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Protection of Bartlett pear against fire blight with deoxyribonucleic acid from virulent and avirulent Erwinia amylovora
Physiological
Plant Pathology
(1975)
7, 153-170
Protection of Bartlett pear against fire blight with deoxyribonucleic acid from virulent and avirulent Erwinia amylovoraf J. L. MCINTYRE Connecticut
Agtiltural
Experiment
Station,
New
Haven,
Connecticut,
U.S.A.
J. Ku6 Department
of Plant Pathology,
University
of Kentucky,
Lexington,
Kentucky,
U.S.A.
and E. B. WILLIAMS Department (Accepted
of Botany and Plant Pathology, for publication
April
Purdue
University,
Lafayette,
Indiana,
U.S.A.
1975)
Sonic&es of virulent and avirulent Erwinia amylovora protect “Bartlett” pear shoots and etiolated seedlings against fire blight. Activity of sonicates is lost after precipitating nucleic acids with protamine sulfate, whereas nucleic acids purified from sonicates protect. Deoxyribonuclease, but not ribonuclease, destroys activity of nucleic acids isolated from sonicates. Activity is associated with the deoxyribonucleic acid (DNA) fraction from linear log sucrose gradients. DNA prepared by the Marmur technique protects more effectively than DNA prepared from sonicates. The former was calculated to have a GC content of G. 55% by centrifugation to equilibrium in cesium chloride and from DNA melting curves. Specificity of the DNA preparations is suggested since DNA from Pseudomonas tabaci, a bacterium which protects against fire blight, did not protect, whereas DNA from virulent E. amylovora protects. Nucleic acid components, polyanions and polycations did not protect. Host response appears necessary for protection since a lag period is required between injection of DNA and virulent E. amylooora, and DNA added to a nutrient medium does not inhibit the growth of E. amylovora or affects its virulence.
INTRODUCTION Protection of plants against bacterial pathogens by treatment of the host with disrupted cells of avirulent mutants [32] and cell-free extracts of pathogens and non-pathogens [S] indicates that protection is not dependent on the presence of bacteria. Protection of “Bartlett” pear and “Jonathan” apple against fire blight with avirulent Erwinia amylovora, Erwinia herbicola and Pseudomonas tabaci has been reported [9, II, 271, and protection of “Bartlett” pear against fire blight with cell-free sonicates of virulent and avirulent E. amylovora has been demonstrated [26, 271. The purpose of this paper was to determine the nature of the protectant in sonicates. t Research supported in part by a grant from the Herman Frasch Foundation, USDA Cooperative Agreement 12-14-lOO-12,625 (34) and Cooperative State Research Service, USDA, Agreement 316-15-51. Journal paper 75-11-30 of the Kentucky Agricultural Experiment Station, Lexington, Kentucky 40506 and 5878 of the Purdue Agricultural Experiment Station, Lafayette, Indiana 47907, U.S.A.
154
J. L. Mclntrye,
MATERIALS
Bacterial
AND
J. KuC and
E. B. Williams
METHODS
isolates and sonicate preparation
Avirulent Erwinia amylovora (E8) was obtained from R. N. Goodman, University of Missouri. The virulent isolate (challenge) of Erwinia amylovora (Burr) Winslow et al. was isolated and all cultures were maintained and sonicates prepared as described [27]. The basis for classifying the avirulent bacterium used in th.is study as E. amylovora has been reported [25]. Plant
material
Actively growing clonal Pyrus communis L. “Bartlett” at least 30 cm tall were used in all experiments with green tissue. Plants were grown individually in clay pots containing soil and maintained in a greenhouse at 20 to 26 “C and relative humidity greater than 90%. Etiolated Bartlett open-pollinated seedlings were grown in a mixture of vermiculite and soil (6 : 4) for IO days at 19 “C in the dark. Seedlings, 4.5 to 5.5 cm tall, were removed from the soil, washed with tap water and distilled water, and placed on moist filter paper at the bottom of a Pyrex baking dish which was then covered with an inverted baking dish. Dishes were either placed under fluorescent lights or wrapped with aluminum foil and kept in the dark. Etiolated pear seedlings were utilized to assay for the protectant. Seedlings were injected with 10 ~1 protectant [27] in 0.05 M-phosphate buffer, pH 6.8 [1211 and challenged 18 h later with IO2 cells of E. amylovora in 0.05 M-phosphate buffer, pH 6.8. A time study indicated that optimum protection occurred when challenge followed protectant by 18 h [25] and IO2 cells challenge caused rapid symptom development ( ~48 hj in controls which facilitated the detection of protectant. Control plants were injected with 10 ~1 0.05 M-phosphate buffer, pH 6.8. To obtain increased uptake and better distribution of protectant, an assay was developed using etiolated seedlings from which the radicle was excised. Seedlings were maintained in the laboratory under fluorescent lights (Champion F9 OT 17/W) at c. 22 “C for the duration of the assay. Five ml phosphate buffer or buffer containing protectant was placed into 120 mm tall Coplin staining dishes. Seedlings were placed into the solution and the staining dishes were covered with Saran Wrap. After 24 h seedlings were washed with sterile water, 0.5 mm of the hypocotyl base was cut and discarded, and the treated seedlings were placed in Coplin dishes containing lo4 cells/ml challenge in 5 ml phosphate buffer. After 2 h, seedlings were washed as before and either returned to the solution containing protectant or buffer or placed onto moist filter paper at the bottom of a Pyrex baking dish. The seedlings replaced into protectant or buffer were removed after 12 h$ rinsed and the hypocotyl base was trimmed (0.5 mm) to remove tissue where slight toxic effects from buffer often appeared. These seedlings were then placed into baking dishes as described. When protection occurred by the above assays, the presence of protectant was verified in actively growing pear shoots. Pear shoots were injected with protectant by the “drop method” [27], and O-5 or 24 h later injected with IO3 cells E. amylovora. Precipitation
of nucleic acids
Nucleic acids were precipitated from 5 ml of sonicate (c. 1500 pg protein/ml sonicate) with 1 ml of 1 O/Oprotamine sulfate. The protamine sulfate-treated preparations were
Protection
of Bartlett pear against fire blight
155
held at 37 “C for 30 min and precipitate was removed by centrifugation at 8000 rev/ min and 4 “C. The supernatant was assayed by the injection method for its ability to protect and its protein content was determined. Purification
of nucleic acidr from sonicates
Nucleic acids were purified from sonicates of 2 to 3 g (wet weight) bacterial cells with Kirby phenol [18]. Due to shearing by sonication, the nucleic acids could not be spooled on a glass rod after precipitation with two volumes of cold 95% ethanol. Therefore, nucleic acids were collected by centrifugation at 8000 rev/min and 4 “C and the final pellet, obtained after two additional treatments with phenol and three ethanol precipitations, was dried with nitrogen gas and suspended in 1 ml phosphate buffer. The preparation was scanned from 220 to 320 nm to determine 2601280 and 2601230 ratios, and to determine the amount of nucleic acid from the 260 nm peak height. An extinction coefficient of 25 was used to estimate nucleic acid and ribonucleic acid (RNA) and a value of 20 was used for deoxyribonucleic acid (DNA). Protein and carbohydrate were determined by the Lowry and anthrone [30] techniques, respectively. The nucleic acids were tested by the injection method for their ability to protect. Enzyme
treatments of nucleic ads
The nucleic acid pellet obtained after the first ethanol precipitation was dried, suspended in 5 ml phosphate buffer containing 1 nnr-magnesium chloride [29] and treated for 30 min at 22 “C with 500 pg/ml deoxyribonuclease 1 (DNase) (beef pancrease, ICN Nutritional Biochemicals, Cleveland, Ohio). Similarly the pellet from the first ethanol precipitation was suspended in 5 ml SSC buffer (O-15 M-sodium chloride, 0.015 M-sodium citrate, pH 7*2), and treated for 30 min at 37 “C with 50 pg/ml ribonuclease A (RNase) (beef pancrease, ICN Nutritional Biochemicals, Cleveland, Ohio) and 5 units/ml RNase Tl (Aspergillus oryzae, ICN Nutritional Biochemicals, Cleveland, Ohio) which were previously treated to destroy DNase activity [34]. Enzyme was removed by treating with an equal and then one-quarter volume of Kirby phenol. The final ethanol precipitate (RNA or DNA) was suspended in 1 ml phosphate buffer. The suspension was scanned under U.V. and its protein and carbohydrate content and ability to protect (injection method) were determined. Sucrose gradient
centrafugation
Linear log sucrose gradients [4,16] were prepared with RNase-free sucrose (Schwartz/ Mann, Orangeburg, New York) in cellulose nitrate centrifuge tubes for use with a Beckman SW 41 rotor and gradients were held at 4 “C for 24 h prior to use. Nucleic acid preparations were adjusted to c. 100 pg/ml and, after removing O-5 ml from the top of the gradient, O-5 ml nucleic acid was layered onto the gradient. Purified tobacco or brome mosaic virus (100 pg/ml) were degraded in bentonite [3] and layered onto gradients as described. Standards and samples were centrifuged in a Beckman L2-65 ultracentrifuge for 7.5 h at 38 000 rev/min and 14 “C. One-half ml fractions were collected and absorbance at 258 nm determined with an Isco Density Gradient Fractionator with ultraviolet analyzer. Fractions under peaks were pooled and the DNA and/or RNA was precipitated by gentle mixing with two volumes of
