identification several pathogenesis-related proteins in ... · cladosporium fulvum (cooke) (syn....

Plant Physiol. (1989) 89, 945-9510032-0889/89/89/0945/07/$01 .00/0

Received for publication July 15, 1988and in revised form October 10, 1988

Identification of Several Pathogenesis-Related Proteins inTomato Leaves Inoculated with Cladosporium fulvum (syn.

Fulvia fulva) as 1,3-fl-Glucanases and Chitinases'

Matthieu H. A. J. Joosten and Pierre J. G. M. De Wit*Department of Phytopathology, Wageningen Agricultural University, P.O. Box 8025, 6700 EE Wageningen,

The Netherlands

ABSTRACT

Inoculation of tomato (Lycopersicon esculentum) leaves withCladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) re-sults in a marked accumulation of several pathogenesis-related(PR) proteins in the apoplast. Two predominant PR proteins werepurified from apoplastic fluid by ion exchange chromatographyfollowed by chromatofocusing. One protein (molecular mass [Mr]35 kilodaltons [kD], isoelectnc point [pl] -6.4) showed 1,3-#-glucanase activity, while the other one (M,26 kD, pl -6.1) showedchitinase activity. Identification of the products that were releasedupon incubation of the purified enzymes with laminarin or regen-erated chitin revealed that both enzymes showed endo-activity.Using antisera raised against these purified enzymes from tomatoand against chitinases and 1,3-fl-glucanases isolated from otherplant species, one additional 1,3-,8-glucanase (Mr33 kD) and threeadditional chitinases (Mr 27, 30, and 32 kD) could be detected inapoplastic fluids or homogenates of tomato leaves inoculatedwith C. fulvum. Upon inoculation with C. fulvum, chitinase and 1,3-,#-glucanase activity in apoplastic fluids increased more rapidlyin incompatible interactions than in compatible ones. The role ofthese hydrolytic enzymes, potentially capable of degrading hy-phal walls of C. fulvum, is discussed in relation to active plantdefense.

The accumulation of PR2 proteins upon infection withviroids, viruses, bacteria or fungi has been described forseveral plant species (28). PR proteins share a number ofcharacteristics such as a relatively low mol wt, accumulationin the apoplast, a high resistance to proteolytic activity, andextreme isoelectric points (28).The accumulation of PR proteins in tobacco leaves (cv

Samsun NN) showing a hypersensitive response to TMV iswell documented. The major acidic PR proteins could beresolved on native polyacrylamide gels and were referred toas proteins PR-la, - lb, - lc, -2, -N, -0, -P, -Q, -R, and -S, inorder of decreasing mobility (23). Although much informa-tion on the induction and physical properties ofthese proteinsis available, a biological function of some of these proteins

' This research was carried out in the framework of contract BAP-0074-NL ofthe Biotechnology Action Programme ofthe Commissionof the European Communities.

2 Abbreviations: PR, pathogenesis related; TMV, tobacco mosaicvirus.

has been reported only recently. PR proteins -P and -Q wereidentified as chitinases and -2, -N, and -O as 1,3-,B-glucanases.In addition to these acidic proteins, two basic chitinases andone basic 1,3-fl-glucanase were identified (15, 17).

Also, in another species of the Solanaceae, the occurrenceof PR proteins has been reported. Kombrink et al. (16)identified several PR proteins in potato leaves which wereinoculated with Phytophthora infestans. Six chitinases andtwo 1,3-f-glucanases were found in apoplastic fluid as well asin homogenates of infected leaf tissue.

In contrast to P. infestans which ramifies through the tissueinter- as well as intracellularly, Cladosporium fulvum, thecausal agent oftomato leafmold, is a biotroph that is confinedto the apoplast (6). The interchange of proteins and othersubstances between the two organisms in the apoplast ulti-mately determines the outcome ofthe interaction (compatibleor incompatible). A marked accumulation of several apoplas-tic proteins occurs in tomato leaves after inoculation with C.fulvum. In addition to the presence of proteinaceous race-specific elicitors (9, 25) and a compatible interaction-specificprotein (14), the accumulation of various PR proteins in theapoplast has been reported (10, 1 1). Thus far the biologicalfunction of these tomato PR proteins was unknown.Here we describe the purification of two predominant PR

proteins of tomato one of which is a 1,3-f3-glucanase and theother a chitinase. Using antisera raised against 1,3-f3-glucan-ases or chitinases from tomato or other plant species, oneadditional 1,3-f3-glucanase and three additional chitinasescould be detected in C. fulvum inoculated tomato leaves.Evidence is presented for a differential accumulation ofboth enzymes in compatible and incompatible C. fulvum-tomato interactions.

