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Inhibition of polygalacturonase from Verticillium dahliae by a polygalacturonase inhibiting protein from cotton
Phytochemistry 57 (2001) 149–156 www.elsevier.com/locate/phytochem
Inhibition of polygalacturonase from Verticillium dahliae by a polygalacturonase inhibiting protein from cotton Jacinda T. James, Ian A. Dubery * Department of Chemistry and Biochemistry, Rand Afrikaans University, PO Box 524, Auckland Park, 2006, South Africa Received 26 April 2000; received in revised form 20 July 2000
Abstract An extracellular endo-polygalacturonase (PGase) [E.C. 3.2.1.15] was isolated from 18-day-old culture filtrates of Verticillium dahliae and partially purified using gel permeation chromatography. The band responsible for PGase activity was electrophoretically characterized as having a molecular mass of approximately 29 500 and an isoelectric point of 5.4. Kinetic studies indicate a Km of 3.3 mg ml 1 and Vmax of 0.85 mmol reducing units min 1 ml 1 with polygalacturonic acid as substrate. Polygalacturonase inhibitor protein (PGIP) in cotton seedlings was induced by 5 mM salicylic acid and immunochemical analysis indicated high levels in the hypocotyl tissues. PGIP was purified from roots and stems using affinity chromatography with endo-PGase from Aspergillus niger as an immobilised ligand. The purified PGIP contained monomeric and dimeric molecules with molecular masses of 34 and 66 kDa respectively. Purified cotton PGIP inhibited endo-polygalacturonase from A. niger in a non-competitive or mixed manner with an inhibition constant, KI of 15 nM. The isolated V. dahliae PGase was, however, inhibited in a positive cooperative manner, indicative of allosteric interactions between the enzyme and the inhibitor protein. In addition to reducing the reaction rate, decreased substrate affinity may contribute to the accumulation of elicitor-active oligouronides. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Gossypium hirsutum; Malvaceae; Cotton; Verticillium dahliae; Polygalacturonase; Polygalacturonase inhibiting protein
1. Introduction Verticillium dahliae is a destructive soil-borne fungal pathogen that causes vascular wilt diseases on more than 160 plant species including important crop plants such as cotton, tomatoes and potatoes. V. dahliae penetrates the host through the roots and spreads systemically through the xylem, leading to the appearance of wilt symptoms (Daayf et al., 1995). The plant cell wall plays a complex role in resistance by presenting a physical barrier to the infecting pathogen. Polysaccharides capable of regulating gene expression and defense responses, as well as enzymes and proteins involved in host defense mechanisms, are localized in the cell wall (Karr and Albersheim, 1970). The importance of polygalacturonases (PGases) in fungal pathogenesis is now well established for certain plants
* Corresponding author. Tel./fax: +27-11-489-2401. E-mail address: [email protected] (I.A. Dubery).
(Karr and Albersheim, 1970; Jones et al., 1972). Research on fungal endopolygalacturonases has demonstrated that these enzymes release elicitor-active oligogalacturonide fragments from the plant cell wall rather than being elicitors of defense themselves. Polygalacturonase inhibiting proteins (PGIPs) have been found to be present in the cell wall of most dicotyledonous plants, and are specific, reversible, saturable, high-affinity ‘‘receptors’’ for fungal, but not plant endopolygalacturonases (Cervone et al., 1989, 1990). PGIPs are structurally related to several resistance gene products and belong to a super-family of leucine-rich repeat proteins specialized for recognition of non-self molecules and rejection of pathogens (Jones and Jones, 1997). The receptor-like leucine-rich molecular structure of the PGIP protein provides a molecular basis for the proposed role of PGIP as a secreted receptor component of the cell-surface signaling system involved in recognition events between plant and fungi (De Lorenzo et al., 1993; Jones and Jones, 1997). Although PGIPs, which have a broad specificity against fungal endoPGase (Cervone et al., 1990), can reduce the activity of
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these enzymes in vitro (Cervone et al., 1987); PGIPs from different plants inhibit PGase from a single fungal species to different extents (Yao et al., 1995). Furthermore, the inhibition of PGases by PGIPs can be highly specific (Leckie et al., 1999). Different isoforms of PGIP from a single plant source can differentially inhibit PGases from several fungal species or PGase isozymes from a single fungal pathogen. The possibility exists that different PGIP members encode PGIPs with distinct regulation and specificity (Desiderio et al., 1997). Formation of the endo-PGase-PGIP complex in vitro results in an alteration of the balance between the release of elicitor-active oligosaccharides and the depolymerisation of the active oligogalacturonides into inactive molecules, thus favouring the accumulation of elicitor active molecules at the site of infection and the resulting activation of the defense responses of the host (Cervone et al., 1989; Bergmann et al., 1994). This intriguing concept of plant-derived elicitors for activating pathogen defense is very likely to function in many plant-pathogen interactions. In this cotton:V. dahliae study we have characterized PGIP from the host and extracellular PGase from the pathogen and report on the kinetics of interaction between the inhibitor protein and its ligand.
2. Results and discussion 2.1. Polygalacturonase purification and characterization PGase (E.C. 3.2.1.15) was isolated from 18-day-old cultures. The maximum PGase levels were determined by the agarose diffusion assay (Dingle et al., 1953) and correlated to a growth curve. Mycelial growth peaked at days 14–15, decreasing thereafter. Maximum PGase production was obtained from day 17 to 20 when the glucose in the medium was consumed and growth conditions became limiting. The main fraction of V. dahliae PGase activity was partially purified by means of (NH4)2SO4 precipitation and chromatography on a Sepharose Cl-6B gel filtration column (Fig. 1) with a yield of 4.1%. In addition to the main peak of PGase activity, several smaller activity peaks of higher molecular mass, possible aggregates, were observed. The purification procedure is summarized in Table 1. The final specific activity of the preparation varied between 100 and 200 U mg 1. Although it is known that PGase play a central role in pathogenesis (Cervone et al., 1986), the amount of information on it’s structure and function is minimal, due to the technical difficulties in preparing suitable quantities of homogenous PGase. Most of the physiological studies involving PGase have been performed with preparations containing contaminating proteins or enzymes.
Fig. 1. Elution profile of a Sepharose Cl-6B gel filtration column to purify PGase from Verticillium dahliae. The (NH4)2SO4 precipitate fraction was loaded onto the column (602.5 cm) at a flow rate of 20 ml h 1, eluted with dH2O and 3.5 ml samples were collected. The fractions were monitored spectrophotometrically at 280 nm (&). PGase activity (-*-), is expressed as A410.10 4 min 1 ml 1.
Table 1 Purification of extracellular endo-polygalacturonase from Verticillium dahliae culture filtrates Purification step
Vol. Protein concentration (ml) (mg ml 1)
57.1 Culture filtratea (NH4)2SO4 precipitationa 3020 Sepharose Cl-6B column 104.8 Post-column 145.6 concentration
Total protein Yield (mg) (%)
1350 77.1 10 30.2 43 4.5 23 3.4
100 39.2 5.8 4.1
a Final specific activity varied between 100 and 200 U mg 1. Activity could not be reproducibly assayed in crude extracts.
