Effect of the Specific Toxin in Helminthosporium victoriae Host

Platnt Phlysiol. (1968) 43, 21-28

Effect of the Specific Toxin in Helminthosporium victoriaeon Host Cell Membranes'

K. R. Samaddar and R. P. SchefferDepartment of Botany and Plant Pathology, Michigan State University,

East Lansing, Michigan 48823

Received July 31, 1967.

Abstract. Helminthosporium victoriae toxin, which affects only hosts of the toxin-producingfungus, causes loss of electrolytes from roots, leaves, and coleoptiles of treated plants. Roothair cells lost the abi,lity to plasmolyze after 20 minutes exposure to toxin in solution;comparable resistant cells retained plasmolytic ability during 3 hours exposure. Toxinstopped uptake of exogenous amino acids and Pi by susceptible but not by resistant tissue.Incorporation of 32p into organic-P and 14C-amino acids into protein was blocked in susceptiblebut not in resistant tissue. Apparent free space increased in susceptible but not in resistantroots. The increase was evident within 30 minutes, and reached 80 % free space after 2 hoursexposure to toxin. When cell wall-free protoplasts were exposed to 0.16 ,ug toxin/ml, proto-plasmic streaming stopped and all plasma membranes of susceptible protoplasts broke within1 hour. Resistant protoplasts were not affected significantly. Data support the hypothesis ofa primary lesion of toxin in the plasma membrane. Effects on synthesis could result fromlack of transport of exogenous solutes to sites of synthesis. It is possible that all otherobserved effects of toxin are secondary to membrane damage.

"Host-specific toxinis" that are determinanits ofpathogenicity are now known from at least 5 dif-ferent planit infecting fungi. These com,poundsreproduce all visible and all known biochemicalsymptoms of infection. In each case, loss of abilityof a funiiguts isolate to produice its toxinl has resultedin loss of pathogenicity. In each case, the sub-stance is toxic only to the host of the toxinl pro-ducing fuingigus (23). These are usefuil models forthe studv of disease, because many possible com-plexities of host-parasite interaction are bypassed.

One suich suibstance, produced by Heluninthjo-sporiumzictoriaz e Mieehan and Mturphy (HV-toxin),is toxic to suisceptible oat cualtivars, but is harmlessto resistant oat cultivars and to all other non-hostplants tested (15). The toxin is composed of acyclic secondary amine (C17H,9NO) known as"victoxinine" anid a peptide containing aspartic acid,glutamic acid, glycine, valine, and leuicine. Thecomplete toxini has a M.W. between 800 and 2000but the exact structure is still unknown, largelybecauise of problems with lability (15).

Amon.g the many cellular responses that occuirquickly after HV-toxin treatment are: increased0., utptake (21) : decreased incorporation of '4Camino acids anid uridine into trichloroacetic acidlinsoluible celluilar fractions (23); and rapid loss ofelectrolytes (24). Several lines of evidence suggestthat these effects are secondary. Oxygen and Pi

1 Publislhed with the approval of the Director, Michi-gan Agricultural Experiment Station, as Journal articleNo. 4157. Aided by grants GB-1448 and GB-6560Xfrom the N'ational Science Foundationi.

uptake by isolated mitochondria were not affected(4, 21, 23). Toxin uptake appears to be a simpleprocess, not affected by wide ranges lof temperatureand by metabolic inhibitors. The peptide resultingfrom toxin breakdown inhibited toxin uptake, sug-gesting a competition for receptor sites present instusceptible cells (22).

Data to date suiggest thsat the toxini cauises aprimary lesion in the plasma membrane of suiscep-tible cells (23). The 'basis of resistance may be alack of receptors or sensitive sites in the membrane.The possibility that the ini;tial toxin lesion is in theplasma membrane was examined in this stuidy bythe tuse of intact tisstues and cell free systems. Theeffects of toxini oni ion leakage from tissules, cellplasmolysis, uliptake of soltites, apparent free space,and behavior of cell1 wall free protoplasts weredetermined. The data indicate that toxinl affectsmembrane physiology and transport systems ofsusceptible bu,t not resistant cells. An abstract ofpart of the work was published (19).

Materials and Methods

Toxini suisceptible (cv. Park) and resistant (cV.Clinton) oat seedlings were uised in most experi-ments. Seeds were germinated on moist filterpaper and seedlings were grown in the laboratoryat 21 to 220 in White's nutrient solutions (14), *orin vermiculite plus White's nutrients. In somecases larger plants grown in the greenhouse weretused. The inhibition of seedling root growth in adiltution series of toxin was tused as the standardbioassay (15).

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PLANT PHYSIOLOGY

Toxi:n elilted from an alumina coluimn, followiingthe method of Pringle and Bratun (14), was rela-tively stable at pH 3.5, and the concentrated solu-tion was stored at 4°. This preparation gavecomplete inhibitioni of root growth of stusceptibleoat see(lliings at a concentration o/f 0.0016 tug/mlbuit ha(l no effect oIn growth of resistant seedlingroots at 160 ug/ml. Fuirther purification, achieve'dby passnin this soluttion throuigh a Bio-Ge'l P-2coltumn, gave a more toxic buit less stable prepara-tioni.

