Plant Physiol. (1 993) 103: 67-72

Density Cradient Study of Victorin-Binding Proteins in Oat (Avena sativa) Cells

Kazuya Akimitsu', 1. Patrick Hart*, and Jonathan D. Walton

Department of Botany and Plant Pathology (K.A, L.P.H, J.D.W.), Pesticide Research Center (K.A., L.P.H.), and Department of Energy-Plant Research Laboratory (J.D.W.), Michigan State University,

East Lansing, Michigan 48824

Victorin-binding proteins (VBPs) in oat (Avena sativa) cells were identified using native victorin and anti-victorin polyclonal anti- bodies. Homogenates of oat tissues were fractionated in continuous or discontinuous sucrose density gradients or with an aqueous two- phase method, and covalent binding sites of victorin were detected by western blotting. In a 20 to 45% (w/w) sucrose continuous density gradient, the 100-kD VBP was located in fractions of 37 to 44% sucrose, with a peak at 39% sucrose. Based on marker enzyme assays, plasma membranes peaked at 39 to 41% sucrose, mito- chondria peaked at 41%, but Colgi and endoplasmic reticulum were in lower density fractions, peaking at 28 to 29% and 22 to 24% sucrose, respectively. l h e 100-kD VBP was not found in plasma membranes purified by the aqueous two-phase method or in mitochondria purified by discontinuous density gradient centrif- ugation. Victorin binding to 65- and 45-kD proteins was deteded in all fractions in the continuous sucrose density gradients. l h e 65- and 45-kD proteins were both detected in purified plasma mem- branes, but only the 65-kD protein was detected in purified mito- chondria. l h e subcellular location of VBPs was the same in sensitive and resistant oat cells.

~

Victorin is a host-specific toxin produced by the oat (Avena sativa) pathogen Cochliobolous victoriae (Meehan and Mur- phy, 1946; Scheffer, 1983). Victorin is toxic only to oats derived from cv Victoria, and the sensitivity of Victoria-type oats to victorin is controlled by the dominant allele of a single gene, V b (Scheffer, 1983).

It has been proposed that the product of the V b gene is a victorin receptor and that resistance results from the lack of the receptor (Scheffer, 1983). Wolpert and Macko (1989) reported that victorin radiolabeled with lz5I Bolton-Hunter reagent bound covalently in vivo to a 100-kD protein only in susceptible cultivars of oat, although in vitro the lZ5I-labeled victorin bound to the 100-kD protein in both susceptible and resistant cultivars. Using polyclonal anti-victorin antibodies, we detected covalent binding of native victorin by westem blotting, but binding in vivo and in vitro was the same in susceptible and resistant cultivars (Akimitsu et al., 1992). The relationship between sensitivity to victorin and the covalent binding seen by Wolpert and Macko (1989) and by us (Ak- imitsu et al., 1992) remains to be determined.

Present address: Department of Energy-Plant Research Labora- tory, Michigan State University, East Lansing, MI 48824.

* Corresponding author; fax 1-517-353-1926. 67

The objective of this study was to identify the subcellular location of VBPs. Homogenates of dark-grown or light-grown oat shoots were separated by continuous or discontinuous density gradients or by an aqueous two-phase method and mixed with victorin, and proteins to which victorin bound covalently were identified. The location of the VBPs in cells was determined by comparison of victorin binding and mem- brane-marker enzyme analyses.

MATERIALS AND METHODS

Plant Materials

Park and Korwood were used as victorin-sensitive and victorin-insensitive oat (Avena sativa) cultivars, respectively. For aqueous two-phase experiments, the oats were grown for 9 d in a greenhouse. For continuous and discontinuous den- sity gradients, oat seeds were germinated under sterile con- ditions on four layers of wet cheesecloth in an aluminum box covered with aluminum foi1 and grown for 5 to 7 d at room temperature until the plants were about 5 cm tall.