The apparent molecular mass determined by means of SDS–PAGE (Fig. 2) for V. dahliae PGase was approximately 29.5 kDa which correlated with the value of 31 kDa calculated by gel filtration. Molecular mass values, ranging from 32 to 37 kDa seems to be a characteristic property among endo-PGases from different fungi (Cervone et al., 1986). Using native IEF-gels, the pI value determined for partially purified PGase from V. dahliae was 5.4 (Fig. 3). Polygalacturonases from different fungal sources exhibit a great variability of isozymatic forms, distinguishable by their isoelectric points. Apart from structural and functional differences more information is needed on the regulation of PGases to fully understand their biological significance. 2.2. PGIP induction and purification A variation in the distribution of PGIP throughout the plant has been reported (Salvi et al., 1990), and a study was done to determine the occurrence of PGIP in the different plant organs of cotton seedlings.
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Fig. 2. SDS–PAGE analysis of the Sepharose Cl-6B-purified PGase from Verticillium dahliae. Proteins, stained with silver, are indicated in lane 2. Activity staining of the renatured enzyme, indicated with an asterix, on an agarose overlay gel is shown in lane 3. Standard Mr markers are indicated in lane 1.
Immunochemical detection of PGIP levels in crude Gossypium hirsutum L. homogenates by Western blot analyses, using a primary antibody against a conserved, surface exposed peptide from bean PGIP, indicated that the highest amount of PGIP was induced in the hypocotyls and to a lesser extend in the first leaves (Fig. 4). The prominent band detected by the antibody in hypocotyls had a Mr of 34 kDa that corresponds with the value reported for deglycosylated PGIP (Stotz et al., 1994, Yao et al., 1995). The results also show a reaction with other proteins present in the crude extract. Bergmann et al. (1994) reported the same for Phaseolus vulgaris L. homogenates and attributed the cross-reaction to antibodies interacting with carbohydrate moieties.
Fig. 4. Tissue specific distribution of PGIP in cotton seedlings. Western blot analysis of immunoreactive proteins present in crude extracts of the hypocotyls (roots and stems, lane 1) and first leaves (lane 2) of cotton seedlings following separation by 12.5% PAGE. Shown in lane 3 is affinity purified PGIP from bean pods, used as a positive control. Molecular mass markers are indicated at right.
The root and stem tissues of infected plants play an important role in the defense mechanisms to Verticillium wilt (Lahkim and Nachmias, 1995). PGIP was isolated from cotton hypocotyls by means of (NH4)2SO4 precipitation and affinity chromatography on an Aspergillus niger endo-PGase-Sepharose column as indicated in Fig. 5 (Cervone et al., 1987). PGIP is an inducible protein and mRNA transcripts accumulate in response to wounding, elicitors and fungal infection (Devoto et al., 1997). It is known that endogenous levels of salicylic acid increase both locally and systematically as a consequence of pathogen attack and that the application of salicylic acid can induce defense responses and resistance towards pathogens (Yalpani et al., 1991). Following the procedure of Bergmann et al. (1994), who reported that the treatment of bean seedlings with salicylic acid resulted in an increase in steady-state levels of PGIP, cotton seedlings were sprayed with 5 mM salicylic acid at 24 h intervals over a time period of 72 h in order to induce maximal amounts of PGIP. The kinetics of induction of PGIP in cotton was not investigated; however, the yield of salicylic acid induced PGIP was approximately 1.0 mg protein per g tissue (Table 2)
Table 2 Purification of polygalacturonase inhibitor protein, PGIP, from hypocotyls of cotton, Gossypium hirsutum, cv, OR19 seedlings Purification step
Fig. 3. PAGIEF analysis of the Sepharose Cl-6B–purified PGase from Verticillium dahliae. Proteins were stained with silver (lane 2). Activity staining of the enzyme, indicated with an asterix, on an agarose overlay gel is shown in lane 3. Standard pI markers are indicated in lane 1.
Vol. Total protein Yield Protein (%) concentration (ml) (mg) (mg ml 1)
180.9 Crude preparationa (NH4)2SO4 precipitationa 335.6 Dialysis 238.1 Endo-PGase affinity column 9.52 a
233 42.3 6 2 10 2.38 21 0.2
Activity could not be determined in the crude extracts.
100 4.8 5.6 0.5
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compared to barely detectable levels in noninduced seedlings. PGIP purified from bean hypocotyls was used as a control and produced the same elution pattern as for cotton PGIP (Fig. 5). 2.3. Electrophoretic analyses of cotton PGIP Purified cotton PGIP was characterized by means of SDS–PAGE and Western blot analyses. One prominent band was detected by SDS–PAGE analysis corresponding to apparent molecular masses of 66 kDa (Fig. 6A).
This band was also observed in the Western blot (Fig. 6B), together with the 34 kDa band observed in crude extracts. Yao et al. (1995) reported a 34 kDa polypeptide for apple PGIP and suggested that differential glycosylation accounted for the observed heterogeneity in molecular mass. Molecular masses of 34–45 kDa have been reported for PGIPs from several sources (Cervone et al., 1987; Bergmann et al., 1994; Stotz et al., 1994) as well as higher molecular weight forms (91 kDa, Abu-Goukh et al., 1983). It is possible that cotton PGIP exists as a dimer, binding covalently to negatively charged pectin in the cell wall or to each other through non-specific interactions. Cross-linking during the purification procedure due to reaction with bifunctional terpene aldehydes such as gossypol, could also lead to dimerization (Mahoney and Chan, 1985). 2.4. Inhibition of the PGase from Verticillium dahliae by cotton PGIP
Fig. 5. Elution profile of extracts from cotton roots and stems, induced with 5 mM salicylic acid, on an endo-PGase affinity column (131.5 cm). The post-dialysis PGIP fraction (13 ml) was applied to the column with 20 mM NaOAc pH 5.0 and the PGIP fraction was desorbed at 60 ml h 1 with phosphate buffered saline, pH 7.3. Fractions were monitored spectrophotometrically at A280 (-&-). The PGIP peak (-*-) was identified by the inhibition of A. niger polygalacturonase, using the reducing sugar assay. The third peak was obtained with 0.1 M sodium borate buffer, pH 8.0, used to wash and desorb bound phenolics from the column.
Fig. 6. Electrophoretic and immuno-blot analysis of the protein fractions desorbed from the endo-PGase affinity column. SDS–PAGE followed by silver staining, was performed on 12.5% gels on the PGIP fractions eluted from the colum (part A, lanes 1–6) with subsequent Western blot analysis of these fractions (part B of the figure). The Mrs are indicated on the left.