Ioan Lcakagc. T'he effect of toxini 0o iOln leak-age fromn coleoptile tissuie wvas measured by changesin electrical con(lucti:vity of su spending solutions.One g tissuie samples were vacuutim infiltrated for10 minuiites with toxini, water, or inactivated toxinl.I'he samlle was rinse(d in glass distilled water,enclosed in x-ashed cheesecloth bags, mn(l ilncubatedon a shlaker in 100 ml glass distilled Water at 22°.Condchictivitx of the ambient solution was measuiredwith a io(del RC 16B1 IInduistrial Instrumiiienits con-(llct v ity bridge, uising a dip type electro(le cell(k- 1.0). Specific conductivity is given as re-cip)rocal ohmlls (mhos).

322) [Uptakc (111(i Inlcorporationl. l issIle samil)leserele l)lace(l in flasks containin- 2 ml 1 in.\i acetate

l)llffer (pH 5.5), 155 ttg chloramphenicol aln(d 200 uc32 1). Flasks were incuibate(d at 250 onl a Du)biuoffmetabolic shaker (40 oscillations/nin) for 2 houlrs.Samples were then rinse(d thorouighly wvith cold1 mmr KH.,PO and groutnd in boiling 80 % (v/v)ethaniol. Th'e homogeniate wras centrifutged at10,000 X g for 10 mintutes and the stupernatantconlcentrated to 2 ml. A 50 ,ul aliqulot was spottedoni Whatman No. 1 paper. Marker .32p was -s,pottedoni each paper for tracinig the movement of Pi.Descendinig chromatographs ulsing ni-buityl alcohol:propionic acid water (1246:620:874 v/v) were de-veloped for 40 houirs (2). Radioactive zones weredetected with a Nuiclear Chicago monito,r. Radio-auitographs were made on Eastman Kodak X-rayfilms. Individual radioactive zones were removed,gluted to pllanchets and coulnite(d in a Nuiclear Chicagolow background gas flow couinter. Total uiptakewas calculated by adding the couints of all spotsdevelo,ped from a single origin. The amouint ofradioactivity in the inorgalic and organic P spotswas expressed as percent of total uiptake. Noattempt was made to identify the organic P conm-pounds.

14C-.Al into A-cidl Uptake. Uptake was conisideredas the amouint of labeled compouind taken in fromthe medium, and( retained by tissules after repeatedwashings (18). Roots from 10 seedlings wereincubated in 1.0 ml DL-leucine-1-'4C or DL-valine-U-14C (pH 7.0). Concentrations of the amino aciidsolutions varied from 1 to 50 mm with specificactivities from 0.5 to 1.0 pc per ml. Chloram-phenico,l (15 xg) was added to each flask and themixtuire w%vas inculbated for 1 to 3 houirs at 220 with

gentle shakinig. The roolts were then washlecl forI houtr in ruinning tap water, blotted gently, placedinito tightly stoppered tuibes with 0.5 ml 95 %ethanol, and extracted with shakiing for 12 houirs.Aliquiots of the ethanol extracts were coutintedl onplacnchets in a Nuiclear Chicago gas flow couniiter.

Apparent Fr-ce Space. Apparen,t free sp)ace ofexcised roots was determined( bx the India iniktagging method of Bernsteini and Niemani (3).Roots were rinsed in distilled water, thein equili-brated in 35 m.t mannitol soluitioni (50 ml/g roottissuie) for 0.5 to 2 houirs, w\ith 4 or 5 changes ofsoltition. Duiring the last minuite of equIilibration,1 ml India ink suispension (1 part/10 parts 35 mmmannitol soltitioin) w$as adde(d to each 20 mfl ecqili-brating solutioin. The preparatioln was stirred fora few secondcIs before roots w-ere removed, (lrainiedl,aI(l tranIsfer-re(d to 100 mil glass (listille(I wa\N1ter foiexodi f ftisiOn .

Exo(liffusoioI soluitioIns Were stirred foI 5 mmil-ultes, ani(d samples x-erc taken at interval-s of 5, 10,15, 30, and 60 minutes. Optical d1enlsities of 1 :100dilutitnoii of the equilibrating solutiOInS and exodif-fuslioI media wvere deterin;IIe(d w\ith a colorrimneter at(500 mnp- A coI-rectioIn foI- the v-olu1me of equlli-bratinfg solu1tioIn adhering to root S 111-flaces w\s Cal-cculate(d froIml the o,ptical densitV aid volumic ofe(qilib-ating andI exod1ifftisioi media (3). Man-itol concentrations in equilibratioui and exo(liffui-

siOI media were deterimlined b\ ilomonletric titrationas described by Bultler (6). Mannitol adheringto the root suirface was suibtractedl fromn totalmannitol (tiaintity in the exodiffuision mediuim toobtaini the amouint of solute in frec' space. T'oxillcauises leakage of antthrone positive carbohydrates(4), which might give an error in manniitol deter-minatiolis. Therefore, a correction for toxintreated samples kept in water was applied. Ap-parent free space was calcula,ted fromii the diffuisiblesolute (mg) in the exodiffuision mecdiuim anid theconcenitration (nmg/nml) of the e(luilibrating solt-tion, expressed as percentagc fresh w'eight of r(oottissue.