MATERIALS AND METHODS

Plant, Fungus and Inoculation

The various tomato (Lycopersicon esculentum) cultivarscarrying different genes for resistance to Cladosporiumfulvumwere grown as described by De Wit and Flach (7).

C. fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif) wassubcultured on PDA, and conidial suspensions used to inoc-ulate tomato plants were prepared as described previously (6).Races 4 and 5 of C. fulvum were used to inoculate two near-isogenic lines of tomato carrying the resistance genes Cf4

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(susceptible to race 4 and resistant to race 5) and Cf5 (suscep-tible to race 5 and resistant to race 4).

Preparation of Apoplastic Fluids and Leaf Homogenates

Entire leaves were infiltrated with distilled water invacuo, and the apoplastic fluids were isolated by centri-fugation, according to the method described by De Wit andSpikman (9).Leaf homogenates were obtained by grinding leaves (after

removal of the main veins) in a mortar containing 0.5 M Na-acetate buffer (pH 5.2) (4.0 mL/g fresh weight) and sand. Thehomogenate was centrifuged for 15 min at 8,000g, followedby 30 min at 35,000g. The supernatant was collected andanalyzed by PAGE.

Determination of Protein

Protein was determined using the method described byBradford (4), with BSA as a standard.

PAGE

PAGE under low pH, nondenaturing conditions on 10%(w/v) polyacrylamide slab gels was performed following themethod described by Reisfeld et al. (24). Staining and fixationwas as described by Steck et al. (27).SDS-PAGE on 15% (w/v) polyacrylamide slab gels was

performed as described by Joosten and De Wit (14). Themolecular weight of the various proteins was estimated by co-electrophoresis of marker proteins ranging from 14.2 to 66kD (Sigma).

Western Blotting and Radioimmunoassays

Western blotting, incubation with antisera, and the radio-immunological detection (with ['251]-labeled protein A) of theantigen-antibody complexes were carried out as described byDe Wit et al. (10).

1,3-,i-Glucanase Assay

To assay for 1,3-,B-glucanase activity in apoplastic fluids,proteins in apoplastic fluids were precipitated with 4 volumesof acetone at -20°C. The pellet obtained after centrifugation(5 min, 1,500g) was washed twice with 80% (v/v) acetone toremove any reducing sugars. The pellet was suspended incitric acid-phosphate buffer (pH 5.6) and any insoluble ma-terial was removed by centrifugation (5 min, 15,000g). Ap-propriate dilutions of the supernatant were used as enzymesolutions. At various steps in the purification procedure pro-tein fractions were assayed for 1,3-fl-glucanase activity with-out pretreatment.

1,3-3-Glucanase activity was assayed by measuring the rateof reducing sugar production with laminarin (Koch-Light) asthe substrate. The reaction mixture consisted of 0.4 mL ofMcllvaine's citric acid-phosphate buffer (pH 5.6) containing1 mg/mL laminarin and 0.1 mL of enzyme solution. After15 min of incubation at 37°C, 0.5 mL of the alkaline copperreagent was added (26) and the mixture was heated at 100°Cfor 10 min. After cooling on ice, 0.5 mL of the arsenomolyb-

date reagent (20) was added, followed by 3.0 mL ofH20 afterdevelopment ofthe blue color. The absorbance was measuredat 660 nm (1). Glucose was used as a standard, and enzymeand substrate alone were included as controls. One katal (kat)was defined as the enzyme activity catalyzing the formationof 1 mol glucose equivalents/s.

Chitinase Assay

Assays for chitinase activity in apoplastic fluids were carriedout without pretreatment of the enzyme solution. Regener-ated [3H]chitin, prepared as described by Molano et al. (19),was used as a substrate. The reaction mixture (total volume200 ,L) contained 50 mm phosphate buffer (pH 6.4), 0.02%(w/v) NaN3, 1.7 x 105 cpm of [3H]chitin and apoplastic fluidor purified protein fractions. After incubation at 37°C for 15min, 0.6 mL of 10% (w/v) TCA was added, and the suspen-sion was centrifuged at IOOOg for 5 min. The radioactivity ofthe supernatant (400 ,uL) was determined by liquid scintilla-tion counting after the addition of 4.0 mL of Aqua LumaPlus (Lumac). Chitinase activity was calculated as describedby Boller et al. (2) and is expressed as cpm in the releasedsoluble products.