J. L. McIntyre,
156
J. KuC and
E. B. Williams
95% ethanol and placing in the freezer (-20 “Cj for 6 h. Precipitate was collected by centrifugation at 8000 rev/min and 4 “C for 3 min. The supernatant was decanted, and the precipitate dried with nitrogen gas. Nucleic acids were suspended in 1 ml phosphate buffer and assayed by the injection method for protection. The (S) values of samples were determined by comparing the distance moved from the top of the gradient to distance moved by viral RNA components used as standards. Thirty min prior to layering gradients, purified nucleic acid samples were treated with RNase or DNase. These samples were centrifuged as before and one of the two peaks evident after centrifugation of the nucleic acids remained at the top of the gradient. The (S) value of the remaining nucleic acid was determined, and comparison of these values to those obtained from the nucleic acid preparations permitted identification of the DNA and RNA components of the nucleic acid. DNA and RNA obtained after centrifugation of enzyme-treated nucleic acids were precipitated, suspended and assayed for by the injection method for protection. DNA isolated from gradients was either centrifuged again in linear log sucrose gradients or treated with DNase or RNase and centrifuged. A Beckman 50.1 rotor was used and the samples were centrifuged at 48 000 rev/min for 2.5 h at 14 “C. D.NA preparations
from lysed bacterial cells
To obtain large quantities of DNA, bacteria were grown in 10 1 fermentors at 25 + 1 “C and 5 l/min air flow. Inoculum was grown in 100 ml shake cultures containing medium 924 salt [19] plus additions [IO]. After 24 h, the inoculum was added to fermentors which contained the same medium as the shake cultures. The bacteria were maintained for 24 h in the fermentors, harvested with a Sharples centrifuge and washed with SSC buffer (2 to 3 g cells/25 ml buffer). The bacteria were centrifuged at 10 000 rev/min and 4 “C for 15 min and pelleted cells frozen ( - 20 “C) . Within 24 h of freezing, the cells were thawed at room temperature and then refrozen. The cells then readily lysed upon treatment with sodium dodecyl sulfate (SDS), and DNA was purified by the Marmur technique [24]. The method allowed for the purification of DNA with minimal shearing. Purified DNA was also treated with DNase and enzyme was removed by the Marmur technique. DNA (200 pg/ml) and DNasetreated DNA were scanned under U.V. and tested for protection in etiolated seedlings from which the radicle was removed. DNA was stored at 4 “C under toluene. Eye& of DJVA concentratiorz on protection DIVA (purified from sonicates of avirulent E. amylovora) was adjusted with buffer concentrations of 10, 100, 1000 and 5000 pg/ml. Ten ~1 of each were injected etiolated seedlings and challenged 18 h later with I O2 cells of E. amylovora. Effect of DNA
on growth
and virulence of E. amylovora
to in
in vitro
Ten mg DNA (Marmur technique) were placed in 250-ml flasks with 100 ml mineral medium containing 1 oh sucrose [Z]. Th e fl as k s were inoculated with 2 x lo3 cells of consisted of 10 ml phosphate buffer in place of DNA, virulent E. amylovora. Controls and growth of the bacterium was determined spectrophotometrically at 525 nm. Cells were harvested by centrifugation, suspended in phosphate buffer and injected into etiolated seedlings to determine their virulence.
Protection
of Bartlett
Bacteriophage
pear
against
fire
blight
157
assay
Avirulent E. amylovora was grown in nutrient broth (D&o) and the cultures shaken. When bacteria were in the exponential growth phase (c. 5 x lo* cells/ml), 0.2-ml aliquots were removed and placed into each of 10 sterile culture tubes. Molten H-top agar (45 “C, 2.5 ml) was added to each tube and the contents immediately poured over the surface of an H plate [28]. The agar was allowed to harden and the plates were incubated for c. 10 h at 30 “C. Chloroform lysates of the cultures were prepared and both virulent and avirulent E. amylovora were treated with lysate and plates poured as described by Miller [28]. Plates were incubated overnight at 30 “C and observed for plaque formation. Cesium chloride centriiugation Cesium chloride gradients were prepared for ultracentrifugation with a Beckman SW 50.1 rotor. Sixty per cent cesium chloride (w/w) in O-015 M-sodium citrate, pH 7.2, was prepared and 3.67 ml was pipetted into cellulose nitrate tubes. DNA (1.33 ml, 1 mg/ml) was layered onto the top of the gradients. The samples were centrifuged for 48 h at 44 000 rev/min and 20 “C in the Beckman L2-65 ultracentrifuge and allowed to coast to a stop. A hole was pierced in the bottom of each tube, three drop fractions were collected and the refractive index at 25 “C was determined for each fraction. One-half ml phosphate buffer was added to each fraction and absorbance determined at 260 and 280 nm. GC content of the DNA was estimated by the formula %GC = (BD - 1.660)/0*096 [31], where BD is buoyant density of DNA and GC is mole fraction of guanine plus cytosine in DNA. Fractions containing DNA were pooled and dialyzed for 24 h at 4 “C in 2 1 phosphate buffer changed after 6 and 18 h. DNA was tested for its ability to protect etiolated seedlings from which the radicle was removed. DNA
melting curves
DNA was adjusted to c. 40 pg/ml and 1 ml samples were melted by increasing the temperature from 30 to 100 “C over a lo-min period. GC content was estimated from the formula %GC = ( Tm- 69.3) x 2.44 [29], where Tm is the temperature at which the hypochromic effect is 50% of its maximum. Additional
sources of bacterial
DNA
and com@ounds tested for protective activity
DNA isolated from etiolated “Bartlett” pear seedlings [25] and DNA from E. amylovora, Erwinia herbicola, Pseudomonas tabaci and Xanthomonas campestris (isolated by the Marmur technique) were assayed for activity in etiolated seedlings from which the radicle was removed. All other compounds (bovine serum albumin, apoferritin, salmon sperm DNA, Torula yeast RNA, adenine, adenosine, deoxyadenosine, 5’adenylic acid, cytosine, cytidine, deoxycytidine, 5’deoxycytidylic acid, poly-Dglutamic acid, poly-L-glutamic acid, poly-L-alanine, poly-n-lysine, poly-L-lysine, polyphosphoric acid, polyinosinic acid, ribose, deoxyribose, obtained from ICN Nutritional Biochemicals, Cleveland, Ohio) were placed in phosphate buffer (10 mg/ml) and pH adjusted to 6.8. Ten ~1 was injected into etiolated seedlings, followed 18 h later with 1O2 cells of challenge.
158
J. L. McIntyre,
Injection
J. KuC and
E. B. Williams
of clonal @ear with DNA
DNA (Marmur technique) from virulent and avirulent E. amylouora was injected by the drop method [27] into actively growing shoots of pear. By allowing uptake of many drops of DNA at the site of the puncture, it was possible to get c. 200 LangDNA into shoots. Plants were challenged at the same site either O-5 or 24 h later with an inoculum containing c. lo3 cells.
RESULTS
Injection and vascular uptake are useful for introducing protectant into etiolated pear seedlings. Injection was especially useful when small volumes of protectant were available. As protectant was purified and larger quantities were available, increased uptake and better distribution of protectant, and more permanent protection, were obtained by vascular uptake. Nucleic acids purified from sonicate Precipitation of nucleic acids with protamine sulfate caused a loss of activity in sonicate without appreciable change in protein content (Fig. 1). Protection was associated with nucleic acids purified by Kirby phenol (Fig. 2). U.V. absorbance spectra of the nucleic acids indicated 2601280 ratios of 2-l to 2.2 and 2601230 ratios of 2.2 to 2.4. Protein content of the nucleic acid was c, 5% and carbohydrate content c. 33%.
70 F ; g c
60 50 40
I
I 24
1 Time
1 72
1 (h after
I 120
1
challenge
1 168
1
1
FIG. 1. Protection of pear seedlings against fire blight. Seedlings injected with 10 ~1 phosphate buffer (o----o), sonicate of avirulent E. anlylovora (D-----D) or sonicate from which nucleic acids were precipitated with protamine sulfate (O-O), and challenged 18 h later with IO2 cells of E. amylonora. Sonicate contained 1500 pg protein/ml, and sonicate from which nucleic acids were precipitated contained 1410 pg protein/ml. Fifty seedlings observed per treatment.
Protection
of Bartlett pear against fire blight
24
159
I 72
I
Time (h after
I 120
I
I 168
I
challenge)
FIG. 2. Protection of pear seedlings against fne blight. Seedlings injected with 10 ld phosphate buffer (O-O), sonicate of avirulent E. amylovvra (e-0) or nucleic acids purified from sonicate (O-O) and challenged 18 h later with lo8 cells of E. amylovoru. Sonicate contains 1300 pg protein/ml, and nucleic acid preparation 1000 ng nucleic acid/ml. One hundred seedlings observed per treatment.
100 90
80 70
Time (days
after
challenge)
FIG. 3. Protection of pear seedlings against fire blight. Seedlings injected with 10 ~1 phosphate buffer (O-O), nucleic acid preparation from sonicate of avirulent E. amylooora (O-O) and DNase (O-O) or RNase (A-A) treated nucleic acid preparations. Initial nucleic acid preparation contains 5000 ng nucleic acid/ml, DNase treated preparation 4700 pg RNA/ml and RNase treated preparation 500 pg DNA/ml. Fifty seedlings observed per treatment.
