Purification and characterization of an aspartic proteinase secreted by Botrytis cinerea Pers ex. Pers in culture and in infected carrots

Physiological and Molecular Plant Pathology

289

(1990) 36, 289-302

Purification and characterization of an aspartic proteinase secreted by Botrytis cinerea Pers ex. Pers in culture and in infected carrots SARA MOVAHEDI

and J . B . HEALE

Plant Cell and Molecular Sciences Research Group, London, W8

King's

College London . Kensington Campus, Campden

Hill.

7AH, U .K .

(Accepted for publication January

1990!

A single spore isolate ofBotrytis cinerea, obtained from carrot tissue, when grown in a liquid medium containing gelatin as sole nitrogen source, secreted an aspartic proteinase (E .C . 3 .4 .23) with many similarities to the one extracted from B . cinerea-infected carrot tissue . This enzyme was not detected in healthy tissue . The enzyme from both culture and infected tissue had a pH optimum of 3 . 5 and a pl of 4.0 ; its action was inhibited by pepstatin and diazoacetyl-norleucine methyl ester, known inhibitors of aspartic proteinases, but not by compounds known to inhibit serine-, thiol- or metalloproteinases . The enzyme existed as a single unit with a molecular mass of 3839 kD on sodium dodecylsulphate polyacrylamide gels . It was present in ungerminated spores and was produced during germination before pectic enzyme components, both in culture and in host tissue . Cell death was detected in B . cinerea-infected carrot root tissue before pectic enzyme or necrosis-inducing glycoprotein or polysaccharide production, supporting the view that the aspartic proteinase may be primarily responsible for phytotoxicity .

INTRODUCTION

There have been many demonstrations of proteolytic activity in cultures of fungi pathogenic to plants and also in infected plants [11, 21-24, 26, 27] . However, few studies have provided direct evidence of a role for proteases in infection . In many cases it has not been established whether the enzyme detected in host tissue is of fungal origin, or whether it is produced by the host as a response to infection . Often, distinction has not been made between endopeptidase and exopeptidase activity . In this paper, the term proteinase has been used to indicate endopeptidase activity and the term protease to indicate enzymes which hydrolyse peptide bonds in general [I] . There are several reports of proteinase production by Botrytis cinerea Pers ex . Pers [4, 6, 15, 25-29, 31], but none of them are detailed and no attempt has been made to ascertain the possible role of the enzymes in the host-pathogen relationship . ZalewskaSobczak et al . [31] detected significantly higher levels of protease activity in apple tissue infected with a virulent isolate of B . cinerea than in apple tissue inoculated with an Abbreviations used in text : Apr, Aspartic proteinase ; DAN, diazoacetyl-norleucine methyl ester ; DIFP, di-isopropyl-fluorophosphate ; FPLC, Fast performance liquid chromatography ; PCMB, p-chloromercuribenzoate ; PMSF, phenylmethylsulphonyl fluoride ; PG, Polygalacturonase ; PGL, Polygalacturonate lyase ; PL, Pectin lyase ; PME, Pectin methyl esterase ; PMG, Polymethylgalacturonase . 0885-5765/90/040289+ 14 $03 .00/0

© 1990 Academic Press Limited



290

S . Movahedi and J . B . Heale

isolate of low virulence . The enzyme was also produced earlier than polysaccharidedegrading enzymes both in vitro and in vivo . A more comprehensive study [27] suggested that in vitro cultures of B . cinerea from apple produced an aspartic proteinase . This claim was based upon inhibition of the enzyme by diazoacetyl-norleucine methyl ester (DAN) and an acid pH optimum, but the effect of the more specific inhibitor of aspartic proteinases, pepstatin, was not tested . In view of the occurrence of proteins in plasma membranes, cell walls, and the middle lamellae of plant cells [13], proteolytic enzymes could be important in the primary states of cell damage and/or tissue maceration involved in penetration of the host and in the nutrition of the fungus during infection . These enzymes could also be important in regulating disease development by inactivating host enzymes or hostproduced proteinaceous inhibitors of fungal enzymes which are important in pathogenesis, or by releasing enzymes from inactive precursors . It is necessary to characterize fungal proteolytic enzymes fully before attempting to ascertain their role in pathogenesis and this paper reports the characterization of an aspartic proteinase from B . cinerea with respect to its responses to inhibitors and its enzymatic properties .

