Monoclonal Antibodies to Glycoprotein Antigensof aFungal … · compatible bacteria, that the presence ofthis avirulence gene wasnecessaryforincompatibility in the presenceofplanttissue

Plant Physiol. (1987) 85, 508-5 150032-0889/87/85/0508/05/$0 1.00/0

Monoclonal Antibodies to Glycoprotein Antigens of a FungalPlant Pathogen, Phytophthora megasperma f. sp. glycinea1

Received for publication March 2, 1987 and in revised form June 15, 1987

KEITH L. WYCOFF, JODY JELLISON2, AND ARTHUR R. AYERS*3Department ofCellular and Developmental Biology, Harvard University, Cambridge, Massachusetts 02138(K.L.W., J.J., A.R.A); and Department ofBiology, Cedar Crest College,Allentown, Pennsylvania 18104 (A.R.A.)

ABSTRACT

Genetic studies of plants and their pathogens indicate that dominantalleles for resistance in hosts are complemented by corresponding domi-nant alleles for avirulence in pathogens. Products of these genes have notyet been identified. We have produced murine monoclonal antibodies(mAbs) to extraceliular antigens of the fungal soybean pathogen Phyto-phthora megasperma f. sp. glycinea (Pmg, race 1) as part of a largereffort to identify antigenic determinants associated with particular avi-rulence genes. Thirty-six independent mAbs have been characterized bybinding to Western blots of Pmg extracellular glycoproteins and byenzymewlinked immunosorbent assay with glycoproteins modified bytreatment with periodate, a-mannosidase, and endo-p-N-acetylglucosa-minidas H. The mAbs are predominantly carbohydrate-specific and canbe plaed in six groups based on interactions with Pmg glycoproteins.Binding patterns of various mAbs to Western blots indicate that Pmgproteins may have single or multiple types of attached carbohydrateantigens. Races of Pmg with differing avirulence genes exhibit morecharacteristic differences by Western blot analysis than by protein stain-ing of glycoprotein profiles. Several of the mAbs show much higherreaction levels to glycoproteins from race 1 than from two other races.All of the glycoprotein-specific mAbs cross-react with purified myceLialwalls.

Many host-pathogen interactions exhibit what has been de-scribed as gene-for-gene specificity (11). In these interactions,field isolates ofa pathogen species can be categorized into various'races,' defined by the ability to grow on host cultivars withdifferent resistance genes. When a race is able to grow andproduce the symptoms of disease on a particular host cultivar,the interaction is said to be compatible; when a race makes onlylimited growth, the interaction is incompatible. Each gene fordisease resistance is effective only against races of the pathogenthat have a corresponding gene for avirulence. Thus, for eachdominant resistance gene in the host there is a single, correspond-ing dominant avirulence gene in the pathogen races with whichit is incompatible.One hypothesis for the molecular basis of race-specific plant

'Supported by the Cabot Foundation, National Science Foundation(PCM 83-02789) and United States Department of Agriculture (85-CRCR-1-1632).

2Present address: Forest Biology Department, University of Maine,Orono, ME 04469.

3 Present address: Director Genetic Engineering Technology, CedarCrest College, Allentown, PA 18104.

disease resistance is that the host genes for disease resistance codefor proteins that serve as receptors, or recognition proteins (1 1).These recognition proteins would specifically bind factors, pre-sumably the direct or indirect (enzymic) products of avirulencegenes, produced by incompatible but not compatible pathogenraces. Binding of the correct factor would set in motion thedisease resistance response. The existence of avirulence genes inbacterial plant pathogens has been substantiated by the resultsof Staskawicz et al. (22), who successfully cloned an avirulencegene from the bacterial soybean pathogen Pseudomonas syringaepv glycinea. They showed, through transformation of otherwisecompatible bacteria, that the presence of this avirulence genewas necessary for incompatibility in the presence of plant tissuewith the corresponding resistance gene. The function of theproduct of this avirulence gene is as yet unknown.Root and stem rot of soybean (Glycine max L. merr.), caused

by the fungus Pmg,4 serves as a model host-pathogen system(11). Initial stages of germination and infection of a soybeanseedling by zoospores of compatible and incompatible races ofPmg are visually indistinguishable until 9 to 12 h after inocula-tion (10). After this, growth of the incompatible race slows andeventually stops, while the compatible race continues to grow,ultimately killing the plant. One difference that has been closelyassociated with these two interactions has been the differentialability of the plant to respond to infection by the accumulationoftoxic secondary metabolites known as phytoalexins. It is likelythat the accumulation of phytoalexins plays a crucial role in theinhibition of pathogen growth (1 1). The molecules of host andpathogen that mediate race-cultivar specificity in this systemhave not, however, been identified.There is inconclusive evidence implicating extracellular gly-

coproteins in race-cultivar specificity. Wade and Albersheim (26)isolated ECGP preparations from culture filtrates ofPmg. TheseECGP had no elicitor activity, but ECGP from an incompatiblerace protected soybean seedlings from infection by a normallycompatible race. ECGP from the compatible race had no protec-tive effect. In later attempts to repeat these experiments, however,race-cultivar specificity of protection was not found (5). It ap-pears that ECGP, regardless of race, have the capacity to changenormally compatible interactions to incompatible. In contrast tothe results of Wade and Albersheim (26), Ziegler and Pontzen(27) reported that a purified glycoprotein from compatible, but

4 Abbreviations: Pmg, Phytophthora megasperma f. sp. glycinea;ConA, Concanavalin A; ELISA, enzyme-linked immunosorbent assay;HRP, horseradish peroxidase; AGA, avirulence gene associated; mAb,monoclonal antibody; ECGP, extracellular glycoprotein; DMEM, Dul-becco's Modified Eagle Medium; HT, DMEM supplemented with 15%calf serum and hypoxanthine-thymidine; HAT, HT plus aminopterin;endo-H, endo-fl-N-acetylglucosaminidase H.