J. h. McIntyre,
160
J. Ku6
and
E. B. Williams
Treatment of nucleic acids with DNase or RNase indicated that activity was associated with DNA (Fig. 3), and U.V. absorbance spectra and protein and carbohydrate content were similar to those obtained with nucleic acid preparations. Linear log sucrose gradient centrifugation of nucleic acids prepared from sonicates indicated the presence of two compounds absorbing at 258 nm (Fig. 4). They were identified as RNA and DNA (Fig. 4) after treatment with DNase or RKase. The (S) values were calculated to 4.8 S for RNA and 9.0 S for DNA. Activity was associated 0.61 0.5 0.4 0.3 o-2 0-I
06
0.8 0.7 0.6
Depth i cm 1 FIG. 4. Scanning patterns after linear log sucrose gradient centrifugation of (a) 100 ng nucleic acid/ml from avirulent E. nmylovora; (b) 100 pg nucleic acid/ml treated with DNase; (c) 100 pg nucleic acid/ml treated with RNase. Samples centrifuged for 7.5 h at 38 000 rev/min acids precipitated in the Beckman SW 41 rotor at 4 “C. Fractions pooled (Fl to F9) and nucleic to determine if protection occurred with a nucleic acid fraction.
Protection
of Bartlett pear against fire blight
161
with DNA isolated from the gradients (Fig. 5) and this activity was destroyed by treatment with DNase but not RNase (Fig. 5). Precipitation of DNA from sucrose gradients and centrifugation by linear log sucrose gradients with a SW 50.1 rotor gave only one peak of DNA absorbing at 258 nm (Fig. 6). Treatment of the reisolated nucleic acid with RNase had no affect on the position of the DNA (Fig. 6), whereas treatment with DNase caused a loss of the DNA (Fig. 6). DNA prepared by the Marmur technique protected much better than that prepared by sonication (Figs ‘2 and 7). Protection also occurred when this material was injected into clonal pear shoots and challenged 24 h later with 10s cells of E. amylovora
100 (a) 80 60 40 20
ii 5
l;(cl 20
&T
0
I 24
I 48 Time
I 72 (h after
I 96 challenge)
I 120
I 144
FIG. 5. Protection of pear seedlings with nucleic acids precipitated from fractions collected after linear log sucrose gradients. (a) RNA (Fl)’ (O-O) and RNA plus DNA (F2) O-O) fractions from centrifuged nucleic acid preparations from avirulent E. amylouoru; (b) RNA plus degraded DNA (F4) (O-D) and RNA (F5) (O-O) f rom centrifugation of nucleic acids treated with DNase; (c) DNA plus degraded RNA (F7) (O-O) and DNA (F8) (O-O) from centritirgation of nucleic acids treated with RNase. Seedlings injected with 10 ~1 of phosphate buffer (O-O) or nucleic acid sample and challenged 18 h later with 1Oe cells of E. amylovora. Fifty seedlings observed per treatment. * Fl to F9 represent fractions as shown in Fig. 5 and contain 34, 87, 0, 44, 20, 0, 3, 6 and 0 rig/ml nucleic acid. F3, 6 and 9 gave no protection and are not shown.
162
J. L. McIntyre,
J. Ku6
and
E. B. Williams
0.31
Depth
(cm]
FIG. 6. Scanning patterns of DNA isolated from linear log sucrose gradients (38 000 rev/min and 7.5 h, SW 41 rotor) and recentrifuged in linear log sucrose gradients. (a) DNA; (b) DNA treated with RNase; (c) DNA treated with DNase. Samples centrifuged for 2.5 h in Beckman 50.1 rotor at 48 000 rev/min and 14 “C.
TimeCdays
after
challenge)
FIG. 7. Protection ofpear seedlings with DNA (Marmur technique) (O-O) from avirulent E. amylovora. Seedlings from which radicle was excised were placed in 5 ml phosphate buffer (O-O) or 5 ml buffer containing 200 pg DNA/ml for 24 h. Seedlings were challenged by placing them in 5 ml buffer containing 10’ cells/ml of E. amylovora. Fifty seedlings observed per treatment.
Protection
of Bartlett
pear
against
fire
blight
163
(Fig. 8). DNA concentrations as low as 10 pg/ml protected against fire blight and protection increased with increased concentration of DNA (Fig. 9). DNA did not inhibit the growth of E. amylovora in vitro (Table I), and bacteria grown in the presence of DNA were virulent. The phage assay did not indicate infection of avirulent E. amylovora with a bacteriophage.
80 70 s c
60
i
50
2 40
456789
IO
II
12
Time (days after challenge)
FIG. 8. Protection of actively growing pear shoots with DNA (Marmur technique) from avirulent E. amylovora. Shoots injected with phosphate buffer and challenged 0.5 or ‘24 h later (O-O) with 10s cells of E. amylovora or injected with c. 200 pg DNA in phosphate buffer and challenged 0.5 (O-O) or 24 (O-O) h later. Twelve plants observed per treatment.
Density determinations by centrifugation to equilibrium in cesium chloride indicated a GC content of c. 55% (Fig. lo), and this value was verified by melting curve determinations (GC = 54-l) (Fig. 11). DNA isolated from cesium chloride gradients protected against fire blight (c. as in Fig. 7). TABLE
Growth
of virulent
E. amylovora
Number Time
after
1
in the presence of D.NA from of bacterial
avirulent
E. amylovoraa
cells/ml
inoculation W
Control
0 18 24 36 48 60
9.0 1.5 3.5 6.0 1.0x
10s x 104 x 10s x 10s x 10s 106
DNA
(avirulent
2.5 4.0 9.0 1.1 3.0
E. amylovom) 10s x 10” x 10s x 105 x 10s x 10s
a Test: 100 ml mineral medium containing 1% sucrose, 10 ml 0.05 M-phosphate buffer, pH 6.8, containing 10 mg DNA and seeded with 103 cells of virulent E. amylouora. Control: As test with 10 ml of buffer without DNA.
164
J, L. McIntyre,
J. KuC
and E. B. Williams
8
lil I2345678 Time
I, (days
oite:
I chal’enqe
I, 1
FIG. 9. Protection of pear seedlings with DNA from sonicates of avirulent E. amylovora. Seedlings injected with 10 ~1 phosphate buffer (O-O) or DNA [5000 (W----W), 1000 (O-O), 100 (--D--O), 10 (A-A) pg/ml] and challenged 18 h later with lo2 cells of E. amylovora. Fifty seedlings observed per treatment. Bovine serum albumin, apoferriten, salmon sperm DNA, “Bartlett” pear DNA and the compounds listed on page 163 did not protect against fire blight. Some protection of etiolated seedlings was obtained with DNA from virulent E. amylovora, E. herbicola and X. campestris (Table 2). DNA f rom P. tabaci and Torula yeast RNA caused a delay in fire blight symptoms, but this delay was not statistically significant (Table 2). Clonal pear shoots were protected against fire blight by DNA from both virulent and avirulent E. amylovora (Fig. 12) when challenged 24 h after treatment with DNA. Plants were not protected when challenge followed injection of DNA from avirulent E. amylovora by Q-5 h.
PLATE 1. Fire blight symptom expression 12 days after inoculating pear sredlings from which the radicle was removed. Seedling on left treated with phosphate buffer, center and right symptom expression on center seedling and root treated with DNA (See Fig. 7). L imited formation on seedling to the right are evident. Seedlings with restricted fire blight symptoms are considered infected in calculating data presented in figures and tables.
[facing
page 1641
Protection
of Bartlett
pear
against
fire
blight
165
0.113.
-
I.74
04; r-
-
I.72
OJf j-
-
I70
0.1: 5.
-
1.68
O,If I-
-
1.66
0 I: I-
-
I.64
z
0.1; > _
-
I.62
2 (u 5
0.1 I-
-
I.60
0.K )-
-
I.58
f
0.0s I-
-
I.56
s 0.08 I,-
-
I.54
0.07
-
I 52
O.OE
-
I 50
0.0:
-I48
0.04
-
I.46
0.0: 0.02 0.01 IO Fraction
FIG. collected rotor at bottom fraction
14 number
10. Absorbance (258nm) (O-O) and density (25 “C) (O-O) of DNA fractions from cesium chloride gradients. Samples centrifuged for 48 h in a Beckman SW 50.1 44 000 rev/min and 20 “C. Three drop fractions were collected by piercing a hole in the of the centrifuge tube. One-half ml 0.05 M-phosphate buffer, pH 6.8, was added to each for the determination of absorbance.