MATERIALS AND METHODS

Maintenance and culture of pathogen A single spore isolate, isolate 1, of Botrytis cinerea obtained from carrot was maintained by subculturing monthly on potato-carrot-agar to maintain virulence and grown once on glucose-peptone salts-agar (GPSA) to provide sporulating cultures [10] .

Growth of the fungus in liquid culture Preliminary experiments indicated that B . cinerea produced maximum proteinase activity when grown in a non-shaken liquid medium containing, per litre : 4 g glucose, 4 g gelatin, 0 . 75 g MgSO 4 . 7H,O, and 1 . 75 g KH 2 PO 4 . Spores were harvested from 10 to 12-day-old GPSA cultures by agitation with 15 ml 0 . 02 % Tween 80 solution and the suspension filtered through three layers of sterile muslin . The suspension was centrifuged at 950g for 8 min, the pellet resuspended in the liquid medium to give 1 x 108 spores ml - ' and the resulting suspension incubated in 100 ml lots in 200 ml Roux bottles in the dark at 24± 1 ° C for 48 h . After gentle shaking for 5 s, the resulting mass of germ-tubes and young mycelium was filtered through three layers of sterile muslin . The filtrate obtained was passed through 0 . 45 and 0. 22 µm Millipore filters to produce a sterile, cell-free culture fluid, and either stored at -20 °C, or used immediately .

Assay of protein and carbohydrate The Lowry method [14] was used to estimate protein concentration with BSA as the standard . During column chromatography, protein activity was located by absorbance at 280 nm . Carbohydrate was assayed by the anthrone method [5] using glucose as standard .



An aspartic proteinase secreted by Botrytis cinerea

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Assay of enzymic activity Protease activity was assayed spectrophotometrically by measuring the release of red dye from azocasein (3 "-),) at pH 5 . 0 and pH 8.0 . Aspartic proteinase (APr) activity was estimated by measuring the hydrolysis of a standard haemoglobin substrate by a method based on that of Anson [2] . Enzyme solution (0 . 2 ml) was added to 1 . 0 ml of acid-denatured haemoglobin (2'),() , w/v, in 0 . 2 M glycine-HCI buffer pH 3 . 5) and incubated at 30 ° C for 30 min . The reaction was stopped by the addition of '5 ml 5 .0 °-,, (w/v) trichloroacetic acid . The precipitate was then removed by centrifugation at 1000 g for 10 min and the supernatant was filtered through lens paper . Combined enzyme and substrate blank controls were prepared by adding TCA immediately after the enzyme . A sample of the supernatant (2 . 0 ml) was neutralized by the addition of I ml 0 . 5 M NaOH and I ml of this solution assayed for the presence of soluble peptides by the Lowry method [141 using tyrosine as a standard . One unit of enzyme activity was defined as that amount which released 1 µmol tyrosine equivalents min -r . Carboxypeptidase and aminopeptidase activity were measured by the methods of Nakadi et al . [17, 18] . The polysaccharide-degrading activity of enzyme extracts was assayed by determining the release of reducing sugar equivalents from araban, xylan and galactan (Koch Laboratories, Ltd .), carboxymethylcellulose and sodium polypectate (Sigma) and lime pectin (Bulmers Ltd .) . 0-4 ml of solution of the I °-,, of substrate was incubated with 0 . 4 ml buffer (0 . 1 M acetate buffer pH 5-0 or 0 . 1 M Tris-HCI pH 8 . 5) and 0-2 ml enzyme extract . After incubation for 30 min at 30 ° C, the mixture was assayed for reducing sugars by the Nelson-Somogyi method [19] . To determine whether the release of reducing groups from sodium polypectate or pectin was caused by hydrolase (PG or PMG) or lyase (PL or PGL) activity, the absorbance of reaction products at 240 nm was determined . Pectin methyl esterase (PME) activity was measured titrimetrically by the shift in pH of an unbuffered pectin substrate . The substrate (0 . 5 °-,', lime pectin) was adjusted to pH 5 . 0 before addition of enzyme . Phospholipase activity was determined by measuring the decrease in acyl ester concentration in a 1 °:,, aqueous emulsion of soybean lecithin (Sigma) at pH 5 . 0 and pH 8 . 0 . All assays were run in triplicate, and the three results were usually within ±2°„ of the mean value . Pun/cation o] enzymes Jrom culture filtrate Purification steps were all carried out at 4 ° C . Cell-free culture fluid of B . cinerea was concentrated ten times by lyophilization, adjusted to give 90°) ammonium sulphate solution and the precipitate collected . The precipitate was dissolved in 50 mm phosphate buffer pH 7 . 0, desalted on a PD-10 column (G-25, Pharmacia), concentrated ten-fold by lyophilization, dissolved in 1-7 M ammonium sulphate in 0-05 M phosphate buffer pH 7-0 and applied to a hydrophobic interaction chromatography column (Phenyl Superose HR 5/5, Pharmacia) run on an FPLC system (Pharmacia) . Proteinase was eluted from the column with a linear 1 . 7 M-0 M ammonium sulphate gradient . The fractions showing proteinase activity were