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MONOCLONAL ANTIBODIES TO FUJNGAL GLYCOPROTEINS

not incompatible, Pmg races suppressed the activity of a glucanelicitor in soybean. Although the activity was shown to be dueto carbohydrate, the active determinant proved impossible toisolate (E Ziegler, personal communication). Keen and Legrand(12), on the other hand, extracted glycoproteins from Pmg my-celial walls that seemed to be race-specific in their ability to elicitglyceollin accumulation ('race-specific elicitors'). However, theelicitor-containing fractions were not extensively purified andcontained some of the nonspecific glucan elicitor described byother workers (1, 2, 17, 21).

It is our goal to exploit the specificity ofmonoclonal antibodiesto identify the molecules of pathogen origin involved in race-specific interactions. Monoclonal antibodies can be used as mo-lecular probes to identify antigenic determinants of pathogenorigin associated with particular avirulence genes (AGA anti-gens). As an initial effort in this direction, we report here theproduction and characterization of a library of mAbs againstextracellular glycoproteins of Pmg.

MATERIALS AND METHODS

Organisms. The fungal isolates of races 1 and 4 were a gift ofDr. K. Athow (Purdue University) and the field isolate designatedas 'race A' herein was a gift from Dr. J. Paxton (University ofIllinois).

Following the method ofTooley et al. (25), virulence formulae(incompatible/compatible host genes) were used to describe thevirulence of the three Pmg races used here on soybean (Glycinemax L. Merr.) cultivars carrying identified resistance genes (Ta-ble, below). In these formulae, numbers and letters refer to thecorresponding resistance genes identified in the host (i.e. 'lb'refers to Rps,b, '3' refers to Rps3, etc.):

Virulence formulala, lb, ic, lk, 3, 4, 5, 6/ Harlb, 1k, 3, 4, 5, 6 / la, lc, Har3, 4, 6 / la, lb, lc, 1k, 5, Har

race

1

4A

Our isolates of races 1 and 4 gave the expected pattern ofincompatibility and compatibility on the soybean differentialstested (15). Race A did not fit the virulence pattern of any of theraces that have been numerically designated, but was selected foruse because it differed in many avirulence genes from race 1.

Williams (rps,), Williams 79 (Rps,c) and Williams 82 (Rps,k)were from Illinois Foundation Seeds. L77-1863 (Rps,b), PI86.972-1 (Rps3), PI 86.050 (Rps4 + Rps,c), and Altona (Rps6)were a gift of Dr. R. L. Bernard (University of Illinois). Harosoy(Rpsh.,), Harosoy 63 (Rpsla), and L62-904 (Rps5) were a gift ofDr. R. I. Buzzell (Harrow, Ontario). Inoculations were carriedout by introducing mycelia into wounds in hypocotyls, andreactions were rated as compatible or incompatible 4 to 5 d later.The myeloma line used for all fusions, P3X63Ag8653, was a

gift of Dr. Tim Springer, Dana-Farber Cancer Institute.Preparation of Pmg glycoprotein antigens. Cultures of Pmg

were grown in stationary 500 ml jars containing 100 ml ofmodified Erwin's synthetic medium (7). Cultures were incubatedat 19°C until mycelial mats covered the surface of the broth(about 2 weeks). Mycelia were removed by filtration throughglass fiber filters (Whatman GF/A), further clarified by filtrationthrough 0.2 Mm filters, and the filtrate was concentrated 30-foldby ultrafiltration (pmlO Diaflo, Amicon; cut-off 10 kD). Thisconcentrate was used directly or after dialysis against phosphatebuffered saline (PBS; 10 mM sodium phosphate, 150 mM NaCl,pH 7.4) for immunization, but for all other purposes it waslyophylized, resuspended in Tris buffer (5 mM, pH 6.8), dialyzedagainst the same buffer, and brought to a concentration of 2 mgprotein/ml. Yield of protein (19) was typically 8 to 10 mg/jar.These preparations were previously found to contain primarily

glycoprotein (26) and are referred to herein as extracellularglycoprotein.

Production of mAbs. Myeloma and hybridoma cells weremaintained in DMEM supplemented with 15% calf serum, 100Mm hypoxanthine, and 20 Mm thymidine (HT). HAT mediumalso contained 0.4 Mm aminopterin.Female Balb/C mice (Charles River Labs), 6 to 20 weeks old,

were immunized with concentrated fungal glycoproteins. Theinjection schedule was as follows: d 1, 7, and 14, intraperitonealinjection of 0.1 to 0.2 mg protein in 50% Freund's complete (d1) or incomplete (d 7 and 14) adjuvant (total volume = 100 to200 Ml). On d 28, mice were bled and their serum was tested byELISA. Mice with serum reactions above background at dilutionsgreater than 1:10,000 were chosen for fusion and boosted thenext day with 0.2 to 0.4 mg protein with no adjuvant. Mice weresacrificed by cervical dislocation 3 to 4 d after the final boost;spleens were surgically removed and dissociated mechanicallywith two forceps into 10 ml of DMEM. Large pieces of tissuewere allowed to settle out in a 15 ml centrifuge tube, and thesuspended cells were pelleted and resuspended twice by centri-fugation at 400g. Myeloma cells growing exponentially in HTwere washed twice in DMEM to remove serum. Cells were mixedin a ratio of 1:1 to 2:1 spleen to myeloma cells and co-pelletedin a 50 ml conical centrifuge tube. Supernatant was removedand 1 ml of 40% PEG (1300-1600 mol wt, ATCC) in DMEMadjusted to pH 8 was added dropwise over the course of 1 min.After another 60 s, 1 ml ofDMEM was added dropwise over thecourse of 1 min, then 7 ml ofDMEM was added over the courseof 3 min. The cell suspension was mixed by gently stirring withthe pipet throughout the procedure. Finally, 30 ml ofHAT with15% calf serum was added, and the suspension was plated intothree 96-well cell culture plates without feeder cells. Cells weremaintained for 2 weeks, feeding every 3 d with HAT, beforeshifting to HT.Clones were obtained from hybridoma colonies by dilution

cloning, which resulted in less than one cell for every three wellson a 96-well plate. Each positive cell line was cloned at leasttwice and, in most cases, only one clone was saved from eachpositive hybridoma colony. Cell lines were grown in flasks con-taining 15 ml ofHT until cell density reached 1 to 2 x 106 cells/ml, at which time cells were pelleted by centrifugation at 400g.Supernatant medium was saved and stored in aliquots at -20°Cto be used in antibody assays. Cells were frozen at 2 x 107 cells/ml in HT containing 15% DMSO and maintained in liquid N2.Cloned hybridoma lines and the mAbs produced by them aredesignated by a pair of letters (corresponding to the initials ofthe workers who generated them) followed by a number.

