Occurrence and specificity of an endopolygalacturonase inhibitor in Pisum sativum

Physiological

Plant Pathology

(1984)

24,4%59

Occurrence and specificity of an endopolygalacturonase inhibitor in Pisum sativum R. M. HOPFMAN~and J. G. TURNER: School of Biological (Accepted

Sciences, University

of East Anglia, Norwich

NR4 7TJ,

Norfolk,

UK

for publication September 1983)

The endopolygalacturonase (endo-PC) inhibitor from Pisum sat&m (pea) was characterized in terms of its effect on PGs from five pathotypes of Arcocihyta pisi and seven other species of fungi and its occurrence in 14 pea varieties and species from three other genera. Eachpathotypc of Axochyta pisi produced both an exo- and endo-PG and, in each case, the latter enzyme only was affected by the endo-PG inhibitor. Although each of the three other pea pathogens examined here produced an endo-PG, none of these enzymes was sensitive to the endo-PG inhibitor. Of the remaining four fungi, only Phoma sol& produced exclusively exo-PG and Colletotrichum lindcmuthianum and Aspergillus niger produced endo-PGs that were sensitive to the endo-PG inhibitor from pea. The endo-PG inhibitor occurred at highest concentration in the stipules and leaflets of the pea, and more than 75% was recovered in a soluble form in extracts of these organs made in 50 rnM sodium acetate buffer (pH 5.0). Inhibitor was detected in each pea variety tested, although the yield varied considerably. Inhibitor of Axochytapisi endo-PG was similarly extracted from hypocotyls of Phaseolus vulgaris (bean), but was not detected in extracts of cucumber and cabbage seedlings. These results indicate that the endo-PG inhibitor is not directly responsible for the differential resistance of pea varieties to Ascochyta pisi pathovars. In addition, they provide further evidence ofsimilarities between the endo-PGs of C. lindemuthianum and Ascochyta pisi and between the endo-PG inhibitors from pea and bean.

INTRODUCTION

Proteins that inhibit fungal polygalacturonase (PG) have been extracted from the hypocotyl of Phaseolus vulgaris (bean) [I, II], d iseased stone fruits [9], and various vegetables including onion, paprika, white cabbage, cucumber [.5], sweet potato [27] and leaflets of Pisum sativum (pea) [23]. The PG inhibitors from bean hypocotyls and pea leaflets have been studied in some detail and they appear to be quite similar; for example, they are both proteins, molecular weight = 42 000 and inhibit the endo-PG but not the exo-PG from the pathogens of bean and pea, respectively, Colletotrichum lindemuthianum causal agent of the bean anthracnose and Ascochyta+i, causal agent of leaf and pod spot of the cultivated pea. One important difference, however, is that the PG inhibitor from bean hypocotyls is reported to be cell wall-bound whereas between 70 and 90% of that from pea leaflets is soluble in tissue homogenates [I, 131. Not all endo-PGs are equally sensitive to the PG inhibitors. For example, endoPG from Ceratocystti jmbriata, a pathogen of sweet potato, was inhibited four times more strongly by a crude PG inhibitor preparation from sweet potato than the endo-PG tPresent address: Queen Elizabeth College, University :To whom requests for reprints should be sent. 0@%4059/84/010049

+ 11 $03.00/O

of London, 0

London

1984 Academic

WE 7AH. Press Inc.

(London)

Limited

50

R. M. Hoffman

and J. G. Turner

of Fusarium oxysporum f. sp. cubense or a commercial pectinase [17]. Similarly, the endo-PG from C. lindemuthianum was more sensitive to the PG inhibitor from bean hypocotyls than were the endo-PGs from a number of other fungi [I]. The endo-PG inhibitor from bean did not appear to be a determinant of specificity in the hostpathogen interaction, however, because each PG inhibitor preparation from three differential varieties of bean was equally able to inhibit purified endo-PG from the three races of C. lindemuthianum [2]. Anderson & Powelson [3] found that Phoma medicqinis fsp. pinodella “pectinase” was not inhibited by an extract of Austrian winter peas. However, we have previously reported the inhibition of “pectinase” from Ascochyta pisi by the PG inhibitor from the pea variety Small Sieve Freezer [13]. This raises the interesting possibility that either the Phoma medicaginis enzyme is insensitive to the inhibitor, or there is variation in the amount and specificity of the PG inhibitor in different pea cultivars. This problem was investigated in the present study which was intended to further characterize the PG inhibitor from pea in an examination of its activity towards PGs from a variety of fungi and its concentration in different pea cultivars and in other plants.

