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Microbiological Research 163 (2008) 51—62

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Inactivation of polygalacturonase and pectatelyase produced by pH tolerant fungus Fusariummoniliforme NCIM 1276 in a liquid mediumand in the host tissue

Suryakant K. Niturea,�, Ameeta R. Kumarb, Pradeep B. Parabc, Aditi Panta

aDivision of Biochemical Sciences, National Chemical Laboratory, Pune 411008, IndiabInstitute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, IndiacNational Center for Cell Science, University of Pune, Ganeshkhind, Pune 411007, India

Received 4 January 2006; received in revised form 28 February 2006; accepted 21 March 2006

KEYWORDSFusarium monili-forme;Pectate lyase;Polygalacturonase;Pathogenesis

ee front matter & 2006micres.2006.03.004

ing author. Tel.: +91 20893619.ess: skniture@yahoo.co

SummaryFusarium moniliforme NCIM 1276 produced pH dependent an extracellularpolygalacturonase (PG) and pectate lyase (PL) at pH 5 and pH 8, respectively. Inthe extracellular medium about 20.3% PG and 54% of PL protein concentrations werepresent in the active state at pH 5 and pH 8, respectively, whereas in intracellularly,more than 86% of both protein contents remained in the active state at all pH tested.We found two possible reasons, end-product inhibition and effect of environmentalpH on conformation of the proteins after their release into the medium. Additionally,in infected tomato and cauliflower plants, the fungus secreted similar proteins whichwere located near to the epidermal and vascular regions of the hypocotyls. Ininfected tissues, between 26.9% and to 41.5% of PG and only 0.84%–13.4% of PLprotein concentrations were present in active state. Thus, the medium/cell sap pHand concentrations of substrate/end products seem to play an important role infungal invasion during plant pathogenesis are discussed with current literature.& 2006 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights rese

25893300;

m (S.K. Niture).

Introduction

Plant cell wall consists of four major polysac-charides such as cellulose, hemicellulose, ligninand pectin. Pectin a heteropolysaccharide found inthe middle lamella of the plant cell wall composedof a-1-4-linked galacturonate chains with a high

rved.

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S.K. Niture et al.52

percentage of methyl esterification. Hence, pectindegradation can be attained by the combinedaction of several enzymes such as pectin-methy-lesterases and pectin-depolymerases including hy-drolases and lyases. Pectin-depolymerases such aspolygalacturonase (PG) (EC 1.2.3.15) and pectatelyase (PL) (EC 4.2.2.1) act on homo-galacturonicregions of pectin by hydrolytic and trans-elimina-tion cleavage mechanisms, respectively.

Pectinases are the first enzymes to be secretedby fungal pathogens when they attack plant cellwalls (Collmer and Keen, 1986; Idnurm and How-lett, 2001). Pectin degrading enzymes are essentialfor fungal pathogens that do not have specializedpenetration structures as well as for necrotrophicpathogens during the late stages of the invasionprocess (De Lorenzo et al., 1997). Secretion ofpectin degrading enzymes during infection to theplants has been reported from various plant-pathogenic fungi such as Fusarium oxysporum,Botrytis cinerea, Sclerotinia sclerotiorum (Di Pietroand Roncero, 1998; Garcia-Maceira et al., 2001; tenHave et al., 2001; de las Heras et al., 2003; Li etal., 2004), from non-pathogenic fungus RhizoctoniaAG-G (Machinandiarena et al., 2005) and fromseveral yeasts (da Silva et al., 2005). PG producedby bacteria Erwinia carotovora (Lei et al., 1985)and Agrobacterium tumefaciens Biovar 3 (Rodri-guez-Palenzuela et al., 1991) has been known todetermine virulence and cause necrosis in Vignaunguiculata (Cervone et al., 1987). Apart fromacting as virulence factors during infection, endo-PG and PL may also function as virulence determi-nants by releasing their end products (oligo-galacturonides) which act as inducers of plantdefense molecules such as phytoalexins (Davis etal., 1984). PL has been also implicated in patho-genicity from several fungi including Fusariumsolani var pisi and Colletotrichum gloesosporioides(Crawford and Kolattukudy, 1987; Yakoby et al.,2000) and from bacterial genus Erwinia (Lei et al.,1985; Nasser et al., 2005) thereby suggesting that,in nature both PG and PL are agents of plantpathogenecity.

The expression of PL and PG genes in the phyto-pathogenic fungi are subjected to the ambient cell-sap pH or the available carbon source of the culturemedium. For example, during ripening of Perseaamericana cv, Fuerte fruits the pericarp pHregulate PL secretion and affects the pathogenicityof C. gloeosporioides (Yakoby et al., 2000). PGsgenes are also found to be repressed by glucose andinduced by pectin or pectic substrates present inthe medium (Wubben et al., 2000; Fontana et al.,2005; Radoi et al., 2005), moreover, constitutivePG expression also reported in B. cinerea (Van der

Cruyssen et al., 1994). These evidences suggestthat, environmental or host cell sap pH regulatethe expression of pectinolytic enzymes and thus,play an important role against phyto-pathogenicorganisms.