An agarose diffusion assay showed that purified cotton PGIP inhibited the PGase purified from V. dahliae culture filtrates to various degrees when added in different ratios to the reaction mixture (results not shown). A molar ratio of 1(PGase) : 4(PGIP) reduced the activity to near zero but since the inhibition is reversible, a high concentration of PGIP would be needed to completely inhibit the PGase. The inhibition was confirmed with native-PAGE gels and subsequent overlays in the absence and presence of cotton PGIP (overlay gels not shown). 2.5. Kinetic analysis of inhibition PGase from V. dahliae, diluted to a concentration of 30 ng ml 1 in a 400 ml reaction volume, producing a linear response over 30 mins (A410=9.310 3 min 1), was used for kinetic studies. The A410 min 1 was calculated for the inhibited and uninhibited assay and the percentage of inhibition was determined. An inhibition value of 50% was obtained with 7.5 ng ml 1 of purified cotton PGIP, and the activity was reduced to 30% at 9.5 ng ml 1, indicating that cotton PGIP is an effective inhibitor of PGase from V. dahliae. The inhibition of reaction rate by increasing concentrations of purified cotton PGIP was measured at different concentrations of polygalacturonic acid (PGA). Increasing concentrations of PGIP lead to the progressive inhibition of PGase activity (Fig. 7A). Kinetic studies of PGIPs revealed that some inhibit fungal PGases by a competitive type of inhibition whereas others are non-competitive, however this difference might depend on the source of the PGase rather than the source of the PGIP (Jones and Jones, 1997). Mixed or non-competitive inhibition was displayed between cotton PGIP and PGase from A. niger, with a Km of 3.3 mg ml 1, a Vmax of 1.07 mm reducing sugar
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Fig. 8. Inhibition of PGase from Aspergillus niger by cotton PGIP at fixed enzyme concentration (100 ng ml 1) and varying inhibitor concentrations (0–7.2 ng ml 1). The Lineweaver–Burk plot shows the noncompetitive inhibition with several inhibitor concentrations: 2.4 ng ml 1 (&), 4.8 ng ml 1 (~) and 7.2 ng ml 1 (X). The uninhibited assay is denoted by ^. v=Enzyme activity; S=substrate concentration.
Fig. 7. Inhibition of purified PGase from V. dahliae by cotton PGIP at fixed enzyme concentration (30 ng ml 1) and varying inhibitor concentrations: ^, the uninhibited assay; assay with 2.4 ng ml 1 PGIP &; 4.8 ng ml 1 PGIP ~, and 7.2 ng ml 1 PGIP, X. A: Michaelis– Menten plot of PGase activity with different concentrations of PGA together with three different concentrations of PGIP. B: Lineweaver– Burk plot showing the positive allosteric inhibition with several inhibitor concentrations. v=Enzyme activity; S=substrate concentration.
equivalents produced min 1 ml 1 and a Ki of 15 nM (Fig. 8). Non-competitive inhibition is an indication of the inhibitor binding to a site on the PGase molecule different from the active site. Using a mutagenesis approach, Caprari et al. (1996), was able to confirm that the PGIP does not bind to the active site of the PGase from Fusarium moniliforme. Lineweaver–Burk analysis of the data was performed to determine the type of inhibition displayed between PGase from V. dahliae and PGIP from cotton (Fig. 7A and B). Vmax for the uninhibited treatment was estimated as 0.85 mmol reducing sugar equivalents produced min 1 ml 1 with a Km of 3.3 mg ml 1. The graphs obtained are not typical Michaelis–Menten curves and the sigmoidal pattern obtained indicates a positive allosteric interaction between PGase, PGIP and the PGA substrate, suggesting that the reaction rate is reduced at low substrate concentrations but not necessarily at others. Cooperative allosteric interactions occur when the binding of one ligand at a specific site is influenced by the binding of another ligand, modulator or effector at a different (allosteric) site on the protein (Palmer, 1995). Previously, no allosteric inhibition of a fungal PGase by
a PGIP have been reported in the few cases where the kinetics of inhibition have been described. The results obtained indicate that the PGase from V. dahliae is a ‘‘K’’-series enzyme, where the presence of the modifier (in this case PGIP), results in a change in Km but not in the apparent Vmax for the substrate. If the binding characteristics alone are affected, Vmax will usually remain unchanged, so the inhibition pattern could be regarded as non-competitive or mixed inhibition. As PGIP does not bind directly to the active site of the enzyme, it could be possible that binding induces a conformational change in the enzyme, resulting in a lower substrate affinity. The sigmoidal binding of a modifier or inhibitor can amplify the regulatory effect (Palmer, 1995), thus reducing the activity of the PGase. This would delay the rapid degradation of PGA to inactive monomers and result in the generation of elicitor-active oligogalacturonides with a higher degree of polymerization. These results are in support of the model proposed for the PGIP:endo PGase interaction (De Lorenzo et al., 1993) where the formation of the enzyme-inhibitor complex in vitro, results in an alteration of the balance between the release of elicitor-active oligosaccharides and the depolymerisation of the active oligogalacturonides into inactive molecules, thereby favouring the accumulation of elicitor active molecules at the site of infection and the resulting activation of the defense responses of the host.
3. Experimental 3.1. Plant material and fungal isolate Seeds of Verticillium resistant cotton cultivar OR19 (obtained from the Agriculture Research Council’s Institute for Tobacco and Cotton, Rustenburg, South
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Africa) were germinated and maintained at 30 C. Roots and stems of 3-week-old plants were used in all experiments. A pathogenic isolate of V. dahliae was obtained from infected Acala1517/70 cotton stems (Meyer et al., 1994).
toethanol. Gels were then washed for 10 min in 20 mM NaOAc, pH 4.5 and enzyme activity localized as described above. The band of activity was compared to the SDS-gel to determine the molecular weight of PGase from V. dahliae.