Prepa ration of Pr-otopltasts. Protopl asts fromcoleoptiles of oa,ts, corn, an(l sorghtim were pre-pared with cellulase by the method of Ruesink aindThimanni ( 17). Cellulase was prepared from cudl-ttire filFtrates of Myroth eciunt zverrutc(ria isolate 460,olbta,ined from Dr. Mary Mandels of the Quia,rter-master Laboratory at Natick, MIassachulsetts. Thefuingtis was grown with continutouis shaking for 14days on a modified Whitaker's salt mediuim (17, 25).Culltuire filtrates were concentrated to 0.1 voluimein a flash evaporator at 370, anid re-filtered. Thefiltrate was fractionated wi,th solid (NH,) 2SO4at 30 (7). The precipitate obta,ined at 35 to 70 %satuirationi was dissolved in a small volume of wateranid desalted in a Sephadex G-25 coluimn. Thecoltumn was prepared, washed, anid deve,loped with0.1 % NaCI soluitions. Maximuim celltlase activitywas in the second brown (1 ml) fraction eluited

-

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SAMADDAR AND SCHEFFER-EFFECT. OF H. VICTORIAE TOXIN

from the column. Aliquots (200 jul) of this frac-tion were stored in separate vials at -200, sinceactivity was lost with repeated thawing andfreezing.

Coleoptiles from seedlings grown in dim redlight were harvested 72 hours afiter germination.Sections 1 mm long were treated with cellulasesolution diluted 1:1 with 1.0 M mannitol bufferedwith 25 mm soditum phosphate buffer (pH 6.5).After 2 hours incubation in the dark at 220, 10volumes of 0.5 M mannitol buffered at pH 6.5 wereadded. Protoplasts settled to the bottom of thetube in 10 minutes, and the supernatant was re-moved with a pipet. Mannitol solution was againadded and the procedLure was repeated to removecellulase.

Results

Effcct of Toxin ont Colcoptile Tissute. Sincecoleoptiles were to be used in some experiments, itwas necessary to see if they have the usuial differ-ential response of resistant and susceptible plantsto toxin. Toxin increases respiration (21) and ionleakage (24) in leaf and root tissues; these re-

sponses were chosen as possible indicators of toxiceffects on coleofptiles.

Seedllings were grown in the dark, in diffuselight, or in red light. Subapical sections (1 cm

long) of coleoptiles were removed and split 3 daysafter germination. Samples (0.5 g) were vactuuiminfiltrated with toxin solution (0.16 ,,g/ml) or

water at pH 6.5 for 10 minutes. Coleoptile tissueof susceptible toxin-treated oats released electro-lytes at a much faster rate than did the controlsand the treated resistant itissue (fig 1). Eighthours after toxin treatment, the specific conduct-ance of the ambient solution of treated susceptibletissue was 5 times greater than that of any othertreatment solution.

Oxygen uptake by toxin treated and controlcoleoptile tissue was determined manometrically.Toxin caused little or no increase in O., uptake bysusceptible coleoptiles in 4 experiments. Increased02 uptake caused by toxin apparently depends on

the type of tissue, while an increase in ion leakageis evident in all susceptible tissues so far tested (15).

Effect of Toxin on Plasmolytic Ability of Cells.Cells with damaged membranes will not plasmolyzewhen placed in hypertonic solutions. Therefore,we can test whether or not toxin destroys or dam-ages the plasma membrane in situ. The selectiveeffects of toxin were compared with the effect of2,4-dinitrophenol (DNP) on cell membranes, usingconcentrations of DNP known to uncouple oxida-tion from phosphorylation. Roots of 4 day oldplants were placed in either toxin solutions (0.16pug/m4), DNP (10 and 100 Mm), or water forvarying times. After treatment roots were quickly

5

0

x

0

I-

U

z0u

4

3

2

0

23

2 4 6 8

TIME AFTER TREATMENT (HR)

FIG. 1. Effect of toxin on loss of electrolytes fromHe/wlinithosporiihn victoriac resistant (Res) and sus-ceptible (Sus) coleoptiles. Tissue samples (0.5 g) wereinfiltrated with toxin solution (0.16 ,ug/ml) for 10minutes, and suspended in glass distilled water. Elec-trolyte loss was determined from conductivity of thewater. * = Susceptible toxin treated tissue; Q =susceptible control; A = resistant toxin treated; and* = resistant control tissue.

rinsed in water, placed in 0.5 M mannitol solutions,and observed under a microscope. Wilthin 20 min-utes after toxin exposure, susceptible root hair cellslost the ability to plasmolyze in hypertonic solutions(table I). Toxin treated resistant cells showedplasmolytic shrinkage even after 180 minutes Qfexposture to toxin. Resistant and susceptible un-treated controls plasmolyzed normally. DNP at100 jM acted mtuch more slowly than toxin and'destroyed the p-lasmolytic ability of both suisceptibleand resistant cells (table I).