MATERIALS

AND

METHODS

Plants

Seeds ofPisum sat&m L. (pea) cv. Small Sieve Freezer were a gift from the Processors and Growers Research Organisation, Peterborough, England. Other pea varieties were generously given by Dr P. Matthews, John Innes Institute, Norwich, England. Other plants were purchased as seed from local seed merchants. All plants were raised from seed in 5 in (12.5 cm) pots containing Levingtons potting compost, and grown in a heated greenhouse (minimum temperature 20°C) receiving supplementary illumination ( 16 h, sodium lamp). Fungi

Representative cultures of the five pathotypes of Ascochyta pisi Lib. were kindly supplied by Mr P. Darby, University of East Anglia. Mycosphaerellapinodes (Berk. and Blox.) Vestergr. was isolated from an infected plant in a local crop of field peas. A culture of Phoma (=Corticum) solani (Prillieux & Delacroix) Bourdot & Galzin was obtained from Dr T. Musa, University of East Anglia. All other fungi were purchased from the Commonwealth Mycological Institute, Kew, England. Cultures of AphanomyceseuteichesDrechsler (I.M.I. 170485ii), A scochytapisi, Aspergillus niger Van Tiegham, C. lindemuthianum (Saccando & Magnus) Briosi & Cavara (I.M.I. 86740), M. pinodes and Phoma medicaginis var. pinodella (L. K. Jones) Baerema (I.M.I. 135520) were subcultured on a modified Coons agar medium [S]. Fusarium oxysporum f.sp. lycopersici (Saccardo) Snyder & Hansen (I.M.I. 173520) was subcultured on potato dextrose agar and Phoma solani was subcultured on prune dextrose agar. Ascochyta pisi and Phoma solani were grown at 15 “C; all other fungi were grown at 25 “C. Polygalacturonase

assay

Polygalacturonase was assayed using a modified Nelson-Somogyi method to determine reducing sugar [16] released from polygalacturonic acid (PGA) (Sigma, 048% w/v)

Endopolygalacturonase

inhibitor

51

in Pisum setivum

in sodium acetate buffer (pH 5.2, 40m~). The reaction mixture (1 ml) was incubated at 30°C and the reaction terminated by addition of an equal volume of the alkaline copper reagent before 5% of the glycosidic bonds in the substrate were hydrolysed. Reducing sugars were determined from a standard curve prepared using galacturonic acid. Activity of PG is expressed as units where 1 unit is the amount of enzyme releasing 1 pm01 galacturonic acid equivalents in 1 h under standard conditions. Percent inhibition (I%) of PG was calculated according to the equation : I(:/,) =(l

-SJlOO

where S, is the percent of glycosidic bonds in the substrate that were hydrolysed by PG in the presence ofinhibitor during t, the time taken for 1y0 of the glycosidic bonds to be hydrolysed in the controls receiving no inhibitor. Each value of S, was estimated from a plot ofS against time; the values for S were calculated from measurements of reducing sugar in the reaction mixture. From similar measurements we calculated that the mean chain length of our PGA was >30 residues. Percent inhibition of PG was also calculated from measurements of viscosity using the equation:

I( y/J = (usI- 5)

