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Effect of the Specific Toxin in Helminthosporium victoriae on Host Cell Membranes' K. R. Samaddar and R. P. Scheffer Department 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-producing fungus, causes loss of electrolytes from roots, leaves, and coleoptiles of treated plants. Root hair 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. Toxin stopped 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 susceptible but not in resistant tissue. Apparent free space increased in susceptible but not in resistant roots. The increase was evident within 30 minutes, and reached 80 % free space after 2 hours exposure to toxin. When cell wall-free protoplasts were exposed to 0.16 ,ug toxin/ml, protoplasmic streaming stopped and all plasma membranes of susceptible protoplasts broke within 1 hour. Resistant protoplasts were not affected significantly. Data support the hypothesis of a primary lesion of toxin in the plasma membrane. Effects on synthesis could result from lack of transport of exogenous solutes to sites of synthesis. It is possible that all other observed effects of toxin are secondary to membrane damage.
"Host-specific toxinis" that are determinanits of pathogenicity are now known from at least 5 different planit infecting fungi. These com,pounds
reproduce all visible and all known biochemical symptoms of infection. In each case, loss of ability of a funiiguts isolate to produice its toxinl has resulted in loss of pathogenicity. In each case, the substance is toxic only to the host of the toxinl producing fuingigus (23). These are usefuil models for the studv of disease, because many possible complexities of host-parasite interaction are bypassed. One suich suibstance, produced by Heluninthjosporiumzictoriaz e Mieehan and Mturphy (HV-toxin), is toxic to suisceptible oat cualtivars, but is harmless to resistant oat cultivars and to all other non-host plants tested (15). The toxin is composed of a cyclic secondary amine (C17H,9NO) known as "victoxinine" anid a peptide containing aspartic acid, glutamic acid, glycine, valine, and leuicine. The complete toxini has a M.W. between 800 and 2000 but the exact structure is still unknown, largely becauise of problems with lability (15). Amon.g the many cellular responses that occuir quickly after HV-toxin treatment are: increased 0., utptake (21) : decreased incorporation of '4C amino acids anid uridine into trichloroacetic acidl insoluible celluilar fractions (23); and rapid loss of electrolytes (24). Several lines of evidence suggest that these effects are secondary. Oxygen and Pi 1 Publislhed with the approval of the Director, Michigan Agricultural Experiment Station, as Journal article No. 4157. Aided by grants GB-1448 and GB-6560X f rom the N'ational Science Foundationi.
uptake by isolated mitochondria were not affected (4, 21, 23). Toxin uptake appears to be a simple process, not affected by wide ranges lof temperature and by metabolic inhibitors. The peptide resulting from toxin breakdown inhibited toxin uptake, suggesting a competition for receptor sites present in stusceptible cells (22). Data to date suiggest thsat the toxini cauises a primary lesion in the plasma membrane of suisceptible cells (23). The 'basis of resistance may be a lack of receptors or sensitive sites in the membrane. The possibility that the ini;tial toxin lesion is in the plasma membrane was examined in this stuidy by the tuse of intact tisstues and cell free systems. The effects of toxini oni ion leakage from tissules, cell plasmolysis, uliptake of soltites, apparent free space, and behavior of cell1 wall free protoplasts were determined. The data indicate that toxinl affects membrane physiology and transport systems of susceptible bu,t not resistant cells. An abstract of part of the work was published (19).
Materials and Methods Toxini suisceptible (cv. Park) and resistant (cV. Clinton) oat seedlings were uised in most experiments. Seeds were germinated on moist filter paper and seedlings were grown in the laboratory at 21 to 220 in White's nutrient solutions (14), *or in vermiculite plus White's nutrients. In some cases larger plants grown in the greenhouse were tused. The inhibition of seedling root growth in a diltution series of toxin was tused as the standard bioassay (15).