Table I. Effects of Toxitt and 2,4-Dinitrophenol onPlasntiolysis of Root-hair Cells in Hypertoanic

Solutions

Exposure time' required to destroyTreatment plasmolysis ability

Susceptible ResistantMilt Mill

Control2 > 180 > 180Toxin, 0.16 ,u/ml 20 > 180DNP, 100 AuM 90 120DNP, 10 lAM 180 > 180

Exposure times were 10, 20, 30, 40, 50, 90, 120, 150,and 180 minutes prior to placing cells in hypertonicsolution.

2 Control roots were placed in water or deactivate&toxin.



Table II. Effect of Toxii oil 32p Uptake anid Inicorporationt by 0at Leaf 7issueToxini conicentrationi was 0.16 ug/nil. Each tissue sample was 0.5 g, pre-treated with toxinl or water for 4

hours and incublated with 3eP for 2 hours. Treatments were duplicated.

Oat type and Radioactivitvtreatmenit Total uptake Iniorganiic P Organiic P

r15 Z!,~~~~~

Susceptible conitrolSuisceptible plus toxinResistanit controlResistanlt pluls toxini

cpill12,8951560

14,40015,800

95551215

10.16011.990

cpIII3245120

35503780

Table III. Effect of Toxin oni Uptake of 14C Amino Acids by Susceptible and Resistanit Oat RootsReactioni mixture (1.0 ml) containied amino acid as indicated; 1 drop of chloralmpheniicol solutiotn (0.5 mg/

nl) 30 mmI phosphate buffer (pH 7.0) 250 mg fresh root tissue. After incubation for 2 hlours at 220 on a shaker,tissue was washed 1 hour withl water anld extracted with 0.5 ml ethaniol. Aliquots (0.1 ml) oin planchets werecounted. Toxini concentration was 0.16 jug/ml. The valine-U-14C was 1.0 jac/mil. The leucine-1-14C was 0.5 ,uc/ml.

Uptake of 14CExposure timiie Treatmiient Valine (50 nim) Leuci.ne (1 inm)

ltli)l SUS RES St S REStcP in c/ mn c'/l1 /'p m

30 Conitrol 9280 10.950 1540Toxii 5j85 11,240 255 ...

60 Conitl-ol 9570 9525 1700 ...

Toxini 580 10,040 270 ...

120 Control 9985 10,320 1960 1815Toxini 460 10.895 190 1795

Effcct of 'loxi' Iitl P Uptake 1n1( Inicorpora-tionl. Transpiring cuttings of resistant and suiscep-tible 10 day old plaints were allowed to take niptoxini solution (0.16 ug/ml) or water for 4 hoturs.Following treatment, 0.5 g leaf samp-les were inlcuI-bated with bnffered 32p as (lescribe(l previously.Radioactivity (cpm) of the ethanol extract wasused to estimate total ntptake. Cotnnits of the sev-eral spots on paper chromatograms gave the (lis-trilbution of P in organic anid inorganic fractions.Toxin treated suisceptible tissnle hadl less 32P niptakethan did stusceptible control tissnie (table II'). Up-'take was not inhibited in resistanit treated tissnies.-Controls and treated resistant tissnies incorporated20 to 30 % of the total 2p ilnto organic P-com-pouinds, while incorporatioin was completely blockedin treated suisceptible tissues. The experiment wasdlone 3 times withl essentially the same resullts.Similar resuilts were obtained with root and coleop-tile tissties.

In another experimenit, the etlhaniol extracts wereadded to paper uintil all spots had approximatelyequal couints. An auitoradiograph w\-as made of the-developed chromatogram. Labeled organic P-com--potninds occuirred in extracts of control and treatedl-resistanit tissuies but little or noine were fotnd( inlextracts of toxin treated susceptible tissuie.

Effect of Toxin ont Active Upta.ke of AmninoAcids. Amino acid uptake and retenitioni in rootstreated with toxin wrere compare(d \-ith tuptake and-retention in uintreated controls. Roots were treated

with toxinl solnition (0.16 ig/mln)iucubated illlabeled amino acid soluitions, washed and extracte(lwith ethaniol. The radioactivity (cpm) in ethanolextracts was tised as a measuire of intracellular freepool amino acids. Suisceptible roots exposed totoxin for only 30 minuites showed a 90 % decreasein the active uptake of labeled amino acids (tableIII). Similar resuilts wvere obtaiined with labele(iv-aline and leuicine at concentrations from 1 to50 nm\I.

