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Partial characterization and aspects of the mode of action of a hypersensitivity-inhibiting factor (HIF) isolated from Phytophthora infestans
Physiological
Plant Pathology
(1979)
15, 127-140
Partial characterization and aspects of the mode of action of a hypersensitivity-inhibiting factor (HIF) isolated from Phy fophthora in festanst N. DOKE, Plant Pathology Laboratory, Facula of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Japan
N. A. GARASand J. KG . Department of Plant Pathology, Unjversity Ixxington, (Acceptedfor
Kentucky
40506,
publication
of Kentucky,
U.S.A.
January
1979)
Factors which
inhibit the hypersensitive reaction of potato tuber tissue (Kennebec, R,) to Phytoplrthora infestam were isolated from mycelia and zoospores of race 1234 (compatible) and race 4 (incompatible) of the fungus. They were partially characterized as glucans containing p-1+3 and g-l+6 linkages and 17 to 23 glucose units. The glucans from both mycelia and zoospores included a non-anionic glucan and an anionic glucan; one or two residues of the latter were esterified with a phosphoryl monoester. Death of host cells, browning and the accumulation of rishitin (hypersensitive reaction) in tuber slices inoculated with race 4 or treated with an elicitor from the fungus were suppressed by pretreatment of slices with the glucans. The glucans from the compatible race were more active in suppressing the hypersensitive reaction than those from the incompatible race. The anionic glucan was more active than the non-anionic glucan. Crude elicitors from races 4 and 1234 lost terpenoid-eliciting activity when mixed with a microsomal fraction prepared from potato tuber tissue. The glucans from the compatible race, but not the incompatible race, markedly reduced the loss resulting from the reaction between the crude elicitor and the microsomal fraction. The data suggest that the compatible interaction between potato tissue and P. infeshzns may be caused by a suppression of the hypersensitive response of the host tissue by watersoluble glucans from the fungus.
INTRODUCTION Potato tissues carrying R genes governing resistance to Phytophthora infestam (Mont) de Bary respond hypersensitively when inoculated with incompatible races of the fungus. All cultivars, even those lacking R genes for resistance, react hypersensitively when treated with cell wall components of the fungus [33]. The hypersensitive reaction is characterized by the loss of electrolytes [19], rapid cell death and tissue browning [I& 281 and the accumulation of terpenoids [17, 23, 30, 331. Compatible races of the fungus penetrate and develop in host tissue for at least 3 days without causing t Journal paper Kentucky 40506. 0048-4059/79/050127+
no.
78-l
l-73
14$02.00/O
of the
Kentucky
Agricultural @ 1979 Academic
Experiment Press Inc.
Station, (London)
Lexington, Limited
128
N. Doke, N. A. Garas and J. Ku6
collapse of host cells [28] or significant terpenoid accumulation [23, 301. Experimental evidence suggests that the lack of response may be caused by the suppression of the hypersensitive reaction [17, 27, 31, 3.71. It was demonstrated that watersoluble high molecular weight compounds from zoospores of compatible races of P. inz~stu~ [.5, 91 and their germination fluids [S, 91 inhibited or delayed the occurrence of the hypersensitive death of potato tissue cells elicited by infection with an incompatible race. Procedures for the isolation of the water-soluble suppressors of the hypersensitive response from mycelia of P. infestam have been described [I,?]. It has also been reported that crude preparations of elicitor from P. infestuns reacted with some components in the microsomal fraction from potato tuber tissue, resulting in loss of the eliciting activity [9, 101. Furthermore, water-soluble extracts from zoospores of germination fluid of P. infestam, which contained a hypersensitivityinhibiting factor (HIF), apparently prevented the binding reaction between crude elicitor and components of host microsomal fraction [9, 101. These preliminary experiments suggested that interaction between elicitor, HIF and some components in the microsomal fraction may be involved in a mechanism of host-parasite interaction in potato late blight. In the present paper, the factor suppressing the hypersensitive reaction of potato to P. infestam was purified, partially characterized and its mode of action investigated, MATERIALS
AND
METHODS
Plants and fungi Kennebec (R,) potato tubers were used in all assays. The tubers were stored at 4 “C until used. Two races of P. infestam, race 1234 (compatible) and race 4 (incompatible), were used for the preparation of HIF. Zoospores of race 4 and crude preparations of elicitor from mycelia of race 1234 or race 4 were used to elicit the hypersensitive response in potato. Microbiological procedures Methods described elsewhere were used for the maintenance of P. infestans and preparation of mycelia [l.?]. Zoospore suspensions were prepared from zoosporangia collected from mycelial mats grown for 11-13 days on lima bean agar media as described previously [S]. After counting zoospores, the zoospore suspension was used as inoculum or quickly frozen at -30 “C for extraction of HIF.
Extraction of HIF Procedures described elsewhere were used for the extraction of HIF from mycelia [I,?]. To extract HIF from zoospores, a thawed suspension of zoospores was homogenized in acetate buffer, pH 4.5 (O-05 M final concentration), in a Polytron homogenizer (Brinkmann Instruments), and centrifuged at 20 000 g for 30 min. The supernatant was treated with phenol and chloroform-methanol as described in the method of extraction from mycelia [12]. Either freeze-dried or non-freeze-dried samples were used for further purification.
A hypersensitivity
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fhytophthora
129
Fractionation of HIF The sample (in 0.01 M Tris-HCl buffer, pH 7.4) was applied to a column (I.8 x 20 cm) of DEAE-cellulose (Whatman) which had been equilibrated with 0.01 M Tris-HCl buffer, pH 7.4. The column was washed with 100 ml of the buffer and the bound material was eluted from the column in buffer containing 0.1 M NaCl at a flow rate of 3.5 ml per 10 min. Fractions, each of four ml, were collected and their carbohydrate content and ultraviolet absorbance were determined. The fractions containing anionic and non-anionic carbohydrate were pooled separately and both were dialyzed against distilled water, concentrated by ultra-filtration using an Amicon UM 2 filter, and applied in 2.0 ml of 0.01 M Tris-HCl buffer, pH 7.4, to Sephadex G 75 columns (1.4 x 50 cm equilibrated with 0.01 M Tris-HCl buffer). The columns were eluted with the buffer and 2 ml fractions were collected. The carbohydrate content and U.V. absorbance of each fraction were determined. Assay of HIF activity Potato tuber discs (18 mm in diameter, 5 mm thick) were prepared from the central parenchymatous tissue of potato tubers as described earlier [6]. Six h after the discs were cut, 0.1 ml of solution containing glucans (mixture of anionic and non-anionic glucans which were mixed after separation by Sephadex gel filtration) dissolved in 0.01 M Tris-HCl buffer, pH 7.4, or the buffer as a control, was applied to the upper surface of each disc. The treated surfaces of discs were inoculated with zoospores of race 4 (5 to 8 x lo5 zoosporangia per ml) or treated with a crude preparation of elicitor (equivalent to 10 mg dry wt of fungus per ml) 12 h after treatment with glucans or water. The discs were incubated on moist filter paper in a Petri dish at 18 “C. All assay procedures were carried out under aseptic conditions. The percentage of dead cells that had been infected with the fungus was determined 18 h after inoculation according to the method of Doke & Tomiyama [6]. Intensity of browning elicited by infection or treatment with crude elicitor was estimated 48 h after inoculation or treatment; discs in each treatment were divided into four groups depending on the intensity of browning and assigned values 0 to 3 (non-browned, 0; most strongly browned, 3). Rishitin and lubimin were extracted from the upper 1 mm of tissue discs 72 h after inoculation or treatment with elicitor, and determined by gas chromatography as described elsewhere [IZ]. Preparation of crude elicitor and microsomalfraction Crude elicitor was prepared from mycelia of P. infestanr as described elsewhere [12]. To prepare the microsomal fraction, cylinders, 18 mm in diameter, were cut from the central parenchymatous tissue of potato tubers which were held at room temperature for 24 h before cutting. Tissue discs, 3 mm thick, were cut from the cylinders, washed thoroughly with a large volume of distilled water, and then incubated on moist filter paper in a Petri dish at 18 “C for 18 h. One hundred g of the discs were dipped in 150 ml of 0.05 M Tris-HCl buffer, pH 7.4, containing O-5 M sucrose, 0.01 M MgCI, and 0.1 y0 of ascorbic acid in an ice bath and infiltrated under vacuum for 15 min. The discs were then placed in a cooled Waring blender bowl and homogenized for 1 min. The homogenate was filtered through four layers of cheese
130
N. Doke, N. A. Garas and J. Ku6
