Proc. Natl. Acad. Sci. USA Vol. 90, pp. 9533-9537, October 1993 Biochemistry Purification and characterization of a soluble salicylic acid-binding protein from tobacco (monoclonal antibody/pathogenesis-related proteins/plant defense mechanism/plant signal transduction/systemic acquired resistance) ZHIXIANG CHEN, JOSEPH W. RICIGLIANO, AND DANIEL F. KLESSIG* Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, NJ 08855-0759 Communicated by Charles S. Levings III, July 12, 1993 (received for review May 11, 1993) ABSTRACT Previously, we identified a soluble salicylic SA in the induction of defense responses is provided by the acid (SA)-binding protein (SABP) in tobacco whose properties transgenic tobacco plants which contain and constitutively suggest that it may play a role in transmitting the SA signal express the nahG gene encoding salicylate hydroxylase from during plant defense responses. This SA-binding activity has Pseudomonas putida (21). In these transgenic plants, induc- been purified 250-fold by conventional chromatography and tion of SAR by inoculation with tobacco mosaic virus was was found to copurify with a 280-kDa protein. Monoclonal blocked, presumably due to the destruction of the SA signal antibodies capable of immunoprecipitating the SA-binding by the hydroxylase. activity also immunoprecipitated the 280-kDa protein, indicat- We have been interested in identifying cellular compo- ing that it was responsible for binding SA. These antibodies also nent(s) which directly interact with SA, as a first step to recognized the 280-kDa protein in immunoblots ofthe partially elucidate the mechanism(s) of action of SA in plant signal purified SABP fraction or the crude extract. However, when transduction. We have detected and partially characterized a the crude extract was prepared in the presence of antioxidants, soluble SA-binding protein (SABP) in tobacco leaves (12). only a 57-kDa protein was recognized. Since the SABP has a Here, we report the successful purification of SABP by using native molecular mass of 240 kDa, it appears that the SABP is conventional chromatography and monoclonal antibodies a complex which contains a 57-kDa subunit and perhaps one or (mAbs). The isolated SABP has also been further character- more additional proteins which are covalently crosslinked in ized to assess its functional relevance to plant defense the absence of antioxidants. The ability of a variety of phenolic mechanisms induced by SA. compounds to compete with SA for binding to the SABP was both qualitatively and quantitatively correlated with their MATERIALS AND METHODS biological activity in inducing defense-related genes. Moreover, the inducibility of the pathogenesis-related (PR)-1 genes by SA Materials. [7-14C]SA (55 Ci/mol; 1 Ci = 37 GBq) was from was proportional to the abundance of the SABP in different New England Nuclear. SA and other phenolic compounds organs. These correlations are consistent with a role for the were from Sigma or Aldrich. DEAE-Sephacel, Sephacryl SABP in perceiving and transducing the SA signal in plant S-300, and protein A-Sepharose were from Pharmacia. Blue defense. dextran-agarose and other general chemicals were from Sigma. Assays. [14C]SA binding was assayed with spin-column Plants, like other organisms, use environmental signals to exclusion chromatography (12). Protein concentrations were make appropriate adaptive responses. An excellent example determined according to Bradford (13) with the Bio-Rad of this is the induction of defense responses by plants upon protein assay kit. infection by microbial pathogens (1, 2). In the well- Purification Procedure. Tobacco (Nicotiana tabacum cv. characterized tobacco/tobacco mosaic virus system, the Xanthi nc) leaves (200 g) were sliced and homogenized with resistance response consists of both local and systemic a Polytron homogenizer (Brinkmann) in 1 liter of binding components. The local response, referred to as the hyper- buffer [20 mM citrate (pH 6.5)/5 mM MgSO4/1 mM EDTA/ sensitive response, involves the formation ofnecrotic lesions 10% (vol/vol) glycerol/phenylmethylsulfonyl fluoride at 30 and the restriction of virus proliferation to small zones Ztg/ml] with 2% (wt/vol) polyvinylpolypyrrolidone. The ho- around the sites of infection (3). In addition, plant defense mogenate was filtered through four layers of cheesecloth and genes, including five or more families of unrelated pathogen- then clarified by centrifugation at 40,000 x g for 40 min. The esis-related (PR) genes, are activated in both the inoculated resulting supernatant was loaded onto a DEAE-Sephacel and the uninoculated portions of the plant (4). The systemic column (2.5 x 15 cm) that had been equilibrated with binding expression of PR genes correlates with the establishment of buffer. After loading, the column was washed with binding systemic acquired resistance (SAR) (5), in which the entire