Analysis of 1,3-ft-Glucanase and Chitinase Products

The products released by incubation of laminarin or chitinwith the purified 1,3-fl-glucanase or chitinase were analyzedwith a HPLC system equipped with a refractive index detector(Waters). Aminex HPX-87P and HPX-42A columns (300 x7.5 mm, temperature 85°C) were used for separating mono-saccharides and oligosaccharides, respectively.

Purification of a 1,3-j8-Glucanase and a Chitinase fromApoplastic Fluids

Four hundred mL of apoplastic fluid, originating from theinteraction Cf5/race 5, obtained 14 d after inoculation, wasconcentrated to about 100 mL by freeze-drying and clarifiedby centrifugation (10 min, 1000g). Proteins were precipitatedin 70% (v/v) acetone at -20°C, and the pellet obtained aftercentrifugation (5 min, 1000g) was air dried. The pellet wassuspended in 30 mL of 25 mm Na-acetate buffer (pH 4.5) andcentrifuged for 5 min at 2000g. The clear supernatant wasapplied to a CM-Sephadex C-25 column (1.6 x 7.5 cm; 15mL bed volume) equilibrated with 25 mm Na-acetate buffer(pH 4.5), and the column was washed with 40 mL of thesame buffer at a flow rate of 15 mL/h to remove the nonboundcompounds. The bound proteins were eluted with 0.5 M NaClin 25 mm Na-acetate buffer, at the same flow rate. The eluatewas dialyzed against distilled water, freeze-dried, and theresulting residue was dissolved in 20 mL of 25 mm imidazole-HC1 buffer (pH 7.4) and applied to a Polybuffer Exchanger94 column (Pharmacia, 1.2 x 20 cm, 25 mL bed volume)equilibrated with 25 mm imidazole-HCl buffer (pH 7.4).Elution with 200 mL of Polybuffer 74-HCI (pH 4.0) at 35mL/h resulted in a pH gradient from pH 7.0 to pH 4.0. Theeluent was monitored at 280 nm and the collected fractions(1.8 mL) were analyzed by SDS-PAGE. A Sephadex G-75column (1.3 x 80 cm, 100 mL bed volume), equilibrated with

946 JOOSTEN AND DE WIT



IDENTIFICATION OF PATHOGENESIS-RELATED PROTEINS IN TOMATO LEAVES

a b c d e m

Uq 66

35- -m _433-

26- _-

_ew 45

o_ 36

am 29q ~ 24

H20, was used to separate purified proteins from polybuffer.The column was eluted with H20 at a flow rate of 4 mL/h.

Antiserum Preparation

Fifteen hundred ,ug of purified 1,3-f3-glucanase and 300 ygof purified chitinase were used to raise polyclonal antibodiesin rabbits (14). This antiserum was used for immunologicaldetection of proteins on Western blots.

RESULTS

Purification of a 1,3-ft-Glucanase and a Chitinase fromApoplastic Fluid

_ 20.1 Preliminary immunoblotting experiments with partially pu-rified proteins (Mr 33 kD, and 35 kD, respectively) isolatedfrom apoplastic fluids of C. fulvum-infected tomato leaves

gb u Eli revealed that these proteins were detected by antiserum raiseda_ 14.2 against PR-Q, a l,3-fl-glucanase from tobacco (15). Similarly,40 another partially purified protein (Mr 26 kD) reacted with

antiserum raised against PR-P, a chitinase from tobacco (17).These results prompted us to assay for 1,3-f3-glucanase andchitinase activity during the further purification of theseproteins.SDS-PAGE of apoplastic fluid from the interaction Cf5/

A race 5, obtained 14 d after inoculation resulted in the proteinprofile presented in Figure IA, lane a. The 26 kD protein(cross-reacting with the antichitinase serum from tobacco)and the 33 kD and 35 kD proteins (cross-reacting with the

a b c d e anti-1,3-,B-glucanase serum from tobacco) are clearly visibleon the Coomassie brilliant blue-stained gel. These proteinsbound to a CM-Sephadex C-25 column at pH 4.5 and couldbe eluted with 0.5 M NaCl in 25 mm Na-acetate buffer (pH4.5) (Fig. IA, lane b). In the subsequent chromatofocusingstep most of the proteins which were eluted from the CM-Sephadex C-25 column did not bind to the column at pH 7.4