Screening of Monoclonal Antibodies. ELISAs were initiated bybinding glycoproteins (10-100 Mg/ml) in coating buffer (10 mmNaCO3, pH 9.6) to microtiter plates (Immulon I protein bindingpolystyrene, Dynatech) for 1 to 2 h at room temperature orovernight at 4°C. The plates were rinsed three times and allowedto sit 15 min in blocking buffer A. In initial screening ELISAsblocking buffer A consisted of PBS containing 0.1% (w/v) NaN3and 1% (v/v) horse or calf serum; the serum was used because itwas inexpensive, but it gave high backgrounds with some mAbs.In all other ELISA experiments, serum was replaced by 1% (w/v) BSA. Supernatants (containing mAbs) from hybridoma cellcultures were added to the wells and allowed to bind for 1 to 3h at 37°C or overnight at 4C. The plates were rinsed three timeswith blocking buffer A and then a detecting antibody (goat anti-mouse IgG-alkaline phosphatase conjugate, Sigma), diluted1:1000 in blocking buffer A, was added. Detecting antibody wasbound for a minimum of 3 h at 37°C or overnight at 4°C followedby three rinses in blocking buffer A. The alkaline phosphatasereaction was performed by addition of one 5 mg tablet of p-nitrophenyl phosphate (Sigma) per 5.0 ml of developing buffer

race

509

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Plant Physiol. Vol. 85, 1987

(10% diethanolamine, 0.1 mg/ml MgC2 -6H20, 0.2% NaN3, pH9.8). ELISA results were quantitated using a Bio-Tek EIA readerwith a 405 nm filter and interfaced with an Apple Macintoshcomputer.

Binding of mAbs to fungal mycelial walls was quantitated inan analgous manner to the ELISA above. Purified fungal myce-lial walls (1) were suspended in coating buffer at 500 ,tg dryweight/ml and allowed to bind to plates for 1 h. The assay wascompleted as described above for the soluble glycoproteins.

Probing of Western Blots. Samples containing 20 to 40 ,ug ofglycoprotein were separated by SDS-PAGE (14) using 12% gels,then transferred to nitrocellulose in a Bio-Rad Transblot appa-ratus, containing running buffer (25 mm Tris-HCl, 192 mMglycine, 20% methanol, pH 8.3), at 70 V for 3 h or 30 V overnight(3). Blots were stained with Ponceau S to locate lanes, then cutinto strips, each of which displayed the entire spectrum ofseparated proteins. Strips containing mol wt standards and asample ofthe glycoproteins were directly stained for protein withamido black (16). Strips to be probed with mAbs were incubatedfor 1 h in blocking buffer B (PBS, 5% calf serum, 0.1% Tween20). They were then incubated for 1 h at 25°C with cell culturesupernatants diluted 1:5 (v/v) in blocking buffer B. The stripswere rinsed five times for 5 min each in blocking buffer B, thenincubated for 3 h at 25°C or overnight at 4°C in goat anti-mouseIgG-HRP conjugate diluted 1:1000 in blocking buffer B. Thestrips were then rinsed twice for 20 min each with blocking bufferB then twice with PBS. The presence ofbound HRP was detectedusing a developing solution consisting of 30 mg of 4-chloro-1-naphthol and 50 ,l of 30% H202 in 10 ml methanol and 50 mlPBS.Some strips were stained with ConA and HRP by a modifica-

tion ofthe method ofFaye and Chrispeels (9). Strips were blockedwith blocking buffer C, consisting of TBS (20 mM Tris-HCl, 500mM NaCl, pH 7.4, + 0.5% [w/v] BSA) and 0.1% (v/v) Tween,then incubated for 1 h with 25 ug/ml ConA (type IV, Sigma) inblocking buffer C. They were washed four times with TBS +0.1% Tween and then incubated for 1 h with 50 ,ug/ml HRP(Sigma) in blocking buffer C. They were then washed four times

with TBS + 0.1% Tween and two times with TBS. The presenceofbound HRP was detected as described above.Monoclonal antibodies were tested for isotype using a com-

mercially available kit (ICN Immunobiologicals), utilizing anOuchterlony-type assay.Chemical and Enzymic Modifications of Glycoproteins. Gly-

coproteins were treated to modify carbohydrate groups afterbinding to microtiter plates. Microtiter plates coated with gly-coproteins at 10 Ag/ml were blocked for 20 min with blockingbuffer D (PBS + 0.5% BSA [Ig free, Sigma]) and then treated asfollows:

(a) Periodate Oxidation. Plates were washed three times andwells filled with 100 Al sodium acetate buffer (50 mM, pH 4.5).In wells to be treated by periodate oxidation, acetate buffer wasreplaced by 100 ,u of 20 mM sodium metaperiodate in sodiumacetate buffer (50 mM, pH 4.5). Plates were then incubated for20 min to 4 h at 4°C, washed with blocking buffer D, and probedwith mAbs, as described above, or ConA, as described below.

(b) Mannosidase Digestion. Plates were washed three timeswith sodium acetate buffer (50 mm, pH 4.5) and wells filled with100 Al a-mannosidase buffer (100 mm sodium acetate, pH 4.5 +5 mM ZnCl2 + 0.5% BSA [Ig free, Sigma]). In wells to be treatedby a-mannosidase, this was replaced by 100 1l of jack bean a-mannosidase (Sigma), 2.7 units/ml in a-mannosidase buffer.Plates were then incubated for 1 to 24 h at 37°C, washed withblocking buffer D, and probed with mAbs as described above, orConA, as described below.