292

S . Movahedi and J . B . Heale

desalted, lyophilized, dissolved in glycine-HCl buffer pH 3 . 5 and applied to a gel filtration column (Superose-12, Pharmacia) run on FPLC . The same purification procedure was used in the case of extracts trom carrot tissue . Extraction of enzymes from carrot tissue

Carrots (cv . Chantenay Red-Cored) were grown as described previously [9] and stored at 2 °C until required . They were allowed to lose 8-10 11 0 fresh weight at room temperature in order to predispose them to infection [IOJ . They were then surfacesterilized in 1 °,, sodium hypochlorite solution for 1 min, rinsed three times in sterile distilled water, and cut into 0 . 5-0 •7 cm thick slices . Three slices were placed on moist filter paper in each sterile Petri dish in an illuminated double chamber system [8] . The slices were inoculated 1 h after slicing . Infected tissue, and healthy tissue from uninoculated carrots, was sliced, ground with fine acid-washed sand, and extracted with 50 mm phosphate buffer pH 5 . 6

(1 g tissue to 2 ml buffer), containing 1 mm dithiothreitol (to prevent oxidation), 0 . 1 M NaCl (to desorb proteins from cell walls) and 10",, (w/v) polyvinyl pyrrolidone (to absorb phenols) . The proteins in the supernatant after centrifugation for 10 min at 4 ° C (18000g) were precipitated by making up to 90% saturation with ammonium sulphate . The precipitate was redissolved in 50 mm phosphate buffer pH 7 . 0, desalted on a PD-10 column and lyophilized before further purification by hydrophobic interaction chromatography and gel filtration . Polyacrylamide gel electrophoresis

Freeze-dried proteinase preparations were dissolved in sample buffer [62 . 5 mm TrisHCI, pH 6. 8, 10%%, (v/v) sucrose, 2" ( , (v/v) 2-mercaptoethanol, 6 1'/0 (w/v) SDSJ, heated to 100 ° C for 4 min, cooled to ambient temperature and clarified by centrifugation . Samples were subjected to electrophoresis on a linear 10 % (w/v) acrylamide gradient in 0 . 375 M Tris-HCI, pH 8 . 8 . according to the method described by Harries [7] . A 2. 5 % acrylamide stacking gel was used in 0 . 125 M Tris-HCl, pH 6 . 8 . Gel slabs were fixed in 5 °,, methanol/ 10 0/0 glacial acetic acid and stained with 0 . 125 Coomassie blue R-250 . Destaining was achieved using 50 11,, methanol in 10 0 0 acetic acid . Isoelectric focusing

LKB Ampholine PAG plates, pH 3 . 0-9 . 5, were used . Gels were focused on an LKB multiphor for 1 . 5 h . An LKB 2971 power source was used and focusing was conducted at 30W, 1500 V and 50 mA . Samples were focused alongside standards (pH 3 . 5-9 . 3, Pharmacia) . Protein bands in the gels were stained with Coomassie blue R250 and proteolytically active bands were detected by layering gels onto a medium containing 3 0,0 (w/v) agar and 101/1 , ( w/v) gelatin in 0 . 05 M citrate phosphate buffer pH 4 .0 . pH and temperature characteristics

Haemoglobin (2 %, w/v) was prepared in 50 mm buffers as follows : glycine-HCl buffers were used for values less than pH 3 . 0, citrate-phosphate buffers for values between pH 3 . 0-7 . 0 and Tris-HCI buffers for pH values above 7 . 0 . Enzyme activity was expressed as a percentage of the maximum activity attained before assay . The pH stability was



An aspartic proteinase secreted by Botrytis cinerea

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determined by measuring haemoglobin hydrolysis at pH 3 . 5 after preincubation for 12 h at defined pH . The thermal tolerance of enzymes was examined by heating 0. 5 ml samples of enzyme solution at a specified temperature in thin-walled glass tubes for 10 min, then chilling to 0 ° C prior to measurement of activity at 30 ° C . Inhibition of proteinase activity Stock inhibitor solutions used were : phenylmethylsulphonyl fluoride (PMSF) and diisopropyl-fluorophosphate (DIFP) dissolved in propan-2-ol, p-chloromercuribenzoate (PCMB) dissolved in 10 mm NaOH, EDTA and o-phenanthroline in water, pepstatin

in 50°,,(v/v) aqueous methanol, and diazoacetyl-norleucine methyl ester (DAN1 in methanol . 1 mm cupric acetate was included in the DAN assay . The inhibitor solutions were diluted with assay buffer and added to enzyme solution (1 :1) at a final concentration of' I mm . The mixtures were incubated at 30 °C for 30 min and enzyme activity was measured relative to an uninhibited control . Controls without inhibitor, but containing solvents used to dissolve inhibitors, were included ; enzyme activity was not affected . In all studies of inhibition of enzyme activity, replicate experiments agreed within ±3 of enzyme activity . Estimation of cell viability

A 0 . 1 °--,, aqueous solution of Evans Blue

(Gurr) was applied to sections (200 ltm thickness, 3-4 cell layers thick) cut vertically downwards from the upper, treated surface of the slices on microscope slides and 10 min later, coverslips were added . Observations were made 2-3 h later [9] . Twenty four sections were assessed for cell death per treatment . Cell death was defined as the ability of cells to take up this stain and one cell layer was defined as being killed when more than 50 ° , of the cells in that layer were stained . Phytotoxic activity Extracts from B . cinerea-infected carrot root slices were prepared at 2 h intervals after inoculation . Proteins in the extracts were precipitated by taking to 90 0 0 saturation with ammonium sulphate . The precipitate and the supernatant were separately redissolved in 50 mm phosphate buffer pH 7 . 0, desalted on a PD-10 column and lyophilized before further purification by gel filtration chromatography . The carbohydrate and protein contents of fractions exhibiting phytotoxic activity were determined and fractions were tentatively described as proteinaceous, carbohydrate or glycoprotein according to their protein and carbohydrate composition and the extent of their sensitivity to protein and carbohydrate denaturing treatments as described previously [3] . The presence of glycoprotein in phytotoxin fractions was confirmed by the method of Zacharius et al . [30] . The extracts were also assayed for various enzymic activities as described above . RESULTS Proteinase activity in cultures and from infected carrot tissue Preliminary results using casein, bovine serum albumin, peptone and gelatin as substrates indicated that B. cinerea produced maximum proteolytic activity in culture



294

S . Movahedi and J . B . Heale

I I I I I 24 48 72 Culture age (h)

1

96

FIC . .1 Time course of production of proteinase by B. cinerea in culture . The pathogen was grown in a medium containing 0 - 4' ) ,, gelatin as a protein source . Proteinase activity in the culture filtrate was estimated by measuring the release of tyrosine equivalents using the Lowry assay .

0

12 24 36 48 60 Time after inoculation of carrot tissue (h)

Fic . 2 . 'Time course of the production of proteinase in B. cinerea-inoculated carrot tissue . Proteinase activity was assayed as described in Fig . 1 .

when grown on a medium supplemented with 0 . 4 °,%, gelatin . Maximum activity in this medium occurred after 2 days growth at 24 °C (Fig. 1) . Proteinase was detected in distilled water or Tween 80 leachates of spore suspensions before any morphological changes associated with germination were detected . A suspension of 1 x 10 6 spores ml - ' yielded an average of 0-1-0-2 units proteinase ml -1 . From day four on, no activity was detected in the culture medium . The production of proteinase in infected host tissue correlated well with that in culture, with peak activity 48 h after inoculation (Fig . 2) . Soft water-soaked lesions were apparent on the surface of carrot slices by 24 h after inoculation and by 48 h after inoculation the whole surface of slices was soft and macerated with some grey mycelium being visible .



An aspartic proteinase secreted by

295

Botrytis cinerea

Peak I

0-16I •5 i

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Elution volume (mL)

Fie . 3 . Purification of aspartic proteinase from culture filtrates of B. cinerea on Phenyl-Superose see Materials & Methods for details) . The protein (A 280. proteinase activity (-S-S-~-) and the (NH4 ) 2 SO 4 gradient (----) are plotted against elution volume . Fractions of 0 .5 ml (0. 1 ml at protein peaks) were collected and assayed for enzymic activity . Proteinase activity was eluted in peak 1 .

1-20

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Elution volume (mL) Purification of the aspartic proteinase fractions from culture filtrate of B. cinerea on Superose-12 gel filtration column . Protein concentration (A280 - -) and proteinase activity -~-~-) are plotted against the elution volume . [This step in purification followed hydrophobic interaction chromatography on a Phenyl-Superose column (Fig . 3) .] FIG . 4 .



296

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o/ B . cinerea

aid from B .

cinerea

in/ecled carrel

lissae

Step in purification

(a) Culture filtrates Specific activity (units mg- ' ;

Yield

Purification factor

1000 74. 1 62. 2 51 . 4

1.0 2. 1 5. 5 8. 6

Crude culture filtrate (NH 4 ) 2 SO 4 ppt . HIC fraction' Gel filtration fraction'

0 . 36 0 . 75 1 .97 3 . 10

Step in purification

(b) Infected carrot tissue" Specific activity Yield (units mg - ') (°-(,)

Purification factor

Crude tissue extract (NH 4 ) 2 SO 4 ppt . HIC fraction" Gel filtration fraction'

0 . 20 0 . 24 1 . 37 1 . 63

1 .0 1 .2 69 8.2

1000 77 . 6 62 - 1 49 . 6

'Culture filtrate of isolate 1 of B . cinerea grown in a medium containing 0-4'),1(, gelatin for 2 days at 24± 1 °C . 'Extracts from B. cinerea-infected carrot tissue, 2 days after inoculation . "The fraction obtained after hydrophobic interaction chromatography on Phenyl-Superose (Fig . 3) . 'The fraction obtained after gel filtration on Superose-12 (Fig . 4) .

Purification of the proteinase

Typical elution patterns of the proteinase from the Phenyl-Superose and Superose- 12 columns are illustrated in Figs 3 and 4 . The protein content and the activities of the proteinase at various stages of the purification procedure are given in Table 1 . The proteinase appeared as a single symmetrical peak during gel filtration and one peak during hydrophobic interaction chromatography, indicating that it was probably a single enzyme . The purified proteinase preparation did not contain detectable carboxy- or amino-peptidase, cellulase, phospholipase, xylanase, arabinase, galactanase, pectin methyl esterase, pectin lyase, polygalacturonate lyase, polymethylgalacturonase or polygalacturonase activities . Identification of the B . cinerea proteinase in diseased carrots Proteinase activity from infected carrot tissue co-eluted with the B . cinerea proteinase from culture on the Superose-12 gel filtration column and was assumed therefore to have been produced by the fungus . The pH values for maximum activity of proteinase from infected carrots were very similar to those obtained from enzyme produced in mineral-salts gelatin culture (Fig . 5) . Extracts from uninfected carrots showed no proteolytic activity . The acid pH optimum of the extracted and purified proteinase coincided with the change in pH that occurred during infection, from about 6 . 0 in healthy carrot tissue to about pH 3 . 5 in diseased tissue, 3 days after inoculation .



An aspartic proteinase secreted by Botrytis cinerea

297

(b)

(a) „ 100 100 •

c 80 \ E • E 60\ a \ • o \ \ ô 40 c \ \-° 20• • â 0\ 0 IIIA I I I I I , I i 9 .+1, +_J 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 pH pH FH; . 5 . The effect of pH on hydrolysis of haemoglobin by the purified aspartic proteinase from B . cinerea . (a) proteinase purified from culture filtrates of B . cinerea ; (b) proteinase purified from extracts of B . cinerea-infected carrot root tissue . \

E E 80 E 0 60 40 ô 20 â 0

100 •-•-•-- \ 80 E 60 .nÉ 0 wrn 40 0 c U a-v 20 0 I i I i _j_620 30 40 50 60 Temperature (C) Fto .6. Temperature stability of aspartic proteinase purified from culture filtrate of B. cinerea . The stability was examined by incubating the enzyme solutions at different temperatures for 10 min. The activity remaining after the treatment was assayed under standard conditions . A final piece of evidence that the acid proteinase produced in culture and in infected carrots was probably the same enzyme was the similarity in the two isoelectric points obtained, both being between 4 .0 and 4 .2 . Estimation of molecular mass Purified proteinase was resolved on SDS-polyacrylamide gels . This revealed a single component of molecular mass 38-39 kDa . The elution time of the proteinase from the gel filtration column was compared with proteins of known molecular mass (cytochrome c : 12 .4 kDa ; a-lactalbumin : 14 .2 kDa ; chymotrypsinogen A : 25 kDa ; carbonic anhydrase : 29 kDa ; pepsin : 34 .7 kDa ; egg albumin : 45 kDa ; BSA : 66 kDa) . Four independent analyses gave an estimate of



S . Movahedi and J . B . Heale

298 (ABLE

2

h flee/ si iuhihaw % ou 13 . ci

uerca proteinase

Activity renrtiuing alter inhibitor treatment iunits ml -t +SD )

Inhibitor' Control DAN Pepstatin EDTA o-phenanthroline I'MSF DI FP Iodoacetamide PCMB

activity remaining relative to control

Specific for type of proteinase

Cul turc filtrate enzyme"

Inletted carrot enzyme''

Culture filtrate enzyme"

infected carrot cnzyme''

Aspartic Aspartic MetalloMetalloSerine Serine Cysteine Cysteine

5 .00+0120 0 . 75±0 . 015 0 5. 11+0 . 097 4. 92±0 . 090 4 .82+ _ 0096 4. 65+0081 5-01+0020 4-69+0 .014

5 . 00+0 . 075 0. 85±0 . 010 0 5. 02+0 . 091 4. 85±0 . 081 4 . 91+0 . 052 4. 75+0 .091 4-90+0 .015 4. 78+0 .062

100 15 0 102 98 96 93 100 94

100 17 0 100 97 98 95 98 96

"DAN -diazoacetyl-norleucine methyl ester ; PMSF - phenvlmethylsulphonyl fluoride ; DIFP-di-isopropyl-fluorophosphate ; PCMB -p-chloromercuribenzoate . "Aspartic proteinase purified from culture filtrates of B . cinerea . 'Aspartic proteinase purified from B . cinerea-infected carrot root tissue . The enzyme/inhibitor solutions (final concentration I mm? were incubated at 30 'C for 30 min and the enzyme activity was measured relative to an uninhibited control . The proteinase activity of the control was 5 units ml - '. Results are the mean of three replicate readings . There was no significant difference between inhibitor data of proteinase preparations from either source (P < 0 . 001, t-test} .

40 kDa for the molecular mass of' the proteinase . This value agreed well with that estimated by SDS-PAGE and indicated that the enzyme is a monomer in its native state .

pH and temperature characteristics The proteinase had a pH optimum of 3 . 5 (Fig . 5) ; it was relatively stable over a wide range of pH, although it was rapidly inactivated above pH 7 . 0 and at values less than pH 2 . 0 . The temperature stability was analysed at pH 3 . 0 over a temperature range from 20 ° to 100 ° C . The proteinase was completely denatured by heating for 10 min at 70 ° C (Fig . 6) . It had optimal activity against acid denatured haemoglobin at pH 3 .5 and 40 ° C and still expressed 10-15 °;,, of its optimum activity at 0 °C .

Inhibition of activity The effects of eight selected inhibitors on the proteinase activity were tested . The experiments were normally repeated two or three times using independently purified preparations of the proteinase from extracts of infected host or culture filtrates ; representative results are presented in Table 2 . Iodoacetamide did not inhibit proteinase activity while PCMB was only slightly inhibitory . Thus, thiol, groups do not



An aspartic proteinase secreted by Botrytis cinerea

299

1 i i 12 24 36 Time after inoculation of carrot tissue (h) FIG . 7 . Time course of cell death, assayed using Evans blue, in carrot tissue inoculated with B .

cinerea . Bars indicate standard deviation for the number of cell layers killed above that observed in controls. Arrows indicate the first instance of detection of polygalacturonase (PG), pectin lyase (PL), pectin methyl esterase (PME), polysaccharide (PS) and glycoprotein (GP) components . Aspartic proteinase (APr) was detected at time zero as a result of enzyme diffusing from ungerminated spores . No proteinase activity was detected in healthy carrot tissue .

appear to be necessary for activity . Phenanthroline and EDTA, inhibitors of metalloproteinases, had no effect on the hydrolysis of haemoglobin at pH 3 . 5 and neither did PMSF and DIFP, inhibitors of serine proteinases . Pepstatin and DAN, specific inhibitors of aspartic proteinases, caused 100% and 85% inhibition of the B . cinerea proteinase activity respectively . Therefore, the proteinase should be defined as an aspartic proteinase (APr) .

Time offirst detectable activity of necrosis-inducing components in infected carrot tissue

Numbers of dead cells in infected carrot slices greater than that occurring in the uninoculated control slices were first observed 3 h after inoculation with spore suspensions . An average of two additional cell layers were dead at this stage (Fig . 7) . Only the proteinase-containing fraction from such carrot root extracts prepared up to 8 h after inoculation expressed such phytotoxic activity . Pectin lyase activity was not detected until 10 h after inoculation, and the phototoxic glycoprotein and polysaccharide components previously reported [3] were not detected in carrot root tissue in the present study until at least 18 h after inoculation (Fig . 7) . Polygalacturonase and pectin methyl esterase activities were not detected here until at least 8 and 12 h respectively after inoculation . MPP 36



300

S . Movahedi and J . B . Heale

DISCUSSION

In this investigation, an extracellular proteinase, secreted by an isolate (isolate 1) of B . cinerea growing in a liquid medium containing gelatin, has been purified to homogeneity by hydrophobic interaction chromatography and gel filtration and shown to have a molecular mass of 40 kDa, a pH optimum of 3 . 5 and an isoelectric point of 4 . 0 . Urbanek & Kaczmarek [27] purified an aspartic proteinase (APr) from culture filtrates of an isolate of B. cinerea obtained from apples which had a pH optimum of 2 . 5-3 . 0 and was inactivated by DAN, a somewhat less specific inhibitor of APr than pepstatin which was utilized in this investigation . The proteinase we have isolated from Botrytis-infected carrot tissue co-eluted with the enzyme produced in culture from a gel filtration column, had similar optimum pH, pI and sensitivity to inhibitors . No proteinase activity could be detected in healthy carrot tissue and inhibitor studies indicated that the enzymes detected in vitro and in planta are both aspartic proteinases (APrs) . These results strongly suggest that APr activity in diseased tissue is of fungal origin . Fungal APs have been reported to have molecular masses in the range 30-45 kDa and isoelectric point below 5 . 1 [20] . These values are consistent with our observations . The isolate of the pathogen we used (isolate 1) apparently secreted only one type of proteinase into the medium . It is possible that variability exists between different isolates of B . cinerea with respect to proteinase activity both in terms of number and types of proteinase secreted . The isolate of B . cinerea used by Shepard & Pitt [25] secreted four uncharacterized proteinases, with isoelectric points of 2 . 5, 4. 5, 8 . 4 and 12 . 4, into the culture medium . These enzymes were closely associated with three phospholipase enzymes . The isoelectric points of the first two proteinases suggest that they may also be of the APr type . Tseng & Lee [26] reported that protease activity produced by an isolate of B . cinerea had an optimum pH of 8 . 5 . Since their investigation was carried out using crude culture extracts, it is not known if 'a single enzyme was involved . Urbanek & Kaczmarek [27] also detected a carboxypeptidase in culture filtrates from 3-day-old cultures of B . cinerea with a pH optimum of 4 . 7-5 . 0 . An investigation of the range and nature of proteolytic enzymes produced (in vitro and in planta) by different isolates of this pathogen and a possible correlation between proteinase activity and virulence could shed further light on the role of this enzyme in B . cinerea host interactions . The precise functions of proteolytic enzymes in pathogenesis is not yet clear . It has been suggested that they might facilitate pathogen spread in host cells by inactivating host proteins related to defence such as enzymes and proteinaceous inhibitors . The optimal proteinase enzyme activity of the pathogen on gelatin, a hydroxyproline-rich protein, indicates that the proteinase is capable of degrading extensin and related host cell wall proteins . It would be interesting to determine whether the purified APr from B . cinerea can degrade carrot extensin proteins . B . cinerea is an important post-harvest pathogen and frequently infects fruits and vegetables in cold-stores . The purified Apr retained 10-15 % of optimum activity at 0 °C and so at cold-store temperatures it could contribute significantly to necrosis caused by this pathogen . High activity of APR in B . cinerea-infected carrot tissue and a pH activity range for



An aspartic proteinase secreted by Botrytis cinerea

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the enzyme that encompasses values encountered in the diseased host point to a role in pathogenesis . Further, development of APr activity in infected tissues coincided with rates of host cell death and occurred before that of other cell wall degrading enzymes and the necrosis-inducing, polysaccharide or glycoprotein components reported earlier [3], suggesting that it may be involved in the primary stage of infection . The apparent absence of aspartic proteinase inhibitors in the plant kingdom [12] is a major factor which would allow unimpeded activity in the host . An accompanying paper [16] presents results of a more comprehensive study of the roles of the APr and pectic enzymes produced by B. cinerea during the infection process, employing six different plant hosts and eight isolates of the pathogen .

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