glycosidic enzyme activity in peatissue andpea ... · acids) were applied in 5-,ul volumes, which...

Plant Physiol. (1980) 66, 199-2040032-0889/80/66/0199/06/$00.00/0

Glycosidic Enzyme Activity in Pea Tissue and Pea-Fusarium solaniInteractions',,2

Received for publication September 10, 1979 and in revised form March 3. 1980

EVERETT J. NICHOLS, JEAN M. BECKMAN, AND LEE A. HADWIGERDepartment of Plant Pathology, Washington State University, Pullman, Washington 99164

ABSTRACT

Membrane barriers which prevent direct contact between Fusariumsolani and pea endocarp tissue prevent fungal spores from inducing phy-toalexin production. Conversely, preinduced host resistance responses arenot readily transported from the plant across the membrane barrier toFusarium macroconidia.

Crude enzyme extracts from pea endocarp tissues partially degradeFusarium solani f. sp. phaseoli cell wails. Activities of the glycosidicenzymes, chitinase, 8-1,3-glucanase, chitosanase, ,8-D-N-acetylglucosamin-idase, fi-D-N-acetylgalactosaminidase, 8-D-glucosidase, a-D-glucosidase,and a-D-mannosidase, were detected in pea endocarp tissue. If pods are

challenged with Fusarium spores or chitosan, the chitinase activity of theinfected tissue remains higher than water-treated pods 0.5 to 6 hours aftertreatment. The f8-1,3-glucanase activity increases within 6 hours in bothinoculated and control tissue. Chitosanase activity was lower in tissuetreated with Fusarium solani f. sp. pisi, f. sp. phaseoli or chitosan than inwater-treated control tissue. Thus, the pea tissue contains glycosidicenzymes with the potential to degrade the major compounds of the Fusar-ium cell walls.

Some of the cytological and regulatory features which charac-terize the pea-Fusarium interaction as compatible or incompatibleare detectable within 6 h after inoculation (1 1). In the incompatibleinteraction, Fusarium solani f. sp. phaseoli alters the structure ofplant nucleus and enhances the labeling of chromatin-associatedRNA within 25 min (12). Increased levels ofPAL activity and theinitiation of pisatin synthesis are detectable within 6 h (23) and,finally, the growth of form phaseoli is completely suppressedwithin 5-6 h (23) after inoculation.

In the compatible interaction between F. solani f. sp. pisi andpea pod tissue, the pea nucleus is highly disorganized within 25min (12), and phytoalexin synthesis is initiated within 6 h. How-ever, the growth of this fungus is reduced but not terminated (23).Inasmuch as the events which dictate the specificity of the

interaction occur in a rapid sequence almost immediately follow-ing inoculation, information on the initial processing and/orexchanges of molecules at the host-parasite interface becomesessential. The requirement for processing fungal compoundswhich induce phytoalexin synthesis was suspected since phyto-alexin inducers are more potent in aged F. solani cultures than inyoung actively growing cultures (5). Also the phytoalexin-inducingactivity, which is difficult to obtain in quantity from fungal spores

1 This research was supported in part by National Science FoundationGrant PCM-7712924.

2 Scientific Paper No. 5439, College of Agriculture Research Center,Washington State University, Pullman, Washington.

(5), becomes readily available to plant tissues immediately afterinoculation (23).

Cytological observations of cell walls from F. solani f. sp.phaseoli in contact with pea pod tissue for 24 h reveal extensivedegradation and loss of cell wall morphology (23). Further, it wasobserved that spores separated from the plant surface by a waterphase grew more than those in contact with the plant surface.These observations led to the present study which examines theenzymic activity in pea pods of Pisum sativum, which might assistin degrading F. solani cell walls, and their possible action inreleasing regulatory components such as phytoalexin inducers.The size and interchange dependence of host and pathogen com-ponents was assessed with selective barriers.

Since many fungi contain chitin and f8-1,3-glucans as well asother polysaccharides in their cell walls (22), it seems reasonableto assume that enzymes which could degrade chitin (a polymer ofN-acetylglucosamine) and laminarin (f8-1,3-glucan) could alsodegrade the fungal hyphae which contain these components.Bacterial chitinase (EC 2.3.1.14) and ,8-1,3-glucanase (EC 3.2.1.6)have been implicated (16, 22) in degradation of F. solani cell walls.The in vivo lysis of Verticillium albo-atrum in tomato plant infec-tions has been observed (6) and related to chitinase and 8-I1,3-glucanase activities in Lycopersicon esculentum (19, 20).

