A single glycoprotein from Puccinia graminis f. sp. tritici cell walls elicits the hypersensitive lignification response in wheat

Physiological and Molecular Plant Pathology (1988) 33, 173-185

A single glycoprotein from Puccinia graminis f . sp. tritici cell walls elicits the hypersensitive lignification response in wheat (i .

KOGEL, B . BEISSMANN, H . J . REISENER

lostitut,fur Biologie III (Plant Physiology), Aachen University of Technology, 5100 Aachen, F.R .G .

and K . H .

KoGELt

Department o% Biochemistry, Max-Planck-Institutfïr Züchtungsforschung, 5000 Köln 30, W.-Germany (Acceptedfor publication January 1988)

Con A-binding glycoproteins were detected in germ tube walls of Puccinia graminis f. sp . tritici and appeared to be potent inducers of the hypersensitive lignification response . This response, which is typical of the large resistance reaction in wheat leaves, was preceded by a increase in extractable phenylalanine ammonia-lyase (PAL) activity, a key enzyme of the phenylpropanoid pathway . Germ tube glycoproteins were isolated from homogenized fungal cell walls by differential centrifugation and affinity chromatography on a Con A-Sepharose column . The most active glycoprotein was purified on a Mono Q anion-exchange column . The relative molecular mass of the molecule was determined in SDS-polyacrylamide gels to be 67 kD . The carbohydrate portion consists mainly of mannose (50°„) and galactose (47%) and contains the active part of the glycoprotein as demonstrated by the inability of pronase and trypsin digestion to influence activity . An elicitor-active glycoprotein with identical molecular mass and Con A-binding properties was isolated from intercellular fluids of rust-infected plants of the susceptible wheat cultivar Little Club indicating that the elicitor is released from fungal cell walls during infection . We discuss the role of Con A-binding proteins as inducers of non-host resistance in cereals .

INTRODUCTION

The term biotic elicitor is used to describe macromolecules of the plant pathogen origin which are able to induce physiological or biochemical responses associated with the expression of resistance . A wide range of fungal and bacterial metabolites, including polysaccharides [14, 35], glycoproteins [8, 37], peptides [7, 25], fatty acids [4] and hydrolytic enzymes [5] have been implicated as elicitors of resistance reactions in host pathogen interactions . The criteria used to assess elicitor activity include the visual estimation of cellular necrosis [1], measurement of electrolyte leakage [12], the determination of extractable activities of induced enzymes [11, 15], and accumulation of phytoalexins [9] or hydroxyproline-rich cell wall glycoproteins [13] . From a biochemical point of view, elicitors may therefore serve as useful tools both in studies of plant metabolic pathways typically activated during the expression of resistance and in studies flu whom correspondence should be addressed . Abbreviations used in text: CEP, crude elicitor preparation; Con A, concanavalin A; PAL, phenylalanine ammonia-lyase ; Pgt elicitor, elicitor of Puccinia graminis f, sp . tritici; SBA, soybean agglutinin; TBS, Tris buffer saline ; - WGA, wheat germ agglutinin . 0885-5765/88/050173+ 14 $03 .00/0 © 1988 Academic Press Limited



174 G . Kogel et al . concerned with the primary recognition phenomenon between a plant and a pathogen . A prerequisite for both kinds of studies is to provide well defined homogeneous elicitor molecules . In wheat, extreme resistance against the obligate parasite Puccinia graminis fI sp . lrilic i (the causal agent of wheat-stem rust disease), determined by the resistance gene Sr5, is expressed as hypersensitive cell death [34] . The first detectable event after infection is the appearance of a yellow autofluorescing halo around the cell wall penetrating haustorium . This is followed by the fluorescence of the whole cell which is caused by the formation oflignin or lignin-like substances [2,33] . Phytoalexins have not been described for the wheat-stem rust system . The very early occurrence of the autofluorescing material close to the interface ofhost and parasite suggests a recognition between fungal elicitor and host receptor at the haustorium-plasma membrane level as a prerequisite forsubsequent plant responses [21 1 . Therefore, several studies have attempted to isolate and characterize elicitor molecules with activities in wheat leaves . It was found that the lignification response in these leaves can be elicited by several abiotic and biotic elicitors [32, 27] . Among them chitosan and epoxystearic acid, isolated from dormant uredospores, are highly active . However, the most probable candidate for the natural elicitor in rust infected wheat leaves is a factor present in the germ tube wall of uredosporelings [22] . A crude extract from these walls, when injected into the intercellular spaces of the primary leaves of wheat, induced lignification . This reaction was preceded by an increase in the activities of phenylalanine ammonia-lyase (PAL) and other enzymes of the phenylpropanoid and lignin pathway : 4coumarate :CoA ligase, cinnamyl alcohol dehydrogenase and peroxidase [28] . The goal of this study was to purify and characterize the natural elicitor of the hypersensitive lignification response from the germ tube walls of P . graminis uredosporelings . This investigation is an essential prerequisite for further studies concerning the specific recognition event between P. graminis and wheat .