Within 4 days of challenge, all control seedlings from which the radicle was excised had fire blight symptoms, and a few days later they were entirely destroyed. In contrast, 12 days after challenging seedlings treated with DNA, c. 50% had fire blight symptoms and only 20 to 30% of those with symptoms were entirely destroyed (Fig. 7). The remaining seedlings with symptoms showed necrosis only at the hypocotyl base (c. 1.0 cm) and this was often dry in appearance as compared to the normal water-soaked appearance of infected tissues (Plate 1). This indicates that not only can DNA totally protect against fire blight, but that it can also impede and even DNA-protected seedlings, from which the stop the progress of disease symptoms. radicle had been excised, were often observed to form roots (Plate 1). Those which did were transplanted into vermiculite and soil (6 : 4) and maintained between baking dishes containing wet filter paper to keep relative humidity at 100%. Seedlings
166
J. L. McIntyre, TABLE
J. Kud
and
E. B. Williams
2
Protection of et&ted “Bartlett” pear seedlings against Jirc blight with DNA (Marmur technique) from virulent E. amylovora. E. herbicola, X. campestris nnd I’. tabaci and Torulayeast RNA”
time
Treatment and of Inoculation
0 Buffer Buffer Buffer E.a. DNA E.h. DNA XL. DNA P.t. DNA T.y. RNA
0/0 Seedlings with symptoms and days after inoculation
(h)
18 BUffiY Nucleic acid9 Vd V V V V V
2
3
4
5 -
6
7
8
0 0 65 40 40 50 45 35
0 0 100 50 60 60 65 65
0 0 100 50 60 80 80 90
0 0 100 50 60 80 90 95
0 0 100 50 70 80 100 95
0 0 100 50 70 80 100 100
0 0 100 50 i0 80 100 100
a Fifty plants with radicles removed per treatment, were placed in pH 6.8, containing c. 200 pg/ml DiYA or Torula yeast RNA. b t Value calculated with paired t-test. Values significant at the c Nucleic acids: DNA from virulent E. amylocora (Ea. DNA), X. camjest& (X.C. DNA), and P. tabaci (P.t. DNA), or Torula. ’ Challenge : V = lo2 cells of E. amylovora.
t value*
g).fp* 22.3”* 9,1** 2.4 2.2
0.05 M-phosphate
buffer;
0.01 (**) level. E. herbicola (E.h. yeast RPA (T.y.
DNA), RN.4).
maintained in this manner for 1 week were challenged by injecting lo2 cells c. 0.5 cm below the cotyledons. Control seedlings showed fire blight symptoms within 2 days of challenge, whereas seedlings protected with DNA showed no symptoms for at least 1 week. After 1 week, symptoms developed in all seedlings. This indicates the lasting nature of protection with DNA and that protection occurs even at sites removed from the point of initial DNA application. DISCUSSION The data indicate that the protectant in sonicates of avirulent or virulent E. amylouora is DNA and protection occurs even at sites removed from the point of DNA appliThere was no indication that avirulent E. amylovora was infected with a cation. bacteriophage, although the assay may not detect a temperate phage [28]. The GC content of the DNA (Marmur) from avirulent E. amylouora was c. 55% as determined by centrifugation to equilibrium in cesium chloride (Fig. 10) and the DNA melting curve (Fig. 11). This corresponds to values reported for DNA from E. amylovora and further suggests the absence of contaminating DNA from a source such as a bacteriophage [33]. DNA melting curves (Fig. 11) showed our samples of DNA were of relatively high molecular weight. There was no evidence of RNA contamination and HC (hypochromicity) = 22.2%. Lack of protection by bovine serum albumin, apoferritin, polypeptides, polyanions, polycations, salmon sperm DNA, “Bartlett” pear DNA, nitrogen bases, ribose, deoxyribose, nucleosides and nucleotides indicates some structural specificity of the DNA is necessary for protection. Increased protection by relatively unsheared DNA (Marmur technique), as compared to sheared DNA isolated from sonicates of the bacterium, further suggests some relationship with size and/or native composition of the DNA from avirulent E. amylovora and protection.
Protection
of Bartlett
pear
against
fire
blight
167
I.0
0.9
O-8
0.7
;: c
0.6
8 N s 0.5 5 D i; z Q 0.4
03
02
01
I
40
50
60 Temperature
FIG. 11. Melting profile of DNA (Marmur) concentration was c. 40 pg/ml in 0.05 M-phosphate from 30 to 100 “C over a IO-min period.
70
80
90
IO6
(“Cl
from avirulent E. amylovora. buffer, pH 6.8. Temperature
Initial DNA increased
Protection against fire blight with DNA from virulent E. amylovora, E. herbicola and X. campestris (Table 2) is suggestive of homology between DNA of these bacteria and avirulent E. amylovora. The homology may influence binding to active or receptor sites. DNA from avirulent E. amylovoragave better protection than did DNA from the other bacteria, suggesting that slight differences in DNA may lessen protection. DNA from an entirely unrelated source (salmon sperm DNA) did not protect. Virulent E. amylovora in the host may initiate changes which are responsible for symptom expression before its DNA can protect. This possibility is strengthened by the requirement for protection of a time period between injection of DNA, as well as avirulent E. amylovora or sonicates, and challenge. Lack of protection by X. campestris [25] may be explained by its inability to grow in Bartlett pear, therefore
168
J. l.. McIntyre,
J. Ku6
and
E. B. Williams
80
IO
456789 Time (days
after
chailenqe
/I
12
)
FIG. 12. Protection of actively growing pear shoots with DNA (Marmur technique) from virulent and avirulent E. amylovora. Shoots injected with phosphate buffer (O-O) or C. 200 pg DNA from virulent (A-A) or avirulent (O-O) E. amylouora and challenged 24 h later with lo3 cells of E. amylouora. Twelve plants observed per treatment.
large cell populations are not present to release DlNA upon cell death and lysis. Factors other than DNA may also play a role in protection by some bacteria. Pseudomonas tabaci produces a toxin [7] which may affect either E. amylovora or sites of E. herbicola produces P-glucosidase [S] which action in the host for E. amylovora. has been suggested hydrolyzes arbutin to hydroquinone and glucose. Hydroquinone to play a role in resistance of pear to fire blight [l3-1.51, and therefore release of this enzyme by the bacterium may also affect protection. Torula yeast RNA may delay symptom expression in a manner unrelated to protection with DNA, such as directly inhibiting E. amylouora or blocking possible active sites for E. amylouora. DNA contamination in the yeast RNA may have also caused this delay. DNA from virulent and avirulent E. amylovora may find practical application as a protectant against fire blight under field conditions. Concentration of DNA was directly related to protection of etiolated seedlings. More permanent protection occurred in etiolated seedlings from which the radicle was removed and the seedlings placed into solutions containing DNA, presumably because this method allowed more uptake and better distribution of DNA, or its degradation products, than did the injection procedure. This study shows that DNA from virulent and avirulent E. amylouora protects Bartlett pear against fire blight, as does DNA from several other bacteria. Mode of action for protection is unknown, but data indicate some specificity for bacterial DNA, and in particular specificity for DNA from E. amylovora. Work in other laboratories [I, 8, 17, 20-23, 351 has indicated uptake and expression of bacterial DNA by plant cells. We are presently exploring the means by which DNA from E. amylovora interacts with the host to protect against the pathogen.
Protection
of Bartlett pear against fire blight
169
The authors wish to thank Dr A. 0. Jackson, Department of Botany and Plant Pathology, Purdue University, for supplying purified virus samples and for valuable advice and assistance during the course of performing these experiments. We also wish to thank Dr Mike Byers, Biology Department, Purdue University, for performing DNA melting curves, and Cita Helkie and Cindy Mayes for technical assistance. REFERENCES 1. ANKER, P., STROUN, M., GAHAN, P., ROSSIER, A. & GUEPPIN, nucleic acids into plant cells and crown gall induction. Systemr, Ed. by L. Ledoux, pp. 187-192. North Holland 2. ARK, P. A. (1937). Variability in the fire blight organism,
H. (1971). Natural release of bacterial In Informative Molecules in Biological Publishing Co., Amsterdam. Erwinia amylovora. Phytopathologr 27,
l-28. 3. BRAKKJZ, M. K. (1971). Degradation of brome mosaic virus and tobacco mosaic virus in bentonite. Virology 46, 575-585. 4. BRAKKE, M. K. & VAN PELT, N. (1970). Linear-log sucrose gradients for estimating sedimentation coefficients of plant viruses and nucleic acids. Anabtical Biochemistry 38, 56-64. 5. CARROL, R. B. & LUKEZIC, F. L. (1972). Induced resistance in alfalfa to Corynebacterium insidiosum by prior treatment with avirulent cells. Phytopathology 62, 555-564. 6. CHATTERJEE, A. K., GIBBONS, L. N. & CARPENTER, F. A. (1968). Some observations on the physiology of Erwinia herbicola and its possible implication as a factor antagonistic to Erw+ia amylovora in the “fire blight” syndrome. Canadian3ournal of Microbiology 15, 640-642. 7. CLAYTON, E. E. (1936). Water soaking of leaves in relation to the development of the wildfire disease in tobacco. Journal of Agricultural Research 52, 239-269. 8. DOY, C. H., GRESSHOF, P. M. & ROLFE, B. G. (1973). Biological and nuclear evidence for the trangenosis of genes from bacteria into plant cells. Proceedings of the .National Academy of Scimce, U.S.A. 70, 723-726. 9. FARABEE, G. J. & LOCKWOOD, J. L. (1958). Inhibition of Erwinia amylovora by bacterium sp isolated from fire blight cankers. Phytojathology 48, 209-2 11. 10. GARDNER, J. M. & Ado, C. I. (1972). Comparative base sequence homologies of the deoxyribonucleic acids of Erwinia species and other Enterobacteriaceae. International 3ourml of Systematic Bacteriology 22, 201-209. 11. GOODMAN, R. N. (1967). The protection of apple stem tissue against Erwinia amylovora infection by avirulent strains and three other bacterial species. Phytojathologr 57, 22-24. 12. GOODMAN. R. N. & SHAFPER. W. H. (1971). nrocedure for evaluating the efficacv ~ , An inoculation 1 of toxicants against Erwinia amylovora. Proceedings Second Workshop Fire Blight Research, Michigan State University. 13. HILDEBRAND, D. C. (1970). Fire blight resistance in Pyrus: hydroquinone formation as related to antibiotic activity. Canadian Journal of Botany 48, 177-181. 14. HILDEBRAND, D. C. & SCHROTH, M. N. (1964). Antibiotic activity of pear leaves against Erwinia amylovora and its relation to 8-glucosidase. Phytopathologv 54, 59-63. 15. HILDEBRAND, D. C. & SCHROTH, M. N. (1964). Arbutin-hydroquinone complex in pear as a factor in fire blight development. Phytofiathology 54, 640-645. 16. JACKSON, B., JACKSON, A. 0. & BRAKKE, M. K. (1973). Pipetting calculations for linear log gradients for Spinco swinging bucket rotors. Special Publication Plant Virology Laboratory, Department of Plant Pathology, University of Nebraska, Lincoln. 17. JOHNSON, C. B., GRIERSON, D. & SMITH, H. (1973). The expression of A plac 5 DNA within cultured cells of a higher plant. Nature, New Biolopv 244, 105-107. 18. KIRBY, K. S. (1965). Isolation and characterization of ribosomal ribonucleic acid. Biochemical 3ourd 96, 266-269. 19. LANGLEY, R. A. & KADO, C. I. (1972). Studies on Agrobacterium tumfaciem: condition for mutogenesis by methyl-N’-nitro-N-nitrosoguanidine and relationships of A. tumefacienr mutants to crown gall tumour induction. Mutation Research 14, 277-286, 20. LEDOUX, L. & HUART, R. (1968). Integration and replication of DNA of M. lysodeikticus in DNA of germinating barley. Nature 218, 1256-1259. 2 1. LEDOUX, L. & HUART, R. ( 1969). Fate of exogenous bacterial DNA in barley seedlings. 3ournal of Molecular Biology 43, 243-262. 22. LEDOUX, L., HUART, R. & JACOB, M. (1971). Fate of exogenous DNA in Arabidopsis thaliani. I. Translocation and integration. European Journal of Biochemistry 23, 96108.