100

100 - UC1 where ust is the relative viscosity of substrate that has been hydrolysed by PG in the presence of inhibitor during t, the time taken for the hydrolysis of 176 of glycosidic bonds of the substrate in controls receiving no inhibitor; vcl is the relative viscosity of this control at time t. Each value for us1and ucl was estimated, respectively, by plotting usand UCagainst time. Viscosimetric measurements were made as described previously [Z3]. The PG activity was further characterized by the value H (Q,,) which we define here as the percent of glycosidic bonds hydrolysed when the viscosity was reduced by 50%. We have taken a value for H (u& <3 as indicative of an endo-PG and H ( v6,J >20 of an exo-PG [4]. Polygalacturonase

inhibitor

assay

Polygalacturonase inhibitor was assayed as described previously [Z3] using a partially purified endo-PG from Ascochytapisi race 3 (referred to now as pathotype 4, according to Darby [7]) except where indicated otherwise. One unit of PG inhibitor is the amount required to reduce by 50% the activity of the endo-PG (10 units ml-r) under standard conditions. Production andpartialpur@ation of Ascochyta pisi PG Ascochyta pisi PG was produced and partially purified

as described previously [23] using diethylaminoethyl-cellulose (DEAE-cellulose) column (DE 32, Whatman, 2.6 x 10 cm) and carboxymethyl-cellulose (CM-cellulose) column (CM 32, Whatman, 1.4 x 6 cm) chromatography. of PG by di$erent&ngi Conical flasks (250 ml) containing citrus pectin (Sigma, 0.2% w/v) in mineral salts

Production

52

R. M. Hoffman

and J. G. Turner

medium [Z3] (100 ml, pH 5*5), were each inoculated with 10 discs (7 mm diameter) cut from the growing edge of mycelial cultures of the various fungi. These cultures were incubated at 25 “C on an orbital shaker ( 100 r min-l) and samples were removed at intervals and assayed for PG. When the PG activity in a flask appeared to approach a maximum the cultures were harvested and the mycelium removed by filtration on asintered glass disc. The Fusariutn culture filtrates were also passed through a Millipore filter (O-22 pm) to remove the microspores. These culture filtrates were used in PG assays. of PG inhibitor frompea and other plants Mature pea plants were harvested at the pod-filling stage, before leaf senescence became detectable, and separated into leaflets, stipules, stem, pericarp of the fruit and seed. A cold mortar and pestle was used to cornminute samples of each organ in acetate buffer [pH 5.2, 50mM, 1 ml g-l fresh wt] containing ascorbic acid ( 1 mM) and insoluble polyvinylpyrollidone (PolyClar AT, BDH, approx. 2% w/v). The homogenate was centrifuged (13 OOOg, 2 min at room temperature) and 1 ml of the supematant wasdesalted by gel filtration on a Sephadex G-50 column (1.4 x 6 cm) equilibrated in acetate buffer (pH 5.2, 50 mM). Crude PG inhibitor emerged in the fraction (2 ml) which was collected after the void volume of the column had eluted, and this was used directly in enzyme assays. The partially purified PG inhibitor used in some experiments was prepared as described previously [13] by loading the supernatant, produced by centrifuging (40 000 g, 10 min) the crude homogenate, onto a CM-cellulose column (CM 32, Whatman, 1.4 x 6 cm) equilibrated in acetate buffer (pH 5.2, 10 mru). The chromatogram was developed in a linear gradient of acetate buffer (10-500 mM) and the PG inhibitor, which eluted in 50 rnru buffer, was collected and used in enzyme assays. Phaseolus vulgaris L. cvs Canadian Wonder and Kievitsboon Koekoek were harvested 16 days after germination. Bras&a oleracea L. (cabbage) cv. Golden Acre and Cucurnis cueurbitae L. (cucumber) cv. Telegraph Improved were harvested 29 days after germination. The first true leaves and hypocotyls of each plant were collected, except for B. oleracea where only leaves were available. Fractions were prepared using a procedure similar to that described above for the crude PG inhibitor from peas, except that the supernatant of the homogenate was frozen, thawed and centrifuged again (13 OOOg, 2 min, room temperature) before the supematant was desalted on a Sephadex G-50 column (1.4 x 6 cm). This freeze-thaw cycle precipitated a green fraction which was particularly evident in the first supematant of the bean leaf homogenate, but did not affect recovery of the PG inhibitor. All work involving extraction and purification of enzymes and inhibitors was done at 4 “C.