3.2. Polygalacturonase purification and characterization 3.4. Enzyme activity determinations Fungal mycelia were cultivated in liquid culture in the dark at 25 C for 18 days, in 1 l flasks, containing 500 ml of medium (Nachmias et al., 1982). A growth curve was determined over a period of 21 days. The extracts were screened by means of an agarose diffusion assay (Dingle et al., 1953) to determine maximum PGase production (Section 3.8). Culture filtrates harvested on day 18 were used for purification of PGase. Following centrifugation at 15 000g, proteins present in the the clear supernatant (1350 ml) was precipitated at 90% (NH4)2SO4 saturation for a minimum of 1 h. The precipitate was collected by centrifugation at 15 000g for 20 min and dissolved in dH2O. The sample was fractionated using gel permeation chromatography on Sepharose CL-6B (2.560 cm) with dH2O as eluant (Meyer et al., 1994) at a flow rate of 20 ml h 1. The fractions obtained after gel filtration that exhibited PGase activity were combined and concentrated in a dialysis bag against glycerol to stabilize the enzyme for storage at 4 C. PGase from V. dahliae was characterized by native PAGE, agarose overlay gels (Kanellis et al., 1991), SDS– PAGE (Laemmli, 1970) and IEF gels (Robertson et al., 1987). The Mr was determined using analytical gel filtration with a Sepharose CL-6B column (2.560 cm) using 0.1 M NaPi buffer, pH 8.0 as eluting buffer (Andrews, 1970). V0 and Vt were determined with blue dextran and DNP-alanine respectively to calibrate the column, together with a set of calibration proteins from 14 to 158 kDa. 3.3. Enzyme staining in gels Enzyme activity of endo-PGases present in the culture filtrates and crude extract were screened by means of an agarose diffusion assay (Dingle et al., 1953). Following PAGE, the gels were incubated in 0.1 M NaOAc, pH 5.0 for 10 min. Agarose gels (285100 mm) were poured onto Gelbond1 film (FMC Bioproducts). The agarose soln. contained 0.8% agarose (w/v), 100 mM HOAc (pH 5.0) and 1 mg ml 1 PGA (w/v). Following incubation, the overlay gels were stained in 0.005% ruthenium red for 2 h and destained in dH2O until clear bands were evident (Kanellis et al., 1991). In order to determine activity following SDS–PAGE, gels were washed twice for 15 min in 0.1 M NaOAc, pH 4.5 containing 0.05 mg ml 1 BSA and 0.1% TritonX100 at room temperature. The enzyme was allowed to renature at 4 C overnight in 0.1 mM NaOAc, pH 4.5 containing 0.05 mg ml 1 BSA and 0.02% b-mercap-
A reducing sugar assay for the determination of released galacturonic oligomers was used with the colour reagent p-hydroxybenzoic acid hydrazide (Kombrink et al., 1988). PGase, either the positive control Aspergillus niger PGase, 100 ng ml 1, or purified PGase from Verticillium dahliae, 30 ng ml 1, was added as a 200 ml aliquot to 200 ml autoclaved PGA (2.5 mg ml 1) in 20 mM NaOAc buffer, pH 5.0. PGase activity was assessed over time (0–60 min) to determine the linear range. Duplicate samples were taken at 4 different time intervals (0, 10, 20 and 30 min). Activity was expressed as A410 min 1 ml 1 or converted to arbitrary units, one unit being equivalent to the release of 1.0 mmol galacturonic acid units min 1 ml 1 at pH 5.0. 3.5. Induction and purification of PGIP PGIP in cotton seedlings was chemically induced with salicylic acid. Seedlings were sprayed with 5 mM sodium salicylate, pH 7.0 for 72 h. Distilled water was used as a control. The extraction procedure of Salvi et al. (1990) was carried out at 4 C with slight modifications. Plant material (hypocotyls, 200 g) was homogenized for 2 min in cold 20 mM NaOAc, pH 5.0 containing 500 mM NaCl (1:2 m/v). Proteinase inhibitor, PMSF (0.1%), was added prior to homogenization. The suspension was vacuum filtered through a number 1 sinter glass funnel and the filtrate centrifuged at 12 000g for 20 min. The proteins in the supernatant were precipitated for 1 h with (NH4)2SO4 at a final saturation of 90%, followed by centrifugation 12 000g for 20 min to collect the precipitate. The precipitate was resuspended in a small volume of 20 mM NaOAc, pH 5.5 and dialyzed with at least 4 changes of the same buffer in a dialysis membrane with a molecular cut-off value of 12–14 kDa. The suspension was centrifuged at 12 000g for 20 min and the PGIP purified further by affinity chromatography on an endo-PGase affinity column as described by Cervone et al. (1987). PGIP activity was measured using the PGase assay described above. One unit was defined as the amount of inhibitor required to reduce the activity of one unit of enzyme by 50% 3.6. Electrophoretic analyses Protein concentration was determined according to Bradford (1976) or the bicinchoninic acid protein assay from Pierce (Rockford, IL). Purified preparations were
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analysed by native PAGE, agarose overlay gels (Kanellis et al., 1991) and IEF gels (Robertson et al., 1987). SDS-electrophoresis was carried out according to Laemmli (1970); gels were stained with either Coomassie R250 or silver (Bristow, 1990). 3.7. Western blot analyses Blotting was performed overnight at 4 C and 36 V using PVDF-Plus membranes and 25 mM Tris (pH 8.2), 192 mM glycine, 20% (v/v) methanol and 0.1% (w/v) SDS as transfer buffer. The primary antibody was raised in rabbits against a synthetic peptide, A-L-L-Q-I-K-KD-L-G-N-P-Y, located near the N-terminus of bean PGIP and coupled to Keyhole Limpet Haemocyanin (Bergmann et al., 1994). Goat anti-rabbit IgG, conjugated to alkaline phosphatase was used as secondary antibody at a dilution of 1:1000 with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate as substrate (Berger et al., 2000). 3.8. Inhibition studies The agarose diffusion assay (Dingle et al., 1953) for PGase activity was performed to test for the presence of PGIP. The assay medium consisted of 100 mM citrate and 200 mM NaPi (pH 5.3) to which final concentrations of 1% agarose, 0.5% ammonium oxalate and 0.1 mg l 1 PGA were added. The assay was carried out with the addition of 50 ml culture medium supernatant into each well and overnight incubation at 30 C. Activity zones were visualized by staining the background with 0.05% ruthenium red. For enzyme assays and kinetic studies the reducing sugar assay as described in Section 3.4 was used. Enzyme was added to different concentrations of cotton PGIP (0–7.2 ng ml 1) to determine the degree of inhibition. PGIP preparations were mixed with PGase and incubated for 10 min prior to the addition of the PGA substrate. Substrate concentrations were varied between 1.5 and 5.5 mg ml 1 to determine the type of inhibition. Duplicate samples were taken at 0, 10, 20 and 30 min and analyzed for galacturonic acid.
Acknowledgements We wish to acknowledge Dr. D. Berger (ARC, Agricultural Research Council) for cooperation and the gift of the anti-PGIP antibody, and the National Research Foundation (NRF) for research funding.
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Suppression of cellulase and polygalacturonase and induction of alcohol dehydrogenase isozymes in avocado fruit mesocarp subjected to low oxygen stress. Plant Physiology 98, 269–274. Karr, A.L., Albersheim, P., 1970. Polysaccharide-degrading enzymes are unable to attack plant cells without prior action by a ‘‘wallmodifying’’ enzyme. Plant Physiology 46, 69–80. Kombrink, E., Schro¨der, M., Halbrock, K., 1988. Several ‘‘pathogenesis-related’’ proteins in potato are 1,3-b-glucanases and chitinases. Proceedings of the National Academy of Sciences of the United States of America 85, 782–786. Leckie, F., Mattei, B., Capodicasa, C., Hemmings, A., Nuss, L., Aracri, B., De Lorenzo, G., Cervone, F., 1999. The specificity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent-exposed beta-strand/beta-turn region of the leucine-rich repeats (LRRs) confers a new recognition capability. EMBO Journal 18, 2352–2363. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 227, 680–685. Lahkim, L.T., Nachmias, A., 1995. Significance of the root system in Verticillium wilt tolerance in potato and resistance in tomato. Israel Journal of Plant Sciences 43, 315–323. Mahoney, N.E., Chan, B.G., 1985. High-performance liquid chromatographic analysis of terpene aldehydes in cotton. Journal of Chromatography 329, 91–98.