NmOW (Fig. IA, lane c). However, the 26 kD and 35 kD proteinsbound to the column and were eluted at pH 6.1 and pH 6.4,respectively. The 33 kD protein was not retained by thecolumn. After gel filtration on Sephadex G-75, the 35 kD and26 kD proteins were purified to apparent homogeneity as far

3-- W * , _as could be judged from SDS-PAGE (Fig. IA, lane d and e).4_ _ _ Electrophoresis of the fractions presented in Figure IA on4t _ a low pH, nondenaturing gel resulted in the protein profiles

presented in Figure lB. A number of proteins can be recog-5~* nized. Protein band 1 represents the compatible interaction-

specific protein which has been reported earlier (14), while6-"- * *protein band 7 represents the necrosis-inducing peptide (25).

Figure 1. Protein profiles of fractions obtained at different stages ofpurification of the 35 kD and 26 kD proteins from apoplastic fluid.PAGE was performed on SDS gel (A) and on low pH, nondenaturinggel (B). Proteins were stained with Coomassie brilliant blue R-250.Shown are profiles of proteins in apoplastic fluid (lane a, 60 Mg ofprotein); proteins bound to Sephadex C-25 and eluted with 0.5 MNaCI (lane b, 35 jig of protein on the SDS gel and 80 MAg on the lowpH gel); proteins not bound to PBE 94 (lane c, 30 Mg) and proteins

B eluted from PBE 94 within the pH gradient (lane d, 4 gg of the 35 kDprotein and lane e, 5 Mg of the 26 kD protein). In A, the lane markedgm' contains Mr markers (Mr given in kD).

7-

947



JOOSTEN AND DE WIT

4.2t

e_ n__ _

...i..M

*.:-"Hj* -

41g I, -.-..

r35J33_32-30-27"-26

_b

A

-3o

ow"~~~~~~~2

Proteins 2, 4, 4', 5, 6, and 7 were not retained by thechromatofocusing column (lane c), in contrast to the 35 kDprotein (lane d, representing the earlier reported protein 3)(10) and the 26 kD protein (lane e). The purified proteinswere assayed for 1,3-I3-glucanase and chitinase activity. The35 kD protein degraded laminarin (1,3-j3-glucanase activity),

Figure 2. SDS-PAGE (A) of proteins in apoplastic fluids (lanes a, c,and e) and leaf homogenates (lanes b, d, and f) from control plants(Cf5, after 7 d), an incompatible interaction (Cf4/race 5, 7 d afterinoculation) and a compatible interaction (Cf5/race 5, 12 d afterinoculation). Two replicas of the gel presented in A were blotted ontonitrocellulose, and the blots were incubated with antiserum raisedagainst the purified 1,3-fl-glucanase from tomato (B) and antiserumraised against PR-P, a chitinase from tobacco (C). The numbersindicate the Mr in kD of the corresponding protein band. From theuninoculated control plants 300 gL of apoplastic fluid and from theincompatible and compatible interactions 100 ML of apoplastic fluidwas applied (100 uL of apoplastic fluid is obtained from 200 mg (freshweight) of leaf tissue). From the leaf homogenates the proteinsextracted from 50 mg (fresh weight) of leaf tissue were electropho-resed. Lanes marked 'm' contain Mr markers (Mr are as given in Fig.1A).

while the 26 kD protein released soluble products from radio-actively labeled regenerated chitin (chitinase activity). Analy-sis of the products released upon prolonged incubation oflaminarin or chitin with the purified 1,3-fl-glucanase or chi-tinase revealed that both enzymes showed endo-activity. Sev-eral oligomers were detected, but only low amounts of the

4m.


Ailiwami.,.Dl'if 0




S 30

Cl)< 20.wcozC-)D 10~-J

C)

o V4 D6 8 10 12

DAYS AFTER INOCULATION

40

Cf4/5 Cf 5/5m 4 6 8 10 12 .14 m 4 6 8 10 2 14

-m -2

W W*~~* " "_ -_ = ~~~-P14

Figure 3. SDS-PAGE of apoplastic fluids obtained 4, 6, 8, 10, 12,and 14 d after inoculation of tomato cultivars Cf4 and Cf5 with race5 of C. fulvum. Each lane contains 200 ,L of apoplastic fluid. Lanesmarked 'm' contain Mr markers (Mr are as given in Fig. 1 A).

monomers were present in the reaction mixtures (results notshown).