MATERIALS AND METHODS Plants, fungus and cultivation conditions

Near isogenic lines of the wheat cultivar Triticum aestivum L . cv . Marquis either carrying the Sr5 gene for rust resistance, or its sr5 allele, were kindly supplied by Dr K . Rohringer, Canada Agriculture Research Station, Winnipeg. For the preparation of intercellular fluids, the completely susceptible wheat cultivar T. aestivum L . cv . Little Club was used . The maintenance ofstock cultures ofPucciniagraminisfs sp . tritici, Erics . & Henn ., race 32, and the methods for growing and inoculating the plants were essentially as previously described [21] . Assay for PAL activity

Samples (50 µl) to be tested for elicitor activity were injected into the intercellular spaces of 7-day-old primary leaves as previously described [21] using a hypodermic syringe . Five primary leaves were harvested after 20 h and identical sections (3 . 5 cm from the injection site to each side) were immediately frozen in liquid nitrogen . PAL activity was determined photometrically [19] . In order to determine specific activities it was necessary to display a dose-response curve for every fraction to be tested by plotting PAL



Glycoprotein elicitor of hypersensitive lignification

175

activity (µkat kg - ' protein) against the logarithm of elicitor concentration . The specific elicitor activity of a sample is calculated from the carbohydrate concentration inducing half-maximal PAL activity . Specific elicitor activity is then expressed as specific enzyme activity per gram of carbohydrate (µkat kg - ' protein g - ' carbohydrate) . Assay for hypersensitive lign fcation response

Leaves were assayed for hypersensitive cell death at different time points after elicitor injection . The yellow autofluorescence of affected cells was observed with a Zeiss epifluorescence photomicroscope [27] using the following filter combination : G 390-440, FT 460, LP 470 . The phloroglucinol stain for lignin was applied as described before [361 . Preparation of the glycoprotein fraction from uredosporelings

Elicitor-active material was prepared from germ tube walls of uredosporelings by a method published previously [27] . This crude elicitor preparation (CEP), previously termed Pgt elicitor fraction, served as the starting material for further purification . The CEP (1-5 mg glucose equivalents) was dissolved in about 3 ml of 20 mM Tris-HCl, pH 7 . 4, containing 0 . 5 M NaCl and I mM each of Mg Cl2 , MnCl 2 and CaCl 2 (starting buffer), and applied to a column of Con A-Sepharose 4B (1 . 5 x 5 . 0 cm, Pharmacia) equilibrated with the same buffer at 4 ° C . The column was washed with starting buffer (10 column volumes) and then eluted with 4% (w/v) ofa-methyl-n-mannoside (Sigma) in starting buffer . The absorbance was recorded at 280 nm . Fractions of 2 ml were pooled, extensively dialysed against distilled water and analyzed for protein by the method of Bradford [3] and for carbohydrate by the anthrone procedure [10] . HPLC-anion-exchange chromatography

Anion-exchange FPLC was carried out on a prepacked (50 x 5 mm i .d .) Mono QHR 5/5 column (Pharmacia) using 25 mM Tris-HCl pH 8 . 5 (buffer A) and 1 M NaCl in the same buffer (buffer B) . The glycoproteins were eluted at a flow rate of I ml min -1 with the fbllowinglinearsaltgradient :0-35% bufferBin40 ml . Fractionsof0 . 5 ml were collected . Gel electrophoresis, blotting and lectin staining