cloth, the filtrate centrifuged at 20 000 g for 40 min, and the resulting supernatant centrifuged at 100 000 g for 90 min. The pellet from the final centrifugation was suspended in 10 ml of 0.01 M Tris-HCl buffer, pH 7.4, containing 0.01 M MgCI, and homogenized with a Polytron homogenizer. This suspension was used as a microsomal fraction of potato tuber tissues. Mixtures
of crude elicitor, glucans and microsomal fraction
Crude elicitor was mixed with the microsomal
fraction,
glucans from mycelia of
P. infestanr or the microsomal fraction which was mixed previously with the glucans
for 30 min. The mixtures contained O-5 ml of crude elicitor (precipitate from 1 g of mycelium sonicated in acetate buffer, pH 4.5, and dissolved in 10 ml of distilled water), 0.5 ml of a suspension of the microsomal fraction, and O-5 ml of the aqueous solution of glucan (3 mg glucan per ml). The mixture was shaken for 30 min at room temperature, and O-1 ml of the mixture was applied to the surface of each potato tuber disc as described above. Intensity of browning and the amount of accumulated rishitin and lubimin were determined 72 h after treatment. Chemical methodr
Glucans were hydrolyzed by heating with 4 N HCl in a sealed tube at 100 “C for 8 h. Partial acid hydrolysis was performed with 1 N HCl at 90 “C for 1 h. The hydrolysates were dried under reduced pressure in the presence of methanol at 30 “C, and the residue free of HCl was dissolved in distilled water. The products were separated by gel filtration on a Sephadex G 15 column (1.8 x 95 cm) equilibrated with distilled water followed by thin-layer chromatography on silica gel G (Analtech, U.S.A.) using chloroform : methanol (6 : 4, v/v) [Z] ; n-butanol : acetone : water (4 : 5 : 1, v/v) [22] ; chloroform : methanol : 17% ammonium hydroxide (40 : 40 : 20, v/v), and phenol : water (75 : 25, v/v) or by thin-layer chromatography on microcrystalline cellulose (Analtech, U.S.A.) using ethylacetate : pyridine : acetic acid : water (5 : 5 : 1 : 3, v/v) [35]. s u g ars were detected after spraying with silver nitrate [22], diphenylamine-aniline [3], potassium permanganate [20] or P-anisidine HCl [II] ; amino acids and aminosugars were detected with ninhydrin [2]. Total carbohydrate content was determined by the phenol-sulfuric acid procedure using D-&COSe as a standard [ll]. Determination of reducing sugars and reducing end groups were made by the Somogyi-Nelson calorimetric method [25, 26-j. Content of protein, of hexosamine and of inorganic and total phosphate was determined by the methods of Lowry et al. [18], Garde11 [13] and Allen [I], respectively. Enzyme
digestion
Anionic and non-anionic glucans were treated with laminarinase (Calbiochem, U.S.A.), a-amylase (Sigma, U.S.A.) and cellulase (Calbiochem, U.S.A.). The anionic glucans were treated with alkaline phosphatase (calf intestine, Calbiochem, U.S.A.) and phosphodiesterase (viper venom, Calbiochem, U.S.A.). Glucans were incubated for 5 h at 30 “C in 4 ml of buffer containing 1 mg of laminarinase, cellulase or a-amylase. Buffers employed were 0.05 M Na-phosphate buffer, pH 5.9 (laminarinase); 0.01 M Tris-HCl buffer, pH 7.4 (a-amylase); 0.01 M Na-phosphate buffer,
A hypersensitivity
suppression
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Phytophthora
131
pH 6.9 and 4.5 (cellulase). After incubation, the reaction mixture was heated at 100 “C for 10 min, mixed with 3 vols of ethanol and centrifuged at 10 000 g for 30 min. The supernatant was evaporated ( ~45 “C) under reduced pressure to dry ness. The products were separated by Sephadex G 15 gel filtration and thin-layer chromatography. The mixture for hydrolysis with alkaline phosphatase contained 1 mg of anionic glucan and 1 mg of alkaline phosphatase in 2 ml of -005 M Tris-HCl buffer, pH 8.8. The mixture was incubated at 30 “C for 3 h. The incubation of glucans (1 mg) with phosphodiesterase (100 units) was done in 2 ml of 0.05 M Tris-HCl buffer, pH 7.4, at 30 “C for 5 h. After digestion with the two phosphatases, the reaction mixtures were passed through a DEAE cellulose column to determine the ratio of non-anionic glucan to anionic glucan, or fractionated by Sephadex G 75 gel filtration to detect changes in molecular size, as described earlier. RESULTS
Fractionation
of hypersensitivity-inhibiting
factor (HIF)
Preliminary investigation indicated that HIF from mycelia of P. infeetum was extracted with acetate buffer, pH 4.5, followed by treatment with phenol : chloroform : methanol [12]. The factor thus extracted was found to be polysaccharide in nature. Fractionation of the polysaccharides extracted from mycelia on a DEAE cellulose column revealed a single large peak of non-anionic polysaccharide and a single smaller peak of anionic polysaccharide (Fig. 1). Fractionation of the preparation from zoospores revealed several incompletely resolved peaks of anionic polysaccharide in addition to the non-anionic polysaccharide peak. There was a significant difference in the anion exchange chromatography profile of soluble polysaccharide from mycelia and zoospores but not between the two races. The proportion of anionic polysaccharide to non-anionic isolated from mycelia was smaller than that from zoospores (Table 1). Fractionation of the glucans of Sephadex G 75 revealed that the polysaccharides from mycelia produced almost symmetrical elution profiles and the anionic polysaccharide fraction was eluted ahead of the non-anionic polysaccharide fraction (Fig. 2). The elution profile of polysaccharide isolated from zoospores was nearly the same as that from mycelia expect for a slightly broader profile of anionic polysaccharide. The anionic and non-anionic glucans from the two races of fungus could not be distinguished on the basis of their elution profiles. The freeze-dried glucans, however, appeared to be consistently different in texture. Properties and characterization
of polysaccharide
Glucans fractionated on DEAE cellulose followed by Sephadex G 75 showed no U.V. light absorption maxima in the region 2 10 to 340 nm and thereby appeared free of nucleic acid and protein. Complete acid hydrolysis of these polysaccharides followed by silica gel G or cellulose thin-layer chromatography revealed glucose as the only monosaccharide. Partial acid hydrolysis revealed a series of oligosaccharides which were fractionated on Sephadex G 15, and the disaccharide fraction was isolated. Thin-layer chromatography of the disaccharide fraction revealed a major spot with the same R, values
N. Doke, N. A. Garas and J. KuC
132
I
Race
1234
66 c
Race
04 M NaCl
0.5
I
M NaCl
-I3
1
4-1
l
4 0. I M NaCl
.
0.5~
1
NaCl 1
4-
. 2-
* .
T i
(1
Jj-.
IO
20
30
40
50
60
IO
20
30
40
50
60
0 0
= 0,153
1
J oio-
0.05
1
’
-
-i IO FIG.
(lower
20
30
40
50
60 Fraction
20
30
1. Fractionation (4 ml fractions) of glucans from mycelia half) of Phytophthora infestanr on DEAE cellulose columns. TABLE
Percentage
of anionic
Source
g&an
Mycelia Zoospores a The percentage of anionic glucan shown in Fig. 1. Values represent for zoospores.
50
60
(upper
half)
and zoospores
1
in total soluble glucanr from infestans
of glucan
40
number
mycelia
Percentage Race 1234 22+11 5&l* 12
and zoospores of Phytophthora
of anionic
glucan@ Race
4
31+7 64+3
was obtained from DEAE-cellulose fractionation as the mean + S.D. of five fractions for mycelia and three
as laminaribiose prepared from the partial hydrolysis of laminarin and a minor spot with the same R, values as gentiobiose. Amino acids and amino sugars were not detected in acid hydrolyzates of the glucans by thin-layer chromatography. The average degree of polymerization of the glucans, estimated by determining the number of reducing end groups, was found to be between 17 to 23 glucose units
A hypersensitivity
suppression
from
fhytophfhora
(a)
40
50
20 number
Fraction
FIG. 2. Fractionation mycelia of Phytophlhora
30
40
(2 ml fractions) of (0) anionic and (0) non-anionic infestanr on Sephadex G- 75 columns. (a) Race 1234;
50
glucans (b) Race
from 4.
with the degree of polymerization being slightly lower for race 4 than that for race 1234 (Table 2). Further evidence for the major linkages of these glucans was obtained by analysis of the products produced after digestion with laminarinase (p-1-+3 glucanase) which hydrolyzed the glucan, and the liberated glucose indicated better than 90% of total
Proper&s
of water-soluble
glucanr from
TABLE
2
mycelia
and zoospores of Phytophthora
infestans Hydrolysisd
Source Mycelia, Mycelia, Mycelia, Mycelia, Zoospores, Zoospores, Zoospores, Zoospores,
race 1234 race 1234 race 4 race 4 race 1234 race 1234 race 4 race 4
Glucan’ Non-anionic Anionic Non-anionic Anionic Non-anionic Anionic Non-anionic Anionic
Average degree polymerizationb 23+1 21+1 18kl 18kl 22*1 22+1 18k2 17+2
of
Glucose phosphate 14+4 11+2 12+3 8+4
to ratioC
with
L
C
A
+ + + + + + + +
-
-
a Glucans were extracted and fractionated by DEAE-cellulose and Sephadex G 75 column chromatography as described in the text. The fraction containing the major peak on Sephadex gel filtration was used to determine the properties of glucans. b Each value represents the meanf S.D. of five and three different fractions for mycelia and zoospores, respectively. c Each value represents the mean f S.D. of four and three different fractions for mycelia and zoospores, respectively. d L, laminarinase; C, cellulase; A, or-amylase; +, hydrolyzed; -, not hydrolyzed.