Meyer, R., Slater, V., Dubery, I.A., 1994. A phytotoxic protein-lipopolysaccharide complex produced by Verticillium dahliae. Phytochemistry 35, 1449–1453. Nachmias, A., Buchner, V., Krikun, J., 1982. Comparison of proteinlipopolysaccharide complexes produced by pathogenic and nonpathogenic strains of Verticillium dahliae Kleb. from potato. Physiological Plant Pathology 20, 213–221. Palmer, T., 1995. Understanding Enzymes, 4th Edition. Prentice Hall, Ellis Horwood, pp. 148–151, 243–245, 262–263. Robertson, E.F., Dannelly, H.K., Malloy, P.J., Reeves, H.C., 1987. Rapid isoelectric focusing in a vertical polyacrylamide minigel system. Analytical Biochemistry 167, 290–294. Salvi, G., Giarrizzo, F., De Lorenzo, G., Cervone, F., 1990. A polygalacturonase-inhibiting protein in the flowers of Phaseolus vulgaris L. Plant Physiology 136, 513–518. Stotz, H.U., Contos, J.J.A., Powell, A.L.T., Bennet, A.B., Labavitch, J.M., 1994. Structure and expression of an inhibitor of polygalacturonases from tomato. Plant Molecular Biology 25, 607–617. Yalpani, N., Silverman, P., Wilson, T.M.A., Kleier, D.A., Raskin, I., 1991. Salicylic acid is a systemic signal an inducer of pathogenesisrelated proteins in virus-infected tobacco. The Plant Cell 3, 809–818. Yao, C., Conway, W.S., Sams, C.E., 1995. Purification and characterization of a polygalacturonase-inhibiting protein from apple fruit. Phytopathology 85, 373–1377.
Inhibition of polygalacturonase from Verticillium dahliae by a polygalacturonase inhibiting protein from cotton Jacinda T. James, Ian A. Dubery * Department of Chemistry and Biochemistry, Rand Afrikaans University, PO Box 524, Auckland Park, 2006, South Africa Received 26 April 2000; received in revised form 20 July 2000
Abstract An extracellular endo-polygalacturonase (PGase) [E.C. 3.2.1.15] was isolated from 18-day-old culture filtrates of Verticillium dahliae and partially purified using gel permeation chromatography. The band responsible for PGase activity was electrophoretically characterized as having a molecular mass of approximately 29 500 and an isoelectric point of 5.4. Kinetic studies indicate a Km of 3.3 mg ml 1 and Vmax of 0.85 mmol reducing units min 1 ml 1 with polygalacturonic acid as substrate. Polygalacturonase inhibitor protein (PGIP) in cotton seedlings was induced by 5 mM salicylic acid and immunochemical analysis indicated high levels in the hypocotyl tissues. PGIP was purified from roots and stems using affinity chromatography with endo-PGase from Aspergillus niger as an immobilised ligand. The purified PGIP contained monomeric and dimeric molecules with molecular masses of 34 and 66 kDa respectively. Purified cotton PGIP inhibited endo-polygalacturonase from A. niger in a non-competitive or mixed manner with an inhibition constant, KI of 15 nM. The isolated V. dahliae PGase was, however, inhibited in a positive cooperative manner, indicative of allosteric interactions between the enzyme and the inhibitor protein. In addition to reducing the reaction rate, decreased substrate affinity may contribute to the accumulation of elicitor-active oligouronides. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Gossypium hirsutum; Malvaceae; Cotton; Verticillium dahliae; Polygalacturonase; Polygalacturonase inhibiting protein
1. Introduction Verticillium dahliae is a destructive soil-borne fungal pathogen that causes vascular wilt diseases on more than 160 plant species including important crop plants such as cotton, tomatoes and potatoes. V. dahliae penetrates the host through the roots and spreads systemically through the xylem, leading to the appearance of wilt symptoms (Daayf et al., 1995). The plant cell wall plays a complex role in resistance by presenting a physical barrier to the infecting pathogen. Polysaccharides capable of regulating gene expression and defense responses, as well as enzymes and proteins involved in host defense mechanisms, are localized in the cell wall (Karr and Albersheim, 1970). The importance of polygalacturonases (PGases) in fungal pathogenesis is now well established for certain plants
* Corresponding author. Tel./fax: +27-11-489-2401. E-mail address: [email protected] (I.A. Dubery).
(Karr and Albersheim, 1970; Jones et al., 1972). Research on fungal endopolygalacturonases has demonstrated that these enzymes release elicitor-active oligogalacturonide fragments from the plant cell wall rather than being elicitors of defense themselves. Polygalacturonase inhibiting proteins (PGIPs) have been found to be present in the cell wall of most dicotyledonous plants, and are specific, reversible, saturable, high-affinity ‘‘receptors’’ for fungal, but not plant endopolygalacturonases (Cervone et al., 1989, 1990). PGIPs are structurally related to several resistance gene products and belong to a super-family of leucine-rich repeat proteins specialized for recognition of non-self molecules and rejection of pathogens (Jones and Jones, 1997). The receptor-like leucine-rich molecular structure of the PGIP protein provides a molecular basis for the proposed role of PGIP as a secreted receptor component of the cell-surface signaling system involved in recognition events between plant and fungi (De Lorenzo et al., 1993; Jones and Jones, 1997). Although PGIPs, which have a broad specificity against fungal endoPGase (Cervone et al., 1990), can reduce the activity of
0031-9422/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0031-9422(01)00024-3
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these enzymes in vitro (Cervone et al., 1987); PGIPs from different plants inhibit PGase from a single fungal species to different extents (Yao et al., 1995). Furthermore, the inhibition of PGases by PGIPs can be highly specific (Leckie et al., 1999). Different isoforms of PGIP from a single plant source can differentially inhibit PGases from several fungal species or PGase isozymes from a single fungal pathogen. The possibility exists that different PGIP members encode PGIPs with distinct regulation and specificity (Desiderio et al., 1997). Formation of the endo-PGase-PGIP complex in vitro results in an alteration of the balance between the release of elicitor-active oligosaccharides and the depolymerisation of the active oligogalacturonides into inactive molecules, thus favouring the accumulation of elicitor active molecules at the site of infection and the resulting activation of the defense responses of the host (Cervone et al., 1989; Bergmann et al., 1994). This intriguing concept of plant-derived elicitors for activating pathogen defense is very likely to function in many plant-pathogen interactions. In this cotton:V. dahliae study we have characterized PGIP from the host and extracellular PGase from the pathogen and report on the kinetics of interaction between the inhibitor protein and its ligand.
2. Results and discussion 2.1. Polygalacturonase purification and characterization PGase (E.C. 3.2.1.15) was isolated from 18-day-old cultures. The maximum PGase levels were determined by the agarose diffusion assay (Dingle et al., 1953) and correlated to a growth curve. Mycelial growth peaked at days 14–15, decreasing thereafter. Maximum PGase production was obtained from day 17 to 20 when the glucose in the medium was consumed and growth conditions became limiting. The main fraction of V. dahliae PGase activity was partially purified by means of (NH4)2SO4 precipitation and chromatography on a Sepharose Cl-6B gel filtration column (Fig. 1) with a yield of 4.1%. In addition to the main peak of PGase activity, several smaller activity peaks of higher molecular mass, possible aggregates, were observed. The purification procedure is summarized in Table 1. The final specific activity of the preparation varied between 100 and 200 U mg 1. Although it is known that PGase play a central role in pathogenesis (Cervone et al., 1986), the amount of information on it’s structure and function is minimal, due to the technical difficulties in preparing suitable quantities of homogenous PGase. Most of the physiological studies involving PGase have been performed with preparations containing contaminating proteins or enzymes.