170
J. L. McIntyre,
23. LEDOUX, 24. 25.
26. 27. 28. 29.
30. 31. 32. 33.
34.
35.
J. Kui:
and
E. 6. Williams
L., HUART, R. &JACOB, M. (1971). Fate of rx~~enous DNA in Arabidopsis thaiiani. II. Evidence for replication and preliminary results at the biological level. In Informative Molecules In BiologicalSystems, Ed. by L. Ledoux, pp. 159-175. North Holland Publishing Co., Amsterdam. MARMUR, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. Journal of‘ Molecular Biology 3: 208-2 18. MCINTYRE, J. L. (1974). Protection of pear against fire blight by bacteria, bacterial sonicates and bacterial deoxyribonucleic acid. Ph.D. Thesis, Purdue University, Lafayette. MCINTYRE, J. L. & WILLIAB~S, E. B. (1972). Protection of Bartlett pear by avirulent Ew,&io spp. and Pseudomonas tabaci. P&ytopatholog)i 62, 777. MCINTYRE, J. L., KuC, J. & WILLIAMS, E. B. (1973). Protection of pear against fireblight by bacteria and bacterial sonicates. Phytofiathology 63, 872.-877. MILLER, J, H. (1972). Preparation and plaque assay of a phage stock. In Ex@&ments in Molecular Genetics, pp. 37-46. Cold Spring Harbor Laboratory, New York. PARISH, J. H. (1972). Principles and Practice of Experiments zvith Nucleic Acids. Longman Group Ltd, London. ROE, J. H. (1955). The determination of sugar in blood and spinal fluid with anthrone reagent. Journal of Biological Chemistry 212, 335-343. SCHILDKRAUT, C. L., MARMUR, J. & DOTY, P. (1962). Determination of the base composition of deoxyribonucleic acid from its bouyant density in CsCI. Journal of Molecular Biology 4; 430-443. SLEESMAN, H. C., PERLEY, J. E. & HOITINK, H. A. J, (1970). Susceptible and hypersensitive reactions in tobacco and their prevention by cell-free extracts of Pseudomonas tabaci and Pseudomonas glycinea. Phytojathology 60, 13 14. STARR, M. P. & MANDEL, M. (1969). DKA base composition and taxonomy of phytopathogenic bacteria and other Enterobacteria. Journal of General MicrobioloD 56, 113-l 23. STERN, H. (1968). Isolation and purification of plant nucleic acids from whole tissues and from isolated nuclei. In Methods in Enzymology, Ed. by L. Grossman & K. Moldave. XII-B 108: 111. STRAUN, M., ANKER, P., GAHAS, P., ROSSIER, 4. 8i GREPPIX, H. J. (1971). Agrobacterium tumefaciens ribonucleic acid synthesis in tomato cells and crown gall induction. 3oumal of Bacteriology 106,
634-639.
Plant Pathology
(1975)
7, 153-170
Protection of Bartlett pear against fire blight with deoxyribonucleic acid from virulent and avirulent Erwinia amylovoraf J. L. MCINTYRE Connecticut
Agtiltural
Experiment
Station,
New
Haven,
Connecticut,
U.S.A.
J. Ku6 Department
of Plant Pathology,
University
of Kentucky,
Lexington,
Kentucky,
U.S.A.
and E. B. WILLIAMS Department (Accepted
of Botany and Plant Pathology, for publication
April
Purdue
University,
Lafayette,
Indiana,
U.S.A.
1975)
Sonic&es of virulent and avirulent Erwinia amylovora protect “Bartlett” pear shoots and etiolated seedlings against fire blight. Activity of sonicates is lost after precipitating nucleic acids with protamine sulfate, whereas nucleic acids purified from sonicates protect. Deoxyribonuclease, but not ribonuclease, destroys activity of nucleic acids isolated from sonicates. Activity is associated with the deoxyribonucleic acid (DNA) fraction from linear log sucrose gradients. DNA prepared by the Marmur technique protects more effectively than DNA prepared from sonicates. The former was calculated to have a GC content of G. 55% by centrifugation to equilibrium in cesium chloride and from DNA melting curves. Specificity of the DNA preparations is suggested since DNA from Pseudomonas tabaci, a bacterium which protects against fire blight, did not protect, whereas DNA from virulent E. amylovora protects. Nucleic acid components, polyanions and polycations did not protect. Host response appears necessary for protection since a lag period is required between injection of DNA and virulent E. amylooora, and DNA added to a nutrient medium does not inhibit the growth of E. amylovora or affects its virulence.
INTRODUCTION Protection of plants against bacterial pathogens by treatment of the host with disrupted cells of avirulent mutants [32] and cell-free extracts of pathogens and non-pathogens [S] indicates that protection is not dependent on the presence of bacteria. Protection of “Bartlett” pear and “Jonathan” apple against fire blight with avirulent Erwinia amylovora, Erwinia herbicola and Pseudomonas tabaci has been reported [9, II, 271, and protection of “Bartlett” pear against fire blight with cell-free sonicates of virulent and avirulent E. amylovora has been demonstrated [26, 271. The purpose of this paper was to determine the nature of the protectant in sonicates. t Research supported in part by a grant from the Herman Frasch Foundation, USDA Cooperative Agreement 12-14-lOO-12,625 (34) and Cooperative State Research Service, USDA, Agreement 316-15-51. Journal paper 75-11-30 of the Kentucky Agricultural Experiment Station, Lexington, Kentucky 40506 and 5878 of the Purdue Agricultural Experiment Station, Lafayette, Indiana 47907, U.S.A.