buffer and a 400-ml gradient from 0 to 1 M KCI in the binding plant exhibits elevated levels of resistance to secondary buffer was applied at aflow rate of0.5 ml/min. Fractions with infection by the same or unrelated pathogens (3). peak binding activity were pooled and loaded onto a Seph- A growing body of evidence has suggested that salicylic acryl S-300 gel filtration column (2.5 x 100 cm) equilibrated acid (SA) is a natural signal in SAR. More than a decade ago, with binding buffer at a flow rate of 0.5 ml/min. The peak application of exogenous SA or its derivative acetylsalicylic fractions from the gel filtration column were pooled and acid was shown to induce PR genes and, at least, partial loaded onto a blue dextran-agarose column (0.5 x 15 cm) resistance to plant diseases (6). More recently, increases in equilibrated with binding buffer. After extensive washing the levels of endogenous SA were observed to correlate with with the binding buffer, the SA-binding activity was step the expression of defense-related genes and the development eluted at a flow rate of 0.1 ml/min with binding buffer ofSAR (7-11). The strongest evidence for the involvement of Abbreviations: PR, pathogenesis-related; SA, salicylic acid; SABP, The publication costs of this article were defrayed in part by page charge SA-binding protein; SAR, systemic acquired resistance; mAb, payment. This article must therefore be hereby marked "advertisement" monoclonal antibody. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 9533 Downloaded by guest on October 11, 2021 9534 Biochemistry: Chen et al. Proc. Natl. Acad. Sci. USA 90 (1993) containing 0.7 M KCl. The peak fractions were again com- 0.1% Tween 20) containing 5% nonfat milk and was washed bined and injected (0.5 ml per run) into a Superose 6 HR 10/30 three times with PBST buffer. Blots were incubated for 1 hr column connected to an FPLC system (Pharmacia). Proteins with diluted hybridoma medium (1:100) in PBST buffer were eluted with binding buffer at a flow rate of0.35 ml/min, containing 0.2% bovine serum albumin and washed three and peak fractions were pooled for further characterization. times with PBST buffer. The antigen-antibody complexes mAb Production. The Superose 6 HR 10/30 peak fractions were detected with a 1:10,000 dilution of horseradish perox- were pooled and submitted for mAb production (14) to the idase-conjugated sheep anti-mouse IgG antibodies and the Hybridoma Laboratory, Department of Molecular Biology, ECL (enhanced chemiluminescence) detection kit from Am- Princeton University. ersham. ELISA. ELISA was performed essentially as described by Analysis of PR-1 Induction. To determine the biological Walker and Huber (15) except that all steps were carried out activity of SA and its analogues for inducing PR-1 gene at room temperature and 0.2% bovine serum album was expression, three leafdiscs (1 cm in diameter) were floated on added to the solution containing goat anti-mouse-alkaline 30 ml of solution containing 10 AM to 1 mM inducer. To phosphatase-conjugated antibodies. analyze the inducibility of PR-1 genes by SA in different Immunoprecipitation. In standard assays, 100-500 ,ul of organs, 6-week-old plants were watered with 1 mM SA. hybridoma culture medium was incubated with 40 ,ul of Tissue was harvested 48 hr after treatment and homogenized protein A-Sepharose (50% slurry) at 4°C for 2 hr. The in 50 mM Tris, pH 8.0/1 mM EDTA/12 mM 2-mercaptoeth- antibody-protein A-Sepharose complexes were pelleted and anol/phenylmethylsulfonyl fluoride at 10 ,ug/ml. After clar- washed three times with RIPA buffer (150 mM NaCl/5 mM ification by centrifugation, the homogenate was fractionated EDTA/1% sodium deoxycholate/0.1% SDS/10 mM Tris, pH by SDS/PAGE, and immunoblot analysis was performed 7.4), and once with binding buffer containing 150 mM KCl with a 1:1,000 dilution of mAb 33G1, which specifically and 0.1% Nonidet P-40. The complexes were incubated at recognizes PR-1 proteins (16). 4°C for 2 hr with 100 ,ul of the partially purified SABP obtained after blue dextran-agarose chromatography. Anti- gen-antibody-protein A complexes were pelleted and super- RESULTS natants were assayed for residual SA-binding activity. The Purification of SABP. During the initial characterization of pellets were washed three times with RIPA buffer before the soluble SABP, we found that the protein exhibited a very SDS/PAGE. high molecular mass (>600 kDa) when analyzed by gel Immunoblot Analysis. Protein samples were fractionated filtration chromatography after 0-35% ammonium sulfate by SDS/PAGE and the separated proteins were electro- precipitation (12). Subsequent fractionation steps resulted in phoretically transferred to a nitrocellulose filter. The filter very inefficient purification because SABP was eluted with was blocked by a 1-hr incubation at room temperature in the majority of the other proteins present.