While searching for high pectinase producersfrom mangrove ecosystems, a fungal strain identi-fied as Fusarium moniliforme was isolated fromdecaying leaves of mangrove plants (Rhizophoraapiculata and Avicennia officinalis) in the saline,detritus-rich mud of a mangrove estuary on thewest coast of India (18155N, 72154E) (Rao, 1996).Under laboratory conditions and in liquid mediumcontaining 1% citrus pectin as carbon source, theorganism produced a single PL at pH 8 and a singlePG at pH 5 (Rao et al., 1996; Niture et al., 2001). Inour earlier study we have shown that, when theisolate was allowed to infect tomato and cauli-flower seedlings, having different cell sap pH, itcaused vascular wilt in both plants. During infectionin the host tissue, it secreted similar PG, PL andanother PG II isoenzyme, which exhibited totallydifferent physico-chemical properties (Niture andPant, 2004). Hence, it can possible that PG and PLmay act as virulence factors during infection in theabove plants and their production as well assecretion may be pH dependent. In the presentstudy, we have investigated the production andinactivation profiles of PG and PL as a functiondifferent pH conditions. These studies have beencarried out in liquid medium as well as in hostplants such as, Lycopersicon esculentum (tomato)and Brassica oleracea botrytis (cauliflower) whichhaving a cell sap pH of 6.4 and 7.7, respectively.

Materials and methods

Pectin (citrus fruit 8% methyl-esterified), poly-galacturonic acid, di, tri, and saturated D -galacturonic acid, Freund’s complete adjuvant,horse radish peroxidase, were purchased fromSigma Chemical Company USA. Tetramethyl benzi-dine/Hydrogen peroxide (TMB/H2O2) was pur-chased from Bangalore Genei Pvt Ltd India. ELISAplates were purchased from Greiner LabortechnikPvt. Ltd. Chandigarh, India. Wheat bran, tomatoand cauliflower seeds were obtained from the localmarket. All chemicals and reagents were ofanalytical grade.

Microorganism

The present fungal strain F. moniliforme wasisolated from decaying leaves of mangrove plants

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Inactivation of polygalacturonase and pectate lyase produced 53

(R. apiculata and A. officinalis) and identified onthe basis of standard morphological characters(Rao, 1996). The organism has been deposited inthe National Collection of Industrial Microorganism(NCIM) at National Chemical Laboratory, Pune,India, as F. moniliforme NCIM 1276. The funguswas routinely maintained on Czapek–Dox agarslants containing 1% (w/v) pectin at 10 1C.

Effect of different sugars on the productionof cell wall degrading enzymes

One milliliter of spore suspension (106 spores) ofF. moniliforme NCIM1276 was inoculated into thesterile fresh medium containing 37.9mM (NH4)2SO4,11.5mM K2HPO4, 14.7mM KH2PO4 and 6.8mM CaCl2in 50ml sterile glass-distilled water supplied with1% of different sugars such as galacturonic acid,polygalacturonic acid, pectin (8% esterified), glu-cose, starch, xylan, cellulose and wheat bran. Afterautoclaving, the medium pH was adjusted to 5 or 8using either sterile 0.1 N NaOH or 0.1 N HCl. Cultureflasks were incubated at 30 1C and 200 r.p.m. on alaboratory shaker for 96 h. The extracellular pecti-nase, celluase, xylanase and amylase enzymeactivities were determined by standard assayconditions as described below.

Effect of pH on extracellular andintracellular production of PG and PL

The basal medium containing 37.9mM (NH4)2SO4,11.5mM K2HPO4, 14.7mM KH2PO4 and 6.8mM CaCl2 in50ml sterile glass-distilled water supplied with 1%pectin and 0.2% glucose was used for the productionof PG and PL. After autoclaving, the medium pH wasadjusted from 2 to 11 with using either sterile 0.1NNaOH or 0.1N HCl. One milliliter of F. moniliformeNCIM1276 spore suspension (106 spores) was inocu-lated into sterile fresh medium. Culture flasks wereincubated at 30 1C and 200 r.p.m. on a laboratoryshaker for 96h. The extacellular PG and PL activitieswere determined by standard assay conditions.Extracellular PG and PL protein concentrations weremeasured by sandwich ELISA as described previouslyby Niture and Pant (2004). The fungal biomass wasdetermined after 96h of fungal growth. The fungalbiomass was harvested by centrifugation; the pelletwas washed twice with sterile distilled water anddried at 80 1C. For determination of intracellular PGor PL activity and protein concentrations, the wetmycelia pellet was washed with sodium phosphatebuffer (10mM; pH 7) containing 150mM NaCl twiceand the pellet was sonicated for 1min in eithersodium acetate buffer (50mM) pH 5 or Tris/HCl buffer

(50mM) pH 8. Cell debris was removed by centrifuga-tion and PG and PL enzyme activities and proteinconcentrations were determined from supernatant bystandard assay conditions.