(c) Endo-H Digestion. Treatment with Endo-H was donebefore binding to plates. Fifty IAI of glycoproteins at 2 mg/mlwere mixed with 50 Al of endo-H (Sigma) at 0.2 unit/ml insodium acetate buffer (200 mm, pH 5.8). Mixture was incubatedat 37°C for 24 h, then bound directly to plates by the usualprotocol. A control mixture consisting of 50 Al of glycoproteins+ 50 ,A of sodium acetate buffer was incubated and plated in thesame way. The plates were blocked with blocking buffer A andprobed with mAbs as described above.ConA (Sigma) was used to determine the effectiveness of

carbohydrate modification procedures. ConA binds to a-linked

4-A, ;.

I 11 III IV V VI P C i Ilil IV V VI P C 11 III IV V VI

FIG. 1. Probing of Western blots of races 1, 4, and A extracellular glycoproteins with archetypal antibodies. Markers indicate molecular weightin kD. Gels were electrophoretically transferred to nitrocellulose paper, then stained for protein with amido black (P), probed with ConA (C), or

probed with one of the six archetypal mAbs (I, KW2; II, KW4; III, MF7; IV, MFl 1; V, KW8; VI, MF6).

P C

510 WYCOFF ET AL.



MONOCLONAL ANTIBODIES TO FUNGAL GLYCOPROTEINS

Table I. Reactions ofmAbs after Carbohydrate or Protein Modifying ProceduresAll of the mAbs are listed, and placed into groups based upon their pattern of binding to Western blots of

Pmg extracellular glycoproteins. Numbers represent level of binding of each mAb in ELISA to glycoproteinstreated with each carbohydrate modifying procedure relative to untreated controls. Periodate reactions weredone for 4 h, a-mannosidase reactions for 24 h, and Endo-H reactions for 24 h. Unless otherwise noted, valuesare the mean of two replicates.

Group mAbI KW2

KW3KW5KW6KW7MF19JJ9JJ25JJ31JJ32JJ35JJ36JJ37JJ38JJ7MF17

II KW4KW9JJ60

III MF7MF15MF16MF18MF24

IV MFIlMF1MF4MF5MF1OMF14MF20MF23MF26

V KW8

VI MF6MF12

CON Aa Mean of 4 replicates.

IsotypeIgMIgMIgMIgMIgMIgMIgMIgMIgMIgMIgMIgMIgMIgMIgMIgGIIgMIgMIgGI

IgGIIgGiIgGiIgGIIgGI

IgGIIgGIIgG1IgGIIgGIIgGIIgGIIgGIIgGIIgMIgAIgA

Periodate

1.03 + 0.17b1.291.411.601.371.291.401.721.431.891.451.551.291.426.930.08

0.56 ± 0.19b1.020.03

0.04 ± 0.02b0.050.190.310.04

1.3 +0.2b2.21.92.22.32.12.01.92.0

2.7 0.7b2.0 ±0.3b2.6

0.15

b Mean of 4 replicates ± SD.

mannosyl or glucosyl residues. Thus, ConA binding to Pmgglycoproteins is an indicator ofthe completeness of modificationof such residues. ConA was dissolved at 100 ,ug/ml in TBS + 1

mM CaCd2 + 1 mm MnC12, and allowed to bind for 1 h. Excesswas washed out and bound ConA was detected using the samesecond antibody used to detect the binding of mAbs. It wasdiscovered that ConA binds the second antibody, and this allowsevery well on a plate to be treated in an identical manner afterbinding of mAbs or ConA.

RESULTSProduction of mAbs. We have produced and partially charac-

terized 36 monoclonal antibodies using glycoproteins from Pmgrace 1 as inject antigen. Initially, hybridomas secreting antibodiesdirected against race 1 glycoproteins were identified by ELISA.Wells were considered positive if they yielded a response at least0.2 times the response given by a 1:10,000 dilution of polyclonal

serum. In three fusions 70 to 90 positive hybridomas wereidentified per fusion, and 8 to 16 per fusion were recovered inclonal form.

Reaction of mAbs to Western Blots. Each mAb was used toprobe Western blots of extracellular glycoproteins from race 1

and was found to give one of six distinct patterns ofbinding (Fig.1). Based on these patterns, each antibody was placed into oneof six 'groups' (Table I). Group I is characterized by binding tomultiple bands from about 24 kD up, corresponding quite closelyto the pattern of staining by ConA. Group II antibodies show apattern similar to group I, but with enhanced binding to a tripletof proteins at about 55 kD and strong binding to an additionalprotein at 30 kD not seen with amido black or ConA staining.mAbs in group III show a pattern nearly identical to group I, butwith weak binding to the 30 kD protein. The binding pattern ofgroup IV mAbs can be simply described as a smear that starts atthe top of the gel and extends down to about 70 kD. Group V

Mannosidase

0.660.560.660.550.620.710.730.710.690.620.690.640.680.691.400.390.760.730.48

0.450.360.470.550.39

0.590.650.420.780.350.620.710.570.47

0.46

0.630.71

0.3

Endo-H'

0.710.680.680.690.780.730.790.790.700.600.750.610.850.810.990.49

0.870.851.11

0.740.790.350.540.72

0.880.860.860.860.920.940.830.800.93

0.85

1.081.16

0.41

Boiling1.31.21.31.51.31.41.11.51.41.81.31.31.31.31.02.0

1.21.00.9

1.81.86.25.12.1

1.30.91.31.21.11.21.01.11.1

3.4

0.090.13

0.88

51



WYCOFF ET AL.

