Suppression of the activity of non-specific elicitor from Cladosporium fulvum by intercellular fluids from tomato leaves

Physiological and Molecular Plant Pathology (1989) 34, 471-482

471

Suppression of the activity of non-specific elicitor from Cladosporium fulvum by intercellular fluids from tomato leaves TOBIN L . PEEVER

and VERNA J . HIGGINS

Department of Botany, University of Toronto, Toronto, Ontario, Canada M5S JAI (Accepted for publication August 1988)

Intercellular fluids from Cladosporium fulvum-infected tomato suppressed the induction of necrosis and the deposition of callose when co-injected into tomato leaflets with non-specific elicitors from culture filtrates and cell walls of C. fulvum . Similar suppressing activity was also detected in some, but not all, intercellular fluid preparations from uninoculated tomato indicating that the suppressing factor is probably of host origin . Heat lability of suppressor activity distinguished it from the specific elicitors which have been previously described in these intercellular fluids . Suppression of non-specific elicitor activity showed no race/cultivar specificity and appeared to be an enzymatic interaction between components of the intercellular fluids and the elicitor . Activity of both crude and purified preparations of the glycoprotein elicitor was suppressed . Differences in necrosis but not electrolyte leakage were observed between leaf discs injected with non-specific elicitor and intercellular fluids containing or lacking suppressor activity . Intercellular fluids from rust-infected bean also suppressed elicitor-induced necrosis . The ability of intercellular fluids to suppress activity of the non-specific elicitor suggests that this and similar elicitors may not exist for long in vivo .

INTRODUCTION

Plants are able to respond to penetration by pathogens in numerous active ways which can result in the failure of disease development . Fungi grown in vitro have been shown to produce several different molecules which induce or "elicit" similar responses in the absence of the pathogen [1, 2, 18, 19] . This correlation has led many researchers to assume that these "elicitors" play a role in plant-pathogen interactions despite any evidence to demonstrate their existence or activity in vivo . The glycoprotein elicitor produced in culture by Cladosporium fulvum Cooke [syn . Fulviafulva (Cooke) Ciferri] induces electrolyte leakage, callose deposition, necrosis and phytoalexin accumulation when applied to tomato tissue [4, 19] . This elicitor can be isolated from the mycelium, cell walls and culture filtrates of all races of the pathogen [4, 20, 191 and is equally active on resistant and susceptible cultivars of tomato . Thus, this elicitor is unlikely to be involved in the induction of resistance unless a specific suppression of activity occurs in a compatible interaction, a possibility suggested by

Abbreviations used in text : NSE, non-specific elicitor ; IF, intercellular fluid .

0885-5765/89/060471 + 12 $03 .00/0 17

© 1989 Academic Press Limited MPP 34



472 T . L. Peever and V . J . Higgins Higgins [16] based on histological data . Clearly if such elicitors are present in planta, their activity must be modified to allow a compatible, biotrophic interaction to occur . Intercellular fluids obtained from tomato leaf tissue infected with C . fulvum contain other elicitors which appear to be race/cultivar specific [6] . These elicitors induce chlorosis and necrosis only when injected into tomato cultivars resistant to the race of Cm fulvum used to produce them ; one of the elicitors has been characterized as a lowmolecular-weight peptide [8] . The elicitors cannot be isolated from the pathogen grown in vitro but they do appear to be fungal products of the interaction [7] . In addition to the elicitors, intercellular fluids contain other proteins, some of which are induced more rapidly in incompatible interactions and which are called pathogenesisrelated (PR) proteins [3, 9, 10] . Two of these proteins have been characterized as ß-l ‚ 3glucanase and chitinase [11] but their role in vivo is unknown . The present study was undertaken to examine the possibility that molecules contained in intercellular fluids from the C fulvum-tomatoo interaction may suppress or modify activity of the non-specific elicitor .