Effcct of Toxin ont Apprenott Fr-ce Spacc of'Tissiuc. Apparent free space in roots is thonlohtto conisist of cell wall and(l intercelilular spaces (5).The possiblility of toxin-ilnduced chainges in apparentfree space in roots was examined as a fuirthermeasuire of membrane damage. The experimeiltwas based oIn the hypothesis that if tox,in disruiptsthe plasma membrane, the harrier for free permea-tion will disappear anid( apparent free space slhoull(dincrease.

Resistanit and( stusceptible oat plants were growiiin staining dishes with removable trays (22).Toxin or other substances were adldled to the solil-tions of some dishes, while other dishes were uisedas uintreated controls. Appareilt free slpace wassimilar with different equilibration and exodiffusiointimes uip to 2 hoturs and with excised roots anldroots of intact plants. In most cases roots withthe several different toxinl treatment times wereexcised and equiilibrated for 1 houir in 35 mm manl-nitol, followed by 1 houlr exodiffulsioIn in glassidistilled water.

24 PvLANiT PHY'SIOL,OGY



SAMADDAR AND SCHEFFER-EFFECT .OF H. VICTORIAE TOXIN25

90

90 ~~~~~~~Susceptible60

30 Resistant

C~C~c C

2 4 6 8HOURS

FiG. 2. Effect of toxin (0.16 /Ag/ml) oni apparentfree space (AFS) of susceptible and resistalnt oat roots.Roots were treated whith toxinl, excised, and equilibratedin 35 mnM miannitol for 1 hour, then placed in glass dis-tilled water for exodiffusion. Mannitol in the exodif-fusioni mediumii was determinied after 1 hour. for ap-parent free space calculations. 'Maximium aind minimiium,values are indicated for hour 4.

Conitrol and( treated resistant roots had apparentfree space values ranging from 13 to 20 % and(l aretherefore in good agreement with published valuesfor Gramnineae (3, 6). \Vithin 30 mirinutes, freespace in toxiin treated susceptible roots increased to40 %. As the time of .treatment inicreased, valuesalso increased iunitil after 8 houirs 90 to 100 % ofthe total root -oltume becamne free space (fig 2).Membranes of more cells appear to be destroyed astoxin treatment time increases, withl corresponidinginicreases in free space. There was no chalnige in-apparent free space in treated resistant roots, evenafter 8 houirs. Apparent free space appears tochange more slowfly after toxin treatmenit than (doesthe response as meastured by amino acid ipl)take.However, the apparent free space measuiremenitsare for whole tisstues, whi,le the amino acid uiptakeexperiments may include only the ouiter cells.

The effects of toxin (0.16 pg/ml) were com-pared with the effects of 0.1 mm DNP, 1 mat NaF,anid 1 ImlM NaN3 (table IV). After 2 hours ex-posure to toxinl abotut 80 % of the root voluime instusceptible tissue was freely permeable, while ap-parent free space of resistant roots remained uin-changed. _NaF and NaN. had no effect oni ap-parenit free space, although the concentrations uisedare know-%n to inhibit metabolism. DNP increasedapparenit free space in both susceptible ancd resistantroots, anld therefore appears to affecit the plasmamembranies non-specifically.

Effect of Toxin on Free Protoplasts. Cell wall-free protoplasts (17) were prepared from coleop--tiles of corn, sorghum, and suisceptible and resistant

oats. The spherical protoplasts were enveloped byplasma membranes and showed active proto.plasmicstreaming. The protoplasts were transferred toconcavity microscope slides and treated with variousconcentrations of toxin or inhibitors. The basicreaction mixture was 10 p.l proto,plast suspensionplus 10 jul treatment solution, buffered at pH 6.5.Coverslips were placed over the cell suspensions,and slides were incubated at 220 in a moist chamber.A m-icroscope was used to take zero time counts ofintact 'protoplasts, followed by counts at varyingintervals. Survival percentages were based on1 zerotime counts.

Table IV. Effects of 7'oxini anld V'ariotus MctabolicInhibitors on Apparcnt Free Space of Oat RootsThe data were calculated as % of total root volume.

% Apparent free spaceTreatmenit' Susceptible Resistant

Conitrol 16 ± 4 22 ± 4Toxin, 0.16 A-g/nl 80 ± 5 22 ± 4DNP, 0.1 m L 45 ± 5 45 5NaF, 1 mm 16 ± 4 22 4NaN,, 1 Mat 20 ± 2 20 2I Treatment time.

mNr manniitol, 2distilled water, 1

2 lhours. equilibrium time in 35hour: exodiffusion time in glasshour.

Protoplasmic streaming (cyclosis) stopped inmany protoplasts from susceptible oat plants within10 minutes after exposuire to toxini. Toxin at0.16 jg/ml catused 100 % btursting in 1 houir (tableV). Most broken protoplasts and( the remains oftheir plasma membranes sooni lysed and disappeared,leaving mit,ochondria apparently unharmed. Proto-plasts from corn, sorghum, and resistant oats werenot affected. Cyclosis in the resistanit protoplastsdid not stop and there was no more bursting orlysis than in controls. In several experiments donewith slight variations in proce(utire, free protoplastsfrom susceptible aind resistaiit plants clearly re-tained their specific differential response to HV-toxin. Since free protoplasts lack cell walls, wecan eliminate this struictuire as a necessary site ofaction of the toxin.