N. Doke, N. A. Garas and J. Ku6
134
hydrolysis. Cellulase and a-amylase did not hydrolyze the glucans. The extent of enzymatic hydrolysis was established by Sephadex G 15 gel filtration followed by thin-layer chromatography. Laminarin from Im&m-ia hyjerborea (Sigma, U ,S .A.) also was treated with the above enzymes and its sensitivity to hydrolysis was the same as that of the glucans. Significant amounts of phosphate were found in all anionic glucans; the phosphate content of anionic glucan from mycelia was 14 and 11 glucose residues per mole of phosphate for race 1234 and race 4, respectively (Table 2). In the case of each anionic glucan fraction from zoospores, exact phosphate values were difficult to determine in the small sample available. However, the phosphate content of total anionic glucans from zoospores was 12 and 8 glucose residues per mol of phosphate for race 1234 and race 4, respectively. The anionic glucans from mycelia and zoospores of race 4 contained a slightly higher ratio of phosphate to glucose residues than the anionic glucans from mycelia and zoospores of race 1234, respectively. Alkaline phosphatase converted the anionic glucan to the non-anionic glucan (Fig. 3). The
Fraction FIG. 3. Effect DEAE cellulose) 0, non-treated;
number
of treatment with alkaline phosphatase on the elution profile of anionic glucan prepared from mycelia of Phytophthoru l , treated with alkaline phosphatase.
(4 ml fractions,
infestam race 4.
non-anionic glucan formed after treatment with phosphatase was subjected to Sephadex G 75 gel filtration, and its elution profile corresponded to that of the nonanionic glucan. Treatment of both the anionic and non-anionic glucans with phosphodiesterase did not alter their elution patterns on Sephadex G 75 or DEAE cellulose. Hypersensitivity-inhibiting
activity
The water-soluble glucans (mixture of anionic and non-anionic glucans) were dissolved in 0.01 M Tris-HCl buffer, pH 7.4, and applied to the surface of potato tuber tissue discs before inoculation with an incompatible race of P. infestuns or treatment
A hypersensitivity
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Phyfophfhora
135
with a crude preparation of elicitor from the fungus. More than 90% of the infected cells in the tissue treated with the buffer died 18 h after inoculation, while about 30 and 70% of infected cells died in the case of treatment with the glucans from race 1234 (compatible race) and race 4 (incompatible race), respectively (Table 3). A determination of the percentage of dead cells was not made in the case of treatment with crude elicitor. Control tissue inoculated with the incompatible race or treated with elicitor browned extensively in the surface tissues. Tissue treated with the glucan from the compatible race prior to inoculation with an incompatible race or treatment with elicitor showed a more marked reduction in the intensity of browning than that treated with glucans from the incompatible race (Table 3). Elicitation of rishitin accumulation by both living fungus and elicitor was also suppressed more markedly by treatment with glucan from the compatible as compared with the incompatible race of the fungus (Table 3). TABLE
3
Effect of pretreatment with water-soluble glucans on hyperseCnntioe cell death, tissue browning and accumulation of tmpmoids in potato tuber tksue subsequently infer&d with an incompatible race of Phytophthora infestans or treatcd with crude clicit~ Terpenoid accumulation (ug g fresh wt-l)d Glucans~
buffer (0.01 M, pH Race 1234 Race4 Distilled water Race 1234 Race 4
Elicitationb
% of dead cell@
Infection Infection Infection Elicitor Elicitor Elicitor
9324 30+6 69&20 -
Relative of tissue
intensity browningd
Rishitin
Lubimin
Tris
7.4)
2.3 l-5 l-8 2.5 1.6 2-o
85+15 28+ 12 72518 39 20 32
25+6 16+8 2355 21 18 21
a Water-soluble glucan (1 mg ml-l) was applied to the surface of Kennebec potato tuber slices (R,) 6 h after cutting. b Incompatible race 4 (5 to 8x 106 zoospores per ml) or crude elicitor from compatible race 1234 (l/10 dilution) was applied to potato discs 12 h after treatment with the glucans. c Observed 18 h after inoculation (about 100 infected cells were observed for each treatment) . d Observed 48 h after inoculation or treatment. * Determined 72 h after inoculation or treatment. Values for inoculation represent the mean of three to four different experiments with standard deviations; values for treatment with elicitor are averages of two experiments.
Throughout these experiments, there was a positive correlation between inhibition of cell death, inhibition of tissue browning and inhibition of rishitin accumulation after treatment with the glucans. Cytological observation of tissue treated with glucans indicated that only cells in the first and second layer from the treated surface were protected from cell death and tissue browning 48 h after treatment. At later stages, tissue browning was observed in deeper tissues
136
N. Doke,
N. A. Garas
and
J. KuC
To compare the hypersensitivity-inhibiting activity of non-anionic glucan with that of anionic glucan, a low concentration of glucan was applied to the tissue and the percentage of dead cells elicited by infection with an incompatible race was determined (Table 4). Anionic glucan isolated from both mycelia and zoospores of compatible race (race 1234) inhibited cell death more actively than the non-anionic glucan. Treatment with glucan from the incompatible race (race 4) at 20 pg ml-l caused little or no inhibition of the hypersensitive cell death.
Efect of @treatment with anionic phthora infestans on hypcrsenritive
TABLE 4 or non-anionic glucans from mycelia death of potato tuber cells subsequently race 4 of the fWgus
Glucan.@
Nature
a Glucan applied at a concentration of 20 pg ml-l. b Each value represents the mean of two experiments. observed for each treatment. Cell death was observed
In vitro
interaction
of glucans,
Percentage
Non-anionic Anionic Non-anionic Anionic Non-anionic Anionic Non-anionic Anionic
-Distilled water Mycelia, race 1234 Mycelia, race 1234 Mycelia, race 4 Mycelia, race 4 Zoospores, race 1234 Zoospores, race 1234 Zoospores, race 4 Zoospores, race 4
elicitor,
and
microsomal
and zoospores of Phytoinfected with incompatible
of dead
cellsb
100 86 44 99 97 68 39 89 92
About 100 infected 18 h after inoculation.
cells
were
fraction
In vitro interaction among the glucans, crude elicitor and microsomal fraction from potato tuber tissue was investigated by determining changes in the activity of the elicitor. The necessary controls indicated that the glucan from both incompatible and compatible races and the microsomal fraction did not brown the surface of potato discs or elicit the accumulation of terpenoids (Table 5, Expt I, A, B, C). The crude elicitor elicited both strong tissue browning and accumulation of terpenoids (Expt I, D; Expt II, A). Mixing of glucans (mixture of anionic and nonanionic glucans) from the compatible or incompatible race with crude elicitor did not affect the activity of elicitor significantly (Table 5, Expt I, E, F; Expt II, B, C). Mixing of the elicitor with the potato microsomal fraction, however, markedly reduced its ability to elicit both browning and the accumulation of terpenoids (Expt I, G; Expt II, D). When the microsomal fraction was mixed with the glucan from the compatible race before mixing with the crude elicitor, the eliciting activity was not inhibited appreciably (Expt I, H; Expt II, E). Eliciting activity remained low when glucan from the incompatible race was mixed with the microsomal fraction followed by elicitor (Expt 1, I; Expt II, F). When elicitor from race 1234 was mixed with the microsomal fraction before mixing with glucans, neither the glucan from the compatible race nor that from the incompatible race affected the inhibitory effect of the microsomal fraction. (Table 5, Expt II, G. H).
A hypersensitivity
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137
TABLE Effect
of in vitro interaction of crucie elicitor infestans and a microsomal fraction from
Materials
Experiment I
II
Glucansb A. B. C. D. E. F. G. H. I.
Race 1234 Race4 Distilled water Distilled water Race 1234 Race4 Distilled water Race 1234 Race4
A. B. C. D. E. F. G. H.
Distilled water Race 1234 Race 4 Distilled water Race 1234 Race4 Race 1234 Race 4
+
5
and water-soluble potato tuber tissu
treated
Microsomal fractionc
&cans from Phytophthora on activity of the elicitor
Relative inn,t;n~ +
Elicitord
browning
Terpenoid accumulatiorP (pg g fresh wt-1) Rishitin
Lubimin
Buffer
Distilled
water
0.0
0
Buffer Kennebec Buffer Buffer Buffer Kennebec Kennebec Kennebec
(R,) (R$’ (R, )”
Distilled water Distilled water Race 4 Race 4 Race 4 Race 4’ Race 4 Race 4
0.0 0.0 2.6 1.9 2.4 l-4 1.9 1.5
0 0 144 116 1‘iQ 24 119 21
0 0 0 49 53 56 10 42 11
Buffer Buffer Buffer Kennebcc
(R,)
Race Race Race Race
Kennebec Kennebec Kennebec Kemrebec
(R,)e (R,)’ (R,) (R,)
Race 1234 Race 1234 Race 12346 Race 1234’
2.9 2.9 2.9 1.8 2.8
65 73 68 28 67 35 9 24
23 25 29 21 21 19 7 13
(R,)
1234 1234 1234 1234
2.1 1.8 1.9
D Each value is an average of two determinations. b A mixture of anionic and non-anionic glucans was dissolved in distilled water (3 mg ml-r) 0.5 ml. c Microsomal fraction from 100 g tissue was suspended in 10 ml of 0.01 M Tri.-HCl buffer, pH 7.4, containing O-01 M MgCl,; 0.5 ml. d Crude elicitor, equivalent to 100 mg dry wt of fungus, was suspended in 10 ml of distilled water; 0.5 ml. The crude elicitor is a fraction insoluble in pH 4.5 acetate buffer, prepared from a sonicate of mycelia in 10 ml of distilled water. ‘ Treatments underlined indicate pre-incubation of the components for 30 min. The total mixtures for all treatments were incubated for 30 min at room temperature before application to potato discs.