MATERIALS AND METHODS

Culture offungi Isolates of Cm fulvum races 0 and 4 were obtained from the culture collection at the Department of Botany, University of Toronto, Toronto, Ontario, Canada . Races 2 .4 .5 and 2 .4 .5 .9 were received from P . J . G . M . de Wit, Agricultural University, Wageningen, The Netherlands . Cultures were maintained in sterile soil at 4 ° C, plated out on V8 juice agar, and transferred a maximum of three times prior to use . Sporulating plates of the fungus (7-14 days after transfer) were used for the inoculum in the production of intercellular fluids or for transfer to modified Fries medium to produce non-specific elicitor . Culture of plants Tomato seeds of cultivars Potentate and Purdue-135 were obtained from R . A . Brammall, Simcoe Horticultural Station, Simcoe, Ontario, Canada . Seeds of the cultivars Bonny Best and Sonatine were obtained from McKenzie Seed Co . Ltd, Brandon, Manitoba, Canada and de Ruiter Seeds, Columbus, Ohio, U .S .A ., respectively . Seeds were germinated and grown in a standard soil mix and maintained in growth chambers with a 14 h photoperiod at 15000 lux (200 isE m -2 s-1 ), 85 % RH, and day and night temperatures of 23 and 21 °C respectively . Plants were used at 5-6 weeks of age for all experiments . Production of non-specific elicitor Non-specific elicitor (NSE) was prepared from crude culture filtrates of Cladosporium fulvum race 2 .4 .5 . The fungus was grown for 21 days under a 12 h photoperiod at 1000 lux (14 .p E m -2 s` 1 ) at 25 ° C on modified Fries medium according to the method of Lazarovits & Higgins [19] . Fungal mycelium and medium were filtered through Whatman No . 1 filter paper under vacuum . These filtrates were concentrated to 5 % of their original volume using an Amicon ultra-filtration apparatus with a PM-10 membrane and stored at -20 ° C . Carbohydrate levels in the concentrated filtrates



Suppression of non-specific elicitor activity

473

were determined by the method of Dubois et al. [13] using D-glucose as the standard . Concentrations of elicitor used in subsequent assays were expressed as gg ml -1 glucose equivalents . NSE prepared from cell walls of C. fulvum was the gift of P. J . G . M . de Wit, Wageningen Agricultural University, The Netherlands . Fraction B was prepared according to the method of de Wit & Kodde [5] and freeze-dried . The mannose : galactose : glucose ratio of this preparation was 55-6 :3-9 :8-0 . Solutions of elicitor were made up in distilled water and diluted prior to use . fulvum race 0 was purified by affinity chromatography NSE from culture filtrates of Cm on Con-A Sepharose according to the method of Lazarovits et al . [20] . This was followed by gel chromatography using Sephacryl S200 Superfine (Pharmacia, bed volume 1 . 6 x 39 cm packed in distilled water) . The samples (each 2-5 mg glucose equivalents) were eluted with distilled water (flow rate- 0 . 7 ml min -1 ) and 4. 2 ml fractions collected . Active fractions usually eluted in about fractions 4 to 9 (mannoside from the Con-A column began to be eluted at about fraction 15) and were pooled and stored at -20 ° C . Carbohydrate levels in the pooled fractions were determined using the assay of Dubois et al . [13] with D-glucose as the standard .

Inactivation of non-specific elicitor NSE was inactivated using a modification of the procedure of de Wit & Roseboom [4] who demonstrated the instability of NSE incubated under alkaline conditions . An NSE solution containing 1 . 4 mg ml-1 glucose equivalents in 0 . 5 ml distilled water was added to 0 . 5 M NaOH and incubated for 24 h at 25 ° C. Following incubation, 1 . 0 ml of 1 M HCl was added, mixed thoroughly, and the solution dialysed (2000 mol . wt cut-off, 4 °C, 20 h) . The resulting preparation was termed "inactivated NSE" and stored at - 20 °C .

Production of intercellular fluids from tomato Intercellular fluids (IFs) from tomato leaf tissue infected with Cm fulvum were prepared according to the method of de Wit & Spikman [6] . Leaflets were harvested 10-14 days after inoculation when sporulation was evident over the entire abaxial surface of the leaflets . Leaflets were infiltrated in vacuo, placed in specially designed tubes, and centrifuged at 1650 g (5 ° C, 30 min) . Tomato cultivars Potentate or Bonny Best (no known Cm fulvum resistance genes) and various races of Cm fulvum were used to produce the fluids . Leaflets of healthy, uninfected tomato in the same growth chamber (or in a different chamber) as the infected plants were used to produce control intercellular fluids . Control leaflets were harvested, infiltrated and centrifuged at the same time as the leaflets from infected plants and the fluids stored at -20 °C . Prior to use, IFs were centrifuged at 1500 g (5 ° C, 10 min) and dialysed (2000 mol . wt cut-off, 5 °C, 20 h) .