Chitinase and ,B-glucosidase were first reported by Grassmannet al. (10) in extracts of sweet almond. Since then, chitinase hasbeen found in the seeds of plants belonging to one family in themonocotyledons and to 10 families in the woody and herbaceousdicotyledons (18). Chitinase as well as other glycosidases havebeen isolated and purified from the leaves of Phaseolus vulgaris(2). A lysozyme from the latex of both fig and papaya was isolatedand found to possess extremely high chitinase activity (7, 14).Mandels et al. (15) has found a ,8-1,3-glucanase in callus culturesof bean, lettuce, carrot, and pepper.

Since F. solani cell walls do contain chitin and a glucan with,8-1,3 linkages as their major components (22), we looked forchitinase (EC 3.2.1.14) and 8-1,3-glucanase (EC 3.2.1.6) in thepod tissue of P. sativum L. and for other enzymes which may beinvolved in the degradation of F. solani cell walls. These includechitosanase, fl-D-N-acetylglucosaminidase (EC 3.2.1.30), ,8-D-N-acetylgalactosaminidase (EC 3.2.1.53), 8l-D-glucosidase (EC3.2.1.21), a-D-glucosidase (EC 3.2.1.3), and a-D-mannosidase (EC3.2.1.24), all of which, except a-D-glucosidase and chitosanase,have been isolated from P. vulgaris (2, 3). To the best of ourknowledge, this is the first report of the existence of these enzymesin the pod tissue of P. sativum.

MATERIALS AND METHODS

F. solani f. sp. pisi, strain P-A (ATCC 38136), and F. solani f.sp. phaseoli, strain W-8 (ATCC 38135), were obtained from R. J.Cook and D. J. Burke, respectively. The P. sativum pods werefrom the Alaska-type variety "Dot."

Substrates and Reagents. Chemicals purchased from Sigma199

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NICHOLS, BECKMAN, AND HADWIGER

were p-nitrophenyl-N-acetyl-,8-D-galactosaminide, p-nitrophenyl-

N-acetyl-fi-D-glucosaminide, p-nitrophenyl-f8-D-glucoside, p-ni-

trophenyl-a-D-glucoside, p-nitrophenyl-a-D-mannoside, chitin,

glycol chitosan, p-nitrophenol standard, p-dimethylaminobenzal-dehyde, acetyl acetone, and 3,5-dinitrosalicylic acid. Laminarinwas purchased from the United States Biochemical Corp. and

mixed 3H-amino acids (191 mCi/,ig) were purchased from NewEngland Nuclear Corp.

Interface Barrier Analysis. Freshly harvested immature peapods were washed thoroughly in sterile H20. Nuclepore (polycar-bonate) filters (Nuclepore Corp., Pleasanton, Calif.) with 0.4-,umpores, Millipore (mixed esters of cellulose) pH filters (MilliporeCorp., Bedford, Mass) with 0.3-, 1.2-, and 5.0-,um pores, or stand-ard (boiled) dialysis tubing were utilized as barriers between theendocarp surface and the inoculum suspension. Spores suspendedin Vogel's (24) medium (supplemented with 500 mg/l casaminoacids) were applied in 5-,ul volumes, which moistened the thinfilter (or dialysis tubing) to apparent saturation. The filter fitsecurely to the endocarp surface, and there was liquid continuityfrom that surface through the filter to the spores. The interfacemoisture barrier film was maintained by storing the preparationsin 60-mm Petri plates in a small plastic bag within a larger bag,both of which contained water-saturated paper tissues.

In treatments designed to evaluate the transfer of induced orpreinduced resistance through the barrier, resistance was inducedwith an inoculum of the incompatible pathogen which was allowedto come in direct contact with the endocarp surface. The barrierthen was put in place after the specified incubation period forpreinduction of the resistance response. The organism to be testedthen was placed on top of this barrier.

Inoculation and Treatment of Pea Pods. Immature pea pods (2g for preparations of acetone powders, 1 g for fresh homogenates)approximately 2.0 cm long were inoculated with 500 ,ul of one ofthe following four treatments: sterile distilled H20, chitosan (1mg/ml), F. solani f. sp. phaseoli (nonpathogenic on peas) macro-conidia (3.72 x 10r spores/ml), or F. solani f. sp. pisi (a pathogenof peas) macroconidia (3.62 x 106 spores/ml). After inoculation,samples were incubated at room temperature for the appropriatetime period (0, 0.5, 1, 3, 6, and 24 h), frozen with liquid N2 aftercompletion of the incubation time, and stored at -80 C until allof the temporal sequences were complete. A control sample ofunsplit immature pea pods was also frozen immediately andstored.