SDS-polyacrylamide gel electrophoresis was performed using a modified Laemmli discontinuous buffer sytem [24] with 10% (w/v) acrylamide and 0 . 25% (w/v) bisacrylamide for the separation gel . Proteins were AgNO 3 stained [30] or they were transferred onto a nitrocellulose (NC) filter (Schleicher and Schüll) . The electroblotting procedure was modified [17] using a Trans-Blott cell (Bio-Rad) . The transfer was carried out at 400 mA for 2 h under extensive cooling at 4 ° C . The transfer buffer consisted of 25 mM Tris and 192 mM glycine, pH 8 . 3, with 20% (v/v) methanol . Transfer filters were blocked by incubation in TBS (50 mM Tris-HCl, 200 mM NaCl, ph 7 . 9) containing 4% w/v) bovine serum albumen (BSA) for I h at room temperature with agitation . Con A-binding glycoproteins were detected by the method of Hawkes [16] . In experiments with wheatgerm agglutinin (WGA) peroxidase and soybean agglutinin (SBA) peroxidase (both from Sigma) the transfers were blocked with 0 . 4% hipure liquid gelatine (Worland) in Tris buffer saline (TBS) and then incubated for 1 h in the lectin-peroxidase solution (10 .Lg ml - ' in TBS) . In dot blot binding assays nitrocellulose filters were dotted with 2 pl of samples and tested for lectin binding properties as described above .



176

G . Kogel eta/.

mass Fractions (3-5 mg ofcarbohydrates) ofCEP or ofglycoprotein fraction were dissolved in 10m1 of 10mM sodium citrate, pH 3, containing 20mM NaC1 and applied to a 16 x 700cm column of Ultrogel AcA 44 (LKB) or Biogel A l5 m (Bio-Rad) equilibrated with the same buflèr . For chromatography under denaturing conditions, samples were dissolved at 100 ° C in sample buffer containing 5°; ) SDS and columns were elutcd with the same buffer but containing 0'2° ) SDS . Molecular

determination

by get chromatography

Isolation of elicitor-active glycoproteins from SDS-gels

The CEP was run on SDS-polyacrylamide gels as described above . The gels were then frozen and each lane was cut into equal pieces . The pieces were ground with a mortar and pestle and extracted for 72 h with 10 ml of distilled water . About 100 pi ofn-butanol were added to prevent growth of micro-organisms . Samples were filter-sterilized through Sartorius membrane filters (0 . 45 p.m pore size) to remove polyacrylamide particles . SDS was removed by precipitation of the proteins in 90°, (v/v) acetone . The precipitate was dissolved in 2 ml ofTBS and extensively dialysed against distilled water . Periodate treatment and j3 -elimination

Lyophilized elicitor of P. graminis f. sp . tritici was treated with 002 mi Na10 4 for 22 h at 5 ° C as described in [9] . Excess of Na10 4 was destroyed by adding 02 ml of ethylene glycol to 08 ml sample followed by dialysis for 24 h against distilled water . For ßelimination, a procedure modified after Nakajima et al . [31] was used . Samples containing 2 mg of carbohydrate were lyophilized . After the addition of 800 .p1 of OE 1 N NaOH, the samples were maintained at 20 ° C for 24 h . The reaction was stopped with 1 N HCI (100 pi) . The reaction mixture was then adjusted to pH 9 with 40 mri (NH 4 ) 2 C0 3 and loaded on a DE 32 anion-exchange column (Whatman) . The proteins were eluted with 06 M NaG! and proteins and unbound material were assayed for elicitor activity . Pronase and trypsin treatment