Enzyme assays

PG: The assay measured reducing sugar releasedfrom 0.3% polygalacturonic acid by the method ofNelson (1944) and Somogyi (1952). The reactionmixture (1ml) contained 0.3% polygalacturonicacid with 0.7% NaCl and 0.25% Na-EDTA in 0.1Msodium acetate buffer pH 5.0. The reaction productabsorbance was monitored at 500 nm. One unit ofenzyme activity is defined as the amount ofenzyme, which releases 1 mmol of galacturonic acidper minute at 40 1C at pH 5.0 (Collmer et al., 1988).

PL: The assay measured the increase in absor-bance at 232 nm of 0.24% polygalacturonic acid in60mM Tris/HCl buffer, pH 8.5 with 0.6mM CaCl2 at40 1C. One unit of lyase activity was defined as theamount of enzyme which produces 1 mmol ofunsaturated galacturonide (e ¼ 4600M�1 CM�1) perminute (Collmer et al., 1988).

Xylanase, amylase and carboxymethyl-cellulase(CMCase) enzyme activities were determined byDNSA (3-5 dinitrosalicylic acid) method as de-scribed by Miller (1959).

Protein determination

Protein concentration was determined in accor-dance with Lowry et al. (1951) using bovine serumalbumin as a standard.

Enzyme kinetics and inhibition of PG and PLby pectic substrates

PG and PL produced by F. moniliforme in pectinmedium at pH 5 and at pH 8 were purified asdescribed previously (Rao et al., 1996; Niture etal., 2001). Purified PG (0.14 mM) was incubated withdi-, tri-galacturonic acid (25mM), polygalacturonicacid (3mg) and pectin (3mg) in 50mM acetatebuffer pH 5 at 45 1C for specified times. Similarly,purified PL (0.12 mM) was also incubated with di-,tri-galacturonic acid (25mM), polygalacturonic acid(3mg) and pectin (3mg) in Tris/HCl buffer (50mM)pH 8 at room temperature. The relative rate ofhydrolyses of these sugars by PG and PL wasmeasured by standard assay conditions. In anotherexperiment, PG and PL were incubated with mono-,di-, tri-galacturonic acid (5mM) and pectin (3mg)for 2 h at room temperature and the residualactivities of the modified enzymes were measured

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S.K. Niture et al.54

by standard assay conditions using polygalacturonicacid (PGA) as a substrate. In control reaction sameconcentrations of above sugars were suppliedwithout enzymes and absorbance was subtractedform the respective test reactions. In addition, atsaturated concentration of polygalacturonic acid(30–250 mg PGA), the inhibition of the PG by pectinand PL by saturated D-galacturonic acid was testedat various concentrations of inhibitor, pectin(50–500 mg/ml; 8% methyl-esterified) and saturatedD-galacturonic acid (5–50mM), respectively. Thekinetic constants such as Km, Vmax and kcat andinhibition constant (ki) of PG and PL were calcu-lated by fitting the data to a linear regression onLineweaver-Burk plots using ORIGIN 4.1 (Microcal).

Circular dichroism (CD) studies

Purified PL (2.56mM) and PG(1.82mM) were incu-bated with 100mM of glycine–NaOH buffer pH 2,acetate buffer pH 5, Tris/HCl buffer pH 8 and sodiumbicarbonate buffer pH 10 and 11 for 24h at 30 1C. CDspectra were recorded on a Jasco-710 spectropolari-meter from 190 to 250nm at 25 1C using a 1cm pathlength cell. The CD data were analyzed according tothe method of Sreerama and Woody (1993) aftercorrecting for individual buffer spectra.

Enzyme production on host tissue

Lycopersicon esculentum (tomato) and Brassicaoleracea (cauliflower) seeds were surface sterilizedand planted in sterile White’s Basal medium contain-ing 0.8% Gelrite for one month at 25 1C under constantlight intensity of 2.5W/m2 in sterile tissue culturerooms. One milliliter of Fusarium spore (106) suspen-sion prepared as described earlier was injected with asterile syringe near the roots of the test plantswithout disturbing the plants. Controls were grownunder identical conditions but were not exposed tospores. Samples of infected and control plants wereharvested after 8 days. One gram of plant materialfrom each sample was frozen in liquid nitrogen andthen ground in a mortar and pestle. The resultingpowder was suspended in 50mM sodium acetatebuffer at pH 5 or 50mM Tris/HCl buffer at pH 8containing 0.25M NaCl and centrifuged. The super-natant was assayed for enzyme activity and antibody-specific protein determination.