Production of intercellular fluids from bean IFs were prepared from French bean leaf tissue (Phaseolus vulgaris L . cv Pinto) inoculated with the bean rust fungus (Uromyces appendiculatus (Pers .) Unger var . appendiculatus) . Eleven-day-old bean plants were inoculated with rust uredospores or left uninfected and kept in the dark at 22 °C for 24 h . After 24 h, plants were incubated for a further 5 days under a 16 h photoperiod, 10000 lux (140 µE in - ' s -1 ), 85% RH 17-2



474

T . L . Peever and V . J . Higgins

and 22 °C . Leaves were detached, infiltrated under vacuum with distilled water, placed in specially designed centrifuge tubes, and centrifuged at 1000 g (4 °C, 30 min) . Crude IFs from both healthy and infected bean were 'dialysed (8000 mol wt . cut-off 5 ° C, 20 h) or left undialysed and stored at -20 °C for use in subsequent assays .

Assay of necrosis-inducing activity of non-specific elicitor and the effect of intercellular fluids NSE (40-100 µg ml - ' glucose equivalents) was routinely mixed with IFs at their original concentration and injected immediately into interveinal panels on one side of leaflets of cv . Bonny Best . Controls, consisting of NSE mixed in the same ratio with distilled water, were injected into panels on the opposite side of these same leaflets . Generally, four to five leaflets were used per treatment with 12 to 15 panels injected . NSE was routinely used at a concentration of 40-100 µg ml - ' glucose equivalents (20-50 µl in 1 ml water or IF), a concentration which induced a strong necrotic response 24 h after injection . The level of necrosis observed was separated into four categories ranging from 0 (no visible host response) to 3 (strong necrotic response) . IFs fulvum were from both uninfected tomato and tomato infected with various races of Cm co-injected with NSE from different sources and purified to various degrees .

Qualitative assay of callose Callose deposition in injected tissue was determined with the aniline blue fluorescence method used by Lazarovits & Higgins [19] . Uninjected leaflet panels or panels injected with distilled water, NSE + distilled water, or NSE + IF, were cut from the leaflets 24 h after injection and decolourized in methanol for 48 h at 25 ° C . Five panels per treatment were stained, mounted and examined microscopically for the presence of callose .

Pre-injection of intercellular fluids To test whether IFs could affect NSE activity if they were not mixed prior to injection, IFs were injected at various times before injection of NSE . IF produced with race 2 .4 .5 of Cm fulvum was injected into 48 panels on the right side of four leaflets on each of four Bonny Best plants, and distilled water was injected into panels on the opposite side of these same leaflets . Twenty-four, 48, 72 and 120 h following this initial injection, both sides of 12 of these injected panels were re-injected with NSE (40 gg ml - ' glucose equivalents) and the necrosis assessed 24 h after the final injection . In vitro interaction of non-specific elicitor and intercellular fluids To test for in vitro interactions of NSE and IFs, the heat sensitivity of IF activity and heat stability of NSE were utilized . NSE (60-80 µg ml - ' glucose equivalents) was mixed with IF (race 2 .4 .5) or an equivalent volume of distilled water, and the solutions were filter-sterilized with Millipore GS filters . Each solution was treated in one of four ways prior to injection : (1) treated for 10 min at 25 ° C ; (2) treated for 10 min at 100 °C ; (3) incubated at 25 °C for 20 h with no heat treatment ; or (4) incubated at 25 ° C for 20 h followed by heat treatment for 10 min . In two separate experiments, 21 leaflet panels were injected with each solution . Comparable distilled water+NSE controls were injected opposite the appropriate NSE+IF treatment .



Suppression of non-specific elicitor activity

475

Electrolyte leakage assays

To test whether IFs could suppress the induction of electrolyte loss from NSE-treated leaf tissue at an early period after injection, entire leaflets of cv . Purdue-135 were injected with 40 lag ml-1 glucose equivalents NSE in distilled water, in race 4 intercellular fluids, or in control intercellular fluids . Inactivated NSE at 40 gg m1 -1 glucose equivalents was also tested . Plants were incubated for 5 h following injection, at which time discs were cut from the leaflets, distributed over three flasks/treatment in distilled water (5 ml), and the conductivity of this water measured every hour for 3 h according to the method of Lazarovits & Higgins [19] . Net increases in conductivity in each treatment were calculated by subtracting initial leakage values from actual values recorded at 1, 2 and 3 h . Injection of non-specific elicitor into leaf mould lesion

To test for suppressor activity in planta, isolated, circular lesions of leaf mould caused by Cm fulvum race 2 .4 .5 on leaflets of Bonny Best were injected with 40 µg ml -1 glucose equivalents NSE in distilled water . Approximately 50 lesions were injected from the centre to 5 mm beyond the lesion margin . In this way, both colonized and uncolonized tissue was injected with NSE . Uninfected areas on the same leaflets were injected in a fashion similar to that used for the lesions . Necrosis in the injected areas was assessed 24 h after injection .

RESULTS Assay of necrosis-inducing activity of non-specific elicitor and the effect of intercellular fluids

Co-injection of non-specific elicitor (NSE) and intercellular fluids (IFs) from C fulvuminfected d tomato consistently resulted in complete suppression of NSE-induced necrosis in the injected panels (Table 1) . NSE plus distilled water controls injected into the opposite side of the leaflet always induced high levels of necrosis (data not shown) . IFs isolated using two races of Cm fulvum both suppressed the NSE at high levels (Table 1) . In addition to the races 2 .4 .5 and 2 .4 .5 .9 used for IF production in Table 1, race 0 IF suppressed activity of NSE from crude culture filtrates of race 2 .4 .5 and of fraction B from cell walls of C. fulvum . Several preparations of IFs from uninfected plants incubated with the infected plants also suppressed NSE-induced necrosis, but not as consistently as did fluids from the infected plants (Table 1) . Suppressor activity was also detected in IFs from uninfected tomato grown in a growth chamber separate from that used for the growth of the infected plants (Table 1) . IFs prepared with four races of C. fulvum suppressed NSE-induced necrosis in six cultivars of tomato carrying different genes for resistance to the pathogen (Table 2) . No race/cultivar specificity was observed . Unfortunately, it was impossible to determine whether suppression of NSE occurred in some cultivars because of the concurrent activity in the IFs of specific elicitors . This was the case for the injection of race 0 IFs, together with NSE, into Sonatine, or race 4 fluids together with NSE into both Vinequeen and Sonatine . Both of these combinations gave a strong necrotic response in 24 h .



476

T . L . Peever and V . J . Higgins TABLE I

Degree of suppression of necrosis-inducing activity of non-spec(c elicitor (,VSE)' by intercellular fluid SIF) preparations from Cladosporium fulvum-infected tomato or from uninoculated controls incubated in the same growth chamber or in another chamber

Necrosis rating Source of intercellular fluid (C. fulvum race infecting g plant) 2 .4 .5 -l b 2 .4 .5-2 2 .4 .5-3 2 .4 .5-4 2 .4 .5-5 2 .4 .5-6 2 .4 .5 .9

IF from uninoculated plants IF from fulvuminfected plants 01

0 0 0 0 0 0

A°

Be

0 1 2 2 2 NT 3

0 0 0 N'I' l N'I' NT

aNon-specific elicitor from race 0 culture filtrates purified by affinity and gel chromatography using Con-A Sepharose and Sephacryl S-200 (100 µg ml - ' glucose equivalents) . 'Numbers denote six separate isolations of intercellular fluids produced with race 2 .4.5 . `Average necrosis rating at 24 h for 12-15 injected panels of cv . Bonny Best (average rating for NSE+water controls = 3) . 'Incubated in same growth chamber as the inoculated plants . `Incubated in a growth chamber which contained no inoculated plants . 'Not tested . TABLE

2

Suppression of non-specific elicitor activity in tomato leaflets co-injected with intercellular fluids (IFs) from four races of C . fulvum and non-specific elicitor (NSE)'

Source of intercellular fluids Cultivar injected (Cf gene) Bonny Best (CfO) Potentate (CfO) Vetomold (Cf2) Purdue-135 (Cf4) Vinequeen (Cf 2,4) Sonatine (Cf2, 4, 5, 9)

race 0

race 4

race 2 .4 .5

race 2 .4 .5 .9

Ob 0 1 0 0 3'

1 1 1 1 2d 3

0 0 1 NT 0 NT

0 0 NT' NT 0 0

'Non-specific elicitor, race 0 (as described in Table 1) . 'Average necrosis rating at 24 h for 12 injected panels (average rating for NSE +water controls = 3) . 'Not tested . °Vinequeen reacts to specific elicitors in intercellular fluids of race 4 to give rapid necrosis . 'Sonatine reacts to specific elicitors in intercellular fluids of races 0, 4 and 2 .4 .5 to give rapid necrosis.

IFs from rust-infected bean also suppressed the induction of necrosis by NSE when co-injected into Bonny Best . Deposition of callose wall appositions, another conspicuous feature of NSE activity [19], was also suppressed by IFs from tomato . Leaflet panels injected with NSE diluted with water only (necrosis rating 3) displayed extensive deposition of callose throughout



Suppression of non-specific elicitor activity

477

the injected area . In contrast, panels injected with distilled water or NSE + IF (necrosis rating 0) revealed no evidence of callose deposition .

Pre-injection of intercellular fluids NSE and IFs were generally injected simultaneously after prior mixing . To eliminate in vitro interactions, IFs were injected into leaflet panels followed by injection of NSE at various times after . IF injected up to 72 h prior to NSE still significantly (P = 0 . 01 at 72 h, Student's t-test) suppressed necrosis-inducing activity compared to the water-injected controls (Fig . 1) . By 120 h there was no significant difference (P = 0. 05) between levels of necrosis observed in panels pre-injected with water compared to those pre-injected with IF .

w J

24

48

72

120

Hours between injections FIG . 1 . Necrosis ratings of Bonny Best leaflet panels pre-injected with distilled water (open bars) or race 245 intercellular fluids (shaded bars) followed by injection of all panels with non-specific elicitor (NSE) at various time intervals . Values and error bars represent means and standard deviations of 12 injected panels rated from 0 (no necrosis) to 3 (maximum necrosis) . All values were significantly different at P = 0 .01 except those at 120 h .

In vitro interaction of non-specific elicitor and intercellular fluids The heat lability of suppressor activity was used to distinguish between two possible modes of interaction between NSE and IFs, enzymatic degradation and binding of the suppressor to the elicitor . Heat treatment immediately following mixture of NSE and intercellular fluids eliminated suppressor activity but not NSE activity (Fig . 2) . Following a 20 h incubation between mixing and heat treatment, however, almost complete suppression of NSE-induced necrosis occurred similar to that seen in the unheated treatment (Fig . 2) . Filter-sterilized IF diluted to 50, 25, 13 and 6% of the original concentration and incubated alone for 20 h at 25 °C showed no loss of specific elicitor activity compared to IF incubated 20 h at 0 ° C .

Electrolyte leakage assays Representative experiments showing conductivity changes in bathing solutions containing leaf discs cut from injected tomato are given in Fig . 3 . Significant differences



478

T. L. Peever and V . J . Higgins 3

o, S 2 â o 0 u v Z

I

0 no heat

heattreated

no heat

heattreated

Incubation 20 h

No incubation

FIG . 2 . Test for in vitro inactivation of non-specific elicitor (NSE) in the presence of intercellular fluids (IF) by prolonged incubation under sterile conditions followed by heat inactivation of IF suppressor activity prior to injection . Mixtures of NSE+water (open bars) or NSE+IF (shaded bars) were heat treated (100 °C, 10 min) immediately after mixing (no incubation) or after 20 h incubation at 25 °C . Other comparable mixtures were not heat treated . Assay was by injection on cv . Bonny Best using a necrosis rating from 0 (no necrosis) to 3 (maximum necrosis) . Values and error bars represent means and standard deviations of 21 injected panels .

( b) 10 8

o E

6 4

Z1 U 7 ß O U

2

Ed 10 N

w

8

C -

6

d z

4 2 5

6

7

8

Hours after injection FIG . 3 . Conductivity changes in flasks containing leaf discs of Purdue-l35 injected with distilled water only (0-0), NSE+distilled water (+ -- +), NSE +control intercellular fluids (0-0), or NSE+race 4 intercellular fluids (0---O) . Values and error bars represent means and standard deviations of three flasks/treatment (a) or four flasks/treatment (b) . Leaflets were injected, and following 5 h incubation were removed from the plant and discs taken for the assay . After incubation of the discs for 24 h, only those from the NSE+water and NSE+control intercellular fluids treatments showed necrosis .



Suppression of non-specific elicitor activity

479

in electrolyte leakage were observed between water-injected and NSE-injected tissue after 1 h [Fig . 3 (a)] . Co-injection of NSE with IF from healthy tomato or race 4 IF resulted in similar levels of electrolyte leakage for both treatments [Fig . 3 (b)], such leakage being not significantly different from that seen in the water control . After the conductivity measurements had been completed, the discs were kept in the bathing solutions for 24 h and observed for necrosis . Necrosis was observed in panels injected with the NSE+healthy IF, and in NSE+water treatments, but not in the NSE+race 4 IF injected panels . NSE inactivated by alkaline treatment and injected into leaflets elicited levels of electrolyte leakage that were not significantly different from the levels seen in leaflets injected with water in several experiments (data not shown) . Injection of non-specific elicitor into leaf mould lesions

Injection of isolated, circular lesions of leaf mould with NSE resulted in the development of necrosis surrounding the margin of the lesion but not within its boundaries (Fig . 4) . This result was consistently observed in all of the lesions injected . Control injections of NSE into leaflet tissue not colonized by C . fulvum resulted in necrosis of the entire injected area .

FIG . 4 .

Suppression of NSE-induced necrosis in a leaf mould lesion on tomato caused by C.

fulvum . Healthy leaf tissue, NSE-induced necrotic leaf tissue and non-necrotic tissue within the boundary of a leaf mould lesion are indicated by the letters a, b and c respectively .

DISCUSSION Intercellular fluids (IFs) from tomato infected with C fulvumi suppressed the induction of necrosis by the non-specific elicitor (NSE) when both were injected together . This activity is probably biologically significant as the suppressing factor in the IF preparations is much more dilute than would be found in vivo due to the nature of the isolation and injection procedures . This suppression was not race/cultivar specific . IFs produced with four different races of Cm fulvum suppressed NSE-induced necrosis in all



480

T . L . Peever and V. J . Higgins

cultivars, including those both compatible and incompatible with the race of C . fùlvum used to produce the IFs . The lack of race/cultivar specificity suggests that the suppression of NSE is fundamentally different from that of the cultivar-specific suppressors found in the Phylophthora infestans-potato interaction [14] . The suppressors described in this study may be similar to those of Oku et al . [21] and Heath [15] which are host-specific rather than cultivar-specific . These suppressors allowed increased growth of non-pathogens, and those from Mycosphaerella species also inhibited phytoalexin accumulation induced by non-specific elicitors [211 . IFs also contain race and cultivar specific elicitors [6] which limit the ability to detect suppression of NSE in some incompatible cultivars but not in others . For example, tomato cultivars carrying the C . fulvum resistance gene 2 react much more slowly (i .e ., 3 or 4 days) to the specific elicitors in intercellular fluids than do cultivars carrying gene 9 [17] . Thus, in a Cf 2 plant, suppression of NSE can readily be assayed after 24 h incubation, whereas, in a Cf 9 plant, necrosis due to the specific elicitor activity also occurs within 24 h . The maintenance of specific-elicitor activity in filtersterilized IF incubated alone for 20 h indicates that breakdown of the specific-elicitor did not occur under the same conditions that caused breakdown of the NSE . The presence of suppressor activity in some preparations of IF from uninfected, healthy tomato demonstrates that it is a host-produced factor and may therefore be induced to various degrees by biotic stress such as C. fulvum infection or by unknown abiotic factors (e .g ., ethylene build-up in the growth chambers may vary depending on plant size and density) . A balance between NSE-induced necrosis and NSE suppression in injected tissue may be tipped in favour of suppression on addition of IF from another healthy tomato. Suppressor activity is retained for 72 h after injection of IF into leaflets, indicating that it is not readily degraded in the intercellular space . Several possible modes of interaction between NSE and suppressor are possible ; these modes include the enzymatic breakdown of NSE, binding of the suppressor directly to NSE, or interference by the suppressor with an NSE-binding site . Elimination of NSE-induced necrosis in leaf mould lesions could be due to breakdown or binding of NSE by pathogen or host within the intercellular spaces . The in vitro incubation experiments suggest an enzymatic type of interaction that does not depend upon the presence of the host . The heat lability of the suppressor is also consistent with an enzymatic m echanism . d e Wit & Kodde [5] characterized the NSE as a peptidogalactoglucomannan, and the activity of the NSE has previously been shown to be reduced by treatment with ot-mannosidase, proteinase K [4, 20] and pronase [4] . Two enzymes, ß-1,3-glucanase and chitinase, have been isolated and purified from intercellular fluids [11], and it is possible that the glucanase or other enzymes present in the fluids are responsible for breakdown of the NSE . The nature of the enzymes which may be involved is the subject of continuing study . Injection of NSE into tomato tissue induced electrolyte leakage, as has been observed previously, and this leakage has been correlated to the induction of necrosis [19] . Alkaline incubation of NSE eliminated its ability to induce both responses . NSE mixed with IFs from healthy or Cd fulvum-infected tomato induced similar levels of electrolyte leakage for the first 3 h after injection but only NSE +healthy IF caused necrosis . This suggests that the effects of electrolyte leakage and necrosis may be separated . Continuation of the electrolyte leakage experiments for a longer period may clarify the



Suppression of non-specific elicitor activity

481

relationship between these two variables . The effect of the suppressor on phytoalexin accumulation could also be used as an additional assay of suppressor activity . The NSE is a component of the cell wall of the pathogen [4] and is probably present both in vivo and in vitro but there is no direct evidence as yet that it plays a role in the interaction . Both NSE and the specific elicitors reproduce the callose deposition and necrosis associated with the incompatible interaction . Higgins [16] observed that when conidia were injected into tomato leaf tissue, conidia and young germ tubes elicited callose deposition in both compatible and incompatible hosts . This was interpreted as possibly due to NSE in the fungal cell wall . If, as the data in this study suggest, complete enzymatic degradation of "available" NSE occurs in susceptible hosts, the possibility of isolating NSE from infected plants is unlikely . The observation by Higgins [16] that, in a compatible cultivar, germ tubes of injected conidia soon ceased eliciting callose deposition, could be explained by the enzymatic degradation of the NSE . In the incompatible hosts, the presence of specific elicitor may explain the continued elicitation of the above responses in the absence of NSE . The results of the present study strongly suggest that, in the interaction between Cm fulvum and tomato, the glycoprotein non-specific elicitor plays no role because, in the normal slow infection process, it would be immediately degraded by enzymatic components of the apoplast . The array of non-specific elicitors, particularly the glucans, in other fungal-plant systems may suffer the same fate, at least in their host plants . The inhibition of NSE by IF from rust-infected bean suggests that such enzymes may be common constituents of plants infected by biotrophs . Elicitor breakdown by hydrolytic enzymes is also demonstrated in the recent work of Schmidt & Ebel [22] who used ß-glucohydrolase inhibitors in their soybean membrane preparations in order to prevent breakdown of their elicitor . Thus, non-specific elicitor activity may prove to be an artefact of assay systems which assault the plant tissue with abnormally high concentrations of elicitor rather than with the gradual exposure occurring in natural infections . The authors would like to thank the Natural Sciences and Engineering Research Council of Canada for financial support . Intercellular fluids from rust-infected bean, and fraction B from cell walls of C. fulvum were kindly provided by Aiming Li, University of Toronto and P . J . G . M . de Wit, Wageningen Agricultural University, The Netherlands, respectively .

REFERENCES 1. 2.

3.

4.

ANDERSON-PROUTY, A . J . & ALBERSHEIM, P. (1975) . Isolation of a pathogen-synthesized fraction rich in glucan that elicits a defense response in the pathogen's host . Plant Physiology 56, 286-291 . BOSTOCK, R . M ., LAINE, R . A. & Kuc, J . A . (1982) . Factors affecting the elicitation of sesquiterpenoid phytoalexin accumulation by eicosapentanoic and arachidonic acids in potato . Plant Physiology 70, 1417-1424 . DE WIT, P. J . G . M . & BAKKER, J . (1980) . Differential changes in soluble tomato leaf proteins after inoculation with virulent and avirulent races of Cladosporium fulvum (syn. Fulvia fulva) . Physiological Plant Pathology 17, 121-130 . DE WIT, P . J . G . M . & ROSEBOOM, P . H . M . (1980) . Isolation, partial characterization and specificity of glycoprotein elicitors from culture filtrates, mycelium and cell walls of Cladosporium fulvum (syn . Fulvia fulva) . Physiological Plant Pathology 16, 391-408 .



482 5.

T . L . Peever and V . J . Higgins DE WIT, P . J . G . M . & KDUDE, E .

(1981) . Further characterization and cultivar-specilicity of

glycoprotein elicitors from culture filtrates and cell .walls of Cladosporium . lulvum (syn . Fulvia fùlva ; . Physiological Plant Pathology 18, 297-314 .

J . G . M . & SPIKMAN, G . (1982) . Evidence for the occurrence of race and cultivar specific elicitors of necrosis in intercellular fluids of compatible interactions of Cladosporium,fùlvum and tomato. Physiological Plant Pathology 21, 1-11 .

6.

DE WIT P.

7.