Fig. 1. Elution profile of a Sepharose Cl-6B gel filtration column to purify PGase from Verticillium dahliae. The (NH4)2SO4 precipitate fraction was loaded onto the column (602.5 cm) at a flow rate of 20 ml h 1, eluted with dH2O and 3.5 ml samples were collected. The fractions were monitored spectrophotometrically at 280 nm (&). PGase activity (-*-), is expressed as A410.10 4 min 1 ml 1.
Table 1 Purification of extracellular endo-polygalacturonase from Verticillium dahliae culture filtrates Purification step
Vol. Protein concentration (ml) (mg ml 1)
57.1 Culture filtratea (NH4)2SO4 precipitationa 3020 Sepharose Cl-6B column 104.8 Post-column 145.6 concentration
Total protein Yield (mg) (%)
1350 77.1 10 30.2 43 4.5 23 3.4
100 39.2 5.8 4.1
a Final specific activity varied between 100 and 200 U mg 1. Activity could not be reproducibly assayed in crude extracts.
The apparent molecular mass determined by means of SDS–PAGE (Fig. 2) for V. dahliae PGase was approximately 29.5 kDa which correlated with the value of 31 kDa calculated by gel filtration. Molecular mass values, ranging from 32 to 37 kDa seems to be a characteristic property among endo-PGases from different fungi (Cervone et al., 1986). Using native IEF-gels, the pI value determined for partially purified PGase from V. dahliae was 5.4 (Fig. 3). Polygalacturonases from different fungal sources exhibit a great variability of isozymatic forms, distinguishable by their isoelectric points. Apart from structural and functional differences more information is needed on the regulation of PGases to fully understand their biological significance. 2.2. PGIP induction and purification A variation in the distribution of PGIP throughout the plant has been reported (Salvi et al., 1990), and a study was done to determine the occurrence of PGIP in the different plant organs of cotton seedlings.
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Fig. 2. SDS–PAGE analysis of the Sepharose Cl-6B-purified PGase from Verticillium dahliae. Proteins, stained with silver, are indicated in lane 2. Activity staining of the renatured enzyme, indicated with an asterix, on an agarose overlay gel is shown in lane 3. Standard Mr markers are indicated in lane 1.
Immunochemical detection of PGIP levels in crude Gossypium hirsutum L. homogenates by Western blot analyses, using a primary antibody against a conserved, surface exposed peptide from bean PGIP, indicated that the highest amount of PGIP was induced in the hypocotyls and to a lesser extend in the first leaves (Fig. 4). The prominent band detected by the antibody in hypocotyls had a Mr of 34 kDa that corresponds with the value reported for deglycosylated PGIP (Stotz et al., 1994, Yao et al., 1995). The results also show a reaction with other proteins present in the crude extract. Bergmann et al. (1994) reported the same for Phaseolus vulgaris L. homogenates and attributed the cross-reaction to antibodies interacting with carbohydrate moieties.
Fig. 4. Tissue specific distribution of PGIP in cotton seedlings. Western blot analysis of immunoreactive proteins present in crude extracts of the hypocotyls (roots and stems, lane 1) and first leaves (lane 2) of cotton seedlings following separation by 12.5% PAGE. Shown in lane 3 is affinity purified PGIP from bean pods, used as a positive control. Molecular mass markers are indicated at right.
The root and stem tissues of infected plants play an important role in the defense mechanisms to Verticillium wilt (Lahkim and Nachmias, 1995). PGIP was isolated from cotton hypocotyls by means of (NH4)2SO4 precipitation and affinity chromatography on an Aspergillus niger endo-PGase-Sepharose column as indicated in Fig. 5 (Cervone et al., 1987). PGIP is an inducible protein and mRNA transcripts accumulate in response to wounding, elicitors and fungal infection (Devoto et al., 1997). It is known that endogenous levels of salicylic acid increase both locally and systematically as a consequence of pathogen attack and that the application of salicylic acid can induce defense responses and resistance towards pathogens (Yalpani et al., 1991). Following the procedure of Bergmann et al. (1994), who reported that the treatment of bean seedlings with salicylic acid resulted in an increase in steady-state levels of PGIP, cotton seedlings were sprayed with 5 mM salicylic acid at 24 h intervals over a time period of 72 h in order to induce maximal amounts of PGIP. The kinetics of induction of PGIP in cotton was not investigated; however, the yield of salicylic acid induced PGIP was approximately 1.0 mg protein per g tissue (Table 2)
Table 2 Purification of polygalacturonase inhibitor protein, PGIP, from hypocotyls of cotton, Gossypium hirsutum, cv, OR19 seedlings Purification step
Fig. 3. PAGIEF analysis of the Sepharose Cl-6B–purified PGase from Verticillium dahliae. Proteins were stained with silver (lane 2). Activity staining of the enzyme, indicated with an asterix, on an agarose overlay gel is shown in lane 3. Standard pI markers are indicated in lane 1.
Vol. Total protein Yield Protein (%) concentration (ml) (mg) (mg ml 1)
180.9 Crude preparationa (NH4)2SO4 precipitationa 335.6 Dialysis 238.1 Endo-PGase affinity column 9.52 a
233 42.3 6 2 10 2.38 21 0.2
Activity could not be determined in the crude extracts.
100 4.8 5.6 0.5
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compared to barely detectable levels in noninduced seedlings. PGIP purified from bean hypocotyls was used as a control and produced the same elution pattern as for cotton PGIP (Fig. 5). 2.3. Electrophoretic analyses of cotton PGIP Purified cotton PGIP was characterized by means of SDS–PAGE and Western blot analyses. One prominent band was detected by SDS–PAGE analysis corresponding to apparent molecular masses of 66 kDa (Fig. 6A).
This band was also observed in the Western blot (Fig. 6B), together with the 34 kDa band observed in crude extracts. Yao et al. (1995) reported a 34 kDa polypeptide for apple PGIP and suggested that differential glycosylation accounted for the observed heterogeneity in molecular mass. Molecular masses of 34–45 kDa have been reported for PGIPs from several sources (Cervone et al., 1987; Bergmann et al., 1994; Stotz et al., 1994) as well as higher molecular weight forms (91 kDa, Abu-Goukh et al., 1983). It is possible that cotton PGIP exists as a dimer, binding covalently to negatively charged pectin in the cell wall or to each other through non-specific interactions. Cross-linking during the purification procedure due to reaction with bifunctional terpene aldehydes such as gossypol, could also lead to dimerization (Mahoney and Chan, 1985). 2.4. Inhibition of the PGase from Verticillium dahliae by cotton PGIP
Fig. 5. Elution profile of extracts from cotton roots and stems, induced with 5 mM salicylic acid, on an endo-PGase affinity column (131.5 cm). The post-dialysis PGIP fraction (13 ml) was applied to the column with 20 mM NaOAc pH 5.0 and the PGIP fraction was desorbed at 60 ml h 1 with phosphate buffered saline, pH 7.3. Fractions were monitored spectrophotometrically at A280 (-&-). The PGIP peak (-*-) was identified by the inhibition of A. niger polygalacturonase, using the reducing sugar assay. The third peak was obtained with 0.1 M sodium borate buffer, pH 8.0, used to wash and desorb bound phenolics from the column.
