Sep 11, 1972 - thesized by A. niger, was the gift of Rohm and Haas Company. The C. lindemnuthianum endopolygalacturonase was purified as described (8) ...
Plant Physiol. (1973) 51, 489-491
Host-Pathogen Interactions VI. A SINGLE PLANT PROTEIN EFFICIENTLY INHIBITS ENDOPOLYGALACTURONASES BY COLLETOTRICHUM LINDEMUTHIANUM AND ASPERGILLUS NIGER'
SECRETED
Received for publication September 11, 1972
MINA L. FISHER, ANNE J. ANDERSON, AND PETER ALBERSHEIM Department of Chemistry, University of Colorado, Boulder, Colorado 80302 ABSTRACT
Endopolygalacturonases have been purified from the extracellular enzymes of Colletotrichum lindemuthianum and Aspergillus niger. A protein, purified from Red Kidney (Phaseolus vulgaris) beans for its ability to inhibit the endopolygalacturonase secreted by C. lindemuthianumn, inhibits the A. niger endopolygalacturonase almost as efficientlv as it inhibits the C. linden, uthianumn enzyme.
Plant pathogens have the ability to secrete enzymes capable of degrading the polysaccharides of plant cell walls (1). Of these enzymes, those that degrade pectin are believed to be of key importance in pathogenesis. The pectic-degrading enzymes secreted by a number of pathogens cause maceration of plant tissue, loss in electrolytes, and cell death (4, 6, 10, 14). Also, pectic-degrading enzymes have been detected in lesion areas of infected plant tissue (2, 5, 9, 15). Furthermore, an endopolygalacturonase secreted by Colletotrichum lindemuthianum is the only polysaccharide-degrading enzyme of several that have been tested, which will initiate degradation of isolated plant cell walls (8). Pretreatment of isolated cell walls by endopolygalacturonase is a requirement for effective degradation of the walls by other polysaccharide-degrading enzymes [8, 12]. A polygalacturonase is the first of a series of polysaccharide-degrading enzymes to be secreted into the culture medium when either C. lindemuthianum or Fusarium oxysporum f. sp. lycopersici is grown on isolated plant cell walls [7, 11]. These
early polygalacturonases are completely inhibited by proteins extracted from a variety of plant cell walls including those of Red Kidney hypocotyls (1). The activities of the polygalacturonases secreted by the only other fungi that we have examined, Sclerotium rolfsii (1), and, as reported here, Aspergillus niger, are also inhibited by extracts from Red Kidney bean hypocotyls. This indicates that plants have proteins capable of inhibiting the endopolygalacturonases secreted by a wide variety of fungal pathogens. It seemed unlikely, however, that a unique inhibitor protein would be required for each fungal endopolygalacturonase, since plants would then have to possess an unrealistically large number of different polygalacturonase
tein may inhibit the endopolygalacturonases secreted by more than one pathogen (1). This inhibitor was isolated from Red Kidney bean hypocotyls and was purified 560-fold for its ability to inhibit the endopolygalacturonase secreted by C. lindemuthianum. Although this purified inhibitor has no activity against the polygalacturonase secreted by S. rolfsii, the inhibitor does possess some ability to inhibit the polygalacturonase secreted by F. oxysporum; the purified inhibitor is 40 times more effective against the C. lindemuthianum endopolygalacturonase than against the F. oxysporum enzyme. The results reported in this paper demonstrate that the highly purified inhibitor of the endopolygalacturonase secreted by C. lindemuthianu,m can inhibit, with almost equal efficiency, an endopolygalacturonase secreted by A. niger. MATERIALS AND METHODS
Pectinol R-10, a commercial preparation of enzymes synthesized by A. niger, was the gift of Rohm and Haas Company. The C. lindemnuthianum endopolygalacturonase was purified as described (8) from culture filtrates of the fungus grown on citrus pectin. The inhibitor of the C. lindemuthianum endopolygalacturonase was extracted from the cell walls of 8-dayold Red Kidney (Phaseolus vulgaris) bean hypocotyls and purified as described (1). Assays. Polygalacturonase activity was assayed as described (1). One unit of the polygalacturonase is defined as that amount of enzyme which releases 1.4 ,imoles of galacturonic acid-reducing equivalents in 1 hr at 30 C when the reaction is performed in 50 mm sodium acetate (pH 5.2) and in a total volume of 1 ml. This solution usually contains 800 ,ug of polygalacturonic acid (equivalent to 4.5 /Amoles of galacturonic acid). The polygalacturonic acid was the gift of Sunkist Growers, Inc., Ontario, California. One unit of the endopolygalacturonase inhibitor is defined as that amount which reduces the activity of one unit of the C. lindemuthianum endopolygalacturonase by 50% when assayed under these conditions. Endopolygalacturonase activity was also measured viscometrically as described (8). The substrate consisted of 0.84% (w/v) citrus pectin in 50 mm sodium acetate, pH 5.2, (the gift of Sunkist Growers, Inc.), that had been reduced using NaBH, (100 Ag/lI, pH 10.0, 0 C, 1 hr). Polygalacturonic acid lyase activity was assayed as described (3). Protein was measured by the method of Lowry et al. (13).