Toxi:n elilted from an alumina coluimn, followiing the method of Pringle and Bratun (14), was relatively stable at pH 3.5, and the concentrated solution was stored at 4°. This preparation gave complete inhibitioni of root growth of stusceptible oat see(lliings at a concentration o/f 0.0016 tug/ml buit ha(l no effect oIn growth of resistant seedling roots at 160 ug/ml. Fuirther purification, achieve'd by p assnin this soluttion throuigh a Bio-Ge'l P-2 coltumn, gave a more toxic buit less stable preparationi. Ioan Lcakagc. T'he effect of toxini 0o iOln leakage fromn coleoptile tissuie wvas measured by changes in electrical con(lucti:vity of su spending solutions. One g tissuie samples were vacuutim infiltrated for 10 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 ilncubated on a shlaker in 100 ml glass distille d Water at 22°. Condchictivitx of the ambient solution was measuired with 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 recip)rocal ohmlls (mhos). 322) [Uptakc (111(i Inlcorporationl. l issIle samil)les erele l)lace(l in flasks containin- 2 ml 1 in.\i acetate l)llffer (pH 5.5), 155 ttg chloramphenicol aln(d 200 uc 32 1). Flasks were incuibate(d at 250 onl a Du)biuoff metabolic shaker (40 oscillations/nin) for 2 houlrs. Samples were then rinse(d thorouighly wvith cold 1 mmr KH.,PO and groutnd in boiling 80 % (v/v) ethaniol. Th'e homogeniate wras centrifutged at 10,000 X g for 10 mintutes and the stupernatant conlcentrated to 2 ml. A 50 ,ul aliqulot was spotted oni Whatman No. 1 paper. Marker .32p was -s,potted oni each paper for tracinig the movement of Pi. Descendinig chromatographs ulsing ni-buityl alcohol: propionic acid water (1246:620:874 v/v) were developed for 40 houirs (2). Radioactive zones were detected with a Nuiclear Chicago monito,r. Radioauitographs were made on Eastman Kodak X-ray films. Individual radioactive zones were removed, gluted to pllanchets and coulnite(d in a Nuiclear Chicago low background gas flow couinter. Total uiptake was calculated by adding the couints of all spots develo,ped from a single origin. The amouint of radioactivity in the inorgalic and organic P spots was expressed as percent of total uiptake. No attempt was made to identify the organic P conmpounds. 14C-.Al into A-cidl Uptake. Uptake was conisidered as the amouint of labeled compouind taken in from the medium, and( retained by tissules after repeated washings (18). Roots from 10 seedlings were incubated in 1.0 ml DL-leucine-1-'4C or DL-valine-U14C (pH 7.0). Concentrations of the amino aciid solutions varied from 1 to 50 mm with specific activities from 0.5 to 1.0 pc per ml. Chloramphenico,l (15 xg) was added to each flask and the mixtuire w%vas inculbated for 1 to 3 houirs at 220 with
gentle shakinig. The roolts were then washlecl for I houtr in ruinning tap water, blotted gently, placed inito tightly stoppered tuibes with 0.5 ml 95 % ethanol, and extracted with shakiing for 12 houirs. Aliquiots of the ethanol extracts were coutintedl on placnchets in a Nuiclear Chicago gas flow couniiter. Apparent Fr-ce Space. Apparen,t free sp)ace of excised roots was determined( bx the India inik tagging method of Bernsteini and Niemani (3). Roots were rinsed in distilled water, thein equilibrated in 35 m.t mannitol soluitioni (50 ml/g root tissuie) for 0.5 to 2 houirs, w\ith 4 or 5 changes of soltition. Duiring the last minuite of equIilibration, 1 ml India ink suispension (1 part/10 parts 35 mm mannitol soltitioin) w$as adde(d to each 20 mfl ecqilibrating solutioin. The preparatioln was stirred for a few secondcIs before roots w-ere removed, (lrainiedl, aI(l tranIsfer-re(d to 100 mil glass (listille(I wa\N1ter foi exodi f ftisiOn . Exo(liffusoioI soluitioIns Were stirred foI 5 mmilultes, ani(d samples x-erc taken at interval-s of 5, 10, 15, 30, and 60 minutes. Optical d1enlsities of 1 :100 dilutitnoii of the equilibrating solutiOInS and exodi ffuslioI media wve re deterin;IIe(d w\ith a colorrimneter at (500 mnp- A coI-rectioIn foI- the v-olu1me of equllibratinfg solu1tioIn adhering to root S 111-flaces w\s Calcculate(d froIml the o,ptical densitV aid volumic of e(qilib-ating andI exod1ifftisioi media (3). Manitol concentrations in equilibratioui and exo(liffuisiOI media were deterimlined b\ ilomonletric titration as described by Bultler (6). Mannitol adhering to the root suirface was suibtractedl fromn total mannitol (tiaintity in the exodiffuision mediuim to obtaini the amouint of solute in frec' space. T'oxill cauises leakage of antthrone positive carbohydrates (4), which might give an error in manniitol determinatiolis. Therefore, a correction for toxin treated samples kept in water was applied. Apparent free space was calcula,ted fromii the diffuisible solute (mg) in the exodiffuision mecdiuim anid the concenitration (nmg/nml) of the e(luilibrating solttion, expressed as percentagc fresh w'eight of r(oot tissue.