Victorin and Antibody Preparation

The major form of victorin was isolated from culture filtrates of Cochliobolous victoriae (isolate HVl146A) as described previously (Akimitsu et al., 1992). Anti-victorin antibodies were produced in rabbits immunized with victo- rin-BSA conjugate and purified by immobilized protein A and BSA columns (Akimitsu et al., 1992).

Detection of VBPs by Western Blotting

Protein concentration was measured by the Bradford (1976) method with BSA as the standard. Fractions of mem- brane preparations, separated by continuous or discontinuous density gradients or by an aqueous two-phase method, were mixed with victorin at a final concentration of 1 pg mL-' and analyzed on 6.5% polyacrylamide gels in the buffer system of Laemmli (1970). Protein from each sample (40 pg per gel lane) was loaded. After separation by SDS-PAGE and trans- fer to nitrocellulose filter, proteins bound to victorin were detected with anti-victorin antibodies followed by goat anti- rabbit immunoglobulin G labeled with lZ5I as previously

Abbreviations: GS 11, glucan synthase 11; VBP, victorin-binding protein.

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68 Akimitsu et al. Plant Physiol. Vol. 103, 1993

described (Akimitsu et al., 1992). For the continuous density gradient studies, the intensity of the bands was measured by computer densitometer (Image Quant/Molecular Dynamics Co., Ltd.), and the relative intensity of each band was nor- malized to the intensity of the band in the 44% Suc fraction. The M, of bands was estimated with prestained mo1 wt standards (14,300-200,000).

Continuous Density Cradients

The procedure used for the continuous Suc density gradient was modified from the methods described by Walton and Ray (1981, 1982). Dark-grown oat shoots (15 g) were har- vested and chilled on ice for 10 to 15 min. The shoots were sliced with a razor blade into I-mm-thick sections and then ground in 20 mL of isolation buffer (50 m Mops [pH 7.51, 2 m EDTA, 0.4 M SUC, 5% [v/v] 2-mercaptoethanol). The homogenate was filtered through four layers of cheesecloth and two layers of Kimwipes and centrifuged for 10 min at 1OOOg. An aliquot (10 mL) of each supernatant (total 20 mL) was layered onto two, 20-mL linear 20 to 45% (w/w) Suc density gradients in gradient medium (50 m Mops [pH 7.51, 2 m EDTA, 5 m DTT) and centrifuged for 3.5 h at 80,OOOg in a Beckman SW 27 rotor at 4OC. After centrifuga- tion, 1.5-mL fractions were collected. The Suc percentage (w/w) of each fraction was measured with a refractometer. Aliquots of each fraction were mixed with victorin and in- cubated for 1 h at room temperature with occasional vortex- ing and stored at -8OOC. Victorin binding was detected by westem blotting as described above. The remaining sample was kept at 4OC for 3 to 4 d for latent IDPase assays or stored at -8OOC for other enzyme assays.

lsolation of Mitochondria

Dark-grown oat shoots (20 g) were homogenized in 60 mL of isolation buffer (0.35 M sorbitol, 30 m Mops [pH 7.51, 1 mM EDTA, 0.2% BSA, 5% [v/v] 2-mercaptoethanol) as de- scribed above. The homogenate was centrifuged at 1,OOOg for 2 min. The supernatant was centrifuged for 5 min at lO,OOOg, and the pellet was gently resuspended with 20 mL of washing buffer (0.3 M sorbitol, 20 m Mops [PH 7.21, 1 m EDTA, 0.2% [w/v] BSA, plus 5 m DTT). The solution was centrifuged again at 1,000 and lO,OOOg, as described above, and the pellet was suspended in 2 mL of suspension buffer (0.25 M SUC, 30 mM Mops [pH 6.81, 5 m DTT). The suspension was layered onto a discontinuous density gradient consisting of layers of 0.6, 0.9, 1.2, 1.45, and 1.8 M SUC in 10 m KH2PO4 (pH 7.2), 1% BSA, 5 m DTT and centrifuged for 45 min at 40,OOOg in a Beckman SW 27 rotor. Fractions (1.5 mL) were collected, and the band containing mitochon- drial inner membranes determined by the Cyt c oxidase assay. An aliquot of the mitochondrial fraction was mixed with victorin for 1 h at room temperature with occasional stirring, and binding was detected by westem blotting as described above.