Extraction

RESULTS Sensitivity of PCs from Ascochyta pisi pathotypes to the PC inhibitor from pea LeaJets Polygalacturonase in culture filtrates of Ascothytu pisi pathotype 2 separated into two

fractions during CM-cellulose chromatography (Fig. 1). Viscosimetric assays revealed that the enzyme in fractions 10 to 13 was an exo-PG and the enzyme in fractions 26

Endopolygalacturonase

inhibitor

in Pisum safivum

53

60 50

0

5

IO

15

20

Fraction

25

30

35

0

400

number

FIG. 1. Separation

of PCs of Ascochytn pisi pathotype 2 by CM-cellulose chromatography in acetate buffer at pH 5.2. Polygalacturonase that had bound to the column was eluted in a linear gradient ofincreasing acetate buffer molarity (- -). Each fraction was assayed for protein (0) and for PG (a). TABLE

1

of the PC inhibitor frompea leoJets on PCs from d$mzt

Effect

Inhibition

Fungus

H( so)

Pea pathogens Mycosphaerelln pinodes Phoma mrdicaginis var. pinodclla Aphanomyces eutiches

5.6 3.4 7.4

Other plant pathogens Phomo solani Fusatium oxysporum hp.

lindemulhianum

Non pathogen Aspergillus nigcr

=PCs in culture filtrates were 44 units of the endo-PC inhibitor

Reducing sugar assay

25 -13

5‘ --10 20

0

38

3.5 3.3

5 96

ND 70

1.2

84

ND

to similar activity and assayed in the presence leaflets of pea cv. Small Sieve Freezer.

adjusted

from

ofPG’(%)

15

>30

lycopcrsici

Collelotrichum

Viscosimetric assay

sources

of

to 32 was an endo-PG. This elution profile was therefore very similar to that previously reported for the PC of Ascochytu pisi race 3 (pathotype 4) [13]. Essentially similar results were reproduced during the partial purification of PGs from the other A. pisi pathotypes, 1, 3 and 5. However, the ratio of endo-PG to exo-PG varied between 1.3 : 1 for pathotype 4 to 7 : 1 for pathotype 3. The endo-PGs from all pathotypes were equally sensitive to the PG inhibitor whereas all the exo-PGs were unaffected.

54

R. M. Hoffman TABLE Effect

of the PC

inhibitor

from pea

1eaJlets on

medicaginis

and J. G. Turner

2

endo-PGspartially

pur$ed from cullurefillrates and Ascochyta pisi

uar. pinodella

Enzyme

Inhibitiona

f4%,)

Phoma medicaginis endo-PG-Ab Phoma medicaginis endo-PG-BC Ascochytapisi endo-PG

3.05

of Phoma

(%)

8.6 -3.5

1.55 1.00

100

aDetermined in the presence of44 units of PG inhibitor. bBound to DEAE-cellulose column in acetate buffer (pH 5.2, 5 mM); eluted at 100 mM in buffer gradient (10-500 m*l). CBound to CM-cellulose column in acetate buffer (pH 5.2, 10 mM); eluted at 50m~ in buffer gradient (IO-500 mM). TABLE

of the

Concentration

Organ Seed Pericarp Stem Leaflet Stipule

aPea cv. Small Sieve Freezer. bMean of two determinations; determinations.

Sensitivity of PGs from d@erentfungi

units

3

PC inhibitor

in peaa organs

Inhibitor g-1 fresh wt

111 123 220 373

Relative concentrations

38b &32 +21 &51 &83

values

from

1-O 2.9 3.2 5.8 9.8

other

organs

to the PG-inhibitor from

are

mean

kS.D.