Glycoprotein preparations were dissolved in 50 mri Tris-HCI, pH 78, containing 20 p.g ml' pronase (Calbiochem .) and 1 m CaCl 2 . The mixtures were incubated at 37 ° C for 96 h . The liberation ofamino acids was measured using the method of Moore, Spackman & Stain [291 . For trypsin digestion glycoproteins were dissolved in 50 mr'i (NH 4 ) 2 C0 3 pH 8 •0 containing 20 p.g ml 1 trypsin (Sigma) at 37 ° C for 4 ° C . The enzymes mixtures were then inactivated by incubating at 100 ° C for 15 min . by gas chromatography For gas chromatography the alditol acetate derivates for CEP, GF and FPLC -purifled elicitor fractions were prepared [18] . Separation was performed on a Fractovap 4160 chromatograph (Carlo Erba Strumentazione) using a prepacked capillary column 0V 1 (Machery & Nagel) . Samples were injected in acetone solution run on the following temperature programme : 5 min post-injection hold at 170 ° C, followed by a linear 3 ° C min temperature rise to 195 ° C and a 6 ° C min temperature rise to 260 °C . A nitrogen carrier gas flow rate of25 ml min was maintained . The injection port temperature was 160 ° C and the dual hydrogen flame ionization detector temperature was 260°C . Identfication of sugar components



Glycoprotein elicitor of hypersensitive lignification

177

TABLE I

Specific elicitor activities and lectin binding properties offractions obtained during the purification of the Pgt elicitor from mycelial cell walls of Puccinia graminis uredosporelings

Specific elicitor activity' Fraction CEP" GF' NB" A B C' D E F

Lectin binding activity

sr5

Sr5

Con A

WGA

8 .9 n .d .' 1.2 103 . 5 42 .0 175 . 7 8 .8 15-3 2-2

94 n .d .' 0.8 985 44 .0 183 .4 8.8 16 .9 3-7

+ +

+ +

+ + + + + +

+l +1+ +

SBA

'Activities are expressed as specific PAL activity per g injected carbohydrates in cv . Marquis either bearing the Sr5 gene for resistance or the sr5 gene . Lectin binding activities are expressed on a scale ; +, strong ; + / -, weak; -, none . "CEP: crude elicitor preparation . `GF : glycoprotein fraction . "Fractions NB-B obtained from the FPLC-step . Fraction C is referred to as Pgt elicitor . 'n .d . : not determined because of an excess of a-D-methylmannoside in the fraction .

Preparation of intercellular fluids of wheat leaves

Intercellular fluids of the wheat cv. Little Club were isolated using the method described by Deverall & Deakin [6] . Primary leaves were harvested 6 days after inoculation and typically, 1 . 5 ml of intercellular fluids were obtained from 160 leaves . RESULTS

Elicitor preparation and pur(cation

A crude elicitor preparation (CEP) from mycelial cell walls ofPgraminis f. sp . tritici, race 32, was active as elicitor of PAL activity when injected into 7-day-old wheat primary leaves (Table 1) . The final yield of the CEP was typically 2 mg of polysaccharide per 1 g of germinated uredospores . The presence ofglycoproteins in the CEP was analysed in dot-blot binding assays on nitrocellulose filters by using several peroxidase coupled lectins as probes (Table 1) . The CEP exhibited the highest affinity for Con A, a lectin which specifically binds to Dglucopyranose or D-mannopyranose residues . Significant, but lower, binding was observed with wheat germ agglutinin (WGA), a lectin specific for a-D-N-acetylglucosamine in sugar residues . Peroxidase labelled soybean agglutinin (SBA), a lectin specific for a-D-galactose or galactosamine did not bind to CEP . For further purification the CEP was applied to a Con A-Sepharose affinity column . A large peak, containing mainly protein, eluted in the column volume but the contained material was inactive in bioassays . Elicitor-active glycoproteins specifically eluted with 0 . 2 M a-D-methylmannoside .



178

G . Kogel et al. St

GF

C

kD

FIG . 1 . SDS-polyacrylamide gel electrophoresis of glycoproteins isolated from mycelial walls of

graminis uredosporelings . St, standards ; GF, glycoprotein fraction obtained from CEP by Con A-sepharose affinity chromatography ; C, Pgt elicitor obtained by FPLC anion-exchange chromatography of GF (Fig . 2, peak C) . The peptides were stained with AgNO ., . Puccinia

Gel electrophoresis of this glycoprotein fraction in the presence of SDS and ß-mercaptoethanol yielded several bands over a broad range of molecular masses (20-95 kD) (Fig . 1, lane 2) . Corresponding areas of unstained gels were excised and activity was mainly recovered in the 65-67 kD area . For further purification the glycoprotein fraction was subjected to FPLC-assisted anion-exchange chromatography resulting in a rapid purification of elicitor molecules (Fig . 2) . Besides the non-bound fraction four major peaks and several minor peaks were detected, when the Mono Qcolumn was eluted with a linear NaCl gradient at pH 8 . 5 . Peaks A and C both contained molecules with high elicitor activity . Peaks B, D, E and F appeared to have very low activity (Table 1) .