2.5 -

a

2.0

_1.5

w

I-I0

0.5

0.0 -

A0

0

w

1.0

0.9

0.8

0.7

0.6

2 3TREATMENT TIME (HRS)

<C 0.5

0 0.4-

0.3

0.2

B 0 5 10 15 20 25TREATMENT TIME (HOURS)

FIG. 2. A, Time course of periodate treatment.

for 20min to 4 h with sodium metaperiodate

antibodies and ConA. Values for various treatment

as a fraction of the untreated controls. Group I (0), I ((0),III (X), group IV (U), group V (E), group VI (A),

course of mannosidase treatment. Glycoproteins

with a-mannosidase were probed with archetypal ConA.

Values for various treatment times are expressed

untreated controls. Group I (0), group 11 (0), (X),

(U), group V(O), group VI (A), ConA (A).

consisted of only one mAb (KW8), which

pattern similar to group I from about 60

binding to proteins smaller than this. The

VI (MF6 andMF12) were different from

bound to only one band, at 12 kD. The other

weakly to this protein. Six mAbs representative

groups, referred to below as the six archetypal

used to probe Western blots of race 4 and

as well as race (Fig. 1). The pattern of mAb

and A glycoproteins was similar to binding to

were predominantly quantitative. The most striking

difference was in the binding of the group

showed attenuated binding to proteins larger

ing to proteins smaller than, about 60 kD

The fact that most of the mAbs bound to

that the bands corresponded to the bands

suggested that the mAbs were specific for

than protein antigen domains.

ELISA of Carbohydrate-Modified Glycoproteins.

for carbohydrate was tested by treating glycoproteins

(by periodate oxidation) and enzymically (with a-

mannosidase and endo-H) before probing

archetypal antibodies were used to probe

had been treated with sodium meta-periodate min,or 4 h (Fig. 2A). Binding of each of the mAbs

pretreatment of the glycoproteins; this is as

are glycospecific. Treatment with periodate reduced the bindingof some of the mAbs while binding of others was enhanced (Fig.2A; Table I). Periodate pretreatment had the same effect onglycoproteins from each of the three races tested (data notshown).Most of the antibodies bound less after the glycoproteins were

treated with a-mannosidase for 24 h, and there was some varia-tion between mAbs within a group, suggesting a diversity ofspecificity not obvious from Western blots (Table I). The sixarchetypal mAbs were used to probe glycoproteins exposed to atime course of a-mannosidase treatment (Fig. 2B). Those thatwere affected by a-mannosidase treatment did not show the samekinetics in the reduction of binding as ConA; that is, reductionin mAb binding occurred fairly evenly over the course of thetreatment, whereas reduction in ConA binding occurred rapidlyat first, then more slowly.

Treatment with endo-H for 24 h reduced the binding of ConAby 60%, but the binding of most of the mAbs was affected onlyslightly (TableI). Again, there was considerable variability be-tween different antibodies within the same group. Although mostof the mAbs in group I were reduced by 15 to 30%, MF17 wasreduced by 51%, and JJ7 was not affected at all. In groupIII,MF7, MF15 and MF24 were reduced 21 to 28%, while MF16and MF18 were reduced by 46% and 65%, respectively (TableI). Only JJ7, JJ60, and the two group VI mAbs were unaffectedby endo-H treatment.

Boiling of the extracellularglycoproteins destroyed the bindingof both mAbs in group VI, while all of the other mAbs wereeither unaffected, or had their binding increased by this treat-ment. Again, MF16 and MF18 were different from other mAbsin their Western blot group (groupIII). JJ7 and MF1 7, likewise,seem distinct from other members of groupI.ELISA against Different Races. MAbs were tested in ELISA

against glycoproteins from races 1, 4,and A ofPmg (Fig. 3, A,B, and C). Glycoproteins from each race were bound to assayplates at a concentration previously shown to saturate the platesurface (data not shown) and mAb supernatants were used atsaturating concentrations. All mAbs reacting with race glyco-proteins also reacted with glycoproteins from the other two races.However, some of the mAbs did show quantitative differencesin binding to the three races. These differences correlated withtheir grouping by pattern of binding to Western blots (TableI).For instance, the reaction of mAbs in groups I and II, except forMFM7, was roughly equal to glycoproteins from all three races,while mAbs in group III reacted more than 10-fold more stronglyto race 1 glycoproteins than to glycoproteins from the other tworaces. Mabs in group IV, on the other hand, reacted equally toraces 4 and 1 but much stronger to race A.

Large insoluble mycelial wall fragments fromPmg can bebound to plates in the same manner as soluble proteins. Bindingwas confirmed by direct observation using an inverted micro-scope, and the fragments were not dislodged by the washing stepsused in the ELISA. Each mAb was tested for binding to mycelialwalls of races 1, 4, and A in order to determine if mycelial wallsdisplayed the same antigenic determinants as extracellular gly-coproteins (Fig. 3, A, B, and C). Most mAbs reacted to mycelialwalls of the three races with patterns similar to those observedwith glycoproteins. The most striking difference, however, wasin the antibodies of group III (Fig. 3B). These mAbs showeddramatically lower levels of binding to glycoproteins of race 4and race A when compared to race 1, but they reacted to mycelialwalls of the three races equivalently.Two of the mAbs placed in group I based upon their Western

blot pattern(JJ7 and MFl 7) appeared quite different from therest in their ELISA reactions to glycoproteins and mycelial walls(Fig. 3A).

