Our research is focused on understand, 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 the plant’s immune responses. 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. Furthermore, we are now employing the technology developed and knowledge gain from our work on SA and plant immunity to identifying the targets of aspirin (acetyl SA) and its major metabolite SA in humans.
A. Systemic acquired resistance (SAR) in Arabidopsis, tobacco, and potato
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 (SABPs).
In addition to disease resistance, SA affects many other plant processes, including flowering, seed germination, adventitious root initiation and thermogenesis. Over a 20-year period, we identified four SABPs from tobacco (methyl salicylate esterase/SABP2, catalase, ascorbate peroxidase, and carbonic anhydrase/SABP3) and characterized their potential roles in disease resistance. In order to identify additional SABPs through which SA exerts its many effects, we have developed during the past few years two high throughput screens to identify candidate SABPS (cSABPs). The first utilizes SA analogs 4-azido SA or 3-aminoethyl SA, in conjunction with either a photoaffinity labeling technique or surface plasmon resonance (SPR)-based technology, to identify and evaluate cSABPs from Arabidopsis. Using this screen, multiple proteins, including the E2 subunit of -Ketoglutarate Dehydrogenase and the Glutathione S-Transferases GSTF2, GSTF8, GSTF10, and GSTF11 were identified as SABPs. Their association with SA was further substantiated by the ability of SA to inhibit their enzymatic activity. The photoaffinity labeling and SPR-based approaches are 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 are helping to dissect the SA signaling network in plants (Tian et al., 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 4-azido SA, yielded several dozen cSABPs. While most of these were false positives, at least two SABPs were identified, the thimetoligopeptidases TOP1 and TOP2. Extensive characterization of these two proteins by Sorina Popescu’s group has demonstrated their involvement in defense responses (Moreau et al., 2013).
Following further optimization of the screening conditions, screening of a new 10,000 PMA uncovered 77 cSABPs. Recent characterization of a subset of these cSABPs and some of those from the 4-azido SA crosslinked – immuno-selection screen resulted in identification of an additional nine new SABPs (Manohar et al., 2015). To date 20 new SABPs have been identified in the two screens and approximately 80 cSABPs await further assessment.
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’s group using an in vitro yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts. SA inhibited TBSV replication in all three assays; it did so 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 of virus replication (Tian et al., 2015).
B2. Human SAPBs
Through most of human history medicine has been based on plant remedies. Even in “modern” medicine many drugs have plant origins. SA and its derivatives, collectively termed salicylates, are a prime example. Acetyl SA, commonly called aspirin, has been the most widely used drug worldwide for the past two centuries – even today Americans alone consume 80 million aspirin tablets daily.
During the past two years, more of our focus has been on identifying and characterizing novel human 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 three novel targets that play important roles in several of the most widespread and devastating diseases. One aspirin/SA target is a pro-inflammatory protein associated with sepsis, arthritis, Crohn’s disease, atherosclerosis, and cancer. Another is a key player in diabetes and energy metabolism. The third is involved in hepatitis A, B, and C virus infection and is the key suspect in neurodegenerative diseases including Alzheimer’s, Parkinson’s, and Huntington’s.
Moreover, based on our studies with two of these targets, a synthetic and a natural derivative of aspirin have been identified, which are approximately 100 times more effective than aspirin. In sum, at least three new targets of aspirin/SA have been identified, and two novel derivatives of SA discovered that are far more potent than aspirin. Therefore, the impact on human health could be profound, especially since the use of aspirin is so widespread and generally is well tolerated, particularly at low dosages.
C. CRT1/MORC1 characterization
Over the past several years we have characterized the 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 including i) effector-triggered immunity, ii) PAMP-triggered immunity, iii) SAR, and iv) non-host immunity. CRT1/MORC1 primarily localizes to endosomal-like vesicles. However, 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, 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 the closely related tomato and potato. In contrast to tomato, silencing of CRT1/MORC1 in potato reduces resistance to Phytophthora infestans. 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 acids in the C-t region of these 650 amino acid proteins. We found that this C-t region 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).
D. Modulation of plant immunity by nematode ascarosides
In collaboration with Frank Schroeder, we discovered that one or more ascarosides (e.g. ascr#18) either induce or prime induction of expression of defense genes associated with PAMP-triggered immunity, of the prototypic SA-responsive PR-1 gene, and JA- responsive genes like PDF1.2. Moreover, ascr#18 enhanced resistance in Arabidopsis, tobacco, tomato, potato and barley to viral, bacterial, oomycete, fungal, and nematode pathogens (Manosalva et al., 2015).
Over the past decade we have hosted 10 undergraduate research interns. All have worked closely with senior Post Doctoral Fellows or Research Associates, with most of them studying various aspects of SA-mediated defense signalling in plants. In addition, two helped characterize CRT1/MORC1.