Cross-Reaction of Proteins Present in Leaf Homogenatesand Apoplastic Fluids with Antisera Raised against 1,3-at-Glucanases and Chitinases

SDS-PAGE of proteins in apoplastic fluids and leafhomog-enates obtained from control plants (Cf5), an incompatibleinteraction (Cf4/race 5), and a compatible interaction (Cf5/race 5) resulted in the protein profiles presented in Figure 2A.The differences in protein patterns between the various sam-ples were mainly quantitative, showing relatively highamounts of the 35 kD 1,3-fl-glucanase and 26 kD chitinasein apoplastic fluid obtained from the incompatible and com-patible interactions (lanes c and e, respectively). In leaf ho-mogenates ofboth interactions, the 33 kD and 35 kD proteinsare present in more or less equal amounts (lanes d and f,respectively).

Replicates of the gel presented in Figure 2A were blottedonto nitrocellulose and incubated with different antisera. InFigure 2B the blot was incubated with antiserum raised againstthe purified 1,3-fl-glucanase from tomato. The antiserumdetected the 35 kD 1,3-f3-glucanase and cross-reacted with the33 kD protein. Incubation of the blot with antiserum raisedagainst a 1,3-,3-glucanase (PR-O) from tobacco or antiserumraised against a 1,3-fl-glucanase from potato revealed thatthese antisera had a higher affinity to the 33 kD protein thanto the 35 kD protein (results not shown).

In Figure 2C the blot was incubated with antiserum raisedagainst a purified chitinase (PR-P) from tobacco. The anti-serum detected the 26 kD chitinase and cross-reacted with a27, 30, and 32 kD protein. Antiserum raised against thepurified 26 kD chitinase from tomato or antiserum raisedagainst a chitinase isolated from bean leaves (2) detected the26, 30, and 32 kD proteins, but not the 27 kD protein (resultsnot shown).When samples similar to those presented in Figure 2A were

electrophoresed under low pH, nondenaturing conditions,

14 16

A

4 6 8 10 12 14 16DAYS AFTER INOCULATION B

Figure 4. Time course of 1,3-l-glucanase (A) and chitinase (B)activities in apoplastic fluids of leaves of tomato cultivars Cf4 (U) andCf5 (f) after inoculation with race 5 of C. fulvum and of uninoculatedCf5 plants (0). Apoplastic fluids were obtained 4, 6, 8, 10, 12, and14 d after inoculation, and enzyme activities were determined asdescribed in 'Materials and Methods."

antisera raised against the purified 1,3-f3-glucanase from to-mato and PR-O from tobacco cross-reacted with bands 3 and5 (Fig. 1B) on a western blot (results not shown). Antiseraraised against the purified chitinase from tomato and PR-Pfrom tobacco both detected the chitinase on a western blotand showed low affinity to two proteins near protein band 2(results not shown).

Differential Accumulation of the 1,3-j-Glucanase andChitinase in Apoplastic Fluids of Compatible andIncompatible Interactions of C. fulvum and Tomato

Apoplastic fluids from tomato cultivars Cf4 and Cf5, in-oculated with a conidial suspension of race 5 of C. fulvumwere isolated at 4, 6, 8, 10, 12, and 14 d after inoculation.SDS-PAGE of the apoplastic fluids revealed that, besides P14(11), several proteins accumulated about 4 d earlier in theincompatible interaction (Fig. 3). In this interaction (Cf4/race5) maximal accumulation of the 35 kD 1,3-,B-glucanase and

J1al rI

949




26 kD chitinase had already occurred at 6 d after inoculationand remained more or less constant up to 14 d. The accu-mulation of P14, however, showed a maximum at 8 d afterinoculation and declined afterward. In the compatible inter-action (Cf5/race 5) there was a steady increase of the 1,3-fl-glucanase and chitinase and several other proteins, up to 14d after inoculation.The time course of total 1,3-f3-glucanase activity in apo-

plastic fluid followed the observed accumulation ofthe 35 kD1,3-f3-glucanase (Fig. 4A). In the incompatible interaction, afast increase in 1,3-,B-glucanase activity occurred, reaching amaximum within 6 d, while in the compatible interaction,after a slow initial increase, the maximal level was reached 4d later.The time course of total chitinase activity in apoplastic

fluids also revealed a fast increase in the incompatible inter-action. The increase in chitinase activity in apoplastic fluidsfrom the compatible interaction was lower than was antici-pated from the observed accumulation of the 26 kD chitinaseat later stages of infection (Fig. 4B).Both 1,3-f-glucanase and chitinase activities showed some

increase in uninoculated Cf5 plants which were incubatedunder the same conditions as the inoculated plants. Measure-ments of time dependent 1,3-,B-glucanase and chitinase ac-cumulation in the reciprocal interactions (Cf5/race 4 andCf4/race 4) gave similar results (results not shown).