Aqueous Two-Phase Purification of Plasma Membranes

The aqueous two-phase method using dextran and PEG was based on methods described by Larsson et al. (1987).

Green oat leaves were chilled and ground in 50 m Mops (pH 7.5), 2 mM EDTA, 0.4 M SUC, and 5% (v/v) 2-mercapto- ethanol at a ratio of 1 g of tissue 2.5 mL-' of isolation buffer. The homogenate was filtered through four layers of chleese- cloth and centrifuged at 10,OOOg for 10 min. The superriatant was centrifuged at 50,OOOg for 30 min, and the pellet was resuspended in 10 mL of 0.33 M SUC, 3 mM KCl, !j mM potassium phosphate (pH 7.8). Nine grams of the suspension were added into 27 g of the phase mixture to give a 36-g phase system with a final composition of 6.5% (w/w) dextran T-500, 6.5% (w/w) PEG 3350, 0.33 M SUC, 3 m KC1, 5 mM potassium phosphate (pH 7.8). The phase system was inixed by inverting the tube 30 to 35 times and centrifugecl in a swinging bucket rotor (Beckman SW 27) at 15008 for !I min. The upper layer was washed twice with fresh lower phase and diluted with an equal volume of the isolation buffcbr; the lower layer of the first tube was also washed twice with fresh upper phase and diluted with 9 volumes of the isolation buffer. Both diluted solutions were centrifuged at 100,OOOg for 30 min, and each pellet was resuspended in 0.5 to 1 mL of isolation buffer. Cyt c oxidase and GS I1 assays were performed with preparations stored at -8OOC. To lletect victorin binding, the preparations were used either immedi- ately or after freezing and thawing four times in liquid Nz and water (2OOC) to produce a mixture of inside-out and right-side-out plasma membranes, as described by Palrngren et al. (1990).

Membrane-Marker Enzyme Assays

GS I1 activity was determined by the method of Widelll and Larsson (1990) with some modifications. The GS I1 activity was assayed in 100 pL of buffer (50 m Hepes-KOH [pH 7.251, 0.33 M SUC, 0.8 mM spermine, 16 m cellobiose, 4 mM EGTA, 4 mM CaClZ, 1 m DTT, and 0.01% digitonin). An aliquot (10 pL) from each fraction was added to 100 ,uL of buffer, and the reaction was started by adding 0.025 WCi of [14C]UDP-Glc (specific activity 318 mCi mmol-') and unla- beled UDP-Glc to a final concentration of 2 m. The realction was run at 21OC and stopped after 30 min by adding 95% ethyl alcohol to a final concentration of 66%. The sarnples were filtered through Whatman 3MM under vacuum and washed twice with 1 mL of 66% ethyl alcohol. The l'ilters were washed twice for 1 h each time in 0.35 M ammonium acetate (pH 3.6), 30% (v/v) ethyl alcohol. The filters were dried, and radioactivity was measured in a liquid scintillation counter.

Vanadate-sensitive ATPase activity was determined using the Tris salt of ATP (Hodges and Leonard, 1974; Widell and Larsson, 1990). Vanadate (final concentration 0.1 m:~ was freshly prepared from sodium orthovanadate by boili ng in 50 m Tris-Mes buffer for 2 min and adjusting the pH ío 6.0 with solid Mes.

Cyt c oxidase, latent IDPase, and NADH-Cyt c reductase activities were measured as described by Walton and Ray (1981, 1982).