Fig. 6. Electrophoretic and immuno-blot analysis of the protein fractions desorbed from the endo-PGase affinity column. SDS–PAGE followed by silver staining, was performed on 12.5% gels on the PGIP fractions eluted from the colum (part A, lanes 1–6) with subsequent Western blot analysis of these fractions (part B of the figure). The Mrs are indicated on the left.
An agarose diffusion assay showed that purified cotton PGIP inhibited the PGase purified from V. dahliae culture filtrates to various degrees when added in different ratios to the reaction mixture (results not shown). A molar ratio of 1(PGase) : 4(PGIP) reduced the activity to near zero but since the inhibition is reversible, a high concentration of PGIP would be needed to completely inhibit the PGase. The inhibition was confirmed with native-PAGE gels and subsequent overlays in the absence and presence of cotton PGIP (overlay gels not shown). 2.5. Kinetic analysis of inhibition PGase from V. dahliae, diluted to a concentration of 30 ng ml 1 in a 400 ml reaction volume, producing a linear response over 30 mins (A410=9.310 3 min 1), was used for kinetic studies. The A410 min 1 was calculated for the inhibited and uninhibited assay and the percentage of inhibition was determined. An inhibition value of 50% was obtained with 7.5 ng ml 1 of purified cotton PGIP, and the activity was reduced to 30% at 9.5 ng ml 1, indicating that cotton PGIP is an effective inhibitor of PGase from V. dahliae. The inhibition of reaction rate by increasing concentrations of purified cotton PGIP was measured at different concentrations of polygalacturonic acid (PGA). Increasing concentrations of PGIP lead to the progressive inhibition of PGase activity (Fig. 7A). Kinetic studies of PGIPs revealed that some inhibit fungal PGases by a competitive type of inhibition whereas others are non-competitive, however this difference might depend on the source of the PGase rather than the source of the PGIP (Jones and Jones, 1997). Mixed or non-competitive inhibition was displayed between cotton PGIP and PGase from A. niger, with a Km of 3.3 mg ml 1, a Vmax of 1.07 mm reducing sugar
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Fig. 8. Inhibition of PGase from Aspergillus niger by cotton PGIP at fixed enzyme concentration (100 ng ml 1) and varying inhibitor concentrations (0–7.2 ng ml 1). The Lineweaver–Burk plot shows the noncompetitive inhibition with several inhibitor concentrations: 2.4 ng ml 1 (&), 4.8 ng ml 1 (~) and 7.2 ng ml 1 (X). The uninhibited assay is denoted by ^. v=Enzyme activity; S=substrate concentration.
Fig. 7. Inhibition of purified PGase from V. dahliae by cotton PGIP at fixed enzyme concentration (30 ng ml 1) and varying inhibitor concentrations: ^, the uninhibited assay; assay with 2.4 ng ml 1 PGIP &; 4.8 ng ml 1 PGIP ~, and 7.2 ng ml 1 PGIP, X. A: Michaelis– Menten plot of PGase activity with different concentrations of PGA together with three different concentrations of PGIP. B: Lineweaver– Burk plot showing the positive allosteric inhibition with several inhibitor concentrations. v=Enzyme activity; S=substrate concentration.
equivalents produced min 1 ml 1 and a Ki of 15 nM (Fig. 8). Non-competitive inhibition is an indication of the inhibitor binding to a site on the PGase molecule different from the active site. Using a mutagenesis approach, Caprari et al. (1996), was able to confirm that the PGIP does not bind to the active site of the PGase from Fusarium moniliforme. Lineweaver–Burk analysis of the data was performed to determine the type of inhibition displayed between PGase from V. dahliae and PGIP from cotton (Fig. 7A and B). Vmax for the uninhibited treatment was estimated as 0.85 mmol reducing sugar equivalents produced min 1 ml 1 with a Km of 3.3 mg ml 1. The graphs obtained are not typical Michaelis–Menten curves and the sigmoidal pattern obtained indicates a positive allosteric interaction between PGase, PGIP and the PGA substrate, suggesting that the reaction rate is reduced at low substrate concentrations but not necessarily at others. Cooperative allosteric interactions occur when the binding of one ligand at a specific site is influenced by the binding of another ligand, modulator or effector at a different (allosteric) site on the protein (Palmer, 1995). Previously, no allosteric inhibition of a fungal PGase by
a PGIP have been reported in the few cases where the kinetics of inhibition have been described. The results obtained indicate that the PGase from V. dahliae is a ‘‘K’’-series enzyme, where the presence of the modifier (in this case PGIP), results in a change in Km but not in the apparent Vmax for the substrate. If the binding characteristics alone are affected, Vmax will usually remain unchanged, so the inhibition pattern could be regarded as non-competitive or mixed inhibition. As PGIP does not bind directly to the active site of the enzyme, it could be possible that binding induces a conformational change in the enzyme, resulting in a lower substrate affinity. The sigmoidal binding of a modifier or inhibitor can amplify the regulatory effect (Palmer, 1995), thus reducing the activity of the PGase. This would delay the rapid degradation of PGA to inactive monomers and result in the generation of elicitor-active oligogalacturonides with a higher degree of polymerization. These results are in support of the model proposed for the PGIP:endo PGase interaction (De Lorenzo et al., 1993) where the formation of the enzyme-inhibitor complex in vitro, results in an alteration of the balance between the release of elicitor-active oligosaccharides and the depolymerisation of the active oligogalacturonides into inactive molecules, thereby favouring the accumulation of elicitor active molecules at the site of infection and the resulting activation of the defense responses of the host.
3. Experimental 3.1. Plant material and fungal isolate Seeds of Verticillium resistant cotton cultivar OR19 (obtained from the Agriculture Research Council’s Institute for Tobacco and Cotton, Rustenburg, South
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Africa) were germinated and maintained at 30 C. Roots and stems of 3-week-old plants were used in all experiments. A pathogenic isolate of V. dahliae was obtained from infected Acala1517/70 cotton stems (Meyer et al., 1994).
toethanol. Gels were then washed for 10 min in 20 mM NaOAc, pH 4.5 and enzyme activity localized as described above. The band of activity was compared to the SDS-gel to determine the molecular weight of PGase from V. dahliae.
