the presence of NO, aconitases lose their iron-sulfur cluster in the catalytic center, and hence their enzymatic activity, but gain RNA binding activity. The Arabidopsis aconitase binds the 5’-UTR of the chloroplastic Cu/Zn superoxide dismutase mRNA and appears to have a role in resisting oxidative stress and activating cell death (Moeder et al., 2007).

Most of our research on NO signaling over the past decade has focused on identifying the NO synthase (NOS) responsible for NO synthesis during infection. Unfortunately, its identity is still unknown. Our work on the putative NOS activity of the P protein could not be reproduced. Moreover, our subsequent studies of AtNOS1 (identified by Nigel Crawford’s group) indicate that it is not an NOS, but is a GTPase (Moreau et al., 2008).

Selected Publications

View All Publications    |    All papers on PubMed

Dempsey, D.A. and Klessig, D.F. 2012. SOS - too many signals for systemic acquired resistance?. Trends Plant Sci 17: 538-545

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. 2012. CRT1 is a nuclear-translocated MORC endonuclease that participates in multiple levels of plant immunity. Nature communications 3: 1297

Mang, H.G., Qian, W.Q., Zhu, Y., Qian, J., Kang, H.G., Klessig, D.F. and Hua, J. 2012. Abscisic acid deficiency antagonizes high-temperature inhibition of disease resistance through enhancing nuclear accumulation of resistance proteins SNC1 and RPS4 in Arabidopsis. Plant Cell 24: 1271-1284

Moreau, M., Tian, M. and Klessig, D.F. 2012. Salicylic acid binds NPR3 and NPR4 to regulate NPR1-dependent defense responses. Cell Res 22: 1631-1633

Tian, M., von Dahl, C.C., Liu, P.P., Friso, G., van Wijk, K.J. and Klessig, D.F. 2012. The combined use of photoaffinity labeling and surface plasmon resonance-based technology identifies multiple salicylic acid-binding proteins. The Plant journal : for cell and molecular biology 72: 10271038

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. 2012. Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation. Cell Host Microbe 11: 587-596

Liu, P.P., von Dahl, C.C. and Klessig, D.F. 2011. The extent to which methyl salicylate is required for signaling systemic acquired resistance is dependent on exposure to light after infection. Plant Physiol 157: 2216-2226

Liu, P.P., von Dahl, C.C., Park, S.W. and Klessig, D.F. 2011. Interconnection between methyl salicylate and lipid-based long-distance signaling during the development of systemic acquired resistance in Arabidopsis and tobacco. Plant Physiol 155: 1762-1768

Dempsey, D.A., Vlot, A.C., Wildermuth, M.C. and Klessig, D.F. 2011. Salicylic acid biosynthesis and metabolism. In The Arabidopsis Book American Society of Plant Biologists 0:

Moreau, M., Lindermayr, C., Durner , J. and Klessig, D.F. 2010. NO synthesis and signaling in plants--where do we stand?. Physiol Plant 138: 372-383

Mosher, S., Moeder, W., Nishimura, N., Jikumaru, Y., Joo, S.H., Urquhart, W., Klessig, D.F., Kim, S.K., Nambara, E. and Yoshioka, K. 2010. The lesion-mimic mutant cpr22 shows alterations in abscisic acid signaling and abscisic acid insensitivity in a salicylic acid-dependent manner. Plant Physiology 152: 1901-1913

Manosalva, P.M., Park, S.W., Forouhar, F., Tong, L., Fry , W.E. and Klessig, D.F. 2010. Methyl esterase 1 (StMES1) is required for systemic acquired resistance in potato. Mol Plant Microbe Interact 23: 1151-1163

Liu, P.P., Yang, Y., Pichersky, E. and Klessig, D.F. 2010. Altering expression of benzoic acid/salicylic acid carboxyl methyltransferase 1 compromises systemic acquired resistance and PAMP-triggered immunity in arabidopsis. Mol Plant Microbe Interact 23: 82-90

Liu, P.P., Bhattacharjee, S., Klessig, D.F. and Moffett, P. 2010. Systemic acquired resistance is induced by R gene-mediated responses independent of cell death. Mol Plant Pathol 11: 155-160