Prepa ration of Pr-otopltasts. Protopl asts from coleoptiles of oa,ts, corn, an(l sorghtim were prepared with cellulase by the method of Ruesink aind Thimanni ( 17). Cellulase was prepared from cudlttire filFtrates of Myroth eciunt zverrutc(ri a isolate 460, olbta,ined from Dr. Mary Mandels of the Quia,rtermaster Laboratory at Natick, MIassachulsetts. The fuingtis was grown with continutouis shaking for 14 days on a modified Whitaker's salt mediuim (17, 25). Culltuire filtrates were concentrated to 0.1 voluime in a flash evaporator at 370, anid re-filtered. The filtrate was fractionated wi,th solid (NH,) 2SO4 at 30 (7). The precipitate obta,ined at 35 to 70 % satuirationi was dissolved in a small volume of water anid desalted in a Sephadex G-25 coluimn. The coltumn was prepared, washed, anid deve,loped with 0.1 % NaCI soluitions. Maximuim celltlase activity w as in the second brown (1 ml) fraction eluited
SAMADDAR AND SCHEFFER-EFFECT. OF H. VICTORIAE TOXIN
from the column. Aliquots (200 jul) of this fraction were stored in separate vials at -200, since activity was lost with repeated thawing and freezing. Coleoptiles from seedlings grown in dim red light were harvested 72 hours afiter germination. Sections 1 mm long were treated with cellulase solution diluted 1:1 with 1.0 M mannitol buffered with 25 mm soditum phosphate buffer (pH 6.5). After 2 hours incubation in the dark at 220, 10 volumes of 0.5 M mannitol buffered at pH 6.5 were added. Protoplasts settled to the bottom of the tube in 10 minutes, and the supernatant was removed with a pipet. Mannitol solution was again added and the procedLure was repeated to remove cellulase.
Results Effcct of Toxin ont Colcoptile Tissute. Since coleoptiles were to be used in some experiments, it was necessary to see if they have the usuial differential response of resistant and susceptible plants to toxin. Toxin increases respiration (21) and ion leakage (24) in leaf and root tissues; these responses were chosen as possible indicators of toxic effects on coleofptiles. Seedllings were grown in the dark, in diffuse light, or in red light. Subapical sections (1 cm long) of coleoptiles were removed and split 3 days after germination. Samples (0.5 g) were vactuuim infiltrated with toxin solution (0.16 ,,g/ml) or water at pH 6.5 for 10 minutes. Coleoptile tissue of susceptible toxin-treated oats released electrolytes at a much faster rate than did the controls and the treated resistant itissue (fig 1). Eight hours after toxin treatment, the specific conductance of the ambient solution of treated susceptible tissue was 5 times greater than that of any other treatment solution. Oxygen uptake by toxin treated and control coleoptile tissue was determined manometrically. Toxin caused little or no increase in O., uptake by susceptible coleoptiles in 4 experiments. Increased 02 uptake caused by toxin apparently depends on the type of tissue, while an increase in ion leakage is evident in all susceptible tissues so far tested (15). Effect of Toxin on Plasmolytic Ability of Cells. Cells with damaged membranes will not plasmolyze when placed in hypertonic solutions. Therefore, we can test whether or not toxin destroys or damages the plasma membrane in situ. The selective effects of toxin were compared with the effect of 2,4-dinitrophenol (DNP) on cell membranes, using concentrations of DNP known to uncouple oxidation from phosphorylation. Roots of 4 day old plants were placed in either toxin solutions (0.16 pug/m4), DNP (10 and 100 Mm), or water for varying times. After treatment roots were quickly
TIME AFTER TREATMENT (HR)
FIG. 1. Effect of toxin on loss of electrolytes from
He/wlinithosporiihn victoriac resistant (Res) and susceptible (Sus) coleoptiles. Tissue samples (0.5 g) were infiltrated with toxin solution (0.16 ,ug/ml) for 10 minutes, and suspended in glass distilled water. Electrolyte loss was determined from conductivity of the water. * = 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 minutes after toxin exposure, susceptible root hair cells lost the ability to plasmolyze in hypertonic solutions (table I). Toxin treated resistant cells showed plasmolytic shrinkage even after 180 minutes Qf exposture to toxin. Resistant and susceptible untreated controls plasmolyzed normally. DNP at 100 jM acted mtuch more slowly than toxin and' destroyed the p-lasmolytic ability of both suisceptible and resistant cells (table I).