inhibitors. The properties of a previously characterized inhibitor of a fungal endopolygalacturonase suggest that a single plant pro-
RESULTS AND DISCUSSION 10
Fractionation of the Polygalacturonases from Pectinol Ron DEAE2-Sephadex. Pectinol R-10 contains several physi-
'Supported in part by Atomic Energy Commission Contract "Abbreviation: DEAE: diethylaminoethyl.
AT(1 1-1)-1426. 489
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Physiol, Vol. 51) 1973
was washed with 200 ml of 10 mm sodium acetate, pH 5.2, followed by 400 ml of the same buffer containing 150 mM NaCl. The column was then eluted with a linear gradient formed from 300 ml of 10 mm acetate buffer containing 150 mM NaCl and 300 ml of 10 mm acetate buffer containing 400 mm NaCl. Five-ml fractions were collected in tubes containing 100 ,ul of an 0.5% (w/v) solution of bovine plasma albumin. The albumin was present to stabilize the polygalacturonase. Those fractions containing NaCl were dialyzed for 18 hr against 10 mm sodium acetate, pH 5.2. Each fraction was assayed for polygalacturonase activity and those 0.4fractions which contained this activity were reassayed in the presence of two units of inhibitor (Fig. 1). The inhibitor preparation used for this assay had been purified 120-fold 0 0.30~ from a crude extract of Red Kidney bean hypocotyls by following the procedures described as far as the gradient elution from Sephadex G-25-300 (1). 0.2I--, Lf 1.0 II Fractionation of the A. niger protein on DEAE-Sephadex yields three distinct polygalacturonases, each of which can be completely inhibited by the protein purified from Red Kidney 0.1M 0.55 bean hypocotyls for its ability to inhibit the C. lindemuthianum endopolygalacturonase. Aspergillus niger polygalacturonase I does not bind to the DEAE-Sephadex column in 10 mm acetate I -L I 2Z. buffer, pH 5.2. Polygalacturonase II is eluted from the DEAE210 190 80 60 40 20 10 Sephadex column in the 150 mm NaCl wash, and polyDEAE-SEPHADEX A-25 FRACTION galacturonase III is eluted in the NaCl gradient. Inhibition of FIG. 1. DEAE-Sephadex A-25 chromatography of an extract polygalacturonase I requires an amount of inhibitor 15-fold from Pectinol R-1O. Polygalacturonase was dssayed in the absence (0) and in the presence (0) of two units of inhibitor. See text for greater than that required to inhibit an equivalent activity of conditions of assay and for the detaisN ol the cnromatography. The C. lindemuthianum endopolygalacturonase. Since only two three peaks of endopolygalacturonase activity described in the text units of inhibitor activity were used during the assay of the fractions from this column presented in Figure 1, no inhibition are denoted by I, II, and III. of those fractions containing polygalacturonase I was expected and no inhibition of these fractions was observed. Polygalacturonase II was not examined further. However, the Red Kidney protein was very effective as an inhibitor of polygalacturonase III. This enzyme was further purified as described below. 1.25 Chromatography of A. niger Polygalacturonase Im on BioGel P-150. The DEAE-Sephadex fractions containing polyD VOI galacturonase III (Nos. 195-215) were combined and concentrated to 6 ml using an Amicon ultrafilter with a PSWP -I 1.00 Pellicon membrane under 40 p.s.i. of nitrogen. Two and onehalf ml of this concentrated polygalacturonase preparation were applied to a Bio-Gel P-150 column (1.4 X 91 cm) and
cally separable polygalacturonases; one of these enzymes was extensively purified and a second was partially purified. An extract of Pectinol R-10 was prepared by suspending 450 mg of the dry powder in 15 ml of 10 mm sodium acetate, pH 5.2. The insoluble material was removed by centrifugation for 5 min at 13,000g. The supernatant liquid was applied to a DEAE-Sephadex A-25 column (1.4 X 21 cm) that had been equilibrated with 10 mm sodium acetate, pH 5.2. The column
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P-iSO chromatography of polygalacturonase FIG. 3. A comparison of the ability of the A. niger polygalacIII. Polygalacturonase was assayed in the absence (0) and in the turonase III to hydrolyze the glycosidic linkages of polygalacturonic FIG. 2. Bio-Gel
presence (0) of two units of inhibitor. See text for conditions of assay and for the details of the chromatography.