DISCUSSION Glucans which inhibited the hypersensitive reaction of potato to P. infestans were isolated from both mycelia and zoospores of the fungus. The glucans were watersoluble, had an average degree of polymerization of 17 to 23 glucose units (Table Z), and contained p-1-+3 and p-1-+6 glycosidic linkages. One of the glucans was a nonanionic glucan which is similar to the glucan isolated from P. infestunr [I& 24, 261. Another glucan was anionic, contained phosphorus and was similar to the glucan isolated from P. palmivora [34]. It appears that one or two glucose residues of the anionic glucan are esterified with a phosphor-y1 residue via a monoester linkage since an alkaline phosphatase converted the anionic glucan to the non-anionic glucan. This report appears to be the first report of the isolation of an anionic phosphoglucan from both mycelia and zoospores of P. infestans. Although the anionic
138
N. Doke, N. A. Garas and J. KuC
glucan has nearly the same degree of polymerization as the non-anionic glucan, the former appeared to be eluted earlier than the latter on Sephadex G 75 gel filtration (Fig. 2). It is possible that the faster migration of the anionic glucan was due to ionic interaction with residual anions in the Sephadex gel which block the movement of the polymer into the gel particles as described with the anionic glucan from P. ~almivoru [34]. The elution pattern on DEAE cellulose suggests zoospores may contain several similar but not identical anionic glucans (Fig. 1). There were only slight differences in glucose to phosphorus ratio and the degree of polymerization of glucose (Table 2) between race 1234 and race 4, but the appearance of freeze-dried samples of the glucans from the two races was significantly different, suggesting some differences in the structure of the glucans. Further characterization of the structure of the glucans is necessary. The data indicate that P. infestans contains water-soluble glucans that suppress the hypersensitive response of potato to P. infetans; the glucans from a compatible race being more active than those from an incompatible race (Tables 3, 4, 5). Earlier publications reported that infection with a compatible race of P. infestans inhibited rapid cell death [5, 8, 27, 281 and the accumulation of terpenoids [1.2, 17, 31, 321 from subsequent infection with an incompatible race. High molecular weight water-soluble compounds which inhibited the hypersensitive reaction were extracted from zoospores and germination fluids [S, 8, 9, lo] of compatible races of P. infestans. The compounds appear to be the glucans whose isolation and partial characterization are reported in this paper. Browning and accumulation of terpenoids in tubers have not been reported without hypersensitive cell death. The occurrence of cell death may also be closely involved in the resistant reaction of potato tissue. Treatment of potato tuber slices with elicitor enhanced electrolyte leakage, whereas pretreatment with glucan from a compatible race, but not incompatible race, of the fungus markedly reduced leakage [ 121. Dextran-bound p-chloromercuribenzoic acid, which does not readily penetrate cell membranes, prevented the hypersensitive cell death [7]. Accordingly, the glucans from compatible races may protect cell membranes of potato tissue from damage caused by elicitors. In the present experiments, a microsomal fraction from potato tissue was used to investigate the interaction among elicitor, glucans and cell membranes. Although the elicitor and microsomal fraction are very crude preparations, the in vitro reaction among them suggests that HIF suppresses the reaction between elicitor and cell membranes. Conceivably, elicitor may bind to some substance (receptor) on cell membranes and the glucan may block the binding site of the substances for elicitor. Though the data presented are consistent with the postulated role of the glucans in determining the specificity of interaction between potato and P. infestatu [9, 17, 31, 321, further experiments with many cultivars of potato and races of P. inzstans are needed before a role for the elicitor and glucans can be established. The research reported in this paper was done at the Department of Plant Pathology, University of Kentucky, and has been supported in part by a grant from the Herman Frasch Foundation and grant no. 316-15-51 of the Cooperative State Research Service of the United States Department of Agriculture. We acknowledge the technical assistance of Mrs Barbara Stoner.
A hypersensitivity
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from
fhytophthora
139
REFERENCES 1. ALLEN, R. J. L. (1940). The estimation of phosphorus. Biochemical 3ournal34, 858-865. 2. AMINOFF, D. & MORGAN, W. T. J. (1948). H exosamine components of the human blood group substances. Jvatarc 162,579-580. 3. BAILEY, R. W. & BOURNE, E. J. (1960). Color reaction given by sugars and diphenylamine-aniline spray reagent on paper chromatograms. Journal of Chromatography 4, 206-213. 4. BRENNER, M., NIEDERWEISER, A. & POTOICI, G. (1965). Amino acid and derivatives. In Thin Layer Chromatograghy, Ed. by E. Stahl, pp. 247-269. Springer-Verlag, Berlin. 5. DOKE, N. (1975). Prevention of the hypersensitive reaction of potato cells to infection with an incompatible race of Phytophthora infestam by constituents of the zoospores. Physiologicnl Plant Pathology 7, l-7. 6. DOKE, N. & TOMIYAMA, K. (1975). Effect of blasticidin S on hypersensitive death of potato leaf petiole cells caused by infection with an incompatible race of Phytophthoru infestam. Physiological Plant Pathology 6, 169-175. 7. DOKE, N. & TOMIYAMA, K. (1978). Effect of sulfhydryl-binding components on hypersensitive death of potato tuber cells following infection with an incompatible race of P&ytophthora infestans. Physiological Plant Pathology 12, 133-139. 8. Dorm, N. & TOMNAMA, K. (1977). Effect of high molecular substances released from zoospores of Phytophthora infeskm on hypersensitive response of potato tubers. Phytopathologische +itschrift. 90, 236-242. 9. Doxa, N., TOMIYAMA, K., LEE, H. S., NISHIMURA, N. & MATSUMOTO, N. (1976). Mode of physiological response of potato tissue induced by constituents of Phytophthora infestanr in relation to host-parasite specificity. In Biochemistry and Cytology of Plant-Parasite Interaction, Ed. by K. Tomiyama, J. M. Daly, I. Uritani, H. Oku & S. Ouchi, pp. 157-167. Kodansha Ltd, Tokyo. 10. DOKE, N., TOMIYAMA, K., NISHIMURA, N. & LEE, H. S. (1975). Z n vitro interaction between components of Phytophthora infestans zoospores and components of potato tissue. Annuls of the Phytopathological Socie& of Japan 41, 425-433. 11. DIJBOIS,M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A. & SMITH, F. (1956). Golorimetric method for the determination of sugars and related substances. Analytical Chemistry 28, 350-356. 12. GARAS, N. A., DOKE, N. & Ku6, J. (1979). Suppression of the hypersensitive reaction in potato tubers by mycelial components from Phytofihthora infestans. Physiological Plant Pathology 15,117-126. 13. GARDELL, S. (1953). Separation on Dowex 50 ion-exchange resin of glucosamine and galactosamine, and their quantitative determination. Acta Chemica Scandinavica 7, 207-2 15. 14. HOUGH, L. & JONES, J. K. N. (1962). Chromatography on paper. In Advances of Carbohydrate Chemistry, Ed. by L. Wolfram & R. S. Tipson, Vol. 17, pp. 21-31. Academic Press, New York. 15. HODGESON, W. A., MUNRO, J., SINGH, P. R. & WOOD, F. A. (1969). Isolation from Phytophthora infeskm of a polysaccharide that inhibits potato virus X. Phytopatholop 59, 1334-1335. 16. KITAZAWA, K. & TOMIYAMA, K. (1968). Microscopic observation of infection of potato cells by compatible and incompatible races of Phytophthora infestans. Phytopathologische ,$&.schr$t 66, 317-324. 17. Ku&J., CURRIER,W., ELLISTON, J. & MCINTYRE, J. (1976). Determinants of plant-disease resistance and susceptibility: A perspective based on three plant-parasite interactions. In Biochemistry and Cytology of Plant-Parasite Interaction, Ed. by K. Tomiyama, J. M. Daly, I. Uritani, H. Oku & S. Ouchi, pp. 168-180. Kodansha Ltd, Tokyo. 18. LOWRY, 0. H., ROSENBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. 3ournal of Biochemistry 193, 265-275. 19. MATXJMOTO, N., TOMIYAMA, K. & Dotca, N. (1976). Alteration of membrane permeability of potato-tuber tissue infected by incompatible and compatible races of Phytophthora infestans during the initial phase of infection. Annals of the Phytopathological So&y of Japan 42, 279-286. 20. PACSU, E., MORA, T. P. & KENT, P. W. (1949). G eneral method for paper chromatographic analysis of reducing and nonreducing carbohydrates and derivatives. Science 110, 446-447. 21. PATRIDGE, S. M. & WESTALL, R. G. (1948). Filter-paper partition chromatography of sugars. I. General description and application to the qualitative analysis of sugars in apple juice, egg white and fetal blood of sheep. Biochemical 3ournul42, 238-250. 22. PIFFERI, P. G. (1965). Improved thin-layer chromatographic separation of hexososes and pentoses by using Kiesel gel G. Analytical Chemistry 37, 925. 23. SATO, N., TOMIYAMA, K., KATSUI, N. & MA~AMUNE, T. (1968). Isolation of rishitin from tubers of interspecific potato varieties containing different late-blight resistance genes. Annals of the Phytofiathological Society of Japan 34, 140-142. 24. SINGH, R. P., WOOD, F. A. & HODGESON, W. A. (1970). The nature of virus inhibition by a polysaccharide from Phytojhthora infestmu. Phytojatholoa 60, 1566-1569.