RESULTS

Localization of VBPs in Continuous Density Cradients

Homogenates of dark-grown oat shoots were separated in a 20 to 45% density gradient, and the maximum actiwty of

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Subcellular Localization of Victorin Binding 69

% sucroea;(w/w) ; I . . . %sucrme(w/w) j

36 sucrose ( w h ) % sucrose (wjw)

Figure 1. Victorin-binding intensity and enzyme activity profiles from density gradient separation of membranes from susceptible (left column) and resistant (right column) oats. Victorin binding was detected with anti-victorin antibody and second antibody conju- gated to l2’I. The intensity of bands was quantitated by densitom- etry. The intensity of binding in each fraction was calculated as a percentage of the intensity in the 44% Suc fraction. Van, Vanadate.

membrane-marker enzymes was determined for fractions from susceptible and resistant cultivars of oats (Fig. 1). The peaks for each marker enzyme were: plasma membranes, 39 to 41% (w/w) Suc (both GS I1 and vanadate-sensitive ATP- ase); mitochondrial membranes, 41% (w/w) Suc (Cyt c oxi- dase); Gol@ membranes, 28 to 29% (w/w) SUC (IDPase); and ER membranes, 22 to 24% (w/w) Suc (Cyt c reductase) (Fig.

1). The intensity of victorin binding was determined by densitometry and compared with the profiles of membrane- marker enzyme analysis (Fig. 1). The 100-kD band appeared in the 36 to 44% (w/w) fractions with a peak at 39% in both susceptible and resistant cultivars. These fractions also con- tained a minor peak of IDPase activity, perhaps associated with Golgi vesicles (Leonard, 1984). The intensity of the 100- kD bands was not significantly different between susceptible and resistant oats. The 100-kD binding protein was not detected in the lower density fractions containing Golgi or ER membranes. In contrast, 65- and 45-kD proteins were detected in a11 fractions in both susceptible and resistant cultivars (Fig. 1).

Effect of Victorin on the Activity of Membrane-Marker Enzymes

Continuous density gradient fractions with the highest enzyme activity were mixed with different concentrations of victorin and compared with enzyme activity without victorin. Victorin had no effect on marker enzyme activity in either susceptible or resistant cultivars (Tables I and 11).

Victorin Binding in Mitochondria

Mitochondria were isolated from a homogenate of dark- grown oat shoots by discontinuous Suc density gradient centrifugation. Mitochondria appeared as a band at the 1.8 ~ / 1 . 4 5 M Suc interface, as identified by the Cyt c oxidase assay. The 65-kD band, but not the 100- and 45-kD bands, was detected in mitochondrial fractions of both susceptible and resistant cultivars of oats (Fig. 2).

VBPs in Plasma Membranes Purified by the Aqueous Two-Phase Method

Plasma membranes of light-grown oat shoots were sepa- rated from other subcellular membranes by the aqueous two- phase method (Larsson et al., 1987). The ratio of upper phase

Table 1. The effect of victorin on the activities of marker enzymes in susceptible oat tissues (CV Park) Numbers in parentheses indicate the percentage change of enzyme activity from control by

addition of victorin. Water was added as a control instead of different concentrations of victorin. Units of enzyme activity are indicated as follows: ATPase, pmol of Pi min-’; GS 11, incorporation of I’4C1UDP-Glc (cpm); NADH-Cyt c reductase and Cyt c oxidase, Asso min-’; IDPase, pmol of Pi h-’.

Change in Marker Enzyme Activity

Enzyme Victorin concentration (ng mL-’) Control

o. 1 1 10 1 O00

Vanadate-sensitive ATPase 0.24

GS I I 6788

NADH-Cyt c reductase 0.123

(100)

(99)

(1 02)

(108)

(100)

Cyt c oxidase 0.119

Latent IDPase 0.34

0.22 (92)

(1 04) 7131

0.125

0.107

0.323

(103)

(97)

(94)

0.25 (104) 6377

(93)

(97)

(97)

(98)

0.1 17

O. 107

0.333

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70 Akimitsu et al. Plant Physiol. Vol. 103, 1993

Table II. The effect of victorin on the activities of marker enzymesin resistant oat tissues (cv Korwood)

Numbers in parentheses indicate the percentage change of en-zyme activity from control by addition of victorin. Water was addedas a control instead of different concentrations of victorin. Units ofenzyme activity are indicated as follows: ATPase, ^mol of Pi min"1;CS II, incorporation of [14C]UDP-Clc (cpm); NADH-Cyt c reductaseand -Cyt c oxidase, A550 min~'; IDPase, ^mol of Pi h"'.