Immunolocalization of PG and PL in hosttissue

Control and infected hypocotyls of both tomatoand cauliflower were fixed in 50% ethanol, 5% acetic

acid and 4% formaldehyde in water and dehydrated ina 10–90% ethanol series. The dehydrated sectionswere embedded in Paraffin and transversely sec-tioned with a Leica microtome Model RM 2155. Thesections were blocked with 1% BSA (w/v) in PBS/Tandincubated overnight at 4 1C with diluted primary PGand PL antibodies at a concentration of 1mg/ml in 1%BSA. The sections were washed and again incubatedwith appropriate anti-rabbit antibodies labeled withTexas Red for 3h at 25 1C. The sections were observedunder Leitz-Laborlux Fluorescence microscope usingN2 filter.

Statistical analyses

Each experiment was performed at least 3 timesand the data are shown as mean7SD whereapplicable. Statistically significant differences be-tween groups in each assay were determined usinga Student’s t-test.

Results

Production of different polysaccharidases byF. moniliforme

The present organism was isolated from thedecaying leaves of mangrove plants where its modeof nutrition was saprophytic rather than pathogenic(Rao, 1996). To test whether the organism was ableto produce cell wall degrading enzymes, for this,F. moniliforme was grown on different sugarssubstrates and extracellular cell wall degradingenzyme activities were determined. As shown inTable 1, both acidic and alkaline conditions theorganism produced significant levels of induciblexylanase and amylase enzymes whereas, theproduction of cellulose was lower compared withother enzymes. PG and PL production was inducedby pectin rather than other sugars and the organismproduced higher PG activity at acidic pH and PL atalkaline conditions. Interestingly, when the organ-ism was grown on crude substrate wheat bran, itproduced all enzymes significantly under bothacidic and alkaline conditions (Table 1).

Profiles of PG and PL production at differentpH by F. moniliforme

Previously, we have shown that the presentisolate produced single form of PG and PL in theliquid medium containing 1% pectin at pH 5 and pH8, respectively (Rao et al., 1996; Niture et al.,2001). In this report we have analyzed the extra

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Table 1. Production of different polysaccharidases by F. Moniliforme NCIM 1276

Substrate (1%) PG (U) PL (U) CMCase (U) Xylanase (U) Amylase (U)

At pH 5Galacturonic acid 670.12 3.170.11 ND ND NDPolygalacturonic acid 10.570.3 14.270.1 ND ND 1.2370.1Pectin 11.570.08 2670.12 0.5170.1 0.2570.1 0.6570.11Cellulose 0.6570.1 ND 1.5570.11 3.1270.08 0.2570.1Starch 4.170.22 ND ND ND 19.271.6Xylan 3.270.01 ND 0.6170.11 22.172.3 NDGlucose ND 16.1570.1 ND ND NDWheat bran 2.170.1 7.770.2 2.570.13 28.770.18 1671.5

At pH 8Galacturonic acid 1.2370.1 8.270.12 0.5270.11 ND NDPolygalacturonic acid ND 11070.23 2.170.2 1.270.1 0.3270.02Pectin 2.570.13 18070.51 ND ND 170.11Cellulose ND 50.270.23 0.7570.01 1.970.11 NDStarch 1.4570.13 1570.8 ND ND 2370.1Xylan 0.9570.11 3070.03 ND 5574.2 NDGlucose ND 2.570.1 ND ND NDWheat bran 1.3470.14 8770.010 971.1 6073.1 18.470.1

F. moniliforme was grown on 1% different polysaccharides at pH 5 and pH 8, after 96 h of growth, enzymes activities were calculatedfrom 50ml of extracellular broth. ND – Not detected. Each value represents the mean of replicated values7SE.

Table 2. Effect of different medium pH on extracellular production of PG and PL

pH Total Biomass (mg) PG (U) PG (mg ) Active PG (%) PL (U) PL (mg /ml) Active PL (%)

2 0.3570.02 ND 670.01 ND ND 4570.03 ND3 88.3571.2 0.0570.01 3270.01 0.89 1270.02 10070.2 22.64 211.572.12 1070.01 17570.1 32.2 2570.03 37570.4 12.585 295.372.12 1470.01 38570.2 20.5 3070.02 51071.9 11.156 315.573.2 7.570.02 27570.3 15.3 24570.02 63072.4 73.377 311.4272.1 0.570.01 2570.02 11.2 26070.3 118072.8 41.578 299.2172.8 0.0570.01 2070.01 1.4 40570.4 136075 53.179 191.2371.91 ND 1070.05 ND 35070.2 111070.3 59.4910 89.5670.45 ND 87.01 ND 12570.02 32570.2 72.5911 35.3470.08 ND 570.01 ND 970.01 3070.02 56.6

F. moniliforme was grown at different pH and after 96 h of growth PG and PL activities protein concentrations were determined from50ml of extracellular broth. Percentage active PG and PL fractions were calculated on the basis of specific activity of the purifiedenzyme and sandwich ELISA. Each value represents the mean of replicated values7SE. ND–Not detected.