140
N. Doke, N. A. Garas and J. KuC
25. SOMOGYI, M. (1945). Determination of blood sugar. 3ournal of Biological Chemistry 160, 69-73. 26. SOMOGYI, M. (1952). Notes on sugar determination. Journal of Biological Chemistrb 195, 19-23. 27. TOMIYAMA, K. (1969). Double infection by an,incompatible race of Phytophthora infestuns of a potato plant cell which has previously been infected by a compatible race. Annals of the Phytopathological Society ofJapan 32, 181-185. 28. TOMIYAMA, K. (1967). Further observation on the time requirement for hypersensitive cell death of potatoes infected by Phytophthora infestam and its relation to metabolic activity. Phytopathologische ,Qitschrijt 58, 367-378. 29. TREVELYAN, W. E., PROCTOR, D. P. & HARRISON, J. S. (1950). Detection of sugars on paper chromatograms. Nature 166, 444-445. 30. VARNS, J. L., Ku6, J. & WILLIAMS, E. B. (1971). Terpenoid accumulation as a biochemical response of the potato tuber to Phytophthora infestam. PhytopatholoQ 61, 174-l 77. 31. VARNS, J. L. & Ku6, J. (1971). Suppression of rishitin and phytuberin accumulation and hypersensitive response in potato by compatible races of Phytophthora infestans. Phytopatholou 61, 178-181. 32. VARNS, J. L. & Ku6, J. (1972). Suppression of the resistance response as an active mechanism for susceptibility in the potato-Phytophthora infestans interaction. Phytotoxins in Plant Disease, Ed. by R. K. S. Wood, A. Ballio and A. Graniti, pp. 46-68. Academic Press, London, New York. 33. VARNS, J. L., CURRIER, W. W. & KuC, J, (1971). Specificity of rishitin and phytuberin accumulation by potato. Phytopathologv 61, 968-971. 34. WANG, M. C. & BARTNICKI-GARCIA, S. (1973). Novel phosphoglucans from the cytoplasm of Phytophthora palmiuora and their selective occurrence in certain life cycle stages. 3ournal of Biological Chemidry 248, 4112-4118. 35. WOLFROM, M. L., PATIN, D. L. & LEDERKREMER, R. M. DE (1965). Thin-layer chromatography on microcrystalline cellulose. Journal of Chromatography 17, 488-494. 36. WOOD, F. A., SINGH, R. P. & HODGESON, W. A. (197 1). Characterization of a virus-inhibiting polysaccharide from Phytophthora infestanr. Phytopathologv 61, 1006-1009.
Plant Pathology
(1979)
15, 127-140
Partial characterization and aspects of the mode of action of a hypersensitivity-inhibiting factor (HIF) isolated from Phy fophthora in festanst N. DOKE, Plant Pathology Laboratory, Facula of Agriculture, Nagoya University, Chikusa-ku, Nagoya, Japan
N. A. GARASand J. KG . Department of Plant Pathology, Unjversity Ixxington, (Acceptedfor
Kentucky
40506,
publication
of Kentucky,
U.S.A.
January
1979)
Factors which
inhibit the hypersensitive reaction of potato tuber tissue (Kennebec, R,) to Phytoplrthora infestam were isolated from mycelia and zoospores of race 1234 (compatible) and race 4 (incompatible) of the fungus. They were partially characterized as glucans containing p-1+3 and g-l+6 linkages and 17 to 23 glucose units. The glucans from both mycelia and zoospores included a non-anionic glucan and an anionic glucan; one or two residues of the latter were esterified with a phosphoryl monoester. Death of host cells, browning and the accumulation of rishitin (hypersensitive reaction) in tuber slices inoculated with race 4 or treated with an elicitor from the fungus were suppressed by pretreatment of slices with the glucans. The glucans from the compatible race were more active in suppressing the hypersensitive reaction than those from the incompatible race. The anionic glucan was more active than the non-anionic glucan. Crude elicitors from races 4 and 1234 lost terpenoid-eliciting activity when mixed with a microsomal fraction prepared from potato tuber tissue. The glucans from the compatible race, but not the incompatible race, markedly reduced the loss resulting from the reaction between the crude elicitor and the microsomal fraction. The data suggest that the compatible interaction between potato tissue and P. infeshzns may be caused by a suppression of the hypersensitive response of the host tissue by watersoluble glucans from the fungus.
INTRODUCTION Potato tissues carrying R genes governing resistance to Phytophthora infestam (Mont) de Bary respond hypersensitively when inoculated with incompatible races of the fungus. All cultivars, even those lacking R genes for resistance, react hypersensitively when treated with cell wall components of the fungus [33]. The hypersensitive reaction is characterized by the loss of electrolytes [19], rapid cell death and tissue browning [I& 281 and the accumulation of terpenoids [17, 23, 30, 331. Compatible races of the fungus penetrate and develop in host tissue for at least 3 days without causing t Journal paper Kentucky 40506. 0048-4059/79/050127+
no.
78-l
l-73
14$02.00/O
of the
Kentucky
Agricultural @ 1979 Academic
Experiment Press Inc.
Station, (London)
Lexington, Limited
128
N. Doke, N. A. Garas and J. Ku6
collapse of host cells [28] or significant terpenoid accumulation [23, 301. Experimental evidence suggests that the lack of response may be caused by the suppression of the hypersensitive reaction [17, 27, 31, 3.71. It was demonstrated that watersoluble high molecular weight compounds from zoospores of compatible races of P. inz~stu~ [.5, 91 and their germination fluids [S, 91 inhibited or delayed the occurrence of the hypersensitive death of potato tissue cells elicited by infection with an incompatible race. Procedures for the isolation of the water-soluble suppressors of the hypersensitive response from mycelia of P. infestam have been described [I,?]. It has also been reported that crude preparations of elicitor from P. infestuns reacted with some components in the microsomal fraction from potato tuber tissue, resulting in loss of the eliciting activity [9, 101. Furthermore, water-soluble extracts from zoospores of germination fluid of P. infestam, which contained a hypersensitivityinhibiting factor (HIF), apparently prevented the binding reaction between crude elicitor and components of host microsomal fraction [9, 101. These preliminary experiments suggested that interaction between elicitor, HIF and some components in the microsomal fraction may be involved in a mechanism of host-parasite interaction in potato late blight. In the present paper, the factor suppressing the hypersensitive reaction of potato to P. infestam was purified, partially characterized and its mode of action investigated, MATERIALS
AND
METHODS
Plants and fungi Kennebec (R,) potato tubers were used in all assays. The tubers were stored at 4 “C until used. Two races of P. infestam, race 1234 (compatible) and race 4 (incompatible), were used for the preparation of HIF. Zoospores of race 4 and crude preparations of elicitor from mycelia of race 1234 or race 4 were used to elicit the hypersensitive response in potato. Microbiological procedures Methods described elsewhere were used for the maintenance of P. infestans and preparation of mycelia [l.?]. Zoospore suspensions were prepared from zoosporangia collected from mycelial mats grown for 11-13 days on lima bean agar media as described previously [S]. After counting zoospores, the zoospore suspension was used as inoculum or quickly frozen at -30 “C for extraction of HIF.
Extraction of HIF Procedures described elsewhere were used for the extraction of HIF from mycelia [I,?]. To extract HIF from zoospores, a thawed suspension of zoospores was homogenized in acetate buffer, pH 4.5 (O-05 M final concentration), in a Polytron homogenizer (Brinkmann Instruments), and centrifuged at 20 000 g for 30 min. The supernatant was treated with phenol and chloroform-methanol as described in the method of extraction from mycelia [12]. Either freeze-dried or non-freeze-dried samples were used for further purification.