Table I. Effects of Toxitt and 2,4-Dinitrophenol on Plasntiolysis of Root-hair Cells in Hypertoanic
Exposure time' required to destroy plasmolysis ability Susceptible Resistant Milt Mill Control2 > 180 > 180 Toxin, 0.16 ,u/ml 20 > 180 DNP, 100 AuM 90 120 DNP, 10 lAM 180 > 180 Exposure times were 10, 20, 30, 40, 50, 90, 120, 150, and 180 minutes prior to placing cells in hypertonic solution. 2 Control roots were placed in water or deactivate& toxin. Treatment
Table II. Effect of Toxii oil 32p Uptake anid Inicorporationt by 0at Leaf 7issue Toxini 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
Radioactivitv Iniorganiic P
cpill 12,895 1560 14,400 15,800
Susceptible conitrol Suisceptible plus toxin Resistanit control
Resistanlt pluls toxini
9555 1215 10.160
P Organiic Z!,~~~~~
cpIII 3245 120 3550 3780
Table III. Effect of Toxin oni Uptake of 14C Amino Acids by Susceptible and Resistanit Oat Roots Reactioni 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 were counted. 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 14C Valine (50 nim) Leuci.ne (1 inm) RES SUS St S RES c/ mn c'/l1 tcP in /'p m 1540 10.950 9280 255 ... 11,240 5j85 ... 9525 1700 9570 ... 270 580 10,040 1815 1960 10,320 9985 1795 190 10.895 460
P Uptake 1n1( InicorporaEffcct of 'loxi'itl I tionl. Transpiring cuttings of resistant and suisceptible 10 day old plaints were allowed to take nip toxini solution (0.16 ug/ml) or water for 4 hoturs. Following treatment, 0.5 g leaf samp-les were inlcuIbated with bnffered 32p as (lescribe(l previously.
Radioactivity (cpm) of the ethanol extract was used to estimate total ntptake. Cotnnits of the several spots on paper chromatograms gave the (listrilbution of P in organic anid inorganic fractions. Toxin treated suisceptible tissnle hadl less 32P niptake than did stusceptible control tissnie (table II'). Up'take was not inhibited in resistanit treated tissnies. -Controls and treated resistant tissnies incorporated 20 to 30 % of the total 2p ilnto organic P-compouinds, while incorporatioin was completely blocked in treated suisceptible tissues. The experiment was dlone 3 times withl essentially the same resullts. Similar resuilts were obtained with root and coleoptile tissties. In another experimenit, the etlhaniol extracts were added to paper uintil all spots had approximately equal 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( inl extracts of toxin treated susceptible tissuie. Effect of Toxin ont Active Upta.ke of Amnino Acids. Amino acid uptake and retenitioni in roots treated with toxin wrere compare(d \-ith tuptake and -retention in uintreated controls. Roots were treated
iucubated ill with toxinl solnition (0.16 ig/mln) labeled amino acid soluitions, washed and extracte(l with ethaniol. The radioactivity (cpm) in ethanol extracts was tised as a measuire of intracellular free pool amino acids. Suisceptible roots exposed to toxin for only 30 minuites showed a 90 % decrease in the active uptake of labeled amino acids (table III). Similar resuilts wvere obtaiined with labele(i v-aline and leuicine at concentrations from 1 to 50 nm\I. Effcct of Toxin ont Apprenott Fr-ce Spacc of 'Tissiuc. Apparent free space in roots is thonloht to conisist of cell wall and(l intercelilular spaces (5). The possiblility of toxin-ilnduced chainges in apparent free space in roots was examined as a fuirther measuire of membrane damage. The experimeilt was based oIn the hypothesis that if tox,in disruipts the plasma membrane, the harrier for free permeation will disappear anid( apparent free space slhoull(d
Resistanit and( stusceptible oat plants were growii in staining dishes with removable trays (22). Toxin or other substances were adldled to the soliltions of some dishes, while other dishes were uised as uintreated controls. Appareilt free slpace was similar with different equilibration and exodiffusioin times uip to 2 hoturs and with excised roots anld roots of intact plants. In most cases roots with the several different toxinl treatment times were excised and equiilibrated for 1 houir in 35 mm manlnitol, followed by 1 houlr exodiffulsioIn in glassi distilled water.