154
J. L. Mclntrye,
MATERIALS
Bacterial
AND
J. KuC and
E. B. Williams
METHODS
isolates and sonicate preparation
Avirulent Erwinia amylovora (E8) was obtained from R. N. Goodman, University of Missouri. The virulent isolate (challenge) of Erwinia amylovora (Burr) Winslow et al. was isolated and all cultures were maintained and sonicates prepared as described [27]. The basis for classifying the avirulent bacterium used in th.is study as E. amylovora has been reported [25]. Plant
material
Actively growing clonal Pyrus communis L. “Bartlett” at least 30 cm tall were used in all experiments with green tissue. Plants were grown individually in clay pots containing soil and maintained in a greenhouse at 20 to 26 “C and relative humidity greater than 90%. Etiolated Bartlett open-pollinated seedlings were grown in a mixture of vermiculite and soil (6 : 4) for IO days at 19 “C in the dark. Seedlings, 4.5 to 5.5 cm tall, were removed from the soil, washed with tap water and distilled water, and placed on moist filter paper at the bottom of a Pyrex baking dish which was then covered with an inverted baking dish. Dishes were either placed under fluorescent lights or wrapped with aluminum foil and kept in the dark. Etiolated pear seedlings were utilized to assay for the protectant. Seedlings were injected with 10 ~1 protectant [27] in 0.05 M-phosphate buffer, pH 6.8 [1211 and challenged 18 h later with IO2 cells of E. amylovora in 0.05 M-phosphate buffer, pH 6.8. A time study indicated that optimum protection occurred when challenge followed protectant by 18 h [25] and IO2 cells challenge caused rapid symptom development ( ~48 hj in controls which facilitated the detection of protectant. Control plants were injected with 10 ~1 0.05 M-phosphate buffer, pH 6.8. To obtain increased uptake and better distribution of protectant, an assay was developed using etiolated seedlings from which the radicle was excised. Seedlings were maintained in the laboratory under fluorescent lights (Champion F9 OT 17/W) at c. 22 “C for the duration of the assay. Five ml phosphate buffer or buffer containing protectant was placed into 120 mm tall Coplin staining dishes. Seedlings were placed into the solution and the staining dishes were covered with Saran Wrap. After 24 h seedlings were washed with sterile water, 0.5 mm of the hypocotyl base was cut and discarded, and the treated seedlings were placed in Coplin dishes containing lo4 cells/ml challenge in 5 ml phosphate buffer. After 2 h, seedlings were washed as before and either returned to the solution containing protectant or buffer or placed onto moist filter paper at the bottom of a Pyrex baking dish. The seedlings replaced into protectant or buffer were removed after 12 h$ rinsed and the hypocotyl base was trimmed (0.5 mm) to remove tissue where slight toxic effects from buffer often appeared. These seedlings were then placed into baking dishes as described. When protection occurred by the above assays, the presence of protectant was verified in actively growing pear shoots. Pear shoots were injected with protectant by the “drop method” [27], and O-5 or 24 h later injected with IO3 cells E. amylovora. Precipitation
of nucleic acids
Nucleic acids were precipitated from 5 ml of sonicate (c. 1500 pg protein/ml sonicate) with 1 ml of 1 O/Oprotamine sulfate. The protamine sulfate-treated preparations were
Protection
of Bartlett pear against fire blight
155
held at 37 “C for 30 min and precipitate was removed by centrifugation at 8000 rev/ min and 4 “C. The supernatant was assayed by the injection method for its ability to protect and its protein content was determined. Purification
of nucleic acidr from sonicates
Nucleic acids were purified from sonicates of 2 to 3 g (wet weight) bacterial cells with Kirby phenol [18]. Due to shearing by sonication, the nucleic acids could not be spooled on a glass rod after precipitation with two volumes of cold 95% ethanol. Therefore, nucleic acids were collected by centrifugation at 8000 rev/min and 4 “C and the final pellet, obtained after two additional treatments with phenol and three ethanol precipitations, was dried with nitrogen gas and suspended in 1 ml phosphate buffer. The preparation was scanned from 220 to 320 nm to determine 2601280 and 2601230 ratios, and to determine the amount of nucleic acid from the 260 nm peak height. An extinction coefficient of 25 was used to estimate nucleic acid and ribonucleic acid (RNA) and a value of 20 was used for deoxyribonucleic acid (DNA). Protein and carbohydrate were determined by the Lowry and anthrone [30] techniques, respectively. The nucleic acids were tested by the injection method for their ability to protect. Enzyme
treatments of nucleic ads
The nucleic acid pellet obtained after the first ethanol precipitation was dried, suspended in 5 ml phosphate buffer containing 1 nnr-magnesium chloride [29] and treated for 30 min at 22 “C with 500 pg/ml deoxyribonuclease 1 (DNase) (beef pancrease, ICN Nutritional Biochemicals, Cleveland, Ohio). Similarly the pellet from the first ethanol precipitation was suspended in 5 ml SSC buffer (O-15 M-sodium chloride, 0.015 M-sodium citrate, pH 7*2), and treated for 30 min at 37 “C with 50 pg/ml ribonuclease A (RNase) (beef pancrease, ICN Nutritional Biochemicals, Cleveland, Ohio) and 5 units/ml RNase Tl (Aspergillus oryzae, ICN Nutritional Biochemicals, Cleveland, Ohio) which were previously treated to destroy DNase activity [34]. Enzyme was removed by treating with an equal and then one-quarter volume of Kirby phenol. The final ethanol precipitate (RNA or DNA) was suspended in 1 ml phosphate buffer. The suspension was scanned under U.V. and its protein and carbohydrate content and ability to protect (injection method) were determined. Sucrose gradient
centrafugation
Linear log sucrose gradients [4,16] were prepared with RNase-free sucrose (Schwartz/ Mann, Orangeburg, New York) in cellulose nitrate centrifuge tubes for use with a Beckman SW 41 rotor and gradients were held at 4 “C for 24 h prior to use. Nucleic acid preparations were adjusted to c. 100 pg/ml and, after removing O-5 ml from the top of the gradient, O-5 ml nucleic acid was layered onto the gradient. Purified tobacco or brome mosaic virus (100 pg/ml) were degraded in bentonite [3] and layered onto gradients as described. Standards and samples were centrifuged in a Beckman L2-65 ultracentrifuge for 7.5 h at 38 000 rev/min and 14 “C. One-half ml fractions were collected and absorbance at 258 nm determined with an Isco Density Gradient Fractionator with ultraviolet analyzer. Fractions under peaks were pooled and the DNA and/or RNA was precipitated by gentle mixing with two volumes of
J. L. McIntyre,
156
J. KuC and
E. B. Williams
95% ethanol and placing in the freezer (-20 “Cj for 6 h. Precipitate was collected by centrifugation at 8000 rev/min and 4 “C for 3 min. The supernatant was decanted, and the precipitate dried with nitrogen gas. Nucleic acids were suspended in 1 ml phosphate buffer and assayed by the injection method for protection. The (S) values of samples were determined by comparing the distance moved from the top of the gradient to distance moved by viral RNA components used as standards. Thirty min prior to layering gradients, purified nucleic acid samples were treated with RNase or DNase. These samples were centrifuged as before and one of the two peaks evident after centrifugation of the nucleic acids remained at the top of the gradient. The (S) value of the remaining nucleic acid was determined, and comparison of these values to those obtained from the nucleic acid preparations permitted identification of the DNA and RNA components of the nucleic acid. DNA and RNA obtained after centrifugation of enzyme-treated nucleic acids were precipitated, suspended and assayed for by the injection method for protection. DNA isolated from gradients was either centrifuged again in linear log sucrose gradients or treated with DNase or RNase and centrifuged. A Beckman 50.1 rotor was used and the samples were centrifuged at 48 000 rev/min for 2.5 h at 14 “C. D.NA preparations
from lysed bacterial cells
To obtain large quantities of DNA, bacteria were grown in 10 1 fermentors at 25 + 1 “C and 5 l/min air flow. Inoculum was grown in 100 ml shake cultures containing medium 924 salt [19] plus additions [IO]. After 24 h, the inoculum was added to fermentors which contained the same medium as the shake cultures. The bacteria were maintained for 24 h in the fermentors, harvested with a Sharples centrifuge and washed with SSC buffer (2 to 3 g cells/25 ml buffer). The bacteria were centrifuged at 10 000 rev/min and 4 “C for 15 min and pelleted cells frozen ( - 20 “C) . Within 24 h of freezing, the cells were thawed at room temperature and then refrozen. The cells then readily lysed upon treatment with sodium dodecyl sulfate (SDS), and DNA was purified by the Marmur technique [24]. The method allowed for the purification of DNA with minimal shearing. Purified DNA was also treated with DNase and enzyme was removed by the Marmur technique. DNA (200 pg/ml) and DNasetreated DNA were scanned under U.V. and tested for protection in etiolated seedlings from which the radicle was removed. DNA was stored at 4 “C under toluene. Eye& of DJVA concentratiorz on protection DIVA (purified from sonicates of avirulent E. amylovora) was adjusted with buffer concentrations of 10, 100, 1000 and 5000 pg/ml. Ten ~1 of each were injected etiolated seedlings and challenged 18 h later with I O2 cells of E. amylovora. Effect of DNA
on growth
and virulence of E. amylovora
to in
in vitro
Ten mg DNA (Marmur technique) were placed in 250-ml flasks with 100 ml mineral medium containing 1 oh sucrose [Z]. Th e fl as k s were inoculated with 2 x lo3 cells of consisted of 10 ml phosphate buffer in place of DNA, virulent E. amylovora. Controls and growth of the bacterium was determined spectrophotometrically at 525 nm. Cells were harvested by centrifugation, suspended in phosphate buffer and injected into etiolated seedlings to determine their virulence.