A hypersensitivity
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fhytophthora
129
Fractionation of HIF The sample (in 0.01 M Tris-HCl buffer, pH 7.4) was applied to a column (I.8 x 20 cm) of DEAE-cellulose (Whatman) which had been equilibrated with 0.01 M Tris-HCl buffer, pH 7.4. The column was washed with 100 ml of the buffer and the bound material was eluted from the column in buffer containing 0.1 M NaCl at a flow rate of 3.5 ml per 10 min. Fractions, each of four ml, were collected and their carbohydrate content and ultraviolet absorbance were determined. The fractions containing anionic and non-anionic carbohydrate were pooled separately and both were dialyzed against distilled water, concentrated by ultra-filtration using an Amicon UM 2 filter, and applied in 2.0 ml of 0.01 M Tris-HCl buffer, pH 7.4, to Sephadex G 75 columns (1.4 x 50 cm equilibrated with 0.01 M Tris-HCl buffer). The columns were eluted with the buffer and 2 ml fractions were collected. The carbohydrate content and U.V. absorbance of each fraction were determined. Assay of HIF activity Potato tuber discs (18 mm in diameter, 5 mm thick) were prepared from the central parenchymatous tissue of potato tubers as described earlier [6]. Six h after the discs were cut, 0.1 ml of solution containing glucans (mixture of anionic and non-anionic glucans which were mixed after separation by Sephadex gel filtration) dissolved in 0.01 M Tris-HCl buffer, pH 7.4, or the buffer as a control, was applied to the upper surface of each disc. The treated surfaces of discs were inoculated with zoospores of race 4 (5 to 8 x lo5 zoosporangia per ml) or treated with a crude preparation of elicitor (equivalent to 10 mg dry wt of fungus per ml) 12 h after treatment with glucans or water. The discs were incubated on moist filter paper in a Petri dish at 18 “C. All assay procedures were carried out under aseptic conditions. The percentage of dead cells that had been infected with the fungus was determined 18 h after inoculation according to the method of Doke & Tomiyama [6]. Intensity of browning elicited by infection or treatment with crude elicitor was estimated 48 h after inoculation or treatment; discs in each treatment were divided into four groups depending on the intensity of browning and assigned values 0 to 3 (non-browned, 0; most strongly browned, 3). Rishitin and lubimin were extracted from the upper 1 mm of tissue discs 72 h after inoculation or treatment with elicitor, and determined by gas chromatography as described elsewhere [IZ]. Preparation of crude elicitor and microsomalfraction Crude elicitor was prepared from mycelia of P. infestanr as described elsewhere [12]. To prepare the microsomal fraction, cylinders, 18 mm in diameter, were cut from the central parenchymatous tissue of potato tubers which were held at room temperature for 24 h before cutting. Tissue discs, 3 mm thick, were cut from the cylinders, washed thoroughly with a large volume of distilled water, and then incubated on moist filter paper in a Petri dish at 18 “C for 18 h. One hundred g of the discs were dipped in 150 ml of 0.05 M Tris-HCl buffer, pH 7.4, containing O-5 M sucrose, 0.01 M MgCI, and 0.1 y0 of ascorbic acid in an ice bath and infiltrated under vacuum for 15 min. The discs were then placed in a cooled Waring blender bowl and homogenized for 1 min. The homogenate was filtered through four layers of cheese
130
N. Doke, N. A. Garas and J. Ku6
cloth, the filtrate centrifuged at 20 000 g for 40 min, and the resulting supernatant centrifuged at 100 000 g for 90 min. The pellet from the final centrifugation was suspended in 10 ml of 0.01 M Tris-HCl buffer, pH 7.4, containing 0.01 M MgCI, and homogenized with a Polytron homogenizer. This suspension was used as a microsomal fraction of potato tuber tissues. Mixtures
of crude elicitor, glucans and microsomal fraction
Crude elicitor was mixed with the microsomal
fraction,
glucans from mycelia of
P. infestanr or the microsomal fraction which was mixed previously with the glucans
for 30 min. The mixtures contained O-5 ml of crude elicitor (precipitate from 1 g of mycelium sonicated in acetate buffer, pH 4.5, and dissolved in 10 ml of distilled water), 0.5 ml of a suspension of the microsomal fraction, and O-5 ml of the aqueous solution of glucan (3 mg glucan per ml). The mixture was shaken for 30 min at room temperature, and O-1 ml of the mixture was applied to the surface of each potato tuber disc as described above. Intensity of browning and the amount of accumulated rishitin and lubimin were determined 72 h after treatment. Chemical methodr
Glucans were hydrolyzed by heating with 4 N HCl in a sealed tube at 100 “C for 8 h. Partial acid hydrolysis was performed with 1 N HCl at 90 “C for 1 h. The hydrolysates were dried under reduced pressure in the presence of methanol at 30 “C, and the residue free of HCl was dissolved in distilled water. The products were separated by gel filtration on a Sephadex G 15 column (1.8 x 95 cm) equilibrated with distilled water followed by thin-layer chromatography on silica gel G (Analtech, U.S.A.) using chloroform : methanol (6 : 4, v/v) [Z] ; n-butanol : acetone : water (4 : 5 : 1, v/v) [22] ; chloroform : methanol : 17% ammonium hydroxide (40 : 40 : 20, v/v), and phenol : water (75 : 25, v/v) or by thin-layer chromatography on microcrystalline cellulose (Analtech, U.S.A.) using ethylacetate : pyridine : acetic acid : water (5 : 5 : 1 : 3, v/v) [35]. s u g ars were detected after spraying with silver nitrate [22], diphenylamine-aniline [3], potassium permanganate [20] or P-anisidine HCl [II] ; amino acids and aminosugars were detected with ninhydrin [2]. Total carbohydrate content was determined by the phenol-sulfuric acid procedure using D-&COSe as a standard [ll]. Determination of reducing sugars and reducing end groups were made by the Somogyi-Nelson calorimetric method [25, 26-j. Content of protein, of hexosamine and of inorganic and total phosphate was determined by the methods of Lowry et al. [18], Garde11 [13] and Allen [I], respectively. Enzyme
digestion
Anionic and non-anionic glucans were treated with laminarinase (Calbiochem, U.S.A.), a-amylase (Sigma, U.S.A.) and cellulase (Calbiochem, U.S.A.). The anionic glucans were treated with alkaline phosphatase (calf intestine, Calbiochem, U.S.A.) and phosphodiesterase (viper venom, Calbiochem, U.S.A.). Glucans were incubated for 5 h at 30 “C in 4 ml of buffer containing 1 mg of laminarinase, cellulase or a-amylase. Buffers employed were 0.05 M Na-phosphate buffer, pH 5.9 (laminarinase); 0.01 M Tris-HCl buffer, pH 7.4 (a-amylase); 0.01 M Na-phosphate buffer,
A hypersensitivity
suppression
from
Phytophthora
131
pH 6.9 and 4.5 (cellulase). After incubation, the reaction mixture was heated at 100 “C for 10 min, mixed with 3 vols of ethanol and centrifuged at 10 000 g for 30 min. The supernatant was evaporated ( ~45 “C) under reduced pressure to dry ness. The products were separated by Sephadex G 15 gel filtration and thin-layer chromatography. The mixture for hydrolysis with alkaline phosphatase contained 1 mg of anionic glucan and 1 mg of alkaline phosphatase in 2 ml of -005 M Tris-HCl buffer, pH 8.8. The mixture was incubated at 30 “C for 3 h. The incubation of glucans (1 mg) with phosphodiesterase (100 units) was done in 2 ml of 0.05 M Tris-HCl buffer, pH 7.4, at 30 “C for 5 h. After digestion with the two phosphatases, the reaction mixtures were passed through a DEAE cellulose column to determine the ratio of non-anionic glucan to anionic glucan, or fractionated by Sephadex G 75 gel filtration to detect changes in molecular size, as described earlier. RESULTS
Fractionation
of hypersensitivity-inhibiting
factor (HIF)
Preliminary investigation indicated that HIF from mycelia of P. infeetum was extracted with acetate buffer, pH 4.5, followed by treatment with phenol : chloroform : methanol [12]. The factor thus extracted was found to be polysaccharide in nature. Fractionation of the polysaccharides extracted from mycelia on a DEAE cellulose column revealed a single large peak of non-anionic polysaccharide and a single smaller peak of anionic polysaccharide (Fig. 1). Fractionation of the preparation from zoospores revealed several incompletely resolved peaks of anionic polysaccharide in addition to the non-anionic polysaccharide peak. There was a significant difference in the anion exchange chromatography profile of soluble polysaccharide from mycelia and zoospores but not between the two races. The proportion of anionic polysaccharide to non-anionic isolated from mycelia was smaller than that from zoospores (Table 1). Fractionation of the glucans of Sephadex G 75 revealed that the polysaccharides from mycelia produced almost symmetrical elution profiles and the anionic polysaccharide fraction was eluted ahead of the non-anionic polysaccharide fraction (Fig. 2). The elution profile of polysaccharide isolated from zoospores was nearly the same as that from mycelia expect for a slightly broader profile of anionic polysaccharide. The anionic and non-anionic glucans from the two races of fungus could not be distinguished on the basis of their elution profiles. The freeze-dried glucans, however, appeared to be consistently different in texture. Properties and characterization
of polysaccharide
Glucans fractionated on DEAE cellulose followed by Sephadex G 75 showed no U.V. light absorption maxima in the region 2 10 to 340 nm and thereby appeared free of nucleic acid and protein. Complete acid hydrolysis of these polysaccharides followed by silica gel G or cellulose thin-layer chromatography revealed glucose as the only monosaccharide. Partial acid hydrolysis revealed a series of oligosaccharides which were fractionated on Sephadex G 15, and the disaccharide fraction was isolated. Thin-layer chromatography of the disaccharide fraction revealed a major spot with the same R, values
N. Doke, N. A. Garas and J. KuC
132
I
Race
1234
66 c
Race
04 M NaCl
0.5
I
M NaCl
-I3
1
4-1
l
4 0. I M NaCl
.
0.5~
1
NaCl 1
4-
. 2-
* .
T i
(1
Jj-.
IO
20
30
40
50
60
IO
20
30
40
50
60
0 0
= 0,153
1
J oio-
0.05
1
’
-
-i IO FIG.
(lower
20
30
40
50
60 Fraction
20
30
1. Fractionation (4 ml fractions) of glucans from mycelia half) of Phytophthora infestanr on DEAE cellulose columns. TABLE
Percentage
of anionic
Source
g&an
Mycelia Zoospores a The percentage of anionic glucan shown in Fig. 1. Values represent for zoospores.