Pgt elicitor characterization Gel electrophoresis of peak C (from the FPLC separation profile, Fig . 2) which exhibited the highest elicitor activity revealed a single band when stained with AgNO 3 or Con A corresponding to a molecular mass of67 000 (Fig . 1, lane 3) . Since this glycoprotein is the



Glycoprotein elicitor of hypersensitive lignification

0

10

I j I 40 20 30

1 79

50

Volume (ml)

2 . FPLC anion-exchange chromatography of the glycoprotein fraction (GF) isolated from the mycelial walls of Pucciniagraminis uredosporelings : 200 Vg of GF in 500 µl of Tris-HCl buffer pH 8 . 5 were applied on a Mono QHR 5/5 column . Elution was performed with a linear gradient of 0-0-35 M NaCl . Peak C exhibited very high elicitor activity and is referred to as Pgt elicitor. FIG .

most active elicitor molecule from mycelial walls of P . graminis further studies have been restricted to this molecule which is referred to as Pgt elicitor. In order to determine the molecular mass of the native elicitor, CEP was chromatographed on an AcA 44 gel chromatography column . Molecules with elicitor activity were only found in the void volume of the column, indicating that the molecular mass is higher than 130000 . In the presence of 0 . 2% SDS, smaller elicitor molecules were recovered with a main peak corresponding to a molecular mass of approximately 65 000-69 000 . This suggets that the native elicitor is in the form of a self-associating complex . After chromatography on a Biogel A 1 . 5 m column, these complexes were dissociated into two distinct groups with molecular masses of 5 x 10 5 and over 1 . 5 x 106 , respectively . However, these groups were not distinguishable with respect to their protein band pattern after SDS-gel electrophoresis (data not shown) . Table 2 summarizes the results of experiments carried out to define the portion of the Pgt elicitor responsible for activity . Digestion with pronase or trypsin had no effect on elicitor activity but complete loss of activity was observed after periodate treatment . Reaction with 0 . 1 N NaOH for 24 h (ß-elimination) leads to a partial deglycosylation of the Pgt elicitor as evidenced by a protein band shift in SDS gels (data not shown) and the alteration of the carbohydrate/protein ratio . However, PAL-inducing activity was to some extent retained in the partially modified glycoprotein (Table 2, fraction b) . Gas liquid chromatography analyses demonstrated that glucose, mannose and galactose were the predominant sugars in the polysaccharide moieties of the fungal cell wall glycoproteins (Fig . 4) . on primary leaves of wheat Figure 3 shows the dose dependence of increase in PAL activity in wheat leaves treated with the CEP or the Pgt elicitor . The specific elicitor activity calculated from the half maximum PAL activity is higher (175 . 7 kat kg - ' g -I ) for Pgt elicitor than for CEP Effects of the Pgt elicitor



G . Kogel et al.

180 TAB LE

2

Induction of extractable activities of PAL in 7-day-old primary leaves ofthe wheat cv . Afarquis hearing the Sr5 gene for rust-resistance and the near isogenic susceptible line bearing the sr5 allele after treatment aith chemically or enzymatically modified Pgt elicitor

Composition of Pgt elicitor Treatment Pgt elicitor

carbohydrate

Untreated Pronase Trypsin #-elimination Fraction a' Fraction b" NaIO,

1',, PAL induction

, protein

Sr5

sr5

90 n .d .' n .d .

10 n .d . n .d .

100 92 90

100 93 86

87 70 n .d .

13 30 n .d .

7 30 0

5 35 0

'Fraction did not bind to DE 32 anion-exchange column at pH 9 after#-elimination . b Fraction bound to the anion-exchange column at pH 9 after#-elimination . `n .d . : not determined .