DE WIT P . J . G . M ., HO MAN,

8.

chlorosis and necrosis occurring in intercellular fluids of compatible interactions of Cladosporium fulvum (syn . Fulvia fulva) and tomato . Physiological Plant Pathology 24, 17- 23 . DE WIT P . J . G . M ., HOFMAN, J . E ., VELTHIUS, G . C . M . & Kud, J . A . (1985) . Isolation and

J.

E . & AARTS, J . M . M . J . G . (1984) . Origin of specific elicitors of

characterization of an elicitor of chlorosis of necrosis isolated from intercellular fluids of compatible 9.

interactions of Cladosporium fulvum (syn . Fulvia fulva) and tomato. Plant Physiology 77, 642-647. DE WIT, P. J . G . M . & VAN DER MEER, F . E . (1986) . Accumulation of the pathogenesis-related leaf protein PI 4 as an early indicator of incompatibility in the interaction between Cladosporium fulvum (syn . Fulvia fulva) and tomato . Physiological and Molecular Plant Pathology 28, 203-214 .

10 .

DE WIT, P . J . G . M ., BuURLAGE, M . B . & HAMMOND, K . E . (1986) . The occurrence of host, pathogen and interaction-specific proteins in the apoplast of Cladosporiumfulrum (syn . hulvia fielva)-infected tomato leaves . Physiological and Molecular Plant Pathology 29, 154-172 .

11 .

DE WIT, P. J . G . M .,

IOMA, t . M . J . & JoosTEN,

M. H. A.

(19881 .

Race-specific elicitors and

pathogenicity factors in the Cladosporium fulvum-tomato interaction . In Physiological and Biochemical Plant-Microbial Interactions,

Ed . by N . Keen, T . Kosuge & L . L . Walling, pp . 111

119 . American

Society of Plant Physiologists, Rockville, MD . 12 .

Dow, J . M . & CALLOW,

J.

A.

(1979) . Partial characterization of the glycopeptides from culture

filtrates of Fulvia fulva (Cooke) Ciferri (syn . Cladosporium fulvum) the tomato leaf mould pathogen . Journal of General Microbiology 113, 57-66 . 13 .

DUBOIS, M ., GILLES, K . A ., HAMILTON, J, K ., REBERS, P . A . & SMITH, F . (1956) . Colorimetric method

14 .

for determination of sugars and related substances . Analytical Chemistry 28, 350-356 . GARAS, N . A., DOKE N . & Kud, J . (1979) . Suppression of the hypersensitive reaction in potato tubers by mycelial components from Phytophthora infestans . Physiological Plant Pathology 15, 117-126 .

15 .

HEATH, M . C . (1980) . Effects of infection by compatible species or injection of tissue extracts on the susceptibility of non-host plants to rust fungi . Phytopathology 70, 356-360 .

16 .

HIGGINS, V . J . (1982) Response of tomato to leaf injection with conidia of virulent and avirulent races of Cladosporium fulvum . Physiological Plant Pathology 20,

17 .

HIGGINS, V . J .

& DE WIT, P. J . G . M . (,1 985) .

145-155 .

Use of race and cultivar specific elicitors from

intercellular fluids for characterizing races of Cladosporium fulvum and resistant tomato cultivars . Phytopathology 75, 695-699 . 18 .

KEEN, N . T, (1975) . Specific elicitors of plant phytoalexin production : determinants of race specificity in pathogens? Science 187, 74-75 .

19 .

LAZAROVITS, G . & HIGGINS, V . J . (1979) . Biological activity and specificity of 'a toxin produced by

20 .

Cladosporium fulvum . Phytopathology 69, 1056-1061 . LAZAROVITS, G ., BHULLAR, B . S ., SUGIYAMA, H . J . & HIGGINS, V .

21 .

22 .

J . (1979) . Purification and partial characterization of a glycoprotein toxin produced by Cladosporium fulvum . Phytopathology 69, 1062-1068 . OKU, H ., SHIRAISHI, T . & OucHi, S . (1987) . Role of specific suppressors in pathogenesis of Mycosphaerella species. In Molecular Determinants of Plant Diseases . Ed . by S . Nishimura, C. P . Vance & N . Doke . pp . 145-156 . Japan Scientific Societies Press and Springer-Verlag, Tokyo . SCHMIDT, W . E . & EBEL, J . (1987) . Specific binding of a fungal glucan phytoalexin elicitor to membrane fractions from soybean Glycine max . Proceedings of the !Vational Academy of Sciences U .S.A . 84, 4117-4121 .