50
60
(upper
half)
and zoospores
1
in total soluble glucanr from infestans
of glucan
40
number
mycelia
Percentage Race 1234 22+11 5&l* 12
and zoospores of Phytophthora
of anionic
glucan@ Race
4
31+7 64+3
was obtained from DEAE-cellulose fractionation as the mean + S.D. of five fractions for mycelia and three
as laminaribiose prepared from the partial hydrolysis of laminarin and a minor spot with the same R, values as gentiobiose. Amino acids and amino sugars were not detected in acid hydrolyzates of the glucans by thin-layer chromatography. The average degree of polymerization of the glucans, estimated by determining the number of reducing end groups, was found to be between 17 to 23 glucose units
A hypersensitivity
suppression
from
fhytophfhora
(a)
40
50
20 number
Fraction
FIG. 2. Fractionation mycelia of Phytophlhora
30
40
(2 ml fractions) of (0) anionic and (0) non-anionic infestanr on Sephadex G- 75 columns. (a) Race 1234;
50
glucans (b) Race
from 4.
with the degree of polymerization being slightly lower for race 4 than that for race 1234 (Table 2). Further evidence for the major linkages of these glucans was obtained by analysis of the products produced after digestion with laminarinase (p-1-+3 glucanase) which hydrolyzed the glucan, and the liberated glucose indicated better than 90% of total
Proper&s
of water-soluble
glucanr from
TABLE
2
mycelia
and zoospores of Phytophthora
infestans Hydrolysisd
Source Mycelia, Mycelia, Mycelia, Mycelia, Zoospores, Zoospores, Zoospores, Zoospores,
race 1234 race 1234 race 4 race 4 race 1234 race 1234 race 4 race 4
Glucan’ Non-anionic Anionic Non-anionic Anionic Non-anionic Anionic Non-anionic Anionic
Average degree polymerizationb 23+1 21+1 18kl 18kl 22*1 22+1 18k2 17+2
of
Glucose phosphate 14+4 11+2 12+3 8+4
to ratioC
with
L
C
A
+ + + + + + + +
-
-
a Glucans were extracted and fractionated by DEAE-cellulose and Sephadex G 75 column chromatography as described in the text. The fraction containing the major peak on Sephadex gel filtration was used to determine the properties of glucans. b Each value represents the meanf S.D. of five and three different fractions for mycelia and zoospores, respectively. c Each value represents the mean f S.D. of four and three different fractions for mycelia and zoospores, respectively. d L, laminarinase; C, cellulase; A, or-amylase; +, hydrolyzed; -, not hydrolyzed.
N. Doke, N. A. Garas and J. Ku6
134
hydrolysis. Cellulase and a-amylase did not hydrolyze the glucans. The extent of enzymatic hydrolysis was established by Sephadex G 15 gel filtration followed by thin-layer chromatography. Laminarin from Im&m-ia hyjerborea (Sigma, U ,S .A.) also was treated with the above enzymes and its sensitivity to hydrolysis was the same as that of the glucans. Significant amounts of phosphate were found in all anionic glucans; the phosphate content of anionic glucan from mycelia was 14 and 11 glucose residues per mole of phosphate for race 1234 and race 4, respectively (Table 2). In the case of each anionic glucan fraction from zoospores, exact phosphate values were difficult to determine in the small sample available. However, the phosphate content of total anionic glucans from zoospores was 12 and 8 glucose residues per mol of phosphate for race 1234 and race 4, respectively. The anionic glucans from mycelia and zoospores of race 4 contained a slightly higher ratio of phosphate to glucose residues than the anionic glucans from mycelia and zoospores of race 1234, respectively. Alkaline phosphatase converted the anionic glucan to the non-anionic glucan (Fig. 3). The
Fraction FIG. 3. Effect DEAE cellulose) 0, non-treated;
number
of treatment with alkaline phosphatase on the elution profile of anionic glucan prepared from mycelia of Phytophthoru l , treated with alkaline phosphatase.
(4 ml fractions,
infestam race 4.
non-anionic glucan formed after treatment with phosphatase was subjected to Sephadex G 75 gel filtration, and its elution profile corresponded to that of the nonanionic glucan. Treatment of both the anionic and non-anionic glucans with phosphodiesterase did not alter their elution patterns on Sephadex G 75 or DEAE cellulose. Hypersensitivity-inhibiting
activity
The water-soluble glucans (mixture of anionic and non-anionic glucans) were dissolved in 0.01 M Tris-HCl buffer, pH 7.4, and applied to the surface of potato tuber tissue discs before inoculation with an incompatible race of P. infestuns or treatment
A hypersensitivity
suppression
from
Phyfophfhora
135
with a crude preparation of elicitor from the fungus. More than 90% of the infected cells in the tissue treated with the buffer died 18 h after inoculation, while about 30 and 70% of infected cells died in the case of treatment with the glucans from race 1234 (compatible race) and race 4 (incompatible race), respectively (Table 3). A determination of the percentage of dead cells was not made in the case of treatment with crude elicitor. Control tissue inoculated with the incompatible race or treated with elicitor browned extensively in the surface tissues. Tissue treated with the glucan from the compatible race prior to inoculation with an incompatible race or treatment with elicitor showed a more marked reduction in the intensity of browning than that treated with glucans from the incompatible race (Table 3). Elicitation of rishitin accumulation by both living fungus and elicitor was also suppressed more markedly by treatment with glucan from the compatible as compared with the incompatible race of the fungus (Table 3). TABLE
3
Effect of pretreatment with water-soluble glucans on hyperseCnntioe cell death, tissue browning and accumulation of tmpmoids in potato tuber tksue subsequently infer&d with an incompatible race of Phytophthora infestans or treatcd with crude clicit~ Terpenoid accumulation (ug g fresh wt-l)d Glucans~
buffer (0.01 M, pH Race 1234 Race4 Distilled water Race 1234 Race 4
Elicitationb
% of dead cell@
Infection Infection Infection Elicitor Elicitor Elicitor
9324 30+6 69&20 -
Relative of tissue
intensity browningd
Rishitin
Lubimin
Tris
7.4)
2.3 l-5 l-8 2.5 1.6 2-o
85+15 28+ 12 72518 39 20 32
25+6 16+8 2355 21 18 21
a Water-soluble glucan (1 mg ml-l) was applied to the surface of Kennebec potato tuber slices (R,) 6 h after cutting. b Incompatible race 4 (5 to 8x 106 zoospores per ml) or crude elicitor from compatible race 1234 (l/10 dilution) was applied to potato discs 12 h after treatment with the glucans. c Observed 18 h after inoculation (about 100 infected cells were observed for each treatment) . d Observed 48 h after inoculation or treatment. * Determined 72 h after inoculation or treatment. Values for inoculation represent the mean of three to four different experiments with standard deviations; values for treatment with elicitor are averages of two experiments.
Throughout these experiments, there was a positive correlation between inhibition of cell death, inhibition of tissue browning and inhibition of rishitin accumulation after treatment with the glucans. Cytological observation of tissue treated with glucans indicated that only cells in the first and second layer from the treated surface were protected from cell death and tissue browning 48 h after treatment. At later stages, tissue browning was observed in deeper tissues
136
N. Doke,
N. A. Garas
and
J. KuC
To compare the hypersensitivity-inhibiting activity of non-anionic glucan with that of anionic glucan, a low concentration of glucan was applied to the tissue and the percentage of dead cells elicited by infection with an incompatible race was determined (Table 4). Anionic glucan isolated from both mycelia and zoospores of compatible race (race 1234) inhibited cell death more actively than the non-anionic glucan. Treatment with glucan from the incompatible race (race 4) at 20 pg ml-l caused little or no inhibition of the hypersensitive cell death.
Efect of @treatment with anionic phthora infestans on hypcrsenritive
TABLE 4 or non-anionic glucans from mycelia death of potato tuber cells subsequently race 4 of the fWgus
Glucan.@
Nature
a Glucan applied at a concentration of 20 pg ml-l. b Each value represents the mean of two experiments. observed for each treatment. Cell death was observed
In vitro
interaction
of glucans,
Percentage
Non-anionic Anionic Non-anionic Anionic Non-anionic Anionic Non-anionic Anionic
-Distilled water Mycelia, race 1234 Mycelia, race 1234 Mycelia, race 4 Mycelia, race 4 Zoospores, race 1234 Zoospores, race 1234 Zoospores, race 4 Zoospores, race 4
elicitor,
and
microsomal
and zoospores of Phytoinfected with incompatible
of dead
cellsb
100 86 44 99 97 68 39 89 92
About 100 infected 18 h after inoculation.
cells
were
fraction
In vitro interaction among the glucans, crude elicitor and microsomal fraction from potato tuber tissue was investigated by determining changes in the activity of the elicitor. The necessary controls indicated that the glucan from both incompatible and compatible races and the microsomal fraction did not brown the surface of potato discs or elicit the accumulation of terpenoids (Table 5, Expt I, A, B, C). The crude elicitor elicited both strong tissue browning and accumulation of terpenoids (Expt I, D; Expt II, A). Mixing of glucans (mixture of anionic and nonanionic glucans) from the compatible or incompatible race with crude elicitor did not affect the activity of elicitor significantly (Table 5, Expt I, E, F; Expt II, B, C). Mixing of the elicitor with the potato microsomal fraction, however, markedly reduced its ability to elicit both browning and the accumulation of terpenoids (Expt I, G; Expt II, D). When the microsomal fraction was mixed with the glucan from the compatible race before mixing with the crude elicitor, the eliciting activity was not inhibited appreciably (Expt I, H; Expt II, E). Eliciting activity remained low when glucan from the incompatible race was mixed with the microsomal fraction followed by elicitor (Expt 1, I; Expt II, F). When elicitor from race 1234 was mixed with the microsomal fraction before mixing with glucans, neither the glucan from the compatible race nor that from the incompatible race affected the inhibitory effect of the microsomal fraction. (Table 5, Expt II, G. H).