Acetone Powder Preparation. Acetone powders of the podswere prepared and used as the enzyme source in order to minimizebackground absorbance in the spectrophotometric enzyme assaysof fl-D-N-acetylglucosaminidase, fi-D-N-acetylgalactosaminidase,,8-D-glucosidase, a-D-glucosidase, a-D-mannosidase, chitinase, andchitosanase. Powders were prepared from the above frozen sam-ples by grinding the frozen pods in a mortar and pestle with liquidN2. Cold acetone (10 ml) then was added, and grinding continueduntil the tissue was highly dispersed. This homogenate then wasfiltered through a Whatman No. I filter and washed with one 10-ml aliquot of cold acetone followed by 5 ml cold ethyl ether. Theresulting acetone powders were weighed to determine the equiv-alent weight of fresh tissue (76.5 mg acetone powder/g freshtissue) and stored in the cold until ready for use. The crudeenzyme mixture was prepared by suspending 17 mg acetonepowder in the appropriate volume of the designated buffer, de-pending on the enzyme being assayed. The enzyme activity wasrecovered by thorough buffer extraction of the soluble proteinfrom the acetone powder.

Fresh Homogenate Enzyme Preparations. Homogenates wereprepared by grindingI g frozen pea pods in a mortar and pestlewith 3 ml 50 mm K-acetate buffer, pH 5.0. The homogenates werecentrifuged at 10,000 rpm for 10 min, and the supernate wasretained for subsequent assay of activity (1). The use of soluble

enzyme from fresh pods instead of acetone powders helped elim-inate the background of reducing sugars in the f8-1,3-glucanaseassay which are released from particulate polysaccharides in theenzyme preparations from acetone powders.Enzyme Assays. The ,8-D-N-acetylglucosaminidase, fl-D-gluco-

sidase, a-D-glucosidase, f8-D-N-acetylgalactosaminidase, and a-D-mannosidase activities were assayed using the appropriatep-nitro-phenyl glycopyranoside by the method of Gottlieb (9) modifiedas follows. Each enzyme activity was assayed at the reported pHoptimum for beans (2) in 10 mm sodium citrate buffer at 37 C forvarying time intervals. The controls used were: (a) heat-denaturedenzyme mixture plus substrate and buffer; (b) substrate withbuffer; and (c) crude enzyme mixture and buffer. The reactionmixture consisted of 2.5 ,imol appropriate p-nitrophenyl glycopy-ranoside, 10 ,umol citric acid-sodium citrate, and 17 mg appropriateacetone powder in I ml of 10 mm sodium citrate buffer. Thereactions were stopped by the addition of 625 ,tl 0.2 M Na2CO3-The release of p-nitrophenol was determined by measuring A at430 nm on a Beckman model DUR spectrophotometer. Theamount ofp-nitrophenol released was calculated from a standardcurve (0.05 to 0.5 ,tmolp-nitrophenol). Enzyme activity is reportedas nmolp-nitrophenol released/min * g original fresh weight of thetissue. The a-D-glucosidase, f8-D-N-acetylgalactosaminidase, anda-D-mannosidase activities were only assayed using acetone pow-ders from unsplit, uninoculated pea pods.

Chitinase Assay. The chitinase assay was based on the colori-metric determination of N-acetylglucosamine released from chitinaccording to Pegg and Vessey (20). The substrate for the reactionmixtures consisted of 300 mg finely ground chitin (80 mesh)dispersed in 80 ml 0.2 M sodium citrate buffer, pH 5.5. Thereaction mixtures contained 0.8 ml substrate, 0.2 ml buffer, and17 mg appropriate acetone powder. The reaction mixtures wereincubated at 30 C for 24 h. Triplicate samples from each acetonepowder sample were run. The controls used were: (a) heat-dena-tured crude enzyme mixture plus chitin; (b) chitin and buffer; and(c) crude enzyme mixture and buffer. N-Acetylglucosamine wasdetermined by adding 0.3 ml saturated Na2B407 to I ml each ofthe reaction mixtures and heating to 100 C for 5 min (17, 20). Themixtures were cooled to room temperature, and 9 ml glacial aceticacid and 2 ml Ehrlich's reagent (1.0 g p-dimethylaminobenzalde-hyde in 50 ml glacial acetic acid with 2.5 ml concentrated HCI)were added. These mixtures were centrifuged at 3,000 rpm for 3min. The color of the supernatant was allowed to develop for 30min and was read at 540 nm. The reference blank containedsupernatant of the control reaction mixture with the heat-dena-tured enzyme. The amount of N-acetylglucosamine released wasdetermined from a calibration curve (0.680-4.5,umol N-acetyl-D-glucosamine). Chitinase activity was expressed as nmol N-acetyl-glucosamine released/min* g original fresh weight of the tissue. N-Acetylglucosamine was verified by TLC using butanol-pyridine-water (70:15:15) and butanol-acetic acid-water (120:35:50) as thesolvent systems. The developing reagent for the chromatogramwas p-dimethylaminobenzaldehyde (21).