20

10

0

I

5

10

50

100

Elicitor concentration (µg glucose eq . ml

500 1000

-1 )

FIG . 3 . Dose response curves for the induction of extractable activities of phenylalanine ammonia-lyase (PAL) in wheat cv . Marquis primary leaves bearing the Sr5 gene for rust-resistance . Leaves were injected with varying concentrations of crude elicitor preparation (CEP, •) isolated from mycelial cell walls and pure Pgt elicitor (0) . The leaves were harvested 20 h after injecting the elicitor . Nearly identical curves were obtained with wheat leaves bearing the sr5 gene (data not shown) .

(8 . 9 kat) kg - t g - ') (Table 1) . This demonstrates the high activity of Pgt elicitor . With low elicitor concentrations PAL activity increases with increasing concentration of elicitor but eventually PAL activity reaches a maximum and then further increases in elicitor concentration have no additional effect . A resistance gene specific effect was not observed, as the wheat cv . with the sr5 allele had similar extractable PAL-activities after elicitor treatments as the cv, with the Sr5 resistance gene (Table 1) .



Glycoprotein elicitor of hypersensitive lignification

N

1 81

9

100

Man Glc Gol

o o, 7 N

o

80

7

60-

o m ô

cd

40

â

20

U

Fraction :

F

s

NB

A

B

C

D

E

F

CEP

Sugar composition offractions isolated from mycelial walls ofPuccinia graminis uredosporclings as determined by gas-liquid chromatographic analysis . NB-F : fractions obtained by FPLC anion-exchange chromatography (Fig . 2) . CEP, crude elicitor preparation ; Man, mannose ; Glc, glucose ; Gal, galactose . Values are calculated as percentage of total carbohydrates . FIG . 4 .

The glycoprotein produced a hypersensitive lignification response in epidermal and mesophyll cells which was visible 40 h after injection into wheat primary leaves . This reponse was accompanied by the appearance of yellow autofluorescence of affected cells as revealed by fluorescence microscopy . The same cells were stainable with phloroglucinol/HCl demonstrating the presence of lignin or lignin-like substances . Eliciting activities in intercellularfluids of infected wheat leaves The intercellular fluids from P . graminis-infected leaves of a compatible interaction of the susceptible cv . Little Club contained several Con A-binding glycoproteins which were not present in the uninfected leaves (Fig . 5) . When injected into the primary leaves of the wheat cv . Marquis carrying the Sr5 resistance gene the intercellular fluids exhibited elicitor activity . The most active component had a molecular mass of 67 000 as determined by SDS-gel electrophoresis . DISCUSSION and characterization of the Pgt elicitor FPLC anion-exchange chromatography on Mono Q together with affinity chromatography on Con A-Sepharose provided an excellent method for purifying the Pgt elicitor molecule from the mycelial walls of P. graminis uredosporelings . The FPLCelution profile along with the appearance of a single band on SDS-gels strongly suggests that the Pgt elicitor fraction was highly pure . Interestingly, the Pgt elicitor is a Con A-binding glycoprotein and represents only one of several proteins with very similar properties with regard to molecular mass as revealed by gel electrophoresis of the total glycoprotein fraction (Fig . 1) and lectin binding activities (Table 1) . However, these other molecules have little or no eliciting activity at all (Table 1) . Our data, therefore, demonstrate that Con A-binding activity may be a necessary but not a sufficient criterion for elicitor activity . Purification



1 82

G . Kogel IF,

IF1

et al.

C kD

-92

400 -66

-45

-31

-21

FIG . 5 . Western-blot analysis of Pgt elicitor and intercellular fluids isolated from the susceptible wheat cv . Little Club . After SDS-gel electrophoresis peptides were blotted on nitrocellulose membranes and stained using the Con A-peroxidase method . IF S, intercellular fluids obtained from noninfected leaves ; IF., intercellular fluids obtained from rust infected leaves ; C, Pgt elicitor (see Fig . 2) .

The retention of activity after pronase or trypsin digestion indicates that the carbohydrate moiety contains the active site of the elicitor . This is strengthened by the fact that periodate oxidation destroys activity . Treatment of Pgt elicitor with 0 . 1 N NaOH did not release elicitor-active carbohydrate from the protein core, although partial deglycosylation took place . Release of carbohydrates implies that the Pgt elicitor contains O-glycosidic linkages because only these are cleaved by mild alkaline treatment . The carbohydrate moieties of the Pgt elicitor consists mainly of mannose (50 0/0 ) and galactose (47%) and to a lesser extent glucose (3%) . Apparently, all other elicitor-active FPLC peaks (A and B) have the same low amount of glucose whereas inactive fractions (peak D, E and F) contain a very high portion of glucose . The lack of binding activities for SBA, a lectin which specifically binds to terminal galactose or .N-acetylgalactosamine in sugar side chains, demonstrates that a-1-4 bound galactose does not occupy the terminal positions of the glyco-moieties . Although glycoproteins containing .Nacetylglucosamine were identified, using WGA as a probe in dot-blot binding assays, the gas liquid chromatography analysis does not support this conclusion . We assume, however, that the glycoproteins contain such a small amount of the amino sugar, probably in terminal positions, that it is only detectable in the extremely sensitive dotblot assay .



Glycoprotein elicitor of hypersensitive lignification

183

The 67 kD glycopeptide is the smallest molecule with eliciting activity found so far in cell walls of P. graminis . In the absence of SDS the molecule forms high molecular mass self associating complexes even at low concentrations, as shown by gel chromatography of CEP . The molecular mass of the predominant elicitor molecule determined by gel chromatography ofCEP under denaturing conditions coincided with values obtained by SDS gel electrophoresis . Physiological and biochemical responses of the plant after elicitor treatment "The measurement of extractable PAL activities provided a convenient method for the quantitative determination of elicitor activity . Since previous experiments have shown that elicitor-induced PAL activity is maximal 20 h after inoculation [20] activity was determined at that time-point . Correlation of elicitor activity with PAL induction (either on the mRNA or enzyme level) was practised by several workers [15, 23] . This enzyme is necessary in the biosynthesis oflignin, and a drastic increase of enzyme activity precedes the response to avirulent strains of stem rust in wheat leaves bearing the Sr5 allele for resistance [20] . At the microscopic (cell-collapse, autofluorescence) and biochemical levels (increase in PAL activities), symptoms caused by an avirulent fungus are indistinguishable from those produced by the 67 kD glycoprotein that we have now isolated . However, this glycoprotein does not show cultivar specificity . Increase in PAL activities in wheat cv . Marquis treated with as little as 10 ng of Pgt elicitor per leaf appeared to be independent of the presence or absence of the Sr5 gene for rust resistance . The shapes of the dose-response curve--shown only for wheat bearing the Sr5 gene (Fig . 3) -did not significantly differ between the two cultivars . We do not know yet whether the Pgt elicitor shows specificity for other resistance genes or whether it is rust strain-specific . Alternatively, it may play a role as an inducer of the non-host resistance in wheat . This hypothesis is strengthened by the fact that the Pgt elicitor is also active in several other cereals (e .g . barley ; data not shown) which are nonhosts for P . graminis f. sp . tritici . In several plant-pathogen interactions Con A-binding glycoproteins appear to play a decisive role as effector molecules producing identical or similar disease symptoms as the pathogen they are isolated from ([12, 26] and Kogel & Knogge, unpubl .) . These molecules are not cultivar-specific indicating that their recognition is a very basic phenomenon in plants . Eliciting activity in intercellular fluids of infected wheat leaves Detection of elicitor activity in intercellular fluids from infected leaves implies that the source is either the pathogen or the pathogen-host interaction . Several Con A-binding proteins may be pathogen-induced glycoproteins of plant origin which are exported into the intercellular spaces and may therefore be regarded as a special class of pathogenesisrelated proteins (PR-proteins) . However, Holden & Rohringer [17] found that the glycoprotein pattern of intercellular fluids obtained from wheat leaves infected with the leaf rust fungus, Puccinia recondita, was markedly different from that of intercellular fluids from the same host infected with the stem rust fungus . They proposed, therefore, that the intercellular fluids from rust-infected leaves may be a useful source of surface or extracellular proteins from the parasitic mycelium . The apparently identical molecular mass of the Pgt elicitor isolated from cell walls and the eliciting compound in the intercellular fluids, respectively, suggests that the rust