acid with the ability of this enzyme to reduce the viscose properties of this polymer. See text for experimental details.
Plant Physiol. Vol. 51, 1973
HOST-PATHOGEN
the column eluted with 50 mm sodium acetate, pH 5.2. Fourml fractions were collected in tubes containing 200 ,A of 0.5% (w/v) bovine plasma albumin. The fractions were assayed for polygalacturonase activity in the absence and in the presence of two units of inhibitor (Fig. 2). The fractions containing polygalacturonase activity (Nos. 17-25) were combined and concentrated to 4.0 ml by ultrafiltration as described above. This concentrated polygalacturonase preparation was assayed in the presence and in the absence of a more highly purified preparation of inhibitor. This inhibitor preparation had been purified 190-fold from the crude plant extract by following the full procedure described (1). Properties of Polygalacturonase m. By using the more highly purified inhibitor preparation, 370 jig of protein are required for one unit of inhibition of the A. niger polygalacturonase III. In comparison, 240 ,ug of inhibitor protein are required for one unit of inhibition of the C. lindemuthianum endopolygalacturonase. Thus, the purified protein isolated from Red Kidney bean hypocotyls is only 1.5 times more effective an inhibitor of the endopolygalacturonase secreted by C. lindemuthianum than it is of the polygalacturonase secreted by A. niger. Like the C. lindemuthianum endopolygalacturonase (8), A. niger polygalacturonase III hydrolyzes polygalacturonic acid in an endo- rather than an exo- fashion (Fig. 3). This was shown by observing that the A. niger enzyme reduces the viscosity of a solution of polygalacturonic acid by 50% while hydrolyzing only 0.3% of the glycosidic bonds. Also, like the C. lindemuthianum enzyme (8), A. niger endopolygalacturonase III cleaves the polygalacturonic acid chains by a hydrolytic mechanism rather than by an elimination mechanism. This was demonstrated by following the absorbance at 235 nm during the degradation of polygalacturonic acid by the purified A. niger endopolygalacturonase (3). While the enzyme was producing 1.13 /-tmoles per ml of galacturonic acid-reducing equivalents, there was no measurable increase in absorbance at 235 nm. If the endopolygalacturonase had been a lyase, an increase in absorbance of 5.2 would have been expected. The elution volume of endopolygalacturonase III from the Bio-Gel P-150 column gives an estimate of the molecular weight of the enzyme. This endopolygalacturonase elutes at 86 ml, whereas lysozyme (mol wt = 14,400), which was used as a standard, elutes at 109 ml (8). By using this information and assuming the enzyme behaves as a sphere, the calculated molecular weight of the endopolygalacturonase is approximately 3 1,000. In summary, the importance of pectic-degrading enzymes in plant diseases caused by fungi (see Introduction) stimulated our interest in the proteins associated with plant cell walls that could inhibit polygalacturonases secreted by fungal pathogens. Crude extracts of Red Kidney bean hypocotyls can completely inhibit the polygalacturonases secreted by the three plant pathogens, C. lindemuthianum, S. rolfsii, and F. oxysporum. However, the protein from Red Kidney beans purified for its ability to inhibit the C. lindemuthianum endopolygalacturonase is 40 times more effective an inhibitor of this enzyme than of the polygalacturonase secreted by F.
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ITERACTIONS.