,8-1,3-Glucanase Assay. The,8-1 ,3-glucanase activity was deter-mined by the method of Abeles and Forrence (1). Laminarin as

the substrate and dinitrosalicylic reagent (150 ml 4.5% NaOHadded to 440 ml solution containing 4.4 g 3,5-dinitrosalicylic acidand 127 g K-sodium tartrate-6H20) were used to measure thereducing sugars released. The assays were run at pH 5 in 50 mMK-acetate buffer at the reported temperature optimum (50 C) forbean glucanase (1). Supernatant fractions (0.5 ml) of homogenateswere added to 0.5 ml 2% (w/v) laminarin in water (the laminarinwas dissolved by heating the 2% solution briefly in a boiling waterbath before use) and incubatedI h. The reaction was stopped byadding 3 ml dinitrosalicylic reagent and heating the tubes for 5min at 100 C. The tubes then were cooled to room temperatureand diluted 1:5 with water and A was measured at 500 nm. To

200 Plant Physiol. Vol. 66, 1980



ACTIVITY AT THE PEA-FUSARIUM INTERFACE

determine if exoactivity was present, the glucose oxidase reaction(Glucostat from Worthington Biochemical Corp.) was utilized toascertain that glucose was released from laminarin. Glucanaseactivity was expressed in nmol glucose equivalents released/min.g original fresh weight of the tissue.

Chitosanase Assay. Chitosanase activity was based on the col-orimetric determination of glucosamine released from glycol chi-tosan. The substrate solution was prepared by dissolving I g glycolchitosan in 10 ml 0.1 M HCI with stirring. To this solution, 20 ml0.2 M acetate buffer (pH 5.6) were added and the pH was adjustedto 5.6 with 0.1 M NaOH. The volume was made up to 50 ml withdeionized H20. The reaction mixture consisted of the crude en-zyme source (17 mg acetone powder in 0.5 ml acetate buffer) and0.5 ml substrate solution. This mixture was incubated for 10 minat 40 C. Glucosamine liberated was determined by the method ofRondle and Morgan (21). Chitosanase activity expressed as nmolglucosamine released/min- g of the original fresh weight of tissuewas determined using a standard curve using 5 to 300 ,tg gluco-samine/ml.

Mixed 3H-Amino-Acid Labeling and Purification of Fungal CellWalls. Fusarium solani macroconidia (3 x 107 spores) were sus-pended in Vogel's (24) medium (10 ml) and then centrifuged to apellet in a conical centrifuge tube. The supernatant was removedand replaced with 0.3 ml fresh Vogel's medium. One hundred,Cimixed 3H-amino-acids (191 mCi/,ug) were applied to the resus-pended spores and taken up over a 2-h period. (Because of thehigher specific activity available, the mixed amino acid label wasselected as precursor to the cell wall complex.) The volume wasadjusted to 5 ml with additional medium, and the spore suspensionwas distributed as a film on Petri plates and incubated 48 h atroom temperature at 100%1o humidity to allow randomization oflabel. Following germination and labeling, the spores were re-covered, washed extensively, and partially homogenized at 23,000rpm in a VirTis homogenizer in an extraction buffer designed toextract fungal nuclei (8). Complete cell disruption was accom-plished in 10%1o Triton X-100 by sonication over three 2-minperiods in an ice bucket. Following cell rupture, the cell wallswere recovered and cleaned with five cycles of resuspension in

Table I. Nuclepore Filters and Dialysis Tubing as Barriers to Phytoalexin Elicitation and Transmission of DiseaseResistance Factors