Competitions. Previous analyses of the sugar composition of

512 Plant Physiol. Vol. 85, 1987




1.2

1.0

0.8

REACTIONRELATIVE 0.6TO RACE 1

0.4

0.2

0.0

1.0

0.8

REACTIONRELATIVE 0.6TO RACE

0.4

0.2

0.0

*lUmlU u m In'EJmJmJmImLu.1YCELIAL WALLS

IaI I I IKW KW KIN KW IKW F JJ JJ JJ JJ- JJ JJ JJ JJ JJ

A 2 3 5 6 7 19 9 25 31 32 35 36 37 38 7 17

GROUP MABS

1.4

1.2

1.0

REACTIONRELATIVE 0.8TO RACE 1

0.6

0.4

0.2

0.0

1.4

1.2

1.0REACTION

RELATIVE 0.8TO RACE 1

0.6

0.4

0.2

0.0

KW KW JJMF MF WF MF 1 KW MF S

B 4 9 60 7 15 16 18 24 8 6 12GRIOLI,P " tGRU NVG1PVRLV

REACTIONRELATIVE

1

REACTONRELATIVE

C MF1 MF4 MFS MFlO MFll MF14 MF20 MF23 MF26

GROUP IVMAS

FIG. 3. Relative binding of mAbs to extracellular glycoproteins and

mycelial walls of different races of Pmg. Glycoproteins (40 /Ag/ml) or

mycelial walls (500 ug/ml) from races 1, 4, and A, diluted in coatingbuffer, were bound to microtiter plates. After washing and blocking

----------------------------------

I,1 minji.w. . .1

the extracellular glycoproteins ofPmg have shown that they areprimarily mannose, with a smaller amount of glucose and N-acetylglucosamine (26, 27). Assuming this to be the case for theextracellular glycoproteins as a whole, other mannose-containingglycoproteins of known structure were tested for ability to com-pete for the binding of mAbs in ELISA. Binding of mAbs in thisassay was reduced 80 to 100% when competed with 100 ,ug/mlPmgglycoproteins. Five-fold higher concentrations (weight basis)of yeast mannan, ovalbumin, ovomucoid and yeast invertaseshowed no competition (Table II).

DISCUSSIONPhytophthora megasperma f. sp. glycinea race is thought to

carry several avirulence genes not found in races 4 and A, basedupon interactions with a set of soybean differentials carryingdifferent resistance genes. We assumed that these avirulencegenes ultimately determine the presence of particular antigenicdeterminants recognized by the host plant and attempted toidentify antibodies that would bind to race 1 antigens but not tomolecules from the two other races.The evidence presented here suggests that all of the mAbs

selected, except those in group VI, are carbohydrate rather thanprotein-specific, even though they were raised to a mixture ofglycoproteins. With the exception ofMF6 and MF12, the mAbsbind to multiple protein bands on Western blots. Binding toprotein bands of several different sizes can result from thepresence of common carbohydrate groups on these proteins, orto common protein domains generated by discrete proteolyticcleavage of a precursor protein, or to proteins with alternativepatterns of glycosylation. Although the second and third possi-bilities have not been ruled out, they seem unlikely because ofthe very wide range of molecular weights to which the mAbsbind. In addition, ,Mab-binding bands correspond to bandsstained with ConA, which is specific for a-linked mannosyl andglucosyl residues.The differences in banding patterns with different mAbs, seen

in Western blots, may reflect the variety ofcarbohydrate antigenson extracellular proteins. For instance, Western blots suggest thatmany proteins carry both of the antigens recognized by group Iand group II mAbs, but at least one protein, P30, carries thegroup II antigen without the group I antigen. Many proteinscarry both of the antigens recognized by mAbs of groups I andV, but those smaller than about 60 kD carry only group Iantigens. The similarity of patterns regardless of race suggeststhat none of these mAbs are specific for AGA antigens. The factthat all of the mAbs fall into just a few groups suggests that,rather than representing the full diversity of glycoprotein anti-gens, these mAbs are specific for the immunologically dominantantigens of Pmg.

It should be noted that most of the mAbs within a group areof the same Ig heavy chain class. Preferential induction ofimmunoglobulins of a given isotype by particular antigenic de-terminants has been previously observed (18, 20). In this case,all of the mAbs were either IgM or IgG, (Table I).

Treatments that modify carbohydrates modify binding ofmAbs. The expected activity ofeach ofthe modifying proceduresreveals features ofthe antigenic determinants to which the mAbs

plates with blocking buffer A, hybridoma supernatant solutions diluted1:4 (v/v) in blocking buffer A were bound for 1 h, then detected withgoat anti-mouse alkaline phosphatase conjugate. Antibody bound was

quantitated by measuring OD405. Values for races 4 and A are expressedas a fraction of the value for race I with each antibody, and representthe mean of two replicates. Dotted line represents reaction of eachantibody to race 1 antigen. Dark bars, race 4; light bars, race A. A, GroupI mAbs; B, group II, group III, group V, and group VI mAbs; C, group

IV mAbs.

GLYCOPROTEINS * RACE 4 [] RACE A_ __ __ _ __ __ _ _ __ __ _ __ __ __ _ __ ,_

-~~~~----i

GLYCOPROTEI.JS D RACERACE A

N RAE4

MYCELIAL WALLS 1.2ii5i

513

GLYOOPROTNSr



Plant Physiol. Vol. 85, 1987

Table II. Competitionfor Binding ofmAbs to Glycoproteins in ELISAPmg race 1 glycoproteins were bound to ELISA plates. The six archetypal mAbs were diluted with blocking

buffer A to a concentration that was not saturating in this assay, then mixed with either Pmg 1 extracellularglycoproteins or glycoproteins from other sources. Competing Pmg 1 glycoproteins were at 100 ug/ml (protein);all other proteins were at 500 Ag/ml (dry weight). Plates were incubated at 37°C for 1 h, washed, and probedwith second antibody-alkaline phosphatase conjugate as usual. Values are expressed as a fraction of the OD405of uncompeted controls, and are the mean of two replicates.