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oxysporum. The purified protein has no ability to inhibit the S. rolfsii polygalacturonase (1). This raised the question as to whether a unique inhibitor protein exists for each polygalacturonase secreted by each race of every fungal strain. In the present paper, we demonstrate that a single plant protein can inhibit effectively the endopolygalacturonases secreted by two otherwise unrelated fungi, C. lindemuthianum and A. niger. In our studies we have attempted to inhibit the poly-
galacturonases secreted by a total of four fungi and have found that the polygalacturonases from two of these fungi are inhibited by a single plant protein. Since there are many
fungal polygalacturonases, the possibility that we have selected by chance the only two that are inhibited effectively by this plant protein is remote. This finding suggests that the number of polygalacturonase inhibitor proteins a plant possesses is probably not a very large number, and yet, plants are probably able to inhibit the endopolygalacturonases secreted by a wide variety of plant pathogens. LITERATURE CITED 1. ALBERSHEIM, P. A.N-D A. ANDERSON. 1971. Host-pathogen interactions. III. Proteins from plant cell walls inhibit polygalacturonases secreted by plant pathogens. Proc. Nat. Acad. Sci. U.S.A. 68: 18151819. 2. ALBERSHEINI, P., T. M. JONES, AND P. D. EN-GLISH. 1969. Biochemistry of the cell wall in relation to infective processes. Annu. Rev. Phytopathol. 7: 171-194. 3. ALBERSHEIM, P., H. NLEXOKM, AN D H. DEUEL. 1960. Uber die Bildung von ungesattigten Abbauproduktan durch ein pektinabbauendes Enzym. Helv. Chim. Acta 43: 1422-1426. 4. BATEMAN, D. F. AND R. L. MILLER. 1966. Pectic enzymes in tissue degradation. Annu. Rev. Phytopathol. 4: 119-146. 5. BATEMAN, D. F., H. D. VAN ETTrEN, P. D. ENGLISH, D. J. NEVINS, AND P. ALBERSHEIM. 1969. Susceptibility to enzymatic degradation of cell walls from bean plants resistant and susceptible to Rhizoctonia solani Kuhn. Plant Physiol. 44: 641-648. 6. BYRDE, R. J. W. AND A. H. FIELDLNG. 1968. Pectin methyl-trans-eliminase as the macerating factor of Sclerotinia fructigena and its significance in brown rot of apple. J. Gen. Microbiol. 52: 287-297. 7. ENGLISH, P. D., B. JURALE, AND P. ALBERSHEIM. 1971. Host-pathogen interaction II. Parameters affecting polysaccharide-degrading enzyme secretions by Colletotrichum lindemuthianum grow-n in culture. Plant Physiol. 47: 8.
14.
K. KEEGSTRA, AND P. ALBERSHEIM. 1972. A degrading endopolygalacturonase secreted by Colletotrichum lindernuthianum. Plant Physiol. 49: 293-297. GARIBALDI, A. AND D. F. BATEMAN. 1970. Association of pectolytic and cellulolytic enzymes with bacterial slow wilt of carnation caused by Erwinia chrysanthemi. Phytopathologia Mediterranea IX: 136-144. GARIBALDI, A. AND D. F. BATEMAN. 1971. Pectic enzymes produced by Erwinia chrysanthemi and their effects on plant tissue. Physiol. Plant Pathol. 1:
ENGLISH, P. D., A. MAGLOTHIN, cell wall
9.
10.
25-40. 11. JON-ES, T. M., A. J. ANDERSON, AND P. ALBERSHEIM. 1972. Host-pathogen interactions IV. Studies on the polysaccharide-degrading enzymes secreted by Fusarium oxysporum f.sp. lycopersici. Physiol. Plant Pathol. 2: 153-166. 12. KEEGSTRA, K. 1971. Progress toward the elucidation of the structure of plant cell walls achieved with the assistance of purified degradative enzymes. Ph.D. thesis. University of Colorado, Boulder. 13. LoWRY, 0. H., N. ROSEBROUGH, A. FARR, AND R. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. 14. MOUNT, M. S., D. F. BATEMAN, AND H. G. BASHAM. 1970. Induction of electrolyte loss, tissue maceration, and cellular death of potato tissue by an
endopolygalacturonase-trans-eliminase. Phytopathology 60: 924-932. 15. MULLEN, J. M. AND D. F. BATEMAN. 1971. Production of an endo-polygalacturonate trans-eliminase by a potato dry-rot pathogen, Fusanum roseum "Avenicum" in culture and in diseased tissue. Physiol. Plant Pathol. 1: 363373.