Treatment in contact with barrier upperTreatment in Contact with Pod8 ufcsurface

F. solani f. sp. Pisatinb Growthc Barrier' F. solani f sp Growth on uppersurface

Agdphaseoli 28 R None Nonepisi 26 R to + None Nonepisi 31 R to + Filter phaseoli R to +pisi 37 + Filter pisi ++phaseoli 24 R Filter phaseoli +phaseoli 18 R Filter pisi ++Nonee I Filter phaseoli +++Nonee 4 Filter pisi +++Barrier w/o pod Filter phaseoli +++Barrier w/o pod Filter pisi ++++Nonee 0 Filter NoneNonee 0 Nonepisi 23 + Tubing phaseoli (R to ++)fpisi 16 ++ Tubing pisi ++++fphaseoli 18 R Tubing phaseoli +++fphaseoli 11 R Tubing pisi +++fNonee 4 Tubing phaseoli +++Nonee 1 1g Tubing pisi +++Nonee 0 Tubing NoneBarrier w/o pod Tubing phaseoli +++Barrier w/o pod Tubing pisi ++++phaseoli 15 R Tubing Nonepisi 32 ++ Tubing None

a Pods 2 cm in length were split and the treatment of either 5 ,ul spore suspension (3.5 x 106 spores/ml) or 5pl Vogel's media was applied to the endocarp surface of each half pod. Three h following this treatment, anuclepore filter (0.45-,um pore) or dialysis tubing was applied, followed by the barrier surface treatment.

b Extractable pisatin per half pod (60 mg fresh weight) after 24 h. The figures represent averages of duplicateassays.

c Growth of the macroconidia after 24 h was recorded from the fresh sections of surface cells at high power ina light microscope: R, complete resistance; +, growth occurred which was in excess of the macrospore length,implying incomplete resistance; ++, growth in excess of twice the macroconidia length; +++, very extensivegrowth (seven to 10 times the macrospore length); ++++, mycelia grew in mats, and thus individual lengthswere not measurable.

d The SE from the mean is within I SD for all values oftg of pisatin.'None, Vogel's medium only.fA few spots on the barrier accumulated dark green pigmentation which suppressed macroconidial growth.9 Dialysis tubing inoculated with F. solani f. sp. pisi is partially digested within 24 h, thus destroying the

continuity of the barrier.

Plant Physiol. Vol. 66, 1980 201




sterile H20 and centrifugation at 5,000 rpm for 30 s. Cytologicalexamination indicated the fungal walls were clean.

Fungal Cell Wall Digestion Assay. Purified mixed 3H-labeledamino acid cell walls from F. solani f. sp. phaseoli were used todetermine the release of labeled cell wall components by digestionwith the crude enzyme mixture. A 1.0-ml aliquot of the cell wallsuspension contained 102,857 cpm. A 1.0-ml aliquot of the crudeenzyme mixtures was added to 0.7 ml cell wall suspension andincubated at 37 C for various time intervals. After each timeinterval, 100 ,ul reaction mixture supernatant was streaked onto aTLC sheet and chromatographed in butanol-pyridine-water (70:15:15) or butanol-acetic acid-water (120:30:50). Color was devel-oped with a silver nitrate spray. After chromatography, the sheetswere cut in 1-cm strips and counted in a scintillation counter.["4CJN-Acetylglucosamine was run as a chromatographic stand-ard.

RESULTS AND DISCUSSION

Molecular Sieve Barriers to the Host-Parasite Interaction.Contact between pea endocarp tissue and F. solani f. sp. phaseoliappears to be necessary for the induction of disease resistanceresponses, such as phytoalexin production. Molecular barriers,which physically separate host and parasite, effectively limit thetransfer of disease resistance potential from the host to pathogenand of phytoalexin elicitor components from pathogen to host.This limitation occurs even with large pore (0.4,m) membranefilters which should allow interchange of macromolecules. TableI shows that, when either form of the F. solani is in contact withthe pea endocarp tissue, large quantities of phytoalexin are in-duced. When neither pathogen is in direct contact with the pod,phytoalexin production is extremely low. One exception occurswhen F. solani f. sp. pisi is separated from the pod with dialysistubing. This pathogen eventually digests holes in the tubing, andconsequently some pisatin is induced.