CompetitormAbGroup Ovalbumin Ovomucoid Yeast Yeast Pmg 1

mannan invertase glycoproteinI 1.04 0.93 0.98 0.96 0.02

II 0.89 0.89 0.77 0.98 0III 1.20 1.20 1.10 1.20 0.30IV 0.98 0.98 0.85 0.96 0.30V 0.81 1.00 1.00 0.88 0VI 1.07 0.97 1.03 0.83 0

are binding. MAbs with binding disrupted quickly by periodateare likely to be specific for either terminal mannosyl residues orinternal residues with adjacent unsubstituted hydroxyls. ConAbinding is reduced by 85%, confirming that periodate treatmentaffects residues recognized by this lectin. However, binding ofonly seven of the mAbs was disrupted by periodate: all of groupIII, as well as MF17 (group I) and JJ60 (group II). Further,periodate sensitivity due to specificity of mAbs for terminalresidues would be paralleled by sensitivity to a-mannosidasedigestion, since terminal mannosyl residues would be the firstremoved by this enzyme. This does, in fact, seem to be the case(Table I).By contrast, the binding of most of the mAbs is actually

enhanced by pretreatment ofglycoproteins with periodate. Bind-ing enhancement might be explained by an increase in theaccessibility to antibody binding of some epitopes after cleavageby periodate of sugar residues which normally sterically hinderthat binding.

Disruption of binding by a-mannosidase should depend onthe linkages of the mannosyl residues involved. a-Mannosidaseis an exoenzyme that cleaves a-1,6, a-1,2 and a-1,3 mannosyllinkages, with a-1,6 and a--1,2 linkages cleaved 15 times fasterthan a-1,3. These different rates might yield information aboutwhich mannosyl linkages are involved in the epitopes to whichthe different mAbs bind. Binding of ConA is reduced by a-mannosidase treatment much more quickly and to a muchgreater extent than any of the mAbs (Fig. 2B). Rapid loss ofbinding is consistent with binding ofConA to mannosyl residuesthat are a- 1,6- or a-1,2-linked, while most of the residues re-quired for binding of mAbs are either internal to a-1,3-linkedmannosyl residues or to other residues not cleavable by theenzyme.Endo-H cleaves within the chitobiosyl residue joining carbo-

hydrate to protein in glycoproteins. Endo-H treatment of theglycoproteins reduces ConA binding by 60%, but only threemAbs (MF16, MF17, and MF18) have their binding reduced bymore than 50% by pretreatment of glycoproteins with endo-H.The general resistance to endo-H treatment suggests that thecarbohydrates recognized by ConA and those recognized by mostof these mAbs are not on the same carbohydrate chain at all buton chains sensitive and insensitive to endo-h, respectively.High mannose oligosaccharides on glycoproteins (or glycopep-

tides) are cleaved by endo-H, while most modified chains appearto be resistant (8, 23). In addition, endo-H can act on someproteins only after they have been denatured. If this is so, thenthere may be at least two different types ofcarbohydrate moietieson Pmg glycoproteins: (a) those sensitive to endo-h, carryingmost of the epitopes bound by ConA, MF16, MF17, and MF18,

and a small percentage of the epitopes bound by most of themAbs; (b) those not susceptible to endo-H removal (presumablythe modified chains). The greatest effect of endo-H is on MF16and MF18, two mAbs which are affected in a quantitativelydifferent way than other members ofgroup III (Table I), indicat-ing that they probably recognize a different epitope.The epitope recognized by mAbs in group VI is the only one

sensitive to boiling but is also affected somewhat by a-mannosi-dase and is made more antigenic by periodate. This would seemto indicate that the epitope involves both protein and carbohy-drate.There are a number of hypotheses that could be advanced to

explain why all the mAbs fall into only a few groups and aremostly carbohydrate-specific. First of all, since only the hybrid-omas that gave the strongest response in the screening ELISAwere cloned, mAbs against minor components of the antigenmixture, such as individual proteins, may have been missed. Itis likely that all of the extracellular glycoproteins carry the sameglycosyl groups, thus making these carbohydrates the most abun-dant antigens. However, whatever the epitopes recognized bythese mAbs, they are not common to yeast mannan, yeastinvertase, ovalbumin, or ovomucoid, which are all glycoproteinswith glycomoieties consisting only of mannosyl and N-acetylglu-cosaminosyl residues in various linkages.

All of the antibodies here bind to both extracellular glycopro-teins and mycelial walls from all three of the races tested. Thereare some quantitative differences, but most are slight (e.g. groupI). Group III mAbs showed large quantitative differences inbinding to glycoproteins, but they react nearly equivalently tomycelial walls from all three races. It seems unlikely, therefore,that these mAbs recognize AGA antigens. This may merelyreflect differences in the way particular antigens are processedfor either deposition in mycelial walls or secretion on extracel-lular glycoproteins. Group IV mAbs gave consistently higherreactions to race A, using both glycoproteins and mycelial walls.However, the presence of an AGA antigen in race A, but not inraces 1 and 4, is inconsistent with the pattern of identifiedresistance genes. Since these mAbs were raised to race 1 glyco-proteins, the data may be interpreted as reflecting quantitativedifferences independent of avirulence genes.

This paper is the first report of the use of monoclonal anti-bodies to study molecules ofpotential importance in the soybean-Pmg interaction. Although it was not expected, we found thatmost of the mAbs were carbohydrate-specific, which is encour-aging if this approach is to identify AGA antigens, since evidencefrom this (12, 13, 27) and other systems (6, 24) suggest that race-specific interactions involve either carbohydrates or glycopro-teins from the pathogen.

514 WYCOFF ET AL.




It is possible that some of the mAbs may be against AGAantigens that are found in all three of the races tested. Thesewould include the avirulence genes which correspond to theresistance genes Rps3, Rps4, and Rps6. We have recently acquiredisolates lacking these genes and will be testing the mAbs againstantigens isolated from them.

Because the techniques used in this work generated only alimited diversity of mAbs, we are pursuing other avenues togenerate mAbs against AGA antigens. As these antigens may notbe found on extracellular glycoproteins, we are raising mAbsagainst purified mycelial walls. In addition, mAbs are beingraised using a technique involving immunosuppression withcyclophosphamide. Using this technique, we hope to suppressthe immune response to the immunodominant antigens whichare in common between all races, while selecting for antibodiesagainst race-specific determinants. Finally, since AGA antigensmay be expressed only when the pathogen is in contact with itshost (4), mAbs are being raised against fungal antigens fromcultures grown in the presence of plant extracts.