Kang, H.G., Oh, C.S., Sato, M., Katagiri, F., Glazebrook, J., Takahashi, H., Kachroo, P., Martin, G.B. and Klessig, D.F. 2010. Endosome-associated CRT1 functions early in resistance gene-mediated defense signaling in Arabidopsis and tobacco. Plant Cell 22: 918-936

Jeong, R.D., Chandra-Shekara, A.C., Barman, S.R., Navarre, D., Klessig, D.F., Kachroo, A. and Kachroo, P. 2010. Cryptochrome 2 and phototropin 2 regulate resistance protein-mediated viral defense by negatively regulating an E3 ubiquitin ligase. Proc Natl Acad Sci U S A 107: 13538-13543

Vlot, A.C., Dempsey, D.A. and Klessig, D.F. 2009. Salicylic acid, a multifaceted hormone to combat disease. Annu Rev Phytopathol 47: 177-206

Park, S.W., Liu, P.P., Forouhar, F., Vlot, A.C., Tong, L., Tietjen, K. and Klessig, D.F. 2009. Use of a synthetic salicylic acid analog to investigate the roles of methyl salicylate and its esterases in plant disease resistance. Journal of Biological Chemistry 284: 7307-7317

Kawamura, Y., Takenaka, S., Hase, S., Kubota, M., Ichinose, Y., Kanayama, Y., Nakaho, K., Klessig, D.F. and Takahashi, H. 2009. Enhanced defense responses in Arabidopsis induced by the cell wall protein fractions from Pythium oligandrum require SGT1, RAR1, NPR1 and JAR1. Plant Cell Physiol 50: 924-934

Liu, P.P., Yang, Y., Pichersky, E. and Klessig, D.F. 2009. Altering expression of benzoic scid/salicylic acid carboxyl methyltransferase 1 compromises systemic acquired resistance and PAMP-triggered immunity in Arabidopsis. Mol Plant Microbe In 23: 82-90

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. 2008. Identification of likely orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana. Plant Journal 56: 445-456

Vlot, A.C., Klessig, D.F. and Park, S.W. 2008. Systemic acquired resistance: the elusive signal(s). Curr Opin Plant Biol 11: 436-442

Sudhamsu, J., Lee, G.I., Klessig, D.F. and Crane, B.R. 2008. The Structure of YqeH: An AtNOS1/AtNOA1 ortholog that couples GTP hydrolysis to molecular recognition. Journal of Biological Chemistry 283: 32968-32976

Sekine, K.T., Kawakami, S., Hase, S., Kubota, M., Ichinose, Y., Shah, J., Kang, H.G., Klessig, D.F. and Takahashi, H. 2008. High level expression of a virus resistance gene, RCY1, confers extreme resistance to Cucumber mosaic virus in Arabidopsis thaliana. Mol Plant Microbe In 21: 1398-1407

Moreau, M., Lee, G.I., Wang, Y., Crane, B.R. and Klessig, D.F. 2008. AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase. Journal of Biological Chemistry 283: 32957-32967

Moffett, P. and Klessig, D.F. 2008. Plant resistance to viruses: Natural resistance associated with dominant genes. In Encyclopedia of Virology (Mahy, B.W.J. and van Regenmortel, M. eds). Oxford 0: Elsevier.

Kumar, D. and Klessig, D.F. 2008. The search for the salicylic acid receptor led to discovery of the SAR signal receptor. Plant Signal Behav 3: 691-692

Kang, H.G., Kuhl, J.C., Kachroo, P. and Klessig, D.F. 2008. CRT1, an Arabidopsis ATPase that interacts with diverse resistance proteins and modulates disease resistance to turnip crinkle virus. Cell Host & Microbe 3: 48-57

Jeong, R.D., Chandra-Shekara, A.C., Kachroo, A., Klessig, D.F. and Kachroo, P. 2008. HRT-mediated hypersensitive response and resistance to Turnip crinkle virus in Arabidopsis does not require the function of TIP, the presumed guardee protein. Mol Plant Microbe In 21: 1316-1324