Although pisatin production is probably not the major compo-nent in the disease-resistance response (13), total disease resistanceto the pea pathogen F. solani f. sp. pisi is induced in pea tissuewhen F. solani f. sp. phaseoli spores are applied with or shortlybefore those of the pathogen. Apparently this total resistance isunable to traverse completely the artificial barriers. Both avirulentand virulent forms of F. solani, if applied in a nutrient medium,are able to grow freely when separated by a filter barrier (poresizes from 0.3 to 1.2 ,um) from the pod endocarp tissue; however,the growth of F. solani, when separated from the pea pod by aNuclepore filter, is somewhat better when no host response hasbeen induced in the pea tissue on the opposite side of the barrier.This suggests that, after the resistance response has been activatedby a pathogen which is in direct contact with the plant tissue,some of the products do traverse the barrier. However, the effect

Table II. Comparison of Glycosidic Enzyme Activity in Pea Pod Tissue

Enzyme ActivityaSD

fB-D-N-Acetylglucosaminidase 105.4 1.92fl-D-Glucosidase 94.0 1.19f8-D-N-Acetylgalactosaminidase 20.7 1.85/3-1,3-Glucosidase 421.4 0.87a-D-Glucosidase 57.9 0.96a-D-Mannosidase 155.0Chitinase 3.4 1.30Chitosanase 35.0 1.09

a Activity was expressed as nmol ofp-nitrophenol, glucosamine, glucoseequivalents, or N-acetylglucosamine released/min g of the original freshweight of the tissue (see under "Materials and Methods") and values werean average of three assays.

0

3

..S~~~~~.........

0 2 3 4 5 6 24

HOUR S

FIG. 1. Chitinase activity in pea pod tissue following contact with F.solani macroconidia or chitosan. Freshly excised, unsplit pods (0), splitpods treated with water (0- - -), F. solani f. sp. phaseoli (3.7 x 10Imacroconidia/ml; A A); F. solani f. sp. pisi (3.6 x 10" macroconidia/ml; E-U), or chitosan (1 mg/ml; + 4) were assayed for chitinaseactivity.

is only fungistatic at best. Even the non-pathogen F. solani f. sp.phaseoli is able to grow extensively when physically separatedfrom the responding host. Finally, the growth of F. solani f. sp.phaseoli as germinating macroconidia is often suppressed whenthe barrier contains 5-,Lm pores. Cytological examination showsthat the intact Fusarium mycelia can traverse the 5-,um pores andcome into direct contact with the pea endocarp. The growth ofboth the pathogen and non-pathogen was inhibited (at leasttemporarily in the case of the virulent pathogen) when in directcontact with the pea tissue even though each was applied in anutrient medium adequate for unlimited growth.

Acetic anhydride-treated or boiled dialysis tubing and mixedesters of cellulose or polycarbonate filters were utilized as barriers.The results with all barriers with 1.2 ,tm or smaller pore sizes wereconsistently similar to those of the barriers presented in Table I.Apparently the growth of the pathogen was not unduly influencedby the chemical constituents of the barriers themselves.

Both the pathogen and non-pathogen grew when separatedfrom pea tissue throughout a period when copious quantities ofpisatin were being produced, even though in each case the barrierwas not restrictive (in respect to pore size) to the transport ofpisatin molecules. The 0.4-,um filter was not restrictive size-wise toany soluble molecule of the plant cell. Thus, the initiation ofphytoalexin production and the availability of factors which sup-press growth of the non-pathogen require that the cells of the hostand pathogen mnake physical contact. A preliminary report (I 1)suggests that host cells possess enzymes capable of cleaving thefungal cell wall into fragments (probably chitosan molecules)which can both induce pisatin and inhibit fungal growth.

Cytological observations of the pea-Fusarium interaction sug-gests a type of enzymic action which alters the morphology of thefungal wall (12, 23). The end of the germinating macroconidiawhich comes in contact with the plant surface is usually structur-ally distorted within 20 h after inoculation. Cross-sectional viewsofthe host-parasite interaction in the scanning electron microscope




ACTIVITY AT THE PEA-FUSARIUM INTERFACE

zwA-aot

ai 1.2w

000

0

a

A

OAF0 2 3

H 0U R S

FIG. 2. /?-1,3-Glucanase activity in pea pod tissue following contactwith F. solani macroconidia or chitosan. Freshly excised, unsplit pods(0), split pods treated with water (0- - -), F. solani f. sp. phaseoli (3.7x 106 macroconidia/ml; A A), F. solani f. sp. pisi (3.6 x 106 macroco-

nidia/ml; *-E, or chitosan (I mg/ml; + I) were assayed for ,B-1,3-glucanase.