Toxin concentrations from 1.6 to 1.6 X 10-1ug/ml were uised in another experiment. Againtoxin had a dramatic effect on susceptible butt noeffect on resistant protoplasts (fig 3). Toxin at1.6 X 10-4 ug/ml causedI 50 % buirsting of suscep-tible protoplasts in 1 houir, while 1.6 jig/ml catused100 % bursting. Cyc-losis was not affected in thetoxin treaited resistant andl uintreate(d control proto-plasts.

DNP was used at concentrations (10 anid100 ,.LM) known from preliminary experiments todamage oat cuttings. Again the highly specificeffect of 'toxin was evident (table V). Thirtyminutes after exposuire to toxin (0.16 ,ug/ml), 84 %

25



Table V. Comiparative Effects of Toxini and 2,4-Dinitrophenol on Oat Protoplast SurvivalToxin concentration was 0.16 ,tg/ml.

Protoplast Protoplast survival after'type Treaitmenetl 30 min 60 min 120 min

Susceptible Control 94 90 90(cv. Park) Toxin 16 0 0

DNP 100 /M 82 60 38I)NP 10 Am 88 76 45

Resistant Control 96 92 92(cv. Clinton) Toxin 91 90 90

DNP 100 iu- 80 60 40DNP 10 /AM 87 72 52

Solutions were made with 25 mm phosphate buffer (pH 6.5).2 Calculated as % of intact protoplasts at zero time.

of stusceptible protoplasts were destroyed anid theremainder showed no cyclosis. Within 1 houir allsuiscept,ible protoplasts were broken. DNP actedmore slowly, and affected stusceptible and resistantprotoplasts eqtually. Cyclosis sitopped 15 mintutesafter exposture to DNP, and after 2 hoturs 48 to62 % of the protoplasts had lysed or bturst (table V).

Filipin and ribonuclease are known to catusebuirsting -of free protoplasts (9,17). Therefore,these suibstances were tested for possible differen-tial effects on HV-toxin resistaint and suisceptiblecellls. Filipin at 50 ttg/ml caused 20 to 30 % ofprotoplasts to break in 2 hoturs, while 0.03 % solui-tions of ribonuclease cauised aboult 50 % burstingin 1 houir. Resistant anid suisceptible proto,plastswere affected equially by b,oth filipin and ribo-nmiclease.

Bisuilfite is known from previotus work to reduicethe effect of HV-toxin oni susceptible oat seedlinio.s

808'' '"""""""""""""A A ...................80

> 60

ce * Sus Tox 0

0 40 - * SusCk\

o A Res Tox

20 - Res Ck

-o i66 i640162

TOXIN-pg/mi x1.6FIG. 3. Effect of toxin concentration on survival of

resistant and susceptible protoplasts. Intact protoplastswere counted at zero time and after 1 hour exposureto toxin or water (controls). 0 = Susceptible toxintreated; * = susceptible control; A = resistant toxintreated; and Q = resistant control tissue.

(22). Therefore, the effect of sodium bislitfite oIntoxic actioni agaillst protoplasts was tested. Whentoxin was diluted wvith freshly prepared NaHSO3solution at pH 6.5, toxic effects were delayed.Toxin at 0.16 pg/ml catused 100 % bursting ofprotoplasts in 1 houir. In the presenice of 0.8 miMINaHSO3, only 37 % of the protoplasts Ilysed in1 houir, and onily 56 % in 2 houirs. This concentra-tion of bisuilfite alone di(l not affect protoplastsurvival. Bisuilfite didc not completely couinteractthe effects of HV-toxin, since most of the survivingprotoplasts la,ter collapsed. Cyclosis was vigorousin both water and( NaHSO, controls. The bisuilfiteeffect on toxicity to protoplasts parallels the effecton seedllings (22).

Discussion

II. victoriac causes increased respiration in hosttissule, as do maily oither plant pathogens. HV-toxincan reproduce this effect. The primary lesion wasonce thouight to be a toxin--indtuced uncoupling ofoxidationl from phosphorylation, buit the only evi-dence wa. a lack of response by toxin treate(d tis-sties to knlowrn uincouipling agents (16). Primaryeffects of itoxin clearly are not here, since O., andPi uiptake by isolated mitochondria are not affectedby toxin (4, 20, 21, 23). Slightly decreased P/Oratio,s by mitochondria from plan,ts previoulslytreated with toxin (4, 23) may be a secondary effectof cell breakdown products on thie mitochlondria.Fuirthermore, colleoptile tis,sue and aleuirone cells(20) are toxin-sensitive, but do not respond byincrea,sed gas exchange.