Inactivation of polygalacturonase and pectate lyase produced 55

and intracellular production profiles of PG and PL atdifferent pH. As shown in Table 2, the fungusproduced maximum PG and PL activity and proteincontents (determined by ELISA) at pH 5 and pH 8,respectively. PG activity was not detectable at pH 2and between pH 9 to 11 (Table 2). The productionPL activity and protein concentrations were higherthan PG at all pH tested. Protein concentrationprofiles as determined by ELISA showed that a smallbut measurable amount of PG and PL proteincontents were present the extracellular mediumat all pH, irrespective of the attenuation in activityat pH 2, 3, 10 and 11. The organism produced

maximum biomass between pH 4 and 8 anddegraded maximum pectin (68%) at pH 7 presentin the medium (data not shown). The lower biomassand enzyme concentrations were detected at non-physiological pH such as 2, 3, 9 and 11 indicatingthat the organism may be stressed under theseconditions.

As shown in Table 3, intracellular production ofboth enzymes occurred at all pH tested. Thespecific activities of purified enzymes (PG 178U/mg and PL 530U/mg) (Rao et al., 1996; Niture etal., 2001) allow a calculation of the amount ofenzymatically active protein using the ELISA-

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Table 3. Effect of different medium pH on intracellular PG and PL production by F. moniliforme NCIM 1276

pH Total PG Activity (U) Total PG (mg) Active PG (%) Total PL activity (U) Total PL (mg) Active PL (%)

2 0.8370.02 5.370.12 88.35 1.0170.01 2.170.023 90.103 1.7270.12 10.870.88 89.58 3.1870.08 6.2370.56 96.364 3.570.10 21.270.23 92.8 6.1770.05 12.2370.23 95.215 3.7870.09 23.270.89 91.74 9.3970.02 18.070.88 98.426 3.1670.1 18.070.12 98.75 10.8870.2 21.370.89 96.457 2.9470.2 17.770.67 91.8 11.5270.3 24.3271.05 89.448 2.4470.1 15.3470.67 89.37 11.670.4 24.8570.87 88.079 1.670.05 10.2370.45 87.91 9.0670.5 19.4370.56 88.0410 1.5270.02 9.7870.54 87.3 8.5670.33 17.3470.40 93.1411 1.3870.01 8.2370.12 94.5 4.1370.21 8.5370.23 95.60

F. moniliforme was inoculated into 50ml the pectin medium at different pH and after 96 h growth, the total biomass, intracellular PGand PL activity and PG and PL protein concentrations from 50ml of culture broth were quantified. PG and PL enzyme activities weredetermined under the standard assay conditions. Percentage active PG and PL fractions were calculated on the basis of specific activityof the purified enzyme and sandwich ELISA. Each value represents the mean of replicated values7SE. ND–Not detected.

A

0

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0.15

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0 5 10 15 20 30

Time (minutes)

PG

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PGA

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Di -GA

Tri-GA

PGA

Pectin

B

Figure 1. (A) Rate of hydrolyses of different substratesby purified PG. (B) Rate of hydrolyses of differentsubstrates by purified PL.

S.K. Niture et al.56

derived concentrations of the two proteins and,therefore, comparison between the data on in-tracellular and excreted proteins were carried out.As shown in Table 3, on an average more than 86%of both PG and PL inside the cells were enzymati-cally active, whereas the percentage drops toabout 20.5% in the case of PG and 53.17% in thecase of PL in the extracellular medium at pH 5 andat pH 8, respectively (Table 2). Thus, the fungussecreted a 5-fold more PG in the extracelluarmedium, however, a one fold PG fraction present inthe active state. Similarly, near about 46% of PLprotein content was also present in the inactivestate in the extracellular medium.

In order to assign the possible reasons for thisfinding, the rate of hydrolyses of different oligo-galacturonides and their effect on the catalyticactivity of PG and PL were examined. The rate ofhydrolysis of various pectic sugars using purified PGand PL enzymes were carried out using in vitroenzyme assays. As shown in Fig. 1A, PG hydrolyzedpolygalacturonic acid more efficiently than othersugars. The relative rates of degradation of thesesugars by PG were: polygalacrturonic acid (PGA)100%, tri-galacturonic acid 53%, di-galacturonicacid 45%, and pectin 35%, during a 30min reaction.Further, when purified PG was incubated withsaturated mono, di, and tri-galacturonide at pH 5at 25 1C for 2 h, the enzyme showed 100% of itsactivity (data not shown), but on incubation withpectin, 70% inhibition of PG activity was observedunder standard assay conditions. Pectin inhibitsPG activity competitively with ki of 0.312mg/ml.On the other hand, PL also hydrolyzed polygalac-turonic acid more efficiently and the rate ofhydrolyses was decreased on incubation with pectin

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-9000

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190 200 210 220 230 240 250 260

A

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Figure 2. Far–UV-CD spectra of PL and PG. (A) PL and (B)PG. Purified PL (2.56mM) and PG (1.82mM) wereincubated with different pH for 24 h at 30 1C. Controlsamples of PL and PG were incubated at pH 8 and pH 5,respectively, for 24 h. CD spectra were recorded on aJasco-710 spectropolarimeter from 190 to 260 nm at 25 1Cusing a 1 cm path length cell.