Protection
of Bartlett
Bacteriophage
pear
against
fire
blight
157
assay
Avirulent E. amylovora was grown in nutrient broth (D&o) and the cultures shaken. When bacteria were in the exponential growth phase (c. 5 x lo* cells/ml), 0.2-ml aliquots were removed and placed into each of 10 sterile culture tubes. Molten H-top agar (45 “C, 2.5 ml) was added to each tube and the contents immediately poured over the surface of an H plate [28]. The agar was allowed to harden and the plates were incubated for c. 10 h at 30 “C. Chloroform lysates of the cultures were prepared and both virulent and avirulent E. amylovora were treated with lysate and plates poured as described by Miller [28]. Plates were incubated overnight at 30 “C and observed for plaque formation. Cesium chloride centriiugation Cesium chloride gradients were prepared for ultracentrifugation with a Beckman SW 50.1 rotor. Sixty per cent cesium chloride (w/w) in O-015 M-sodium citrate, pH 7.2, was prepared and 3.67 ml was pipetted into cellulose nitrate tubes. DNA (1.33 ml, 1 mg/ml) was layered onto the top of the gradients. The samples were centrifuged for 48 h at 44 000 rev/min and 20 “C in the Beckman L2-65 ultracentrifuge and allowed to coast to a stop. A hole was pierced in the bottom of each tube, three drop fractions were collected and the refractive index at 25 “C was determined for each fraction. One-half ml phosphate buffer was added to each fraction and absorbance determined at 260 and 280 nm. GC content of the DNA was estimated by the formula %GC = (BD - 1.660)/0*096 [31], where BD is buoyant density of DNA and GC is mole fraction of guanine plus cytosine in DNA. Fractions containing DNA were pooled and dialyzed for 24 h at 4 “C in 2 1 phosphate buffer changed after 6 and 18 h. DNA was tested for its ability to protect etiolated seedlings from which the radicle was removed. DNA
melting curves
DNA was adjusted to c. 40 pg/ml and 1 ml samples were melted by increasing the temperature from 30 to 100 “C over a lo-min period. GC content was estimated from the formula %GC = ( Tm- 69.3) x 2.44 [29], where Tm is the temperature at which the hypochromic effect is 50% of its maximum. Additional
sources of bacterial
DNA
and com@ounds tested for protective activity
DNA isolated from etiolated “Bartlett” pear seedlings [25] and DNA from E. amylovora, Erwinia herbicola, Pseudomonas tabaci and Xanthomonas campestris (isolated by the Marmur technique) were assayed for activity in etiolated seedlings from which the radicle was removed. All other compounds (bovine serum albumin, apoferritin, salmon sperm DNA, Torula yeast RNA, adenine, adenosine, deoxyadenosine, 5’adenylic acid, cytosine, cytidine, deoxycytidine, 5’deoxycytidylic acid, poly-Dglutamic acid, poly-L-glutamic acid, poly-L-alanine, poly-n-lysine, poly-L-lysine, polyphosphoric acid, polyinosinic acid, ribose, deoxyribose, obtained from ICN Nutritional Biochemicals, Cleveland, Ohio) were placed in phosphate buffer (10 mg/ml) and pH adjusted to 6.8. Ten ~1 was injected into etiolated seedlings, followed 18 h later with 1O2 cells of challenge.
158
J. L. McIntyre,
Injection
J. KuC and
E. B. Williams
of clonal @ear with DNA
DNA (Marmur technique) from virulent and avirulent E. amylouora was injected by the drop method [27] into actively growing shoots of pear. By allowing uptake of many drops of DNA at the site of the puncture, it was possible to get c. 200 LangDNA into shoots. Plants were challenged at the same site either O-5 or 24 h later with an inoculum containing c. lo3 cells.
RESULTS
Injection and vascular uptake are useful for introducing protectant into etiolated pear seedlings. Injection was especially useful when small volumes of protectant were available. As protectant was purified and larger quantities were available, increased uptake and better distribution of protectant, and more permanent protection, were obtained by vascular uptake. Nucleic acids purified from sonicate Precipitation of nucleic acids with protamine sulfate caused a loss of activity in sonicate without appreciable change in protein content (Fig. 1). Protection was associated with nucleic acids purified by Kirby phenol (Fig. 2). U.V. absorbance spectra of the nucleic acids indicated 2601280 ratios of 2-l to 2.2 and 2601230 ratios of 2.2 to 2.4. Protein content of the nucleic acid was c, 5% and carbohydrate content c. 33%.
70 F ; g c
60 50 40
I
I 24
1 Time
1 72
1 (h after
I 120
1
challenge
1 168
1
1
FIG. 1. Protection of pear seedlings against fire blight. Seedlings injected with 10 ~1 phosphate buffer (o----o), sonicate of avirulent E. anlylovora (D-----D) or sonicate from which nucleic acids were precipitated with protamine sulfate (O-O), and challenged 18 h later with IO2 cells of E. amylonora. Sonicate contained 1500 pg protein/ml, and sonicate from which nucleic acids were precipitated contained 1410 pg protein/ml. Fifty seedlings observed per treatment.
Protection
of Bartlett pear against fire blight
24
159
I 72
I
Time (h after
I 120
I
I 168
I
challenge)
FIG. 2. Protection of pear seedlings against fne blight. Seedlings injected with 10 ld phosphate buffer (O-O), sonicate of avirulent E. amylovvra (e-0) or nucleic acids purified from sonicate (O-O) and challenged 18 h later with lo8 cells of E. amylovoru. Sonicate contains 1300 pg protein/ml, and nucleic acid preparation 1000 ng nucleic acid/ml. One hundred seedlings observed per treatment.
100 90
80 70
Time (days
after
challenge)
FIG. 3. Protection of pear seedlings against fire blight. Seedlings injected with 10 ~1 phosphate buffer (O-O), nucleic acid preparation from sonicate of avirulent E. amylooora (O-O) and DNase (O-O) or RNase (A-A) treated nucleic acid preparations. Initial nucleic acid preparation contains 5000 ng nucleic acid/ml, DNase treated preparation 4700 pg RNA/ml and RNase treated preparation 500 pg DNA/ml. Fifty seedlings observed per treatment.
J. h. McIntyre,
160
J. Ku6
and
E. B. Williams
Treatment of nucleic acids with DNase or RNase indicated that activity was associated with DNA (Fig. 3), and U.V. absorbance spectra and protein and carbohydrate content were similar to those obtained with nucleic acid preparations. Linear log sucrose gradient centrifugation of nucleic acids prepared from sonicates indicated the presence of two compounds absorbing at 258 nm (Fig. 4). They were identified as RNA and DNA (Fig. 4) after treatment with DNase or RKase. The (S) values were calculated to 4.8 S for RNA and 9.0 S for DNA. Activity was associated 0.61 0.5 0.4 0.3 o-2 0-I
06
0.8 0.7 0.6
Depth i cm 1 FIG. 4. Scanning patterns after linear log sucrose gradient centrifugation of (a) 100 ng nucleic acid/ml from avirulent E. nmylovora; (b) 100 pg nucleic acid/ml treated with DNase; (c) 100 pg nucleic acid/ml treated with RNase. Samples centrifuged for 7.5 h at 38 000 rev/min acids precipitated in the Beckman SW 41 rotor at 4 “C. Fractions pooled (Fl to F9) and nucleic to determine if protection occurred with a nucleic acid fraction.
Protection
of Bartlett pear against fire blight
161
with DNA isolated from the gradients (Fig. 5) and this activity was destroyed by treatment with DNase but not RNase (Fig. 5). Precipitation of DNA from sucrose gradients and centrifugation by linear log sucrose gradients with a SW 50.1 rotor gave only one peak of DNA absorbing at 258 nm (Fig. 6). Treatment of the reisolated nucleic acid with RNase had no affect on the position of the DNA (Fig. 6), whereas treatment with DNase caused a loss of the DNA (Fig. 6). DNA prepared by the Marmur technique protected much better than that prepared by sonication (Figs ‘2 and 7). Protection also occurred when this material was injected into clonal pear shoots and challenged 24 h later with 10s cells of E. amylovora
100 (a) 80 60 40 20
ii 5
l;(cl 20
&T
0
I 24
I 48 Time
I 72 (h after
I 96 challenge)
I 120
I 144
FIG. 5. Protection of pear seedlings with nucleic acids precipitated from fractions collected after linear log sucrose gradients. (a) RNA (Fl)’ (O-O) and RNA plus DNA (F2) O-O) fractions from centrifuged nucleic acid preparations from avirulent E. amylouoru; (b) RNA plus degraded DNA (F4) (O-D) and RNA (F5) (O-O) f rom centrifugation of nucleic acids treated with DNase; (c) DNA plus degraded RNA (F7) (O-O) and DNA (F8) (O-O) from centritirgation of nucleic acids treated with RNase. Seedlings injected with 10 ~1 of phosphate buffer (O-O) or nucleic acid sample and challenged 18 h later with 1Oe cells of E. amylovora. Fifty seedlings observed per treatment. * Fl to F9 represent fractions as shown in Fig. 5 and contain 34, 87, 0, 44, 20, 0, 3, 6 and 0 rig/ml nucleic acid. F3, 6 and 9 gave no protection and are not shown.
162
J. L. McIntyre,
J. Ku6
and
E. B. Williams
0.31
Depth
(cm]
FIG. 6. Scanning patterns of DNA isolated from linear log sucrose gradients (38 000 rev/min and 7.5 h, SW 41 rotor) and recentrifuged in linear log sucrose gradients. (a) DNA; (b) DNA treated with RNase; (c) DNA treated with DNase. Samples centrifuged for 2.5 h in Beckman 50.1 rotor at 48 000 rev/min and 14 “C.
TimeCdays
after
challenge)
FIG. 7. Protection ofpear seedlings with DNA (Marmur technique) (O-O) from avirulent E. amylovora. Seedlings from which radicle was excised were placed in 5 ml phosphate buffer (O-O) or 5 ml buffer containing 200 pg DNA/ml for 24 h. Seedlings were challenged by placing them in 5 ml buffer containing 10’ cells/ml of E. amylovora. Fifty seedlings observed per treatment.
Protection
of Bartlett
pear
against
fire
blight
163
(Fig. 8). DNA concentrations as low as 10 pg/ml protected against fire blight and protection increased with increased concentration of DNA (Fig. 9). DNA did not inhibit the growth of E. amylovora in vitro (Table I), and bacteria grown in the presence of DNA were virulent. The phage assay did not indicate infection of avirulent E. amylovora with a bacteriophage.