Another effect of HV-toxin is to stop the in-corporation of 14C-labeled amino acids into tri-chloroacetic ac;id insoltuble celluilar components (23),stuggesting an effect on protein synthesis. How-ever, ribosomes from oats, when prepared carefuillyto prevent bacterial growth, had suich low syntheticactivity that no conclusions were possible (23).Ribosomes from reticuilocyte cells, which are af-

26 PLANT PH YSIOLOGY



SAMADDAR AND SCHEFFER-EFFECT. OF H. VICTORIAETOXIN2

fected by all known inhibitors of protein synthesis,were not affected by HV-toxin (20). We tenta-.tively concltude that the apparent effect on proteinsynthesis in tissues is another secondary effect. Abreakdown in transport to synthetic sites couldexplain the effect of toxin on incorporation ofamino acidls. Similarly, the lack of P- incorpora-tion into organic compounds could resulit from dis-ruption of transport to the active site of synthesis.Drastic damage to the plasma membranie can resultin ileakage of intracellular ions as well as inhibitionof active tuptake of exogenous solutes. However,there tis no direct evidence rtuling otut ilnterferenicewith energy metabolism in viva as part of theexplanation of inhibited synthesis.

There are several inidications that the site of aprimary lesion of toxin is in the plasma membrane.Previous data were suiggestive buit not conclu1sivle(23). Data presented here are more concluisive,and may be stummarizedl as follows. A) Root hlaircells exposed briefly to traces of toxin cannott beplasmolyzed. This is a knowin effect of menmhranedamage. B) TI'oxiin inhibits or stops membraneregutlated, active tuptake of exogenotus soltutes suichas amino acids (8) and P, after brief expostures.C) Appareint free space in tissues increases aftertoxin treatment. The plasma membrane is con-sidered as ithe permeability barrier in tisstues; dis-ruptions are expected to lead to increasedl apparentfree space. D) The plasma membranes of isolattedsusceptible protoplasts break after brief exposulreto 'toxin. Fturthermore, there is evicdence of mem-brane damage from the electron microscopic workof Ltuke et al. (12); howev'er, these workers usedonly 'tissules that h'ad been exposed to toxin for 24hours.

The experiments with isolated protoplasts are ofspecial interest. Since the 'toxin acts selectvelyon protoplasts without cell walls, we can eliminatethe wall as a necessary lesion site. The stabilityof isolated protoplasts depends on intact membranes,and agents affec't'ing this structure can catuse buirst-ing. Filipin, a polyene antibiotic, breaks Neuiro-spora protoplasts, 'presumably by binding with mem-brane sterols (9). Proteases and lipases breakBacillus ntegaterium protoplasts (10), but have noeffect on Avena protoplasts. Ba'sic proteins suichas ribontuclease, cytochrome C, an!d protamine catuse(dbursting of Avena protoplasts, presuimably afterbinding with the membrane (17). We fotund thatfilipin, ribontuclease, and DNP affect HV-toxinstusceptible and resistant protoplasts, and that thetoxin effects are more drastic than ithose of anyother substance tested. The toxin seems 'to lhavea strong affinity for membranes of sutsceptible cells.

Our working hypothesis is that toxin combineswith or affects an unknown component in thesusceptible cell, resulting in disorganization of thesurface. Such disru'ptions could accotunt for allthe effects of toxin described to date. The resistantcell membrane appears to lack the receptor or

,sensitive site, since such cells do not respond inany observable way. Membrane damage by HV-toxin could lead to the "biochemical symptoms"observed in susceptible cells. This postulation isbased in part on published da'ta for several bio-logical systems. Stuidies on the action of colicinesindicate that membrane damage can lead to tem-porarily increased respiration and collapse of syn-thetic systems (13). Membrane damage may affectmany other celluilar components 'becauise of physicaland metabolic interconnections (1, 11).

HV-toxin and 2 other host-specific determinantsof pathogenicity, 1 fro'm Helminthosporium carbo-tmtn and 1 from Periconia circinata, are being usedas models for the stuidy of disease development anddisease resistance in plants (23). Stusceptibility orresistance to all these diseases is based on reactionwith or lack of effect by specific toxins. Thusresistance and stusceptibility appear to be based onconstituitive factors. The extreme specificity ofthese low molecular weight suibstances is of interestfor other reasons as well. For example, the factthat reactions of isolated mitochondria are notaffected indicates selective effects on the differentmembranes within the stusceptible cell].

Acknowledgment

The authors are grateful to Dr. J. E. Varner of theMISU/AEC Plant Research Laboratory, for valuablesuggestions and use of equipment during this stady.The senior author held a Fulbright Travel grant.

Literature Cited

1. BAIN, J. 'M. AND F. V. MIERCER. 1966. Subcellularorganization of the developing cotyledons ofPisumt sativ ion L. Australian J. Biol. Sci. 19:49-67.