DISCUSSION

We have shown that two of the predominant PR proteinswhich accumulate in the intercellular spaces oftomato leavesafter inoculation with C. fulvum possess hydrolytic activity.A 35 kD protein showed 1,3-fl-glucanase activity, while aprotein of 26 kD, possessed chitinase activity.Young and Pegg (29) isolated three different acidic 1,3-,B-

glucanases from healthy tomato leaf and stem, but none ofthem was purified to homogeneity. They also isolated a con-stitutive chitinase from healthy tomato stem tissue with a Mrof 27 to 31 kD and a pI of 8.5 (22). This enzyme could besimilar or related to the 27 kD or 30 kD chitinase in apoplasticfluids or leaf homogenates, which were detected on nitrocel-lulose blots, but it is different from the 26 kD chitinasebecause of its different pI and Mr.

Legrand and co-workers (17) showed that four chitinases,isolated from TMV-infected tobacco leaves, had differentphysicochemical properties but were serologically closely re-lated. Close serological relationships were also found for thefour different 1,3-f3-glucanases isolated from TMV-infectedtobacco leaves ( 15). All six chitinases that were purified frompotato leaves infected by P. infestans showed strong cross-reactivity with antiserum raised against a chitinase isolatedfrom bean leaves (16). The two different 1 ,3-,3-glucanases thatwere purified were serologically closely related as well. Theseresults suggest that 1,3-fl-glucanases and chitinases within theSolanaceae, and even between different plant families, areserologically closely related. Assuming that this serologicalrelationship also holds for the chitinases and 1,3-f3-glucanaseswhich are induced in tomato by C. fulvum, then at least fourdifferent chitinases and two different 1,3-f3-glucanases arepresent in inoculated leaves. The chitinases that were detected

had a Mr of 26, 27, 30, and 32 kD, while the two 1,3-,B-glucanases had Mr of 33 and 35 kD.

Chitinase and 1,3-,B-glucanase activities increase in tobaccoand potato following infection (15-17). In cucumber, chiti-nase is induced in response to viral, bacterial and fungalinfections (18). In tomato, Verticillium or Fusarium wiltinduced an increase in 1,3-,B-glucanase and chitinase activity,which was always higher in the infected susceptible plants,where far more fungal biomass was present than in resistantones (13, 22). In Verticillium-infected tomato plants, theincrease in 1,3-f3-glucanase and chitinase activity coincidedwith a levelling off and subsequent reduction of the amountof viable pathogen in the tissue (21). The host glycosidasesthat were induced by the fungus were found to partiallydegrade Verticillium albo-atrum mycelial wall preparationsand could therefore provide a host defense mechanism (30).In tomato, C. fulvum, however, induced an increase in 1,3-f3-glucanase and chitinase activity in apoplastic fluids that wasmuch faster in the incompatible than in the compatibleinteraction. In the compatible interaction, it took at least 10d to reach a level of activity comparable to the level reachedat 6 d after inoculation in the incompatible interaction. Inthe compatible interaction, at 6 d after inoculation, fungalgrowth is already far more advanced than in the incompatibleinteraction (1 1), indicating that in the incompatible interac-tion the accumulation of the enzymes discussed above isspecifically induced by an avirulent race of C. fulvum and isnot a result of nonspecific stress caused by colonization oftheintercellular spaces. In the latter case, one would expect more1,3-f3-glucanase and chitinase in the compatible interaction atthis stage of infection. The fast induction of these enzymes,and other PR proteins, in the incompatible interaction, couldtherefore be the result of recognition by the host of a race-specific elicitor produced by the fungus.The rapid accumulation of 1,3-fl-glucanases and chitinases

at the site of penetration in incompatible interactions couldplay an important role as a defense mechanism of the plant.Many ofthese hydrolytic enzymes accumulate in the apoplast,as do most of the other PR proteins. These enzymes couldprotect plants against extracellular fungi like C. fulvum butpossibly also against pathogenic bacteria most of which growin the intercellular space.