A hypersensitivity
suppression
from
Phytophthora
137
TABLE Effect
of in vitro interaction of crucie elicitor infestans and a microsomal fraction from
Materials
Experiment I
II
Glucansb A. B. C. D. E. F. G. H. I.
Race 1234 Race4 Distilled water Distilled water Race 1234 Race4 Distilled water Race 1234 Race4
A. B. C. D. E. F. G. H.
Distilled water Race 1234 Race 4 Distilled water Race 1234 Race4 Race 1234 Race 4
+
5
and water-soluble potato tuber tissu
treated
Microsomal fractionc
&cans from Phytophthora on activity of the elicitor
Relative inn,t;n~ +
Elicitord
browning
Terpenoid accumulatiorP (pg g fresh wt-1) Rishitin
Lubimin
Buffer
Distilled
water
0.0
0
Buffer Kennebec Buffer Buffer Buffer Kennebec Kennebec Kennebec
(R,) (R$’ (R, )”
Distilled water Distilled water Race 4 Race 4 Race 4 Race 4’ Race 4 Race 4
0.0 0.0 2.6 1.9 2.4 l-4 1.9 1.5
0 0 144 116 1‘iQ 24 119 21
0 0 0 49 53 56 10 42 11
Buffer Buffer Buffer Kennebcc
(R,)
Race Race Race Race
Kennebec Kennebec Kennebec Kemrebec
(R,)e (R,)’ (R,) (R,)
Race 1234 Race 1234 Race 12346 Race 1234’
2.9 2.9 2.9 1.8 2.8
65 73 68 28 67 35 9 24
23 25 29 21 21 19 7 13
(R,)
1234 1234 1234 1234
2.1 1.8 1.9
D Each value is an average of two determinations. b A mixture of anionic and non-anionic glucans was dissolved in distilled water (3 mg ml-r) 0.5 ml. c Microsomal fraction from 100 g tissue was suspended in 10 ml of 0.01 M Tri.-HCl buffer, pH 7.4, containing O-01 M MgCl,; 0.5 ml. d Crude elicitor, equivalent to 100 mg dry wt of fungus, was suspended in 10 ml of distilled water; 0.5 ml. The crude elicitor is a fraction insoluble in pH 4.5 acetate buffer, prepared from a sonicate of mycelia in 10 ml of distilled water. ‘ Treatments underlined indicate pre-incubation of the components for 30 min. The total mixtures for all treatments were incubated for 30 min at room temperature before application to potato discs.
DISCUSSION Glucans which inhibited the hypersensitive reaction of potato to P. infestans were isolated from both mycelia and zoospores of the fungus. The glucans were watersoluble, had an average degree of polymerization of 17 to 23 glucose units (Table Z), and contained p-1-+3 and p-1-+6 glycosidic linkages. One of the glucans was a nonanionic glucan which is similar to the glucan isolated from P. infestunr [I& 24, 261. Another glucan was anionic, contained phosphorus and was similar to the glucan isolated from P. palmivora [34]. It appears that one or two glucose residues of the anionic glucan are esterified with a phosphor-y1 residue via a monoester linkage since an alkaline phosphatase converted the anionic glucan to the non-anionic glucan. This report appears to be the first report of the isolation of an anionic phosphoglucan from both mycelia and zoospores of P. infestans. Although the anionic
138
N. Doke, N. A. Garas and J. KuC
glucan has nearly the same degree of polymerization as the non-anionic glucan, the former appeared to be eluted earlier than the latter on Sephadex G 75 gel filtration (Fig. 2). It is possible that the faster migration of the anionic glucan was due to ionic interaction with residual anions in the Sephadex gel which block the movement of the polymer into the gel particles as described with the anionic glucan from P. ~almivoru [34]. The elution pattern on DEAE cellulose suggests zoospores may contain several similar but not identical anionic glucans (Fig. 1). There were only slight differences in glucose to phosphorus ratio and the degree of polymerization of glucose (Table 2) between race 1234 and race 4, but the appearance of freeze-dried samples of the glucans from the two races was significantly different, suggesting some differences in the structure of the glucans. Further characterization of the structure of the glucans is necessary. The data indicate that P. infestans contains water-soluble glucans that suppress the hypersensitive response of potato to P. infetans; the glucans from a compatible race being more active than those from an incompatible race (Tables 3, 4, 5). Earlier publications reported that infection with a compatible race of P. infestans inhibited rapid cell death [5, 8, 27, 281 and the accumulation of terpenoids [1.2, 17, 31, 321 from subsequent infection with an incompatible race. High molecular weight water-soluble compounds which inhibited the hypersensitive reaction were extracted from zoospores and germination fluids [S, 8, 9, lo] of compatible races of P. infestans. The compounds appear to be the glucans whose isolation and partial characterization are reported in this paper. Browning and accumulation of terpenoids in tubers have not been reported without hypersensitive cell death. The occurrence of cell death may also be closely involved in the resistant reaction of potato tissue. Treatment of potato tuber slices with elicitor enhanced electrolyte leakage, whereas pretreatment with glucan from a compatible race, but not incompatible race, of the fungus markedly reduced leakage [ 121. Dextran-bound p-chloromercuribenzoic acid, which does not readily penetrate cell membranes, prevented the hypersensitive cell death [7]. Accordingly, the glucans from compatible races may protect cell membranes of potato tissue from damage caused by elicitors. In the present experiments, a microsomal fraction from potato tissue was used to investigate the interaction among elicitor, glucans and cell membranes. Although the elicitor and microsomal fraction are very crude preparations, the in vitro reaction among them suggests that HIF suppresses the reaction between elicitor and cell membranes. Conceivably, elicitor may bind to some substance (receptor) on cell membranes and the glucan may block the binding site of the substances for elicitor. Though the data presented are consistent with the postulated role of the glucans in determining the specificity of interaction between potato and P. infestatu [9, 17, 31, 321, further experiments with many cultivars of potato and races of P. inzstans are needed before a role for the elicitor and glucans can be established. The research reported in this paper was done at the Department of Plant Pathology, University of Kentucky, and has been supported in part by a grant from the Herman Frasch Foundation and grant no. 316-15-51 of the Cooperative State Research Service of the United States Department of Agriculture. We acknowledge the technical assistance of Mrs Barbara Stoner.
A hypersensitivity
suppression
from
fhytophthora
139
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25. SOMOGYI, M. (1945). Determination of blood sugar. 3ournal of Biological Chemistry 160, 69-73. 26. SOMOGYI, M. (1952). Notes on sugar determination. Journal of Biological Chemistrb 195, 19-23. 27. TOMIYAMA, K. (1969). Double infection by an,incompatible race of Phytophthora infestuns of a potato plant cell which has previously been infected by a compatible race. Annals of the Phytopathological Society ofJapan 32, 181-185. 28. TOMIYAMA, K. (1967). Further observation on the time requirement for hypersensitive cell death of potatoes infected by Phytophthora infestam and its relation to metabolic activity. Phytopathologische ,Qitschrijt 58, 367-378. 29. TREVELYAN, W. E., PROCTOR, D. P. & HARRISON, J. S. (1950). Detection of sugars on paper chromatograms. Nature 166, 444-445. 30. VARNS, J. L., Ku6, J. & WILLIAMS, E. B. (1971). Terpenoid accumulation as a biochemical response of the potato tuber to Phytophthora infestam. PhytopatholoQ 61, 174-l 77. 31. VARNS, J. L. & Ku6, J. (1971). Suppression of rishitin and phytuberin accumulation and hypersensitive response in potato by compatible races of Phytophthora infestans. Phytopatholou 61, 178-181. 32. VARNS, J. L. & Ku6, J. (1972). Suppression of the resistance response as an active mechanism for susceptibility in the potato-Phytophthora infestans interaction. Phytotoxins in Plant Disease, Ed. by R. K. S. Wood, A. Ballio and A. Graniti, pp. 46-68. Academic Press, London, New York. 33. VARNS, J. L., CURRIER, W. W. & KuC, J, (1971). Specificity of rishitin and phytuberin accumulation by potato. Phytopathologv 61, 968-971. 34. WANG, M. C. & BARTNICKI-GARCIA, S. (1973). Novel phosphoglucans from the cytoplasm of Phytophthora palmiuora and their selective occurrence in certain life cycle stages. 3ournal of Biological Chemidry 248, 4112-4118. 35. WOLFROM, M. L., PATIN, D. L. & LEDERKREMER, R. M. DE (1965). Thin-layer chromatography on microcrystalline cellulose. Journal of Chromatography 17, 488-494. 36. WOOD, F. A., SINGH, R. P. & HODGESON, W. A. (197 1). Characterization of a virus-inhibiting polysaccharide from Phytophthora infestanr. Phytopathologv 61, 1006-1009.