suggest fungal and host surfaces are partially digested within 7 hafter contact (12). Substantial enzyme activity was detected innoninoculated pea pods for the enzymes ,B-1,3-glucanase, a-D-mannosidase, and,8-D-N-acetylglucosaminidase (Table II). Otherglycosidic enzymes present in pea pod tissue were fl-D-N-acetyl-galactosaminidase, ,/-D-glucosidase, a-D-glucosidase, chitosanase,and chitinase. Both chitinase and 8-1,3-glucanase activities were

high over a broad pH (4.5 to 5.5) range.The crude enzyme mixture of the pea pod tissue released

significant amounts of reducing sugar when incubated with eitherlaminarin (Table II) or purified Fusarium cell walls (data notshown). However, the glucose oxidase test indicated that glucoseis released from laminarin (a /8-1,3-glucan) but not from thepurified Fusarium cell walls. Using laminarin as a substrate, Abelesand Forrence (1) have shown that the bean glucanase is an

endoenzyme releasing only oligosaccharides from laminarin. The/3-1,3-glucanases of callus tissue (15) were also endoenzymes withthe higher oligosaccharides appearing early in the digestion, sug-gesting a random acting endoenzyme. Clarke and Stone (4),however, reported that the /3-1,3-glucanase isolated from grapevines also releases reducing sugars. Further purification of the pea

enzymes is required to resolve exoactivities due to other enzymescontaminating crude preparations.When the crude enzyme mixture from pea pod tissue was

incubated with the purified tritium-labeled fungal cell walls, thelabeled components released into the supernatant increased withincreasing incubation time (data not shown). Some of the labelappeared to be associated with reducing sugars as they developedcolor with a silver nitrate spray. The control incubated with a

denatured enzyme mixture did not release solubilized components

0

224HOURS

FIG. 3. Chitosanase activity in pea pod tissue following contact with F.solani macroconidia or chitosan. Freshly excised, unsplit pods (0), splitpods treated with water (0- - -4), F. solani f. sp. phaseoli (3.7 x 10'macroconidia/ml; A A), F. solani f. sp. pisi (3.6 x 106 macroconidia/ml; E-U), or chitosan (1 mg/ml; * *) were assayed for chitosan-ase activity.

from the purified fungal cell walls. The dissolution of F. solanicell walls has also been shown using streptomycete chitinase and,B- 1,3-glucanase (22).The function of chitinase in plants is not understood because of

the absence of chitin in higher plants. It seems possible that suchenzymes in peas, by releasing soluble eliciting components fromthe fungal cell wall, can function as a mechanism in eitherinhibiting growth of the fungus or stimulating defense mechanismsin the host. The action of a third enzyme, chitin deacetylase, ifavailable at the interface, could provide a link to the productionof chitosan, which has recently been shown to be a potent inducerof pisatin (11). Chitosanase could function to reduce the chainlength of chitosan components released from the fungal cell.

Since the period crucial to the expression of resistance to F.solani f. sp. phaseoli occurs (23) within 6 h after the fungus contactsthe host tissue, the activity levels of chitinase (Fig. 1), ,B-1,3-glucanase (Fig. 2), and chitosanase (Fig. 3), were followed up to24 h after inoculation. Chitinase activity (Fig. 1) is moderate infreshly excised tissue; however, the activity level is briefly in-creased when the pod is split and treated with water. Treatmentof the pod with macroconidia or the fungal cell wall componentchitosan results in an increase in chitinase activity which is stableover 0.5 to 3 h. The 3- 1,3-glucanase activity increases with alltreatments over time and thus remains available for fungal cellwall digestion. Chitosanase activity (Fig. 3) drops briefly in peatissue in the first hour after inoculation, but part of the activity isrecovered within 6 h.

CONCLUSIONSContact between the Fusarium spore and the pea host appears

to be required to initiate host-parasite interaction. If the initial

Plant Physiol. Vol. 66, 1980 203

1

6 2




step in the induction of host resistance requires partial processingof the fungal spore, the enzymic potential for this processingwhich exists in the plant in the form of various glycosidic enzymesis apparently not readily released or transported through artificialbarriers at the host-parasite interface. Although the precise role ofglycosidic enzymes in disease specificity is not presently clear, itappears that pea tissues do possess enough of these enzymes toalter the invading fungal cell. Therefore, to define the regulatoryfungal compounds active in the host-parasite interaction, one mustlook for fungal compounds generated by enzymatic processes ofthis interaction as well as for those released from pure cultures ofthe fungus.