Change in Marker Enzyme Activity

Enzyme Victorin concentrationContro,

Vanadate-sensitive APTase

GSII

NADH-Cyt c reductase

Cyt c oxidase

Latent IDPase

10

0.26(90)

4095(98)

0.25(100)

0.297(99)

0.473(105)

1000

0.30(103)

4054(97)

0.245(98)

0.294(98)

0.459(102)

0.29(100)

4179(100)

0.25(100)

0.3(100)

0.45(100)

(pure plasma membrane) to lower phase of 4.2 for GS IIactivity was similar to that reported by Larsson et al. (1987).No Cyt c oxidase activity was detected in the plasma mem-brane fraction (upper phase; Table III). In western blots, the100-kD protein was detected in the lower phase, but not inthe upper, plasma membrane phase in both the susceptibleand resistant cultivars (Fig. 3). In contrast, the 65- and 45-kD proteins were identified in both phases (upper and lower)in both susceptible and resistant cultivars (Fig. 3). This vic-torin-binding pattern did not change when the membranepreparation was frozen and thawed four times before addi-tion of victorin (data not shown).

DISCUSSION

A victorin receptor in the plasma membrane has beenhypothesized because early effects of victorin are related tochanges in plasma membrane functions (Hanchey andWheeler, 1968; Scheffer, 1983; Walton and Earle, 1985). Werecently characterized an anti-idiotypic anti-victorin poly-clonal antibody that acts as an agonist and as an antagonistto victorin in the stimulation of callose synthesis in suscepti-ble oat protoplasts (Akimitsu et al., 1993). These results alsosuggested that a victorin receptor exists on the surface of oatprotoplasts. Although VBPs have been identified (Wolpertand Macko, 1989; Akimitsu et al., 1992), their role in victorinsensitivity is not known. Because we did not detect the 100-kD VBP in the purified plasma membrane fraction (Fig. 3),its role in victorin sensitivity remains unresolved. The failureof victorin to bind to the 100-kD protein in purified plasmamembranes was not related to right-side-out or inside-outorientation of the plasma membrane preparation, becausevictorin binding was not detected in either case. If victorintoxicity is dependent on initial binding to a plasma membranereceptor, then the role of the 100-kD protein in the sensitivityto victorin must be complex.

We previously reported that victorin binds to the sameproteins in susceptible and resistant oats (Akimitsu et al.,1992). Therefore, a possible explanation for the specificity ofvictorin was that, although victorin binds to the same pro-teins, these proteins may be in different subcellular mem-branes in susceptible and resistant oat cells. However, ourresults reported here do not support this possibility. The100-, 65-, and 45-kD VBPs were detected from the samemembranes in both susceptible and resistant oats (Figs. 1-3).As reported earlier (Akimitsu et al., 1992), the 65-kD proteinbinds victorin only in in vitro experiments and may, therefore,have little role in sensitivity of oats to victorin.

The subcellular location of the 100-kD VBP is unresolved.The 100-kD protein occurs in the 37 to 44% (w/w) Suefractions, which includes plasma membranes and mitochon-dria (Fig. 1). However, the 100-kD protein is located neitherin purified plasma membranes nor in mitochondria (Figs. 2and 3). Wolpert and Macko (1989) reported that the 100-kDprotein was located only in a microsomal fraction that sedi-mented at high g force (100,000g for 30 min) and not at alow g force (4,000g for 4 min); nor was it in the supernatant,suggesting that it is not in the cell wall and is not a solubleprotein. Although plastid membranes should sediment at thelower g force in differential centrifugation experiments (Priceet al., 1987), there is some possibility that this is the locationof the 100-kD protein because some plastid membranes, suchas thylakoid and prolamellar bodies of etioplast membranes,have a density in Sue of 1.17 g mL"1 (about 39% Sue) andalso are in the lower phase of the aqueous two-phase system(Joy and Mills, 1987; Murakami, 1987). Other possibilitiesinclude Golgi-derived vesicles, reported by some to have ahigh density (Leonard, 1984). The minor peak of IDPaseactivity in fractions containing the 100-kD protein (Fig. 1)might suggest localization in certain Golgi vesicles. Coatedvesicles are also reported to have a high density in higherplants (Robinson and Depta, 1988). However, further exper-iments will be necessary to elucidate the subcellular locationof the 100-kD protein.