Kang, H.G. and Klessig, D.F. 2008. The involvement of the Arabidopsis CRT1 ATPase family in disease resistance protein-mediated signaling. Plant Signal Behav 3: 689-690

Yang, Y., Xu, R., Ma, C.J., Vlot, A.C., Klessig, D.F. and Pichersky, E. 2008. Inactive methyl indole-3-acetic acid ester can be hydrolyzed and activated by several esterases belonging to the AtMES esterase family of Arabidopsis. Plant Physiology 147: 1034-1045

Zhou, F.S., Mosher, S., Tian, M.Y., Sassi, G., Parker, J. and Klessig, D.F. 2008. The Arabidopsis gain-of-function mutant ssi4 requires RAR1 and SGT1b differentially for defense activation and morphological alterations. Mol Plant Microbe In 21: 40-49

Park, S.-W., Kaiyomo, E., Kumar, D., Mosher, S.L., and Klessig, D.F. 2007. Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science 318: 113-116

Kumar, D., C. Gustafsson, D. F. Klessig. 2006. Validation of RNAi Silencing Specificity Using Synthetic Genes: Salicylic Acid-binding Protein 2 is Required for Innate Immunity in Plants. The Plant Journal 45: 863-868

Yoshioka, K., W. Moeder, H.-G. Kang, P. Kachroo, K. Masmoudi, G. Berkowitz, D. F. Klessig. 2006. The Chimeric Arabidopsis CYCLIC NUCLEOTIDE-GATED ION CHANNEL11/12 Activates Multiple Pathogen Resistance Responses. Plant Cell 18: 747-763

Forouhar, F., Y. Yang, D. Kumar, Y. Chen, E. Fridman, S. W. Park, Y. Chiang, T. B. Acton, G. T. Montelione, E. Pichersky, D. F. Klessig, L. Tong. 2005. Crystal structure and biochemical studies identify tobacco SABP2 as a methylsalicylate esterase and further implicate it in plant innate immunity. Proc. Nat'l. Acad. Sci. USA 102: 1773-1778

Menke, F. L. H., J. A. van Pelt, C. M. J. Pieterse, D. F. Klessig. 2004. Silencing of the Mitogen-activated Protein Kinase MPK6 Compromises Disease Resistance in Arabidopsis. Plant Cell 16: 897-907

Liu, P.-P., von Dahl, C. C., Park, S.-W., and Klessig, D. F. 2001. Interconnection between methyl salicylate and lipid-based long-distance signaling during systemic acquired resistance in Arabidopsis and tobacco. Plant Physiol 155: 1762-1768

Features

Features

How do plants acquire immunity to disease?

feature released -2008

How do plants acquire immunity to disease?

When we hear the phrase “acquired immunity,”we usually think of humans and vaccinations, but plants also can acquire immunity (more often referred to as “resistance”) when a pathogen invades the plant. Understanding how this system works could lead to crops that more effectively protect themselves from disease.

Dan Klessig’s laboratory studies this phenomenon. In earlier work, his group and others proved that an aspirinlike compound called salicylic acid (SA) is produced at the site of infection. Some of this disease-fighting hormone activates the plant’s local defenses and some is converted into methyl salicylate (MeSA), an inactive form of SA. Studies by others subsequently showed that SA is required in uninfected distant tissue, as well, for the plant to develop systemic acquired resistance (SAR) against secondary infection, but that SA is not the mobile molecule for SAR. This finding suggested there must be another,mobile molecule that carries the message from the infected tissue to other parts of the plant.

In an important breakthrough this year,Klessig’s team discovered that MeSA is the mobile molecular messenger. His group found that a plant enzyme, SABP2, converts MeSA into SA, and that this activity is required in the distant tissue but is inhibited in the infected tissue. They further showed that MeSA travels through the phloem, or transporting tissue, from the site of infection to the distant tissue. They also demonstrated that an enzyme called SAMT,which converts SA into MeSA, is needed only in the infected tissue. These findings established that after a pathogen attacks, some of the SA synthesized at the site of infection is converted by SAMT into MeSA. MeSA is then transported through the phloem to distant, uninfected parts of the plant where SABP2 converts it back into diseasefighting SA, thereby turning on the plant’s defenses in those tissues. Therefore,MeSA is central to the plant’s SAR.