Inactivation of polygalacturonase and pectate lyase produced 57

and oligo-galacturonides (Fig. 1B). The relativerates of degradation of these sugars by PL were:polygalacturonic acid 100%, digalacturonic acid14%, trigalacturonic acid 53% and pectin 80%,during 2min reaction. When purified PL wasincubated with saturated D-galacturonic acid theenzyme loss its 85% of enzyme activity whereasother substrates did not showed any inhibitoryeffect on PL activity. Saturated D-galacturoniccompetitively inhibits PL activity with ki of18mM. Additionally, the kinetics constants data asshown in Table 4, clearly suggest that, both PG andPL shows high affinity towards PGA compared toother pectic sugars. In contrast, in case of PG theKm value was increased �3 times for pectin andkcat/Km decreased �6 times suggesting that, theenzyme shows very low affinity towards pectin (8%esterified). Similarly, PL also decreased the affinitytowards tri and di-galacturonides (high Km and lowkcat/Km) compared to PGA and pectin (Table 4).From above data it can be possible that un-degraded pectin (32%, data not shown) or highlymethyl-esterified oligogalacturonides which arepresent in the medium may be efficiently bindswith PG and inhibits its activity at pH 5 (Table 2).Similarly, PL also inhibited by D-galacturonic acid(the reaction end product of PG) at its optimal pH8. Lower PG activity was detected (0.05 Unit) at pH8 in liquid medium which degrade the homo-galacturonic regions of pectin present in the brothand produced D-galacturonic acid as end productwhich can then bind to PL and inhibit its activity atpH 8. Although these inactivated proteins weredetected by ELISA in larger quantities at theiroptimal pH, but PG and PL may be complexing withesterified pectin or oligogalacturonides or D-galac-turonic acid resulting in loss of their activities.

Effect of pH on the structural confirmation ofPG and PL

To investigate the effect of medium pH oninactivation PG and PL, the purified proteins were

Table 4. Kinetic constants of PG and PL

Enzyme Substrate Km(mg/ml) Vmax

PG PGA 0.12 111.Tri-GA 0.16 103.Di-GA 0.18 85.Pectin 0.33 52.

PL PGA 1.2 657.Tri-GA 1.8 328.Di-GA 7.82 32.Pectin 1.41 551.

exposed to different pH regimes overnight and CDspectra were recorded. PG and PL incubated withpH 2, 3, 10 and 11 showed marginal conformationalchanges in structure compared with their optimalpH 5 and 8 (Figs. 2A and B). The pH relatedconformational changes are reported in Table 5.The percentage a-helix of PG changed from 16.2%at pH 5 to about 13.8% at pH 2, and the percentageof b-sheet increased from 27.2% to 53% over thesame pH range. The change in hydrogen bonding atnon-optimal pH most probably resulted in change inactivity in the enzyme. Changes in the ratios of a-helix to b-sheet were also observed in PL (Table 5).

(mmol/min/mg) kcat(min�1) kcat/Km

11 4273 3.5� 104

11 3961 2.4� 104

32 3281 1.8� 104

33 2012 0.6� 104

12 7891 6.5� 104

5 3957 2.1� 104

8 395 0.05� 104

21 6638 4.7� 104

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Table 5. Effect of pH on PG and PL structural components

pH Activity (U/ml) a b T O SF

PG2 ND 0.138 0.530 0.155 0.172 0.9953 0.0011 0.140 0.526 0.158 0.166 0.9905 0.28 0.162 0.272 0.269 0.300 1.00310 ND 0.229 0.508 0.110 0.176 1.02311 ND 0.274 0.486 0.116 0.168 1.044

PL2 ND 1.002 0.028 0.004 0.011 1.0443 0.25 0.999 0.007 0.002 0.002 1.0098 8.1 0.257 0.388 0.123 0.231 1.00110 2.52 0.762 0.150 0.039 0.088 1.08811 0.175 1.002 0.013 0.010 0.001 1.017

CD analysis was carried out according to the method of Sreerama and Woody (1993). a–a Helix, b–b Sheet, T–Turn, O–Others structuralcomponent , SF–Summation of total fraction (structural components) of the protein molecule.

S.K. Niture et al.58

At pH 2 and 11, a-helix components of PL wasincreased by a 4-fold and b-sheet components weredecreased by a 13–15-fold compared with optimalpH 8 suggesting, a drastic changes in structuralconfirmation of protein. The percentages of otherminer components also changed in the non-optimalpH in both proteins. These data indicating that, inintracellular the fungus produced both PG and PLall pH tested and more than 86% of PG and PLprotein amounts are present in the active state(Table 3), whereas, when these proteins releasedinto the extracellular medium, the medium non-optimal pH such as 2, 3, 10 and 11 altered theirstructural conformational resulted in loss of theiractivities (Table 2), whereas, at optimum physiolo-gical pH, esterified pectin and galacturonic acid areresponsible for their inactivation.