25 SAMADDAR AND SCHEFFER-EFFECT .OF H. VICTORIAE TOXIN25
oats. The spherical protoplasts were enveloped
plasma membranes and showed active proto.plasmic streaming. The protoplasts were transferred to concavity microscope slides and treated with various concentrations of toxin or inhibitors. The basic reaction mixture was 10 p.l proto,plast suspension plus 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 of intact 'protoplasts, followed by counts at varying intervals. Survival percentages were based on1 zero time counts.
FiG. 2. Effect of toxin (0.16 /Ag/ml) oni apparent free space (AFS) of susceptible and resistalnt oat roots. Roots were treated whith toxinl, excised, and equilibrated in 35 mnM miannitol for 1 hour, then placed in glass distilled water for exodiffusion. Mannitol in the exodiffusioni mediumii was determinied after 1 hour. for apparent free space calculations. 'Maximium aind minimiium ,values are indicated for hour 4.
Table IV. Effects of 7'oxini anld V'ariotus Mctabolic Inhibitors on Apparcnt Free Space of Oat Roots The data were calculated as % of total root volume.
Treatmenit' Conitrol Toxin, 0.16 A-g/nl DNP, 0.1 m L NaF, 1 mm NaN,, 1 Mat I
Conitrol and( treated resistant roots had apparent free space values ranging from 13 to 20 % and(l are therefore in good agreement with published values for Gramnineae (3, 6). \Vithin 30 mirinutes, free space in toxiin treated susceptible roots increased to 40 %. As the time of .treatment inicreased, values also increased iunitil after 8 houirs 90 to 100 % of the total root -oltume becamne free space (fig 2). Membranes of more cells appear to be destroyed as toxin treatment time increases, withl corresponiding inicreases in free space. There was no chalnige in -apparent free space in treated resistant roots, even after 8 houirs. Apparent free space appears to change more slowfly after toxin treatmenit than (does the response as meastured by amino acid ipl)take. However, the apparent free space measuiremenits are for whole tisstues, whi,le the amino acid uiptake experiments may include only the ouiter cells. The effects of toxin (0.16 pg/ml) were compared with the effects of 0.1 mm DNP, 1 mat NaF, anid 1 ImlM NaN3 (table IV). After 2 hours exposure to toxinl abotut 80 % of the root voluime in stusceptible tissue was freely permeable, while apparent free space of resistant roots remained uinchanged. _NaF and NaN. had no effect oni apparenit free space, although the concentrations uised are know-%n to inhibit metabolism. DNP increased apparenit free space in both susceptible ancd resistant roots, anld therefore appears to affecit the plasma membranies non-specifically. Effect of Toxin on Free Protoplasts. Cell wallfree protoplasts (17) were prepared from coleop-tiles of corn, sorghum, and suisceptible and resistant
% Apparent free space Susceptible Resistant
16 80 45 16 20
± ± ± ± ±
4 5 5 4 2
22 ± 4 22 ± 4 45 5 22 4 20 2
Treatment time. 2 lhours. equilibrium time in 35
mNr manniitol, 2 hour: exodiffusion time in glass distilled water, 1 hour.