Klessig notes that MeSA may be only one of several molecular messengers involved in the process. Nonetheless, his group’s discovery of MeSA as a mobile messenger, and the enzymes that regulate its level, explains for the first time how molecular information produced at the site of infection is communicated throughout the plant to provide it with acquired immunity.>

Learning from an “Allergic” Plant.

feature released -2007

Learning from an "Allergic" Plant.Gardeners have long noted a connection between damp weather and plant disease, which scientists have attributed to a constellation of factors, such as more favorable conditions for germination of fungal spores. Dan Klessig’s lab recently found that high humidity actually suppresses a plant’s immune system, making it more vulnerable to pathogens.

Klessig lab members studied a mutant of Arabidopsis whose immune response is always turned on-like having very bad allergies. Though good at warding off infection, the mutant puts so much energy into immunity that it looks sickly and stunted. When grown in chambers with high humidity, Klessig’s group found, the mutant plant’s immune response shut down, enabling it to grow normally.

Currently the lab studies additional mutations in the “allergic” plant itself that turn off its immune response. Studying such mutants should help Klessig develop a picture of the early steps in the signaling system plants use to counter infection.

In separate studies, the Klessig lab modified an Arabidopsis plant to knock out one component of an important signaling pathway. In this pathway, the MAP kinase cascade, a signal such as a hormone from inside or outside of the cell sets off a chain reaction, ultimately kick-starting the cell’s response to the signal. Klessig’s lab found that the elimination of one link in this chain reaction increased the susceptibility of the plant to several different pathogens, suggesting that one of the MAP kinase cascade’s duties is to activate the plant’s immune response. Learning about this pathway could enable scientists to manipulate it to create crop plants that are more resistant to disease.

Sending out an SOS.

feature released -2007

Sending out an SOS.When a virus, bacterium, or other pathogen attacks a leaf, the whole plant goes on heightened alert, readying itself to take on all attackers. Plant researchers have long puzzled over the signal the infected part of the plant uses to warn the rest of impending danger.

The signal would probably travel through the phloem, the specialized system that transports nutrients throughout the plant—but what was the signal? One promising candidate was salicylic acid (SA), a molecule Dan Klessig’s lab showed is involved in the general defense response, which is provoked when part of a plant is threatened. But experiments seemed to rule out SA as the so-called phloem mobile signal.

Klessig’s lab wasn’t looking for the phloem mobile signal when they identified several proteins that interact with SA. Instead, they wanted to learn how SA’s presence gets detected and ultimately affects the expression of defense-related genes -a process known as a signaling cascade. Proteins that bound to SA were likely to be part of this cascade, they thought.

They found several such proteins, including SABP2, which was present in such low levels that it took the lab years to purify enough to clone its gene and determine its three-dimensional structure. When Klessig’s collaborators crystallized SABP2 and used X-rays to reveal its shape, though, they discovered something strange. SA itself was bound to SABP2′s active site-that is, the part of the protein with the ability to chemically alter a molecule and thus pass along a signal. But this didn’t seem to make sense if SABP2 was part of a signaling cascade that started with SA.

It turned out that SABP2′s job was to lop a methyl group off of methyl salicylate, thereby converting it to SA. Klessig hypothesizes that methyl salicylate could be the long-sought phloem mobile signal: methyl salicylate sent through the phloem could be later converted to SA and set off a defense response. His group plans to test this hypothesis this year.

Lab Members

Lab Members

Hyong Woo Choi
Hyong Woo Choi
Postdoc
Office/Lab: 211/206
hc746@cornell.edu
Office:
Lab: 607 254 1255
Murli Manohar
Murli Manohar
Postdoc
Office/Lab: 225/206
mm829@cornell.edu
Office:
Lab: 607 254 1255
Patricia Manosalva
Patricia Manosalva
Research Associate
Office/Lab: 225/206
pmm92@cornell.edu
Office:
Lab: 607 254 1255