LITERATURE CITED

1. ABELES FB, LE FORRENCE 1970 Temporal and hormonal control of ,B-1,3-glucanase in Phaseolus vulgaris L. Plant Physiol 45: 395-400

2. AGRAWAL KML, OM P BAHL 1968 Glycosidases of Phaseolus vulgaris If. J BiolChem 234: 103-1 11

3. BAHL OM P, KML AGRAWAL 1968 Glycosidases of Phaseolus vulgaris 1. J BiolChem 234: 98-102

4. CLARKE AE, BA STONE 1962 /1-1,3-Glucan hydrolases from the grape vine ( Vitisvinifera) and other plants. Phytochemistry 1: 175-188

5. DANIELS DL, LA HADWIGER 1976 Pisatin-inducing compounds in filtrates ofvirulent and avirulent Fusarium solani cultures. Physiol Plant Pathol 8: 9-19

6. DIXON GR, GF PEGG 1969 Hyphal lysis and tylose formation in tomato cultivarsinfected by Verticillium albo-atrum. Trans Br Mycol Soc 53: 109-118

7. GLAZER AN, AO BAREL, JB HOWARD, DM BROWN 1969 Isolation and charac-terization of fig lysozyme. J Biol Chem 244: 3583-3589

8. GOFF CG 1976 Histones of Neurospora crassa. J Biol Chem 251: 413141389. GOTTLIEB C, J BAENZIGER, S KORNFELD 1975 Deficient UDP-N-acetylglucosa-

mine: Glycoprotein N-acetylglucosaminyl transferase activity in a clone ofchinese hamster ovary cell with altered surface glycoproteins. J Biol Chem 250:

3303-330910. GRASSMANN W, L ZECHMEISTER, R BENDER, G TOTH 1934 Uber die Chitinspal-

tung durch Emulsin-praparate. III. Mitteil uber enzymatosche Spaltung vonPolysacchariden. Berichte 67: 1-5

1 1. HADWIGER LA 1979 Chitosan formation in Fusarium solani macroconidia on peatissue. Plant Physiol 63: S-133

12. HADWIGER LA, MJ ADAMS 1978 Nuclear changes associated with the host-parasite interaction between Fusarium solani and peas. Physiol Plant Pathol12: 63-72

13. HADWIGER LA, DC LOSCHKE, JR TEASDALE 1977 An evaluation of pea histonesas disease resistance factors. Phytopathology 67: 755-758

14. HOWARD JB, AN GLAZER 1967 Studies of the physiochemical and enzymaticproperties of papaya lysozyme. J Biol Chem 242: 5715-5723

15. MANDELS M, FW MARRISH, ET REESE 1967 fi-1,3-Glucanases from plant calluscultures. Phytochemistry 6: 1097-1 100

16. MITCHELL R 1963 Addition of fungal cell wall components to soil for biologicaldisease control. Phytopathology 53: 1068-1071

17. MORGAN WTJ, LA ELSON 1934 A colorimetric method for the determination ofN-acetylglucosamine and N-acetylchondrosamine. Biochem J 28: 988-995

18. POWNING RF, H IRZYKIEWICZ 1965 Studies on the chitinase systems in beanand other seeds. Comp Biochem Physiol 14: 127-133

19. PEGG GF 1973 Chitinase and glucanase activities in Verticillium albo-atrum-infected tomato plants. Abst 0968, 2nd Int Cong Plant Pathol, Minneapolis

20. PEGG GF, JC VESSEY 1973 Chitinase activity in Lycopersicon esculentum and itsrelationship to the in vivo lysis of Verticillium albo-atrum mycelium. PhysiolPlant Pathol 3: 207-222

21. RONDLE CJM, WTJ MORGAN 1955 The determination of glucosamine andgalactosamine. Biochem J 61: 586-589

22. SKUJINS JJ, HJ POTGIETER, M ALEXANDER 1965 Dissolution of fungal cell wallsby a Streptomycete chitinase and ,- 1 ,3-glucanase. Arch Biochem Biophys I I 1:358-364

23. TEASDALE J, D DANIELS, WC DAVIS, R EDDY, LA HADWIGER 1974 Physiologicaland cytological similarities between disease resistance and cellular incompati-bility responses. Plant Physiol 54: 690-695

24. VOGEL HJ 1956 A convenient growth medium for Neurospora. Microbiol GenetBull 13: 42




glycosidic enzyme activity in peatissue andpea ... · acids) were applied in 5-,ul volumes, which...

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