Protoplasmic streaming (cyclosis) stopped in many protoplasts from susceptible oat plants within 10 minutes after exposuire to toxini. Toxin at 0.16 jg/ml catused 100 % btursting in 1 houir (table V). Most broken protoplasts and( the remains of their plasma membranes sooni lysed and disappeared, leaving mit,ochondria apparently unharmed. Protoplasts from corn, sorghum, and resistant oats were not affected. Cyclosis in the resistanit protoplasts did not stop and there was no more bursting or lysis than in controls. In several experiments done with slight variations in proce(utire, free protoplasts from susceptible aind resistaiit plants clearly retained their specific differential response to HVtoxin. Since free protoplasts lack cell walls, we can eliminate this struictuire as a necessary site of action of the toxin. Toxin concentrations from 1.6 to 1.6 X 101ug/ml were uised in another experiment. Again toxin had a dramatic effect on susceptible butt no effect on resistant protoplasts (fig 3). Toxin at 1.6 X 10-4 ug/ml causedI 50 % buirsting of susceptible protoplasts in 1 houir, while 1.6 jig/ml catused 100 % bursting. Cyc-losis was not affected in the toxin treaited resistant andl uintreate(d control protoplasts. DNP was used at concentrations (10 anid 100 ,.LM) known from preliminary experiments to damage oat cuttings. Again the highly specific effect of 'toxin was evident (table V). Thirty minutes after exposuire to toxin (0.16 ,ug/ml), 84 %
Table V. Comiparative Effects of Toxini and 2,4-Dinitrophenol on Oat Protoplast Survival Toxin concentration was 0.16 ,tg/ml. Protoplast
94 Control 16 Toxin 82 DNP 100 /M 88 I)NP 10 Am 96 Control Resistant 91 Toxin (cv. Clinton) 80 DNP 100 iu87 DNP 10 /AM Solutions were made with 25 mm phosphate buffer (pH 6.5). 2 Calculated as % of intact protoplasts at zero time.
Susceptible (cv. Park)
of stusceptible protoplasts were destroyed anid the remainder showed no cyclosis. Within 1 houir all suiscept,ible protoplasts were broken. DNP acted more slowly, and affected stusceptible and resistant protoplasts eqtually. Cyclosis sitopped 15 mintutes after exposture to DNP, and after 2 hoturs 48 to 62 % of the protoplasts had lysed or bturst (table V). Filipin and ribonuclease are known to catuse buirsting -of free protoplasts (9,17). Therefore, these suibstances were tested for possible differential effects on HV-toxin resistaint and suisceptible cellls. Filipin at 50 ttg/ml caused 20 to 30 % of protoplasts to break in 2 hoturs, while 0.03 % soluitions of ribonuclease cauised aboult 50 % bursting in 1 houir. Resistant anid suisceptible proto,plasts were affected equially by b,oth filipin and ribo-
nmiclease. Bisuilfite is known from previotus work to reduice the effect of HV-toxin oni susceptible oat seedlinio.s
> 60 ce
0 40 -
0 * Sus Tox * Sus Ck\ A
i64 i66 0162 TOXIN-pg/mi x1.6 FIG. 3. Effect of toxin concentration on survival of resistant and susceptible protoplasts. Intact protoplasts o
were counted at zero time and after 1 hour exposure to toxin or water (controls). 0 = Susceptible toxin treated; * = susceptible control; A = resistant toxin
treated; and Q
resistant control tissue.
Protoplast survival after' 120 min 60 min 90 0 60 76 92 90 60 72
90 0 38 45 92 90 40 52
(22). Therefore, the effect of sodium bislitfite oIn toxic actioni agaillst protoplasts was tested. When toxin w as diluted wvith freshly prepared NaHSO3 solution at pH 6.5, toxic effects were delayed. Toxin at 0.16 pg/ml catused 100 % bursting of protoplasts in 1 houir. In the presenice of 0.8 miMI NaHSO3, only 37 % of the protoplasts Ilysed in 1 houir, and onily 56 % in 2 houirs. This concentration of bisuilfite alone di(l not affect protoplast survival. Bisuilfite didc not completely couinteract the effects of HV-toxin, since most of the surviving protoplasts la,ter collapsed. Cyclosis was vigorous in both water and( NaHSO, controls. The bisuilfite effect on toxicity to protoplasts parallels the effect on seedllings (22).