Profiles of PG and PL production in infectedhost tissue

Next we examined the production and inactiva-tion pattern of PG and PL in infected host tissue.For this purpose, L. esculentum (tomato) and B.oleracea (cauliflower) plants were selected whichhaving acidic and alkaline cell sap pH. The plantswere grown under sterile conditions and infectedwith Fusarium spores for 8 days and control andinfected hypocotyls and roots of the plants wereused for quantification PG and PL levels asdescribed in the method section. As shown inTable 6 and Figs. 3A and C, healthy tissue fromL. esculentum and B. oleracea had an intrinsic lowintracellular activity of both PG and PL. Uponinfection, the immuno-localization studies clearlyshowed that, production of both PG and PL

increased in infected host tissue as compared tocontrol plants (Figs. 3A–D). Both PG and PL werefound to be localized near the epidermal as well asthe vascular regions of hypocotyls sections intomato and cauliflower plants. Upon infection tothe plants by the pathogen, a differential expres-sion of the two Fusarium enzymes was found intomato and cauliflower, possibly depending on cellsap pH. Tomato roots which have a cell sap pH of6.4 exhibited a 77-fold increased in PG activity(Table 6) and a 30-fold increased in PL activity, bothresulting from Fusarium enzymes. The increase ofFusarium PG in tomato hypocotyls was even greaterthan that of Fusarium PL. These data are sub-stantiated by ELISA quantification (Table 6). On theother hand, in cauliflower hypocotyls and roots theFusarium produced more PL than PG. The ratio ofPG:PL in infected tomato was 3.6 and that incauliflower was 0.95. These data suggest that smalldifferences in cell sap pH may influence theenzyme secretion in the host tissue by this strainof F. moniliforme. It was also noted that after 8days of infection, in the host tissue, between26–41% of PG and 0.8–13% of PL protein contentswere found in active state (calculated on the basisof specific activity of the purified PG and PL) (Table6), whereas the majority fractions of PG and PLwere inactivated in the host tissue during infection.

Discussion

A strain of F. moniliforme NCIM1276 was isolatedfrom the decaying leaves of mangrove plants whereits mode of nutrition was saprophytic rather thanpathogenic (Rao, 1996). Previously we have shown

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Table 6. Distribution of PG and PL in control and infected tomato and cauliflower plants

S. no PG (U/g) PG (mg/g) PG Active (%) PL (U/g) PL (mg/g) PL Active (%)

Cont. tomato hypocotyls 0.0270.01 ND — 0.0270.01 ND —Cont. tomato roots 0.0570.01 ND — 0.0470.01 ND —Infec. tomato hypocotyls 2.670.2 35.271.0 41.5 0.0670.02 13.672 0.83Infec. tomato roots 4.3270.1 90.571.6 26.9 1.2170.2 17.071.8 13.4Cont. cauliflower hypocotyls 0.0570.01 ND — 0.0270.01 ND —Cont. cauliflower roots 0.0170.01 ND — 0.0170.005 ND —Infec. cauliflower hypocotyls 0.99270.1 18.370.5 30.5 1.4870.13 25.070.6 11.2Infec. cauliflower roots 2.6370.15 45.270.43 32.7 2.7470.15 78.173.1 6.6

One gram of infected and control tissue from roots and hypocotyls were frozen in liquid nitrogen and ground in a mortar and pestle.The powder of the frozen tissue was dissolved in 0.25M NaCl, and after centrifugation total PG and PL activities were determined fromthe supernatants and reported as U/g of plant material. Antibody specific protein of PG and PL in the supernatant were alsodetermined by sandwich ELISA and reported as mg/g of plant material. Each value represents the mean of replicated values7SE.Con.–Control, Infec.–Infected. ND–Not detected.

Figure 3. Immunolocalization of PG and PL in tomato and cauliflower hypocotyls. PG antibody used. (A) Transversesection (TS) of control tomato hypocotyl region. (B) TS of infected tomato hypocotyl region by F. moniliforme. PLantibody used. (C) TS of control cauliflower hypocotyl region (D) TS of infected cauliflower hypocotyl region by F.moniliforme. C–cambium, VB–vascular bundle, EP–epidermis.

Inactivation of polygalacturonase and pectate lyase produced 59

that the organism was adapted to the estuarineenvironment and not true marine species because itcan grow without salt (NaCl) (Niture and Pant,2004). Since the genus Fusarium is well-knownphytopathogen and caused vascular wilt in severalplants (Raabe et al., 1981), and, in our earlier studywe have shown that, when the isolate was allowedto infect tomato and cauliflower seedlings, having

different cell sap pH, it caused vascular wilt in bothplants (Niture and Pant, 2004). In the presentreport, we studied the production of different cellwall degrading enzymes by the isolate. As shown inTable 1 the organism produced several cell walldegrading enzymes such as cellulase, xylanase,amylase and pectinase under laboratory conditionswhen the organism was grown on appropriate

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substrates, suggesting that, these enzymes plays animportant role in disruption of plant cell wallduring pathogenesis. Similarly, earlier severalstudies showed that, plant pathogenic fungi includ-ing genus Fusarium secreted these cell walldegrading enzymes in host tissue during coloniza-tion (Crawford and Kolattukudy, 1987; Di Pietro andRoncero, 1998; Garcia-Maceira et al., 2001).