80 70 s c
60
i
50
2 40
456789
IO
II
12
Time (days after challenge)
FIG. 8. Protection of actively growing pear shoots with DNA (Marmur technique) from avirulent E. amylovora. Shoots injected with phosphate buffer and challenged 0.5 or ‘24 h later (O-O) with 10s cells of E. amylovora or injected with c. 200 pg DNA in phosphate buffer and challenged 0.5 (O-O) or 24 (O-O) h later. Twelve plants observed per treatment.
Density determinations by centrifugation to equilibrium in cesium chloride indicated a GC content of c. 55% (Fig. lo), and this value was verified by melting curve determinations (GC = 54-l) (Fig. 11). DNA isolated from cesium chloride gradients protected against fire blight (c. as in Fig. 7). TABLE
Growth
of virulent
E. amylovora
Number Time
after
1
in the presence of D.NA from of bacterial
avirulent
E. amylovoraa
cells/ml
inoculation W
Control
0 18 24 36 48 60
9.0 1.5 3.5 6.0 1.0x
10s x 104 x 10s x 10s x 10s 106
DNA
(avirulent
2.5 4.0 9.0 1.1 3.0
E. amylovom) 10s x 10” x 10s x 105 x 10s x 10s
a Test: 100 ml mineral medium containing 1% sucrose, 10 ml 0.05 M-phosphate buffer, pH 6.8, containing 10 mg DNA and seeded with 103 cells of virulent E. amylouora. Control: As test with 10 ml of buffer without DNA.
164
J, L. McIntyre,
J. KuC
and E. B. Williams
8
lil I2345678 Time
I, (days
oite:
I chal’enqe
I, 1
FIG. 9. Protection of pear seedlings with DNA from sonicates of avirulent E. amylovora. Seedlings injected with 10 ~1 phosphate buffer (O-O) or DNA [5000 (W----W), 1000 (O-O), 100 (--D--O), 10 (A-A) pg/ml] and challenged 18 h later with lo2 cells of E. amylovora. Fifty seedlings observed per treatment. Bovine serum albumin, apoferriten, salmon sperm DNA, “Bartlett” pear DNA and the compounds listed on page 163 did not protect against fire blight. Some protection of etiolated seedlings was obtained with DNA from virulent E. amylovora, E. herbicola and X. campestris (Table 2). DNA f rom P. tabaci and Torula yeast RNA caused a delay in fire blight symptoms, but this delay was not statistically significant (Table 2). Clonal pear shoots were protected against fire blight by DNA from both virulent and avirulent E. amylovora (Fig. 12) when challenged 24 h after treatment with DNA. Plants were not protected when challenge followed injection of DNA from avirulent E. amylovora by Q-5 h.
PLATE 1. Fire blight symptom expression 12 days after inoculating pear sredlings from which the radicle was removed. Seedling on left treated with phosphate buffer, center and right symptom expression on center seedling and root treated with DNA (See Fig. 7). L imited formation on seedling to the right are evident. Seedlings with restricted fire blight symptoms are considered infected in calculating data presented in figures and tables.
[facing
page 1641
Protection
of Bartlett
pear
against
fire
blight
165
0.113.
-
I.74
04; r-
-
I.72
OJf j-
-
I70
0.1: 5.
-
1.68
O,If I-
-
1.66
0 I: I-
-
I.64
z
0.1; > _
-
I.62
2 (u 5
0.1 I-
-
I.60
0.K )-
-
I.58
f
0.0s I-
-
I.56
s 0.08 I,-
-
I.54
0.07
-
I 52
O.OE
-
I 50
0.0:
-I48
0.04
-
I.46
0.0: 0.02 0.01 IO Fraction
FIG. collected rotor at bottom fraction
14 number
10. Absorbance (258nm) (O-O) and density (25 “C) (O-O) of DNA fractions from cesium chloride gradients. Samples centrifuged for 48 h in a Beckman SW 50.1 44 000 rev/min and 20 “C. Three drop fractions were collected by piercing a hole in the of the centrifuge tube. One-half ml 0.05 M-phosphate buffer, pH 6.8, was added to each for the determination of absorbance.
Within 4 days of challenge, all control seedlings from which the radicle was excised had fire blight symptoms, and a few days later they were entirely destroyed. In contrast, 12 days after challenging seedlings treated with DNA, c. 50% had fire blight symptoms and only 20 to 30% of those with symptoms were entirely destroyed (Fig. 7). The remaining seedlings with symptoms showed necrosis only at the hypocotyl base (c. 1.0 cm) and this was often dry in appearance as compared to the normal water-soaked appearance of infected tissues (Plate 1). This indicates that not only can DNA totally protect against fire blight, but that it can also impede and even DNA-protected seedlings, from which the stop the progress of disease symptoms. radicle had been excised, were often observed to form roots (Plate 1). Those which did were transplanted into vermiculite and soil (6 : 4) and maintained between baking dishes containing wet filter paper to keep relative humidity at 100%. Seedlings
166
J. L. McIntyre, TABLE
J. Kud
and
E. B. Williams
2
Protection of et&ted “Bartlett” pear seedlings against Jirc blight with DNA (Marmur technique) from virulent E. amylovora. E. herbicola, X. campestris nnd I’. tabaci and Torulayeast RNA”
time
Treatment and of Inoculation
0 Buffer Buffer Buffer E.a. DNA E.h. DNA XL. DNA P.t. DNA T.y. RNA
0/0 Seedlings with symptoms and days after inoculation
(h)
18 BUffiY Nucleic acid9 Vd V V V V V
2
3
4
5 -
6
7
8
0 0 65 40 40 50 45 35
0 0 100 50 60 60 65 65
0 0 100 50 60 80 80 90
0 0 100 50 60 80 90 95
0 0 100 50 70 80 100 95
0 0 100 50 70 80 100 100
0 0 100 50 i0 80 100 100
a Fifty plants with radicles removed per treatment, were placed in pH 6.8, containing c. 200 pg/ml DiYA or Torula yeast RNA. b t Value calculated with paired t-test. Values significant at the c Nucleic acids: DNA from virulent E. amylocora (Ea. DNA), X. camjest& (X.C. DNA), and P. tabaci (P.t. DNA), or Torula. ’ Challenge : V = lo2 cells of E. amylovora.
t value*
g).fp* 22.3”* 9,1** 2.4 2.2
0.05 M-phosphate
buffer;
0.01 (**) level. E. herbicola (E.h. yeast RPA (T.y.
DNA), RN.4).
maintained in this manner for 1 week were challenged by injecting lo2 cells c. 0.5 cm below the cotyledons. Control seedlings showed fire blight symptoms within 2 days of challenge, whereas seedlings protected with DNA showed no symptoms for at least 1 week. After 1 week, symptoms developed in all seedlings. This indicates the lasting nature of protection with DNA and that protection occurs even at sites removed from the point of initial DNA application. DISCUSSION The data indicate that the protectant in sonicates of avirulent or virulent E. amylouora is DNA and protection occurs even at sites removed from the point of DNA appliThere was no indication that avirulent E. amylovora was infected with a cation. bacteriophage, although the assay may not detect a temperate phage [28]. The GC content of the DNA (Marmur) from avirulent E. amylouora was c. 55% as determined by centrifugation to equilibrium in cesium chloride (Fig. 10) and the DNA melting curve (Fig. 11). This corresponds to values reported for DNA from E. amylovora and further suggests the absence of contaminating DNA from a source such as a bacteriophage [33]. DNA melting curves (Fig. 11) showed our samples of DNA were of relatively high molecular weight. There was no evidence of RNA contamination and HC (hypochromicity) = 22.2%. Lack of protection by bovine serum albumin, apoferritin, polypeptides, polyanions, polycations, salmon sperm DNA, “Bartlett” pear DNA, nitrogen bases, ribose, deoxyribose, nucleosides and nucleotides indicates some structural specificity of the DNA is necessary for protection. Increased protection by relatively unsheared DNA (Marmur technique), as compared to sheared DNA isolated from sonicates of the bacterium, further suggests some relationship with size and/or native composition of the DNA from avirulent E. amylovora and protection.
Protection
of Bartlett
pear
against
fire
blight
167
I.0
0.9
O-8
0.7
;: c
0.6
8 N s 0.5 5 D i; z Q 0.4
03
02
01
I
40
50
60 Temperature
FIG. 11. Melting profile of DNA (Marmur) concentration was c. 40 pg/ml in 0.05 M-phosphate from 30 to 100 “C over a IO-min period.
70
80
90
IO6
(“Cl
from avirulent E. amylovora. buffer, pH 6.8. Temperature
Initial DNA increased
Protection against fire blight with DNA from virulent E. amylovora, E. herbicola and X. campestris (Table 2) is suggestive of homology between DNA of these bacteria and avirulent E. amylovora. The homology may influence binding to active or receptor sites. DNA from avirulent E. amylovoragave better protection than did DNA from the other bacteria, suggesting that slight differences in DNA may lessen protection. DNA from an entirely unrelated source (salmon sperm DNA) did not protect. Virulent E. amylovora in the host may initiate changes which are responsible for symptom expression before its DNA can protect. This possibility is strengthened by the requirement for protection of a time period between injection of DNA, as well as avirulent E. amylovora or sonicates, and challenge. Lack of protection by X. campestris [25] may be explained by its inability to grow in Bartlett pear, therefore
168
J. l.. McIntyre,
J. Ku6
and
E. B. Williams
80
IO
456789 Time (days
after
chailenqe
/I
12
)
FIG. 12. Protection of actively growing pear shoots with DNA (Marmur technique) from virulent and avirulent E. amylovora. