Proc. Natl. Acad. Sci. USAVol. 85, pp. 782-786, February 1988Botany

Several "pathogenesis-related" proteins in potato are1,3-,f-glucanases and chitinases

(Solanum tuberosum/Phytophthora infestans/fngal elicitor/extraceilular enzymes/cell-wail degradation)

ERICH KOMBRINK, MARTIN SCHRODER, AND KLAUS HAHLBROCKMax-Planck-Institut for Zuchtungsforschung, Abteilung Biochemie, D-5000 Koin 30, Federal Republic of Germany

Communicated by Paul K. Stumpf, October 2, 1987 (receivedfor review July 29, 1987)

ABSTRACT Chitinase {poly[1,4-(N-acetyl-j-D-gluCOSami-nide)Jglycanohydrolase, EC 3.2.1.14} and 1,3-fi-glucanase(1,3-fi-D-glucan 3-glucanohydrolase, EC 3.2.1.6) activities in-creased rapidly in potato (Solanum tuberoswm) leaves inocu-lated with the pathogenic fungus Phytophthora infestans ortreated with fungal elicitor. The enzyme activities were re-solved into a total of two distinct 1,3-13-glucanases and sixproteins with chitinase activity. By several criteria, all of theseproteins are classified as "pathogenesis-related" proteinswhose biochemical functions have so far been unknown. Someof them constitute a major portion of the proteins accumulat-ing in the intercellular space of infected potato leaves and areassumed to play an important role in pathogen defense.

"Pathogenesis-related" (PR) proteins have been describedin plants infected with various types of potential pathogens:fungi, bacteria, viruses, and viroids (1). The most widelyused operational definition ofPR proteins is that of polypep-tides with relatively low molecular weights (Mr, 10,000-40,000) that accumulate extracellularly in infected planttissue, exhibit high resistance to proteolytic degradation,and often, but not always, possess extreme isoelectric points(1). PR proteins have been studied in several systems withrespect to physical properties, relationship to the corre-sponding mRNAs and cDNAs, and gene activation followingpathogen infection or elicitor treatment (1-9). However, thebiochemical functions of PR proteins have not been re-ported.We have investigated the proteins accumulating in the

intercellular space of potato leaves following inoculationwith Phytophthora infestans or treatment with P. infestans-derived elicitor. By this and other criteria, they are classifiedas PR proteins. Several of these proteins were purified andfound to be 1,3-p3-glucanases (1,3-f3-D-glucan 3-glucanohy-drolase, EC 3.2.1.6) and chitinases {poly[1,4-(N-acetyl-P-D-glucosaminide)]glycanohydrolase, EC 3.2.1.14}, suggestingthat they are involved in the degradation of fungal andbacterial cell walls.

MATERIALS AND METHODSPlant, Fungus, and Elicitor. Potato plants (Solanum tube-

rosum L. cv. Datura) were grown from tubers in a green-house under controlled conditions (10). The third and fourthleaves of 8-week-old plants were either inoculated with P.infestans spores or treated with elicitor derived from P.infestans culture filtrate (10, 11). The elicitor was applied insterile aqueous solution (100 ,ug/ml glucose equivalents)through the petioles. Analogous treatments with water aloneserved as controls where indicated (11).

Intercellular Washing Fluid and Leaf Extract. The intercel-lular washing fluid of potato leaves was isolated according tode Wit and Spikman (12) with minor modifications. Leaveswere vacuum-infiltrated with distilled water at room temper-ature for 30 min. The intercellular washing fluid was col-lected by centrifugation of the leaves at 2200 x g for 10 minin specially designed tubes (12) and stored at - 20'C. Crudeleaf extracts were prepared by grinding 1 g of tissue in liquidnitrogen and extracting the fine powder with 2 ml of buffer(0.2 M Tris HCl, pH 7.8/14 mM 2-mercaptoethanol) in thepresence of 0.1 g of Dowex 1X2 equilibrated with the samebuffer. The clear supernatant obtained by centrifugation(20,000 x g, 20 min) was used for enzyme assays andelectrophoresis.Enzyme Assays and Protein Determination. 1,3-p-Glu-

canase activity was measured as described (13), except that4-hydroxybenzoic acid hydrazide (Sigma Chemie, Munchen,F.R.G.) was used as a color reagent to determine reducingsugars (14). Chitinase and lysozyme activities were deter-mined by using published procedures (13, 15, 16). Reactionproducts were analyzed by thin-layer chromatography onSili-Gel 60 plates (Merck, Darmstadt, F.R.G.). 1,3-,8-Glucanase products were separated in the solvent systemn-butanol/acetic acid/H20 (4:1:1) and detected on plates bythe aniline/diphenylamine color reagent (17). Chitinaseproducts were characterized as described (15). Protein wasmeasured according to Bradford (18) using the Bio-Rad(Munchen, F.R.G.) dye reagent and bovine serum albuminas a standard.Enzyme Purification. All operations were carried out at

0C-40C. 1,3-,3-Glucanase was purified from the intercellularwashing fluid of P. infestans-infected leaves [280 ml,adjusted to 50 mM sodium citrate (pH 5); 160 mg of protein].Acidic proteins were removed by adsorption to DEAE-cellulose. About 10 g of DEAE-cellulose was stirred in theintercellular washing fluid for 1 hr at 40C and removed bycentrifugation. The clear supernatant, adjusted to pH 4.2,was passed through a CM-cellulose column (2.5 x 15 cm)equilibrated with 50 mM sodium citrate (pH 4.2). Afterwashing with the same buffer, bound proteins were elutedwith a linear NaCl gradient (0-500 mM). Fractions contain-ing 1,3-f3-glucanase activity were pooled, dialyzed against 50mM sodium formiate (pH 4.2), and subjected to cation-exchange fast protein liquid chromatography using a Mono SHR 5/5 column (Pharmacia) equilibrated with the samebuffer. Bound proteins were eluted with a linear NaClgradient (0-500 mM) in 50 mM sodium formate (pH 4.2) andyielded two 1,3-p-glucanase fractions.

Chitinase was purified from leaves treated for 24 hr withelicitor, following the procedure described by Boller et al.(15) except that the leaves (60-100 g) were homogenized in aWaring Blendor with 2 vol of 100 mM sodium acetate (pH 5).Affinity chromatography on regenerated chitin (15, 19),

Abbreviation: PR protein(s), pathogenesis-related protein(s).

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preceded by heat denaturing and fractionating (NH4)2SO4precipitation of the crude cell extract, yielded a single sharppeak of chitinase activity. This fraction was dialyzed (50 mMpotassium phosphate, pH 6.8/100 mM NaCl) and concen-trated to 2 ml with an Amicon ultrafiltration system fittedwith a Diaflo YM-10 membrane. The concentrated solutionwas applied to a Bio-Gel P-60 column (1.5 x 98 cm)equilibrated with 50 mM potassium phosphate, pH 6.8/100mM NaCl/0.02% NaN3, and the column was washed withthe same buffer at 5 ml/hr. Chitinase activity eluted in twobase-line separated peaks (A and B). The most activefractions of each peak were combined, dialyzed against 20mM sodium formate (pH 4.2), and separately subjected tocation-exchange fast protein liquid chromatography on aMono S HR 5/5 column (Pharmacia). Proteins of fraction Awere eluted with a linear gradient of 0-500 mM NaCl (25 ml)in 20 mM sodium formate (pH 4.2) and yielded two peakswith chitinase activity. A complex salt gradient in thesodium formate buffer was required to separate the otherfour chitinases present in fraction B: 0-40 mM NaCI (2 ml),40 mM NaCI (8.5 ml), 40-250 mM NaCl (16.5 ml), 250-500mM NaCl (3 ml).

All purified 1,3-p-glucanase and chitinase isoforms ap-peared homogeneous on NaDodSO4/polyacrylamide gelsstained with Coomassie brilliant blue R-250 or AgNO3.

Antisera. Rabbit antisera were produced by standardprocedures (20) using 100 ,g of protein for each of the twopurified 1,3-p-glucanase fractions for the first injection and50 ,ug of protein each for subsequent booster injections.Bean chitinase antiserum was a kind gift of T. Boller (Basel,Switzerland).

Polyacrylamide Gel Electrophoresis and Immunoblotting.Discontinuous NaDodSO4/polyacrylamide gel electrophore-sis was carried out according to Laemmli (21) using 0.8-mmslab gels containing 12.5% acrylamide. Details have beendescribed elsewhere (22). Immunoblots were prepared es-sentially as described by Towbin et al. (23). Proteins weretransferred electrophoretically at 30 V (-200 mA) overnightto nitrocellulose sheets (BA 85; Schleicher & Schull, Dassel,F.R.G.) in 25 mM Tris glycine (pH 8.3) (23) or 25 mMethanolamine glycine (pH 9.5) (24), each containing 20%methanol. Peroxidase-conjugated swine antibody to rabbitIgG (Dakopatts, Hamburg, F.R.G.) was used as a secondantibody. Blots were developed in a mixture containing0.018% 4-chloro-1-naphthol, 6% methanol, 0.012% H202 inphosphate-buffered saline (137 mM NaCI/3 mM KCI/9 mMsodium phosphate, pH 7.4). The peroxidase reaction wasterminated after 5-10 min by several washes with water.

RESULTSProtein Accumulation in Infected and Elicitor-Treated Po-

tato Leaves. The amount of extractable protein from theintercellular space increased rapidly following inoculation ofpotato leaves with. P. infestans zoospores. The proteinconcentration in the intercellular washing fluid increasedfrom -0.1 to 1 mg/ml within 3-5 days postinoculation.Analysis by NaDodSO4/polyacrylamide gel electrophoresisrevealed a massive accumulation of at least nine majorproteins in the Mr range of -10,000 to 40,000 (Fig. 1). Otherproteins, particularly those of higher molecular weight,stayed constant or decreased in concentration. Some pro-teins accumulating in the intercellular washing fluid werealso present in the cells. Changes in their concentrationswere less obvious because of the abundance of constitutivelyexpressed intracellular proteins.

Fig. 2 shows the changes in protein content of the inter-cellular washing fluid from detached leaves treated forvarious periods of time with P. infestans elicitor. Compari-

-92.5-66.2

-45.0

- "I_-

-31.0

-21.5

0 12 24 36 48 60 72 84110

Time post inoculation (h )

FIG. 1. Accumulation ofPR proteins in the intercellular space ofpotato leaves inoculated with P. infestans spores. Detached leaveswere sprayed with a spore suspension (5 x 104 spores per ml of P.infestans, race 1) and the intercellular washing fluid was isolated atthe times indicated. After concentration, samples equivalent to 1001. of intercellular washing fluid were separated by NaDodSO4/polyacrylamide gel electrophoresis and proteins were stained withCoomassie brilliant blue. Protein in the intercellular washing fluidincreased from 10 ,ug/ml (0 hr) to 70 jig/ml (110 hr).

son with Fig. 1 demonstrates that elicitor treatment and P.infestans infection induced similar changes.

Increases in 1,3-(3-Glucanase and Chitinase Activities. Theactivities of 1,3-3-glucanase and chitinase were low inhealthy leaves but were stimulated strongly by infection orelicitor treatment (Fig. 3). Both enzyme activities increasedmore rapidly in elicitor-treated than in P. infestans-inoculated leaves, probably because of the rapid uptake anddistribution of elicitor throughout the leaf, as opposed to thetime lag caused by germination and tissue penetration of thefungus. Despite this difference in timing, the enzyme activ-ities reached similar levels in infected and elicitor-treatedleaves.

Mr x 10_3

-92.5-66.2

-45.0

-31.0

4..-* -21.5

_ -14.4

0 24 48 72 48c

Time after onset oftreatment ( h )

FIG. 2. Accumulation ofPR proteins in the intercellular space ofpotato leaves treated with P. infestans elicitor. The elicitor wasapplied through the petiole by placing the leaf in the elicitor solution(50 ,ug/ml carbohydrate equivalents) for 24 hr and then in water. Ina control experiment (lane 48c) leaves were treated with waterinstead of elicitor solution. Samples equivalent to 100 ,l of intercel-lular washing fluid were subjected to NaDodSO4/polyacrylamidegel electrophoresis and proteins were stained with Coomassie bril-liant blue. Protein in Qhe intercellular washing fluid increased from12 zg/ml (0 hr) to 100 IZg/ml (72 hr).

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INFECTION

E

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.C

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ELICITOR

0 24 48 72 96 0 24 48 72

Time after onset of treatment (h)

FIG. 3. Time course for the increase in 1,3-,B-glucanase (BGL, Aand A) and chitinase (CHN, e and o) activities in potato leaves afterinfection with P. infestans spores (Left) or treatment with P.infestans elicitor (Right). Open symbols, control experiment withwater. Cell extracts were prepared from leaves after intercellularwashing fluid isolation. Enzyme activities are based on protein (cellextract) or intercellular washing fluid (IWF) volume as constantparameters, respectively. Chitinase activity is expressed in unitsdefined as soluble products (1000 cpm/min) released.

Purification and Properties of the Enzymes. Several pro-teins from the intercellular washing fluid of P. infestans-infected leaves (% hr postinoculation) were purified toapparent homogeneity. The predominant protein band at Mr

36,000 (Fig. 1) had 1,3-j3-glucanase activity and wasresolved by cation-exchange fast protein liquid chromatog-raphy into two distinct proteins. Each of them appeared as asingle spot on NaDodSO4/polyacrylamide gels stained withCoomassie brilliant blue or AgNO3. They differed slightly inrelative molecular weight [Mr, =36,000 and =36,200 (Fig.4A)] and charge [pI, 9.6 and 9.8, respectively (Fig. 4B)].

Reaction products obtained after prolonged incubation oflaminarin with each of the two purified enzymes were aseries of oligosaccharides differing in size, whereas glucosewas not detectable. These results indicate that both enzymesare endo-1,3-J3-glucanases.

In contrast to 1,3-f3-glucanase, chitinase activity wasassociated with less abundant proteins. Several isoforms ofthis enzyme were purified from total extracts-i.e., intercel-lular washing fluid and intracellular fluid-of whole leavesstimulated for 24 hr with elicitor. An early efficient purifica-tion step was affinity chromatography on regenerated chitin,from which chitinolytic activity eluted as a single sharp peakconsisting of several proteins. Further purification on asizing column (two peaks) followed by fast protein liquidchromatography using two different salt gradients for elution(two and four peaks; Fig. 5) yielded six apparently pureproteins with chitinase activity. The relative molecularweights, given in the order of elution, were 38,000 and 38,700for the large isoforms and 33,200, 32,600, 34,300, and 33,200for the small ones. All isoforms are basic proteins with pIvalues above 7. The six purified chitinases represented mostof the chitinolytic activity detected in potato leaf tissue.Preliminary results indicate that most of the isoforms accu-mulate in the intercellular washing fluid after P. infestansinfection or elicitor treatment, whereas a small number,probably two, are localized intracellularly. Differential stain-ing with concanavalin A on protein blot filters suggests thatonly the latter are glycosylated.The soluble products formed from [3H]chitin after pro-

longed incubation with the individual chitinases were ana-lyzed by thin-layer chromatography. Oligomers of 2-5 units(chitobiose, -triose, -tetraose, and -pentaose) in variablerelative amounts were the main products of all six isoforms.Free N-acetylglucosamine was found maximally in 7% of thetotal products after 14 hr of incubation, and acetate was notdetectable. Thus, all six enzymes are endochitinases withoutdeacetylase activity. The chitinase fraction eluted from thechitin affinity column was positive in an assay for lysozymeactivity.

Serological Relationships. Antisera raised in rabbits againsteach of the two 1,3-f3-glucanases from potato leaves andantiserum against chitinase from bean leaves (kindly pro-vided by T. Boller) were used to study the serologicalrelationships of the purified proteins. Both 1,3-f3-glucanaseswere selectively recognized on immunoblots by the twoanti-1,3-f3-glucanase antisera, and all six chitinases showedstrong cross-reactivity with the bean anti-chitinase anti-serum (Fig. 6). No immunological cross-reactivity betweenthe 1,3-f3-glucanases and chitinases was observed.

A B

MrI1O3 1 2 1 2 pI

92.5-66.2-

45.0-

31.0-

21.5-

14.4-

6.9

-7.3

-8.3___-9.45

-10.65

FIG. 4. Analysis on NaDodSO4/polyacrylamide (A) and isoelec-tric focusing (B) gels of the two 1,3-,B-glucanases (lanes 1 and 2)purified to apparent homogeneity from potato leaves. Proteins (6 ,ugper lane) were stained with Coomassie brilliant blue. The positionsof M, and pI marker proteins are indicated.

DISCUSSIONBy all conventional criteria (1), the 1,3-f3-glucanases andchitinases described here are PR proteins. They constitutenearly all detectable activities of the two enzymes and someof the most abundant PR proteins in this system. Hence,1,3-,B-glucanase and chitinase activities are two major func-tions of PR proteins in potato. This conclusion seems to bemore generally applicable in view of similar independentobservations made recently by Legrand et al. (25) withregard to PR protein accumulation in tobacco mosaic virus-infected tobacco leaves. Beyond this, however, biochemicalfunctions of PR proteins have not been reported. Althoughthe stimulation of 1,3-,B-glucanase and chitinase activities ininfected plant tissue has been observed in several species(26-28), the identity with PR proteins has, to our knowledge,not been recognized previously. In any case, our resultssuggest that the operational, and under certain conditionsmisleading, term PR protein will be eliminated more andmore and replaced by precise functional names.

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0

Lfl'V

-c

I0

II

C0

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-

a6c

(1

0 5 10 15 0 5 10 15 20 25

Elution volume (ml)

FIG. 5. Cation-exchange chromatography on a Mono S column of two chitinase fractions (A and B) obtained by Bio-Gel P-60chromatography. Chitinase activity (0) is expressed in units as defined in Fig. 3. The absorbance at 280 nm (arbitrary units, ) and the NaClconcentration (---) are also shown.

The similarity between the accumulation patterns of PRproteins in infected and elicitor-treated potato leaves sup-ports the previous assumption that elicitor treatment closelymimics a true infection (11) and, moreover, demonstratesthat the accumulated PR proteins are encoded and synthe-sized by the host and not by the pathogen.

All detected isoforms of the two lytic enzymes werepurified to apparent homogeneity. Their properties, includ-ing relatively low molecular weights, basic nature, low pHoptima for catalytic activity, high temperature stability, andendolytic substrate cleavage, were similar to those of 1,3-/-glucanases and chitinases from other plant sources (27, 29,30). The results of immunoblotting experiments suggest thatthe 1,3-f3-glucanase (data not shown) isoforms possess sim-ilar antigenic sites; the same is true for the chitinase iso-forms.The predominant extracellular localization of 1,3-,B-

glucanases and chitinases is an observation that, so far as weknow, has not been reported previously, although otherhydrolases as well as some of the classical PR proteins areknown to occur in intercellular spaces (1, 31-35). Thus, bothenzymes must be secretory proteins. This is in agreementwith recently published sequence data for a cDNA comple-

1 2 3 4 S 6 Mrx10-3-92.5-66.2

-45.0__

-31.0

-21.5

-14.4

FIG. 6. Immunoblot analysis of the six chitinases purified toapparent homogeneity from potato leaves. Proteins (0.3 ,ug per lane)were separated by NaDodSO4/polyacrylamide gel electrophoresis,transferred to nitrocellulose, and probed with antiserum raisedagainst bean leaf chitinase at a 1:1000 dilution. Lanes 1-6 corre-spond to the activity peak numbers obtained by cation-exchangechromatography (Fig. 5).

mentary to bean chitinase mRNA whose product contains a27-residue amino-terminal leader peptide (36). The synthesisof chitinase as a precursor peptide was interpreted as beingconsistent with its previously established vacuolar localiza-tion in bean leaves (37). Now, in view of the extracellularlocalization of at least some of the chitinases in potatoleaves, the questions arise as to whether different isoformsaccumulate at different intra- and extracellular locationsand, if so, how this is regulated.The participation of hydrolytic enzymes, such as 1,3-,f-

glucanase and chitinase, in the defense response of plants topathogens has long been proposed, because many fungi andbacteria contain 1,3-j3-glucans, chitin, or other possiblesubstrates of these enzymes as cell-wall components (38).The capability of the enzymes to degrade cell walls ofpotential pathogens has been established (27), and it hasrecently been demonstrated that chitinase can effectivelyrestrict growth of a test fungus (39). This may also apply tobacteria, since most, possibly all, plant chitinases havelysozyme activity (ref. 15 and this paper).The occurrence of hydrolytic enzymes or PR proteins is,

however, not restricted to pathogen-infected tissue, but ithas also been observed under other conditions of stress-e.g., wounding, plasmolysis, or treatment with chemicals,and during flower development or senescence (22, 30, 31,40-44). This raises the questions of their possible dual ormultiple functions in stress and development and the mech-anisms involved in the differential expression of the respec-tive genes. In this connection, an example of in situ RNAhybridization by using cDNA specific for a PR protein-encoding mRNA has recently demonstrated the rapid acti-vation of the corresponding gene(s) around the necrotic areaof a fungal infection site in a parsley leaf (45).

We thank Ms. Brigitte Hebborn for technical assistance, T. Bollerfor bean chitinase antiserum, J. Parker for critical reading and I.Muller for typing the manuscript, and M. Legrand and B. Fritig forcommunicating recent results prior to publication. This work wassupported by the Max-Planck Society and Fonds der ChemischenIndustrie.

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