LITERATURE CITED

1. AYERS AR, J EBEL, B VALENT, P ALBERSHEIM 1976 Host-pathogen interactions.X. The fractionation and biological activity of an elicitor isolated from themycelial walls of Phytophihora megasperma var sojae. Plant Physiol 57:760-765

2. AYERS AR, B VALENT, J EBEL, P ALBERSHEIM 1976 Host-pathogen interactions.XI. Composition and structure of wall-released elicitor fractions. PlantPhysiol 57: 766-774

3. BURNETTE WN 1981 "Western blotting": electrophoretic transfer of proteinsfrom sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellu-lose and radiographic detection with antibody and radioiodinated protein A.Anal Biochem 112: 195-203

4. DE WIT PJG, AE HOFFMAN, GCM VELTHUIS, JA Kuc 1985 Isolation andcharacterization of an elicitor of necrosis isolated from intercellular fluids ofcompatible interaction of Cladosporium fulvum (syn. fulvia fulva) and to-mato. Plant Physiol 77: 642-647

5. DESJARDINS AE, LM Ross, MW SPELLMAN, AG DARVILL, P ALBERSHEIM 1982Host-pathogen interactions. XX. Biological variation in the protection ofsoybeans from infection by Phytophthora megasperma f.sp. glycinea. PlantPhysiol 69: 1046-1050

6. DOKE N, NA GARAS, J Kuc 1980 Effect on host hypersensitivity ofsuppressorsreleased during the germination of Phytophthora infestans cytospores. Phy-topathology 70: 35-39

7. ERWIN DC, H KATZNELSON 1961 Suppression and stimulation of mycelialgrowth of Phytophthora cryptogea by certain thiamine-requiring and thia-mine synthesizing bacteria. Can J Microbiol 71: 945-950

8. FAYE L, KD JOHNSON, MJ CHRISPEELS 1986 Oligosaccharide side chains of

glycoproteins that remain in the high-mannose form are not accessible toglycosidases. Plant Physiol 81: 206-21 1

9. FAYE L, MJ CHRISPEELS 1985 Characterization of N-Linked oligosaccharidesby affinoblotting with concanavalin A-peroxidase and treatment of the blotswith glycosidases. Anal Biochem 149: 218-224

10. HAHN MG, A BONHOFF, H GRISEBACH 1985 Quantitative localization of thephytoalexin glyceollin I in relation to fungal hyphae in soybean roots infectedwith Phytophthora megasperma fsp. glycinea. Plant Physiol 77: 591-601

11. KEEN NT 1983 Specific recognition in gene-for-gene host-parasite systems.Adv Plant Pathol 1:35-82

12. KEEN NT, M LEGRAND 1980 Surface glycoproteins. Evidence that they mayfunction as the race specific phytoalexin elicitors of Phytophthora mega-sperma f.sp. glycinea. Physiol Plant Pathol 17: 175-192

13. KEEN NT, M YOSHIKAWA, MC WANG 1983 Phytoalexin elicitor activity ofcarbohydrates from Phytophthora megasperma f.sp. glycinea and othersources. Plant Physiol 71: 466-471

14. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

15. LAVIOLETTE FA, KL ATHOW 1983 Two new physiologic races of Phytophihoramegasperma f.sp. glycinea. Plant Dis 67: 497-498

16. MATUS A, G PEHLING, M ACKERMANN, J MAEDER 1980 Brain postsynapticdensities: their relationship to glial and neuronal filaments. J Cell Biol 87:346-349

17. OSSOWSKI P, A PILOTrI, PJ GAREGG, B LINDBERG 1984 Synthesis of a gluco-heptaose and a glucootaose that elicit phytoalexin accumulation in soybean.J Biol Chem 259: 11337-11340

18. PERLMUTTER RM, D HANSBURG, DE BRILES, RA NicoLOrri, JM DAVIE 1978Subclass restriction of murine anti-carbohydrate antibodies. J Immunol121:566-572

19. PETERSON GL 1977 A simplification of the protein assay method of Lowry et.al. which is generally applicable. Anal Biochem 83: 346-356

20. ScoTT MG, JB FLEISCHMAN 1982 Preferential idiotype-isotype associations inantibodies to dinitrophenyl antigens. J Immunol 128: 2622-2628

21. SHARP JK, B VALENT, P ALBERSHEIM 1984 Purification and partial character-ization of a ,-glucan fragment that elicits phytoalexin accumulation insoybean. J Biol Chem 259: 11312-11320

22. STASKAwIcz BJ, D DAHLBECK, NT KEEN 1984 Cloned avirulence gene ofPseudomonas syringae pv. glycinea determines race-specific incompatibilityon Glycine max (L.) Merr. Proc Natl Acad Sci USA 81: 6024-6028

23. TARENTINO AL, TH PLUMMER JR, F MALEY 1974 The release of intactoligosaccharides from specific glycoproteins by endo-,B-N-acetylglucosamin-idase H. J Biol Chem 249: 818-824

24. TEPPER CS, AJ ANDERSON 1986 Two cultivars of bean display a differentialresponse to extracellular components from Colletotrichum lindemuthianum.Physiol Mol Plant Pathol 29: 411-420

25. TOOLEY PW, CR GRAU, MC STOUGH 1982 Races ofPhytophthora megaspermaf.sp. glycinea in Wisconsin. Plant Dis 66: 472-475

26. WADE M, P ALBERSHEIM 1979 Race-specific molecules that protect soybeansfrom Phytophthora megasperma var. sojae. Proc Natl Acad Sci USA 76:4433-4437

27. ZIEGLER E, R PONTZEN 1982 Specific inhibition of glucan-elicited glyceollinaccumulation in soybeans by an extracellular mannan-glycoprotein of Phy-tophthora megasperma f.sp. glycinea. Physiol Plant Pathol 20: 321-331

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Monoclonal Antibodies to Glycoprotein Antigensof aFungal … · compatible bacteria, that the presence ofthis avirulence gene wasnecessaryforincompatibility in the presenceofplanttissue