2. BENSON, A. A., J. A. BASSHAMN, M. CALVIN, T. C.GOODALE, V. A. HASS, AND WV. STEPKA. 1950.The path of carbon in photosynthesis. V. Paperchromatography and radioautography of theproducts. J. Am. Chem. Soc. 72: 1710-19.

3. BERNSTEIN, L. AND R. H. NIEMAN. 1960. Ap-parent free space of plant roots. Plant Physiol.35: 589-98.

4. BLACK, H. S. AND H. WV-HEELER. 1966. Biochem-ical effects of victorin on oat tissues and mito-chondria. Am. J. Botany 53: 1108-12.

O. BRIGGs, G. E. AND R. N. ROBERTSON. 1958. Ap-parent free space. Ann. Rev. Plant Physiol. 8:11-30.

6. BUTLER, G. W. 1953. Ion uptake by young wheatplants. II. The apparent free space of wheatroots. Physiol. Plantarum 6: 617-35.

7. GREEN, A. A. AND W. L. HUGHES. 1955. Proteinfractionation on the basis of solubility in aqueoussolutions of salts and organic solvents. In:Methods in Enzymology. I. S. P. Colowick andN. 0. Kaplan, eds. Academic Press, New Yorkp 76-77.

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PLANT PHYSIOLOGY

8. HOKIx-, L. E. AND M. R. HOKIN. 1963. Biologicaltransport. Ann. Rev. Biochem. 32: 533-78.

9. KINSKY, S. C. 1962. Effect of polyene antibi-otics onl protoplasts of Neurospora crassa. J. Bac-teriol. 83: 351-58.

10. LANDMAN, 0. E. AND S. SPIEGELMIAN. 1955. Eil-zyme formationi in protoplasts of Batcilluts miiega-tctriuiiml. Proc. Natl. Acad. Sci. 41: 698-704.

11. Lips, S. H. AND H. BEEVERS. 1966. Compartmnen-tationi of organlic acids in corn roots. II. Thecytoplasmic pool of mialic acid. Planit Physiol.41: 713-17.

12. LUKE. H. H., H. E. X\NARMKE, AND P. HANCHEY.1966. Effects of thle pathotoxini victorini on ultra-structure of root and leaf tissue of .Izve'a species.Phytopatlhology 56: 1178-83.

13. No-MuRA\, M\f. 19604. AMechanism of action of coli-cinies. Proc. Natl. Acad. Sci. 52: 1514-21.

14. PRINGLE, R. B. AND A. C. BRAUIN. 1957. Theisolationi of the toxin of Heliniithosporiiui -tori(Ic. Phytopathology 47: 369-71.

15. PRINGLE. R. B. AND R. P. SCHIEFFER. 1964. Host-specific plant toxinls. Annii. Rev. Phytopathology2: 133-56.

16. ROMANKO. R. R. 1959. A physiological basis forresistance of oats to Victoria hlight. Phytop-ith -oio-v 49: 32-36.

17. RUESINK. A. W\. AND K. V. THIM\AN N-. 1965.Protoplasts from1 tlhe Avcina coleoptile. Proc.Natl. Acml. Sci. 54: 56-64.

18. SACHER, J. A. 1966. Permeability clharacteristicsand amino acid incorporation during senescence(ripening) of banana tissue. Plant Physiol. 41:701-08.

19. SA-MADDAR. K. R. AND R. P. SCHEFFER. 1966.Effect of Hciii iithosporiu nt victoriae toxin onprotoplasts froim Azvc'na coleoptiles. Phytopath-ology (Abstract) 56: 898.

20. SA-MADDAR, K. R. 1968. Mechanism of action ofthe primary determinant of pathogenicitv fromhIclillinlthlospori-iunli victoriac. Ph.D. thesis, 'Michi-galn State University.

21. SCHEFFER, R. P. AND R. B. PRINGLE. 1963. Res-piratory effects of the selective toxinl of Ifclmiin-thlosporimn v.ictoriae. Phytopathology 53: 46548.

22. SCHEFFER, R. P. AND R. B. PRINGLE. 1964. Up-take of Helmtinithosporiuin/li victoriac toxinl bv oattissue. Phytopathology 54: 832-35.

23. SCHEFFER. R. P. AND R. B. PRINGLE. 1967. Path-ogen produce(l determillants of disease anld theireffects on host plants. In Tile Dynn:rmic Roleof 'Molecular Constituents in Plant-Parasite Inter-action. C. J. 'Mirocha andl 1. Uritani, e(ds. BrucePublislhing Company. St. Patul. 'Minnesota. p 217-36.

24. \WHEELER. H. AND H. S. BLACK. 1963. Effects ofHcl/iiintliospomttiniil victoriac and victorin uponpermeability. Aim. J. Botany 50: 686-93.

25. \HIIITAKER, D. R. 1953. Purificationi of M11vru-tlec-izelivcrruicaria cellulase. Arclh. Bioch1emn.Biophys. 43: 253-68.

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Effect of the Specific Toxin in Helminthosporium victoriae Host