In hyphal tips of many fungi, 1,3-fl-glucans and chitin areexposed at the surface (3) and could be attacked directly by1,3-,3-glucanases and chitinases. The oligosaccharides that arereleased from the fungal cell wall as a result of this hydrolyticactivity could function as elicitors of various plant defenseresponses (5, 8). Fungal growth could also be inhibited by adisturbance of the balance between synthesis and hydrolysisof hyphal wall material, caused by the extracellular presenceof the hydrolytic enzymes (12). In order to test these hy-potheses, in future experiments, 1,3-j3-glucanases and/or chi-tinases will be tested for their ability to hydrolyze myceliumand isolated cell walls of C. fulvum. It will also be testedwhether oligosaccharides released from this substrate are ableto induce plant defense responses.

ACKNOWLEDGMENTS

We thank B. Fritig (Strasbourg), E. Kombrink (Koln), T. Boller(Basel), and D. Peters (Wageningen) for providing antisera and Mrs.

950 JOOSTEN AND DE WIT




J. E. Hofman, Mrs. G. Booij, and Mrs. J. C. De Wit for theircontribution to the experimental work.

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1. Ashwell G (1957) Colorimetric analysis of sugars. Meth Enzymol3: 73-105

2. Boller T, Gehri A, Mauch F, Vogeli U (1983) Chitinase in beanleaves: induction by ethylene, purification, properties, andpossible function. Planta 157: 22-31

3. Boller T (1987) Hydrolytic enzymes in plant disease resistance.In T Kosuge, EW Nester, eds, Plant-Microbe Interactions:Molecular and Genetic Perspectives, Vol 2. Macmillan, NewYork, pp 385-413

4. Bradford M (1976) A rapid and sensitive method for the quan-titation of microgram quantities of proteins utilizing the prin-ciple of protein dye binding. Anal Biochem 72: 248-250

5. Darvill AG, Albersheim P (1984) Phytoalexins and their elici-tors-a defence against microbial infection in plants. AnnuRev Plant Physiol 35: 243-275

6. De Wit PJGM (1977) A light and scanning-electron microscopicstudy of infection of tomato plants by virulent and avirulentraces of Cladosporium fulvum. Neth J Plant Pathol 83: 109-122

7. De Wit PJGM, Flach W (1979) Differential accumulation ofphytoalexins in tomato leaves, but not in fruits after inocula-tion with virulent and avirulent races of Cladosporiumfulvum.Physiol Plant Pathol 15: 257-267

8. De Wit PJGM, Roseboom PHM (1980) Isolation, partial char-acterization and specificity of glycoprotein elicitors from cul-ture filtrates, mycelium and cell walls of Cladosporiumfulvum(syn. Fulviafulva). Physiol Plant Pathol 16: 391-408

9. De Wit PJGM, Spikman G (1982) Evidence for the occurrenceof race and cultivar-specific elicitors of necrosis in intercellularfluids of compatible interactions of Cladosporiumfulvum andtomato. Physiol Plant Pathol 21: 1-11

10. De Wit PJGM, Buurlage MB, Hammond KE (1986) The occur-rence of host, pathogen and interaction-specific proteins in theapoplast of Cladosporium fulvum (syn. Fulvia fulva) infectedtomato leaves. Physiol Mol Plant Pathol 29: 159-172

11. De Wit PJGM, Van der Meer FE (1986) Accumulation of thepathogenesis-related tomato leaf protein P14 as an early indi-cator of incompatibility in the interaction between Cladospor-iumfulvum (syn. Fulviafulva) and tomato. Physiol Mol PlantPathol 28: 203-214

12. Farkas V (1979) Biosynthesis of cell walls of fungi. MicrobiolRev 43: 117-144

13. Ferraris L, Abbattista Gentile I, Matta A (1987) Activation ofglycosidases as a consequence of infection stress in Fusariumwilt of tomato. J Phytopathol 118: 317-325

14. Joosten MHAJ, De Wit PJGM (1988) Isolation, purification

and preliminary characterization of a protein specific for com-patible Cladosporiumfulvum (syn. Fulviafulva)-tomato inter-actions. Physiol Mol Plant Pathol 33: 241-253

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