200-

97-

68-

43-

jj ijiji fij

Figure 2. Victorin binding in mitochondria purified from suscepti-ble (S) and resistant (R) oat shoots. Mitochondria were purified ona discontinuous density gradient. Values at left are the molecularmasses of standard proteins in kD.

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Subcellular Localization of Victorin Binding 71

Table III. Enzyme activity profiles of the plasma membrane fraction isolated by the aqueous two-phase method from susceptible and resistant oat tissues

Microsomal Fraction

Enzyme

GSIIC

Cyt c oxidased

Sb

86480.03

Rb

86540.024

Upper

S"

75320

R"

54960

Phase Ratio'Lower

Cb

Sb

18300.03

Rb

1324 4.10.024

Rb

4.2

' Ratio of upper to lower phases. b S, Susceptible oat (cv Park); R, resistant oat (cv Kor-wood). c Incorporation of [14C]UDP-Clc per 10 L of each sample (cpm). d The change in A5501 min after the substrate was added to 10 ^L of each sample.

The 45-kD protein occurred in the upper and lower phaseof the aqueous two-phase system (Fig. 3), in all fractions ofthe continuous density gradient (Fig. 1), but not in the puri-fied mitochondria fraction (Fig. 2), suggesting that the proteinis associated with more than one cell membrane, includingthe plasma membranes. There were no obvious changes inmarker enzyme activities between victorin-treated enzymepreparations and nontreated preparations (Tables I and II).Victorin binding does appear to be important for toxicitybecause chemically reduced victorin protects against victorin(Wolpert et al., 1988; Wolpert and Macko, 1989) and anti-idiotypic antibodies mimic the effect of victorin (Akimitsu etal., 1993). However, how binding relates to the specificity ofvictorin remains unknown.

Most biologically active molecules act initially by bindingto a protein, which is, therefore, called a receptor. However,a receptor may or may not be the same factor that controlsthe specificity of a ligand. Binding of victorin may be neces-sary for toxicity, but binding alone may not be sufficient forsubsequent toxic effects. We found that victorin binds to thesame proteins localized in the same subcellular sites in bothsusceptible and resistant oats (Akimitsu et al., 1992; thisstudy) and we hypothesize that another factor, the product

KWMF

200-

97-

68-

43-

Figure 3. Victorin binding in plasma membranes purified by theaqueous two-phase method from susceptible (P, cv Park) andresistant (KW, cv Korwood) light-grown oat shoots. MF, Microsomalfraction; U, upper fraction of the two phases, which contains theplasma membranes (Table III); L, lower phases. Values at left arethe molecular masses of standard proteins in kD.

of the Vb gene, controls the sensitivity to victorin afterbinding. The situation with victorin may be similar to tumornecrosis factor. Receptors for tumor necrosis factor have beendetected in a wide variety of normal tissues and cell linesthat are both sensitive and resistant to tumor necrosis factor(Kull et al., 1985; Tsujimoto et al., 1985; Creasey et al., 1987).

ACKNOWLEDGMENTS

The authors gratefully acknowledge discussions and suggestionsby Dr. Glenn Hicks, Michigan State University, Dr. Thomas K.Hodges, Purdue University, Dr. Christer Larsson, University of Lund,Sweden, and Dr. Robert T. Leonard, University of California atRiverside. The authors also thank Dr. Thomas C. Newman and Dr.Masaru Takagi, Michigan State University, for their assistance inoperation of the computer densitometer.

Received March 8, 1993; accepted May 12, 1993.Copyright Clearance Center: 0032-0889/93/103/0067/06.

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