3.2. Polygalacturonase purification and characterization 3.4. Enzyme activity determinations Fungal mycelia were cultivated in liquid culture in the dark at 25 C for 18 days, in 1 l flasks, containing 500 ml of medium (Nachmias et al., 1982). A growth curve was determined over a period of 21 days. The extracts were screened by means of an agarose diffusion assay (Dingle et al., 1953) to determine maximum PGase production (Section 3.8). Culture filtrates harvested on day 18 were used for purification of PGase. Following centrifugation at 15 000g, proteins present in the the clear supernatant (1350 ml) was precipitated at 90% (NH4)2SO4 saturation for a minimum of 1 h. The precipitate was collected by centrifugation at 15 000g for 20 min and dissolved in dH2O. The sample was fractionated using gel permeation chromatography on Sepharose CL-6B (2.560 cm) with dH2O as eluant (Meyer et al., 1994) at a flow rate of 20 ml h 1. The fractions obtained after gel filtration that exhibited PGase activity were combined and concentrated in a dialysis bag against glycerol to stabilize the enzyme for storage at 4 C. PGase from V. dahliae was characterized by native PAGE, agarose overlay gels (Kanellis et al., 1991), SDS– PAGE (Laemmli, 1970) and IEF gels (Robertson et al., 1987). The Mr was determined using analytical gel filtration with a Sepharose CL-6B column (2.560 cm) using 0.1 M NaPi buffer, pH 8.0 as eluting buffer (Andrews, 1970). V0 and Vt were determined with blue dextran and DNP-alanine respectively to calibrate the column, together with a set of calibration proteins from 14 to 158 kDa. 3.3. Enzyme staining in gels Enzyme activity of endo-PGases present in the culture filtrates and crude extract were screened by means of an agarose diffusion assay (Dingle et al., 1953). Following PAGE, the gels were incubated in 0.1 M NaOAc, pH 5.0 for 10 min. Agarose gels (285100 mm) were poured onto Gelbond1 film (FMC Bioproducts). The agarose soln. contained 0.8% agarose (w/v), 100 mM HOAc (pH 5.0) and 1 mg ml 1 PGA (w/v). Following incubation, the overlay gels were stained in 0.005% ruthenium red for 2 h and destained in dH2O until clear bands were evident (Kanellis et al., 1991). In order to determine activity following SDS–PAGE, gels were washed twice for 15 min in 0.1 M NaOAc, pH 4.5 containing 0.05 mg ml 1 BSA and 0.1% TritonX100 at room temperature. The enzyme was allowed to renature at 4 C overnight in 0.1 mM NaOAc, pH 4.5 containing 0.05 mg ml 1 BSA and 0.02% b-mercap-
A reducing sugar assay for the determination of released galacturonic oligomers was used with the colour reagent p-hydroxybenzoic acid hydrazide (Kombrink et al., 1988). PGase, either the positive control Aspergillus niger PGase, 100 ng ml 1, or purified PGase from Verticillium dahliae, 30 ng ml 1, was added as a 200 ml aliquot to 200 ml autoclaved PGA (2.5 mg ml 1) in 20 mM NaOAc buffer, pH 5.0. PGase activity was assessed over time (0–60 min) to determine the linear range. Duplicate samples were taken at 4 different time intervals (0, 10, 20 and 30 min). Activity was expressed as A410 min 1 ml 1 or converted to arbitrary units, one unit being equivalent to the release of 1.0 mmol galacturonic acid units min 1 ml 1 at pH 5.0. 3.5. Induction and purification of PGIP PGIP in cotton seedlings was chemically induced with salicylic acid. Seedlings were sprayed with 5 mM sodium salicylate, pH 7.0 for 72 h. Distilled water was used as a control. The extraction procedure of Salvi et al. (1990) was carried out at 4 C with slight modifications. Plant material (hypocotyls, 200 g) was homogenized for 2 min in cold 20 mM NaOAc, pH 5.0 containing 500 mM NaCl (1:2 m/v). Proteinase inhibitor, PMSF (0.1%), was added prior to homogenization. The suspension was vacuum filtered through a number 1 sinter glass funnel and the filtrate centrifuged at 12 000g for 20 min. The proteins in the supernatant were precipitated for 1 h with (NH4)2SO4 at a final saturation of 90%, followed by centrifugation 12 000g for 20 min to collect the precipitate. The precipitate was resuspended in a small volume of 20 mM NaOAc, pH 5.5 and dialyzed with at least 4 changes of the same buffer in a dialysis membrane with a molecular cut-off value of 12–14 kDa. The suspension was centrifuged at 12 000g for 20 min and the PGIP purified further by affinity chromatography on an endo-PGase affinity column as described by Cervone et al. (1987). PGIP activity was measured using the PGase assay described above. One unit was defined as the amount of inhibitor required to reduce the activity of one unit of enzyme by 50% 3.6. Electrophoretic analyses Protein concentration was determined according to Bradford (1976) or the bicinchoninic acid protein assay from Pierce (Rockford, IL). Purified preparations were
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analysed by native PAGE, agarose overlay gels (Kanellis et al., 1991) and IEF gels (Robertson et al., 1987). SDS-electrophoresis was carried out according to Laemmli (1970); gels were stained with either Coomassie R250 or silver (Bristow, 1990). 3.7. Western blot analyses Blotting was performed overnight at 4 C and 36 V using PVDF-Plus membranes and 25 mM Tris (pH 8.2), 192 mM glycine, 20% (v/v) methanol and 0.1% (w/v) SDS as transfer buffer. The primary antibody was raised in rabbits against a synthetic peptide, A-L-L-Q-I-K-KD-L-G-N-P-Y, located near the N-terminus of bean PGIP and coupled to Keyhole Limpet Haemocyanin (Bergmann et al., 1994). Goat anti-rabbit IgG, conjugated to alkaline phosphatase was used as secondary antibody at a dilution of 1:1000 with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate as substrate (Berger et al., 2000). 3.8. Inhibition studies The agarose diffusion assay (Dingle et al., 1953) for PGase activity was performed to test for the presence of PGIP. The assay medium consisted of 100 mM citrate and 200 mM NaPi (pH 5.3) to which final concentrations of 1% agarose, 0.5% ammonium oxalate and 0.1 mg l 1 PGA were added. The assay was carried out with the addition of 50 ml culture medium supernatant into each well and overnight incubation at 30 C. Activity zones were visualized by staining the background with 0.05% ruthenium red. For enzyme assays and kinetic studies the reducing sugar assay as described in Section 3.4 was used. Enzyme was added to different concentrations of cotton PGIP (0–7.2 ng ml 1) to determine the degree of inhibition. PGIP preparations were mixed with PGase and incubated for 10 min prior to the addition of the PGA substrate. Substrate concentrations were varied between 1.5 and 5.5 mg ml 1 to determine the type of inhibition. Duplicate samples were taken at 0, 10, 20 and 30 min and analyzed for galacturonic acid.
Acknowledgements We wish to acknowledge Dr. D. Berger (ARC, Agricultural Research Council) for cooperation and the gift of the anti-PGIP antibody, and the National Research Foundation (NRF) for research funding.
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