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elicitor is released into the intercellular spaces during infection . It might be that diffusibility is a prerequisite for its effectivity . Deverall & Deakin [6] recently found chemically undefined eliciting activity in intercellular fluids from P . recondita-infected wheat, ev . Chinese Spring, causing paleness, chlorosis and necrosis in uninfected primary leaves . Since the activity could not be correlated with the presence of the Lr20 gene for resistance, they conclude that the intercellular fluids are neither rust strain- nor gene-specific in their eliciting activity . De Wit et al . [7] isolated a peptide showing specific elicitor activity (apparent molecular mass 5500) from the intercellular fluids of a compatible Cladosporium fulvum-tomato interaction which turned out to be determined by the virulence gene present in the fungal race . The question whether eliciting activity associated with intercellular fluids is only effective in the presence of a specific resistance gene (e .g . the Sr5 gene) is under investigation . The presence but ineffectiveness of elicitor molecules in the infected cv . Little Club, which lacks any resistance gene, may indicate that the eliciting activity of the intercellular fluids needs a resistance gene product (receptor?) to be active . On the other hand, the fact that no specificity is observed with the Pgt elicitor isolated from mycelial cell walls of uredosporelings, so far, points to the possibility that in wheat-stem rust interactions suppressors may act as factors of race-cultivar specificity . This work was supported by the Deutsche Forschungsgemeinschaft . We thank Mrs I . Eickmeyer for excellent technical assistance and Dr E . Ziegler for developing the temperature programe for optimal sugar separation by GC . REFERENCES 1 . ANDERSON-PROUTY, A. J . & ALBERSHEIM, P . (1975) . Host-pathogen interactons : VII . Isolation of a pathogen-synthesised fraction rich in glucan that elicits a defense response in the pathogens host. Plant Physiology 56, 286-291 . 2 . BEARDMORE, J ., RIDE,J. P . & GRANGER, J . W . (1983) . Cellular lignification as a factor in the hypersensitive resistance of wheat to stem rust . Physiological Plant Pathology 22, 209-220 . 3 . BRADFORD, M . M . (1976) . A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254 . 4 . BosTOCK, R . M ., KU, J . A. & LAINE, R . A . (1981) . Eicosapentaenoic and arachidonic acids from Phytophthora infestans elicit fungitoxic sesquiterpenes in the potato . Science 212, 67-69 . 5 . COLLMER, A . & KEEN, N . T . (1986) . The role of pectic enzymes in plant pathogenesis . Annual Review of Phytopathology 24, 383-409 . 6 . DEVERALL, B . J . & DEAKIN, A . L . (1985) . Assessment of Lr20 gene-specificity of symptom elicitation by intercellular fluids from leaf rust-infected wheat leaves . Physiological Plant Pathology 27, 99-107 . 7 . DE WIT, P . J . G . M ., HOFFMAN, J . E ., VELTHUIS, G . C . M . & KUC, J . A. (1985) . Isolation and characterization of an elicitor of necrosis isolated from intercellular fluids of compatible interactions of Cladosporium fulvum (syn. Fulvia fulva) and tomato . Plant Physiology 77, 642-647 . 8 . DE WIT, P . J . G . M . & KODDE, E . (1981) . Further characterization and cultivar specificity ofglycoprotein elicitors from culture filtrates and cell walls of Cladosporium fulvum (syn . Fulvia fulva) . Physiological Plant Pathology 18, 297-314 . 9 . 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 . 10 . DISCHE, Z . & BORENFREUND, E . (1951) . Anew spectrophotometric method for the detection and determination of ketosugars and trioses . journal of Biological Chemistry 192, 583-587 . 11 . DIXON, R. A ., DEY, P . M . & WHITEHEAT, I . M . (1981) . Purification and properties of chalcone isomerase from cell suspension cultures ofPhaseolus vulgaris . Biochimica et Biophysica Acta 715, 25--33 . 12 . Dow, J . M . & CALLOW, J . A . (1979) . Leakage of electrolytes from isolated leaf mesophyll cells of tomato induced by glycopeptides from culture filtrates of Fulvia fulva (Cooke) Ciferri (syn . Cladosporium fulvum) . Physiological Plant Pathology 15, 27-34 .



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