of three

pea leajets

Polygalacturonase in the culture filtrates of different fungi grown on citrus pectin as carbon source was partially characterized by determining the percent hydrolysis of glycosidic bonds when the substrate viscosity was reduced by 50%, referred to here as the H(uSO) value (Table 1). These values varied between 1.2 for PG in Aspergillus niger culture filtrates to more than 30 for the PG in Phoma solani culture filtrates. We conclude therefore that PG activity in culture filtrates of the remaining five fungi was due to mixtures of endo-PG and exo-PG in differing proportions. Of the pea pathogens listed in Table 1, none apparently produced an endo-PG that was significantly inhibited by the partially purified PG inhibitor from pea leaflets. By contrast, we have previously shown that PG in similar culture filtrates of Ascochyta pisi was 47% inhibited by a saturating titre of the pea PG inhibitor [13]. This value is comparable with figures in Table 1 for the inhibition of PG determined with the aid of the reducing sugar assay. Amongst the other fungi, C. lindemuthianum and Aspergillus niger alone produced an endo-PG that was significantly inhibited by pea PG inhibitor.

Endopolygalacturonase

inhibitor

in Pisum sativum TABLE

Concentra&

of PG inhibitor

JIIa

Variety

Rondo Dwarf Sugar Dik Tram

Host

198 691 406 iai 193 241 403 395 228 250

Small Sieve Freezer Keeran pea Pisum aroense Pisum humuli

Frazer Vitalis Piszm aroense Pisum jomardii

Inhibit& units g-l fresh wt

reactionb 1 I 1 1 1 2 2 3 4 4 4 5 5 5

423

Pisum elatius Pisum abyssinicum

of d$erent pea varieties

in lea&s

1097 502 461

Pisum aroense

55

4

56 588 625 <40

800 253 908 1250 555 334 208 90 392 58

aLine number in the pea collection ofthe John Innes Institute, Norwich. bHost reaction to ,4wxcochyta pisi pathotype 2 [7]; reaction types are graded (hypersensitive) to 5 (tilly susceptible). CInhibitor was determined using endo-PG from Ascochytapisi pathotype 2.

from

1

TABLET Quantitation

of inhibitorfrom

pea varieties using endo PC

from Ascochyta

pisi and Aspergillus

niger

PC inhibitor units gm’ fresh wt Variety Pisum humuli Pisum arcease

Rondo Dwarf

Sugar

Pisum elatius

Small Sieve Freezer Keeran pea

JIIa

Ascochytapi&

241 228 502 461 198 406 181

=Line number in the pea collection bRati of PG inhibitor determined using Ascochytapisi endo-PG. CInhibitor assayed using endo-PG

Aspergillus

335 392 586 624 a00 906 1250

ofthe John Innes using Aspergillus from

Ascochytapisi,

niger

Ratiob

162 186 356 302 400 38.5 356

Institute, Norwich. niger endo-PG to that pathotype

0.48 0.47 0.61 0.48 0.50 0.42 0.28

determined

4.

The PG-inhibitor used in these studies was assayed during the various stages in its partial purification by its effect on a partially purified endo-PG from Ascochytapisi. It is unlikely that during this purification we missed other PG inhibitors because we have found that the PG inhibitor in crude pea leaflet homogenates exhibits exactly the same specificity as the partially purified PG inhibitor: neither inhibited PG in filtrates of

56

R. M. Hoffman

and J. G. Turner

cultures ofPhoma solani, F. oxysponrm Esp. lyc~persiciand M. Pinodes and both inhibited endo-PG ofAscochyta+-i and Aspergillus niger. These observations support the idea that the only type of PG-inhibitor in pea leaflets is, in fact, the one that we have partially purified [23]. The results in Table 1 are from experiments with crude enzyme preparations. An attempt was made to purify partially endo-PG from Phoma medicaginis in order to establish whether or not its insensitivity to the PG inhibitor was a property of the enzyme itself Two endo-PGs from Phoma medicaginis were identified (Table 2). One (endo-PG-A) bound to the DEAE-cellulose at pH5.2, whereas the other (endoPG-B) was retained only on the CM-cellulose at this pH. Both endo-PGs eluted as a single peak during development of the respective chromatograms in a gradient of acetate buffer ( 10-500 mr.4). Both endo-PGs from Phoma medicaginis were essentially insensitive to the PG inhibitor in assay conditions which gave lOOo/oinhibition of the Ascochyta pisi endo-PG (Table 2). Quantitative determination in other genera