Under laboratory conditions the organism was ableto grow in the wide range of pH, whereas it producedthe maximum biomass and pectin degrading enzymesbetween pH 4 and 8 in the pectin medium (Tables 2and 3). Using PG and PL specific antibodies and ELISAwe quantified the extra and intracellular enzymesconcentrations produced by the isolate at differentpH. The intracellular production profile suggestedthat the organism produced both enzymes at all pHtested and more than 86% (calculated on the basis ofspecific activity of the purified PG and PL) ofintracellular PG and PL protein contents were presentin the active state (Table 3). Interestingly, in theextracellular medium the active percentage of PGand PL was reduced to about 20.5% and 53.17%,respectively, at pH 5 and 8 (Table 2). Incubation ofthe purified PG and PL proteins with pectin andgalacturonic acid at their optimal pH, both enzymeslost their 70–85% residual activities. Pectin (8%esterified) and D-galacturonic acid competitivelyinhibit PG and PL activities at their optimal pHrespectively. Whereas kinetics constants data sug-gested that, both PG and PL have a lower affinitytowards pectin (8% esterified) and di-galacturonicacid respectively compared with other sugars (Figs.1A and B, Table 4). Recently, Bonnin et al. (2002)have reported that, methyl-esterified oligogalactur-onides efficiently binds to the active site of the endo-PG of F. miniliforme, and the modified protein lost itscatalytic as well as substrate binding ability. Similarresults were found here, in culture broth, it is likelythat both un-degraded residual pectin as well as thehydrolyzed end product D-galacturonic acid may beresponsible for the inactivation these enzymes atoptimal pH. Generally, competitive inhibitors inhibitenzyme activity at optimum pH (Keyhani andSattarahmady, 2002). Therefore at optimal pH of 5and 8, both PG and PL protein contents showinglesser activity compare with other pH values such aspH 4, 6 and 10 (Table 2) in extracellular medium.Although these proteins are detected in largerquantities by ELISA at their optimal pH, but PG andPL may be complexing with esterified pectin orgalacturonic acid resulting in loss of their activities.Whereas at non-optimal pH (in acidic and alkalinerange) CD data clearly suggest that both enzymesundergo conformational changes (Fig. 2, Table 5)resulting in their inactivation. Thus, F. moniliforme

secreted excess amount of both PG and PL in themedium however, medium extreme pH as well assubstrate/end products inactive both of these en-zymes in the extracellular medium.

Similarly, when the isolate was allowed to infecttomato and cauliflower plants under laboratoryconditions, the fungus secreted both enzymes inhost tissue which are localized at the epidermal aswell as vascular regions of the hypocotyls (Fig. 3).During infection, Fusarium produced more PG intomato hypocotyls which having acidic cell sap pH6.4. Whereas, PL production was enhanced incauliflower plant which having alkaline cell sappH 7.7 (Table 6) suggesting that, the local cell sappH also regulate the production of PG and PL in hosttissue during infection of Fusarium. In contrast,earlier several reports suggested that host cell sappH, long-chain oligogalacturonides and plant PGinhibiting proteins act as defense factor againstphyto-pathogenic organisms because these factorscontrols PG and PL activities directly or indirectlyin the host tissue during infection (Davis et al.,1984; Dean and Timberlake, 1989; Wijesundera etal., 1989; Cervone et al., 1997; Ridley et al., 2001).Our data showed that, in the host tissue, 26–41% ofPG and only 0.8–13% of PL protein concentrationsare present in the active state (Table 6) suggestingthat these defense factors may be responsible forcontrolling PG and PL activities during infection. Itis also possible that during infection of Fusarium inplants other than tomato or cauliflower, if the localhost tissue pH is non-optimal with respect to eitherPG or PL, then it is likely that though the proteinsmay be produced they may be in an inactive state.

In conclusion, F. moniliforme NCIM 1276 producesPG and PL in liquid medium at all pH tested as well asin the infected host tissue. The activity of theseenzymes was found to be regulated by pH as well asoligo-galacturonides released either in the culturebroth or in the host tissue. During infection control-ling of PG and PL activities by host cell sap pH oroligo-galacturonides would be best mechanism tocontrol Fusarium colonization or infection. It isimportant to know the exact mechanism for inactiva-tion of these enzymes so that strategies for effectivebio-control of phytopathogens such as Fusarium sp.against various agricultural crops and post harvestdecay may be envisaged.

Acknowledgment

SKN had a research fellowship from CSIR NewDelhi, India. We are also thankful Dr. R.S. NadgaudaPTC department (NCL Pune) for her help in theplant tissue culture experiments.

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