Discussion II. victoriac causes increased respiration in host tissule, as do maily oither plant pathogens. HV-toxin can reproduce this effect. The primary lesion was once thouight to be a toxin--indtuced uncoupling of oxidationl from phosphorylation, buit the only evidence wa. a lack of response by toxin treate(d tissties to knlowrn uincouipling agents (16). Primary effects of itoxin clearly are not here, since O., and Pi uiptake by isolated mitochondria are not affected by toxin (4, 20, 21, 23). Slightly decreased P/O ratio,s by mitochondria from plan,ts previoulsly treated w ith toxin (4, 23) may be a secondary effect of cell breakdown products on thie mitochlondria. Fuirthermore, colleoptile tis,sue and aleuirone cells (20) are toxin-sensitive, but do not respond by increa,sed gas exchange. Another effect of HV-toxin is to stop the incorporation of 14C-labeled amino acids into trichloroacetic ac;id insoltuble celluilar components (23), stuggesting an effect on protein synthesis. However, ribosomes from oats, when prepared carefuilly to prevent bacterial growth, had suich low synthetic activity that no conclusions were possible (23). Ribosomes from reticuilocyte cells, which are af-
2>7 SAMADDAR AND SCHEFFER-EFFECT. OF H. VICTORIAE TOXIN2 fected by all known inhibitors of protein synthesis, were not affected by HV-toxin (20). We tenta.tively concltude that the apparent effect on protein synthesis in tissues is another secondary effect. A breakdown in transport to synthetic sites could explain the effect of toxin on incorporation of amino acidls. Similarly, the lack of P- incorporation into organic compounds could resulit from disruption of transport to the active site of synthesis. Drastic damage to the plasma membranie can result in ileakage of intracellular ions as well as inhibition of active tuptake of exogenous solutes. However, there tis no direct evidence rtuling otut ilnterferenice with energy metabolism in viva as part of the explanation of inhibited synthesis. There are several inidications that the site of a primary 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 hlair cells exposed briefly to traces of toxin cannott be plasmolyzed. This is a knowin effect of menmhrane damage. B) TI'oxiin inhibits or stops membrane regutlated, active tuptake of exogenotus soltutes suich as amino acids (8) and P, after brief expostures. C) Appareint free space in tissues increases after toxin treatment. The plasma membrane is considered as ithe permeability barrier in tisstues; disruptions are expected to lead to increasedl apparent free space. D) The plasma membranes of isolatted susceptible protoplasts break after brief exposulre to 'toxin. Fturthermore, there is evicdence of membrane damage from the electron microscopic work of Ltuke et al. (12); howev'er, these workers used only 'tissules that h'ad been exposed to toxin for 24 hours. The experiments with isolated protoplasts are of special interest. Since the 'toxin acts selec tvely on protoplasts without cell walls, we can eliminate the wall as a necessary lesion site. The stability of isolated protoplasts depends on intact membranes, and agents affec't'ing this structure can catuse buirsting. Filipin, a polyene antibiotic, breaks Neuirospora protoplasts, 'presumably by binding with membrane sterols (9). Proteases and lipases break Bacillus ntegaterium protoplasts (10), but have no effect on Avena protoplasts. Ba'sic proteins suich as ribontuclease, cytochrome C, an!d protamine catuse(d bursting of Avena protoplasts, presuimably after binding with the membrane (17). We fotund that filipin, ribontuclease, and DNP affect HV-toxin stusceptible and resistant protoplasts, and that the toxin effects are more drastic than ithose of any other substance tested. The toxin seems 'to lhave a strong affinity for membranes of sutsceptible cells. Our working hypothesis is that toxin combines with or affects an unknown component in the susceptible cell, resulting in disorganization of the surface. Such disru'ptions could accotunt for all the effects of toxin described to date. The resistant cell membrane appears to lack the receptor or
,sensitive site, since such cells do not respond in any observable way. Membrane damage by HVtoxin could lead to the "biochemical symptoms" observed in susceptible cells. This postulation is based in part on published da'ta for several biological systems. Stuidies on the action of colicines indicate that membrane damage can lead to temporarily increased respiration and collapse of synthetic systems (13). Membrane damage may affect many other celluilar components 'becauise of physical and metabolic interconnections (1, 11). HV-toxin and 2 other host-specific determinants of pathogenicity, 1 fro'm Helminthosporium carbotmtn and 1 from Periconia circinata, are being used as models for the stuidy of disease development and disease resistance in plants (23). Stusceptibility or resistance to all these diseases is based on reaction with or lack of effect by specific toxins. Thus resistance and stusceptibility appear to be based on constituitive factors. The extreme specificity of these low molecular weight suibstances is of interest for other reasons as well. For example, the fact that reactions of isolated mitochondria are not affected indicates selective effects on the different membranes within the stusceptible cell].
Acknowledgment The authors are grateful to Dr. J. E. Varner of the MISU/AEC Plant Research Laboratory, for valuable suggestions and use of equipment during this stady.
The senior author held a Fulbright Travel grant.
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