of the Ascochyta pisi endo-PG inhibitor

in di$erent pea varieties and

There was wide variation in the distribution of inhibitor of endo-PG from Ascochyta 4 between the different organs of the pea cultivar Small Sieve Freezer (Table 3). Highest concentrations occurred in leaflets and in stipules, the organs normally colonized by Ascochytapisi. The reactions of 14 pea variaties to Ascochytapisi pathotype 2 were compared with the concentration of the inhibitor of the endo-PG from this pathotype in the leaflets of each variety (Table 4). Although there was considerable variation in the concentration of inhibitor in different varieties, there was no correlation between these values and the plants’ resistance to Ascochytapisi. When this survey was repeated using endo-PG from Aspergillus niger in the inhibitor assay, the calculated inhibitor concentrations were approximately one-half of those recorded in Table 4 (Table 5). There were two exceptions to this general rule. The endo-PG inhibitor in leaflets of Keeran pea was a less effective inhibitor of the Aspergillus niger enzyme than was expected from its effect on the Ascochytapisi enzyme. By contrast, the endo-PG inhibitor in leaflets ofthe pea cultivar Rondo was a more effective inhibitor of the Aspergillus niger enzyme than was expected. These data suggest that there may be qualitative variation in the endo-PG inhibitor in different pea varieties. Ofthe otherplants tested, PG inhibitor was detected only in the two bean varieties. Inhibitor was extracted from the hypocotyl tissues into 50 mM acetate buffer and the yield was 430 i 9 units g-l fresh wt (mean & S.D. ; three determinations) from cv. Canadian Wonder and 539 & 117 units g-l fresh wt from Kievitsboon Koekoek. Smaller amounts, less than 40 units g-1 fresh wt, were detected in extracts of the leaves of each of these two cultivars. The inhibitor was quantitated using endo-PG from Ascochytapisi pathotype 2. We found that the inhibitor had no effect on the exoPG of Ascochyta pisi and was inactivated by heat (80 “C, 10 min).

pisi pathotype

DISCUSSION

Polygalacturonase inhibitor was detected in all of the organs of the pea plant which were examined. There was quantitative variation however, and concentrations of the

Endopolygalacturonase

inhibitor

in Pisum sathum

57

PG inhibitor were highest in the stipules and leaves and lowest in the seed. By contrast, a PG inhibitor from Phaseolus vulgaris occurred at higher concentrations in the hypocotyl than in the leaves. Leaflets from each of the 14 pea varieties which were examined contained the PG inhibitor but again there was considerable quantitative variation, ranging between less than 40 units g-l fresh wt in Dik Trom to 1250units g-l fresh wt in Keeran pea. There was no correlation between PG inhibitor concentration and the reaction of these varieties to the isolate of Ascochyta pisi which produced the endo-PG used in the inhibitor assay. Also, endo-PGs from representative cultures of each of the five pathotypes OfAscochytapisi were all inhibited by the PG inhibitor from the pea cultivar Small Sieve Freezer, which was reported to react differentially with these pathotypes [7]. We conclude therefore that the PG inhibitor in pea leaflets is not the determinant of race specificity in the leaf spot disease of peas caused by Ascochyta pisi. Although there was some evidence for qualitative differences in the biological activity of the PG inhibitor, as revealed by apparent differences in the sensitivity of endo-PGs from Ascochyta pisi and Aspergillus niger to the PG-inhibitors from different pea varieties, this also could not account for the specificity of the associations between Ascochyta pisi pathotypes and pea varieties. It is unlikely that the PG inhibitor from pea is a lectin, as has been suggested for the inhibitor from bean [I], because pea lectin occurs predominantly in the seed [8] which contains the lowest levels of the inhibitor. Also, one of us has found that activity of the inhibitor is unaffected by methyl a-n-glucopyranoside which binds strongly to and inactivates the pea lectin [22] (R.M.H., unpublished results). An extract of Austrian winter peas which should have contained the PG inhibitor was reported not to affect activity of pectinase from Phoma medicaginis fsp. pinodella [3]. By contrast, pectinase ( = “polymethylgalacturonase”) of Ascochytapisi was sensitive to the PG inhibitor [13]. Attempts to purify the Ascochyta pisi pectinase were unsuccessful and we now believe that this pectinase activity was at least partly due to the action, in concert, of pectin esterase and endo-PG on pectin. Likewise, we have found that Phoma medicaginis produces two endo-PGs which, with a pectin esterase, could have given rise to the previously reported pectinase activity of this fungus and, significantly, neither of these endo-PGs was inhibited by the pea PG inhibitor. In fact, of the four hmgal pathogens of pea which were examined, only Ascochytapisi produced a PG which was inhibited by the pea PG inhibitor. Amongst the other finrgi examined, only endo-PGs from C. lindemuthianum and Aspergillus niger were inhibited by this PG inhibitor. It is interesting that the endo-PGs of both C. lindemuthianum and Aspergillus niger were previously found to be inhibited by the Phaseolus vulgaris PG inhibitor [IO] and we show here that an extract of Phaseolus vulgaris hypocotyls also inhibited the endo-PG of Ascochyta pisi. These observations support the idea that the PG inhibitors from pea and Phaseolus vulgaris are very similar indeed : both are proteins, mol wt 42 000 and inhibit the endo-PGs of C. lindemuthianum, Aspergillus niger and Ascochyta pisi. Similarly, endo-PGs of C. lindemuthianum and Ascochyta pisi share a number of properties [23]. The PG inhibitor from Phaseolus vulgaris was reported to be bound to the cell wall [I], although we found that it is released into solution when bean hypocotyls are homogenized in 50m~ buffer. Possibly only a small proportion of the Phaseolus vulgaris PG inhibitor

58

R. M. Hoffman

and J. G. Turner

binds tightly to the cell wall, as has been reported for the pea PG inhibitor [13]. In neither case is it known if the inhibitor is bound to the wall in vivo or whether this binding only occurs after the tissue is cornminuted. Polygalacturonase inhibitors from other plants may show a specificity different from the inhibitors from pea and Phaseolus vulgar-is. For example, cucumber tissues contain a protein that inhibits endo-PG from Cladosporium cucumerinum [25], but extracts of cucumber tissues exhibited no inhibitory activity toward the Ascophyta pisi endo-PG. The function of the proteinaceous PG inhibitors in pea leaflets remains a mystery. Although it is unlikely that they are involved in determining specificity in the leaf spot disease caused by Ascochyta pisi, these inhibitors may affect symptom development. For example, tissue collapse occurred around droplets containing endo-PG from Phoma medicaginis var. pinodella that had been placed on leaflets of pea cultivar Rondo, whereas similar treatments with endo-PG from Ascochyta pisi produced no effect (unpublished results). This difference is easily reconciled with the fact that endo-PG from Phoma medicqinis is unaffected but that from Ascochyta pisi is inhibited by the PG inhibitor from pea leaflets. Another way in which the PG inhibitor may be involved in disease is suggested by the finding that products of the action of endo-PGs on plant cell walls are elicitors of phytoalexin synthesis [14]. Host protein that inhibits fungal endo-PG may therefore reduce elicitor production, and, in consequence, prevent phytoalexin synthesis. Pathogens which produce PGs that are sensitive to these inhibitors may therefore be at an advantage by not eliciting phytoalexin synthesis. On the other hand, it is quite possible that the PG inhibitor does not function in the disease but is involved in regulation ofplant enzymes. In support of this idea we have, during preliminary attempts to isolate the inhibitor, identified a pea PG in extracts from cell walls of healthy stems which was found to be sensitive to the PG inhibitor isolated from the same tissue (unpublished results). We gratefully acknowledge financial support from the Agricultural in the form of a grant to Dr J. G. Turner.

Research Council

REFERENCES 1. ALBERSHEIM, P. & ANDERSON, A. J. (1971). Proteins from plant cell walls inhibit polygalacturonases secreted by plant pathogens. Proceedings of the National Academy of Sciences 66, 1815-1819. 2. ANDERSON, A. J. & ALBERSHEIM, P. (1972). Host-pathogen interactions. V. Comparison of the abilities of proteins isolated from three varieties of Phareolus vulgaris to inhibit the endopolygalacturonases secreted by three races of Collelotrichum lindemuthianum. Physiological Plant Pathology 2, 339-346. 3. ANDERSON, A. J. & POWELSON, M. L. (1979). Production of plant cell wall degrading enzymes by Phoma medicaginis var. pinodelln. Phytopathology 69, 372-375. 4. BATEMAN, D. F. & BASHAM, H. G. (1976). Degradation of plant cell walls and membranes by microbial enzymes. In Physiological Planf Pathology, vol. 4, Ed. by R. Heitefuss & P. H. Williams, pp. 316-355. Springer-Verlag, Berlin. 5. BOCK, W., DONOOWSKI, G., GOBEL, H. & KRAUSE, M. (1975). Nachweis der Hemmung mikrobieller pektin-und pektatlyase durch pflanzeneigene Inhibitoren. Die Nahrung 19, 41 l-416. 6. COONS, C. H. (1916). Factors involved in the growth and pycnidium formation of Plenodomus fu‘uscomaculans. Journal of Agricultural Research 5, 7 13-769. 7. DARBY, P. (1982). Interactions between Ascochyta pin’ (Lib) pathotypes and Pirum genotypes. PhD thesis, University of East Anglia, Norwich, UK.

Endopolygalacturonase 8.

inhibitor

in Pisum sativum

59

G., KOSTIC, J. V. & KOCOUREK, J. (1970). Studies on phytohemagglutinins. and characterisation of hemagglutinins from the pea (Pisum sat&m L.). Biochimica

ENTLICHER,

III.

Isolation

ef Biophysics

Acta

221,272-281.

9. FIEL~NG,

A. H. (1981).

Natural

inhibitors

of fungal

polygalacturonases

in infected

fruits.

Journal

of

General Microbiology 123, 377-38 1. 10. FISHER, M. L., ANDERSON, A. J. & ALBERSHEIM,

1 I. 12.

13. 14. 15.

P. (1973). Host-pathogen interactions. VI. A single plant protein efficiently inhibits endopolygalacturonases secreted by Colletdrichum lindemuthionum and Aspcrgillus niger. Plant Physiology 51, 489491. GOBEL, H. & BOCK, W. (1978). Isolierung und Charackterisierung eines Pektinase-Inhibitors aus grunen Bohnen (Phaseolur vulgaris). Die Nahmng 25809-818. GOLDSTEIN, I. J. & HAYES, C. E. (1978). The lectins: carbohydrate-binding proteins of plants and animals. In Advances in Carbohydrate Chemistry and Biochemistry, Vol. 35, Ed. by R. S. Tipson & D. Horton, pp. 127-340. Academic Press, New York, San Francisco. HOFFMAN, R. M. & TURNER, J. G. (1982). Partial purification ofproteins from pea leaflets that inhibit Ascochytapisi endopolygalacturonase. Physiological Plant Pathology 20, 173-187. LEE, S.-C. & WEST, C. A. (1981). Polygalacturonase from Rhitopus stolonifer, an elicitor of casbene synthetase activity in castor bean (Ricinus camunis L.) seedlings. Plant Physiology 67, 633-639. SKARE, N. H., PAUS, F. & RAA, J. (1975). Production of pectinase and cellulase by Cladosporium cucumerinum with dissolved carbohydrates and isolated cell walls of cucumber as carbon sources. Physiologia

Plantamm

33, 229-233.

16. SPIRO, R. G. (1966). Analysis of sugars found in glycoproteins. In Methods in Enzymology, vol. VIII, Ed. by E. F. Neufeld & V. Ginsburg, pp. 3-26. Academic Press, New York. 17. URITANI, I. & STAHMANN, M. A. (1961). Pectolytic enzymes of CerotocystisJimbriata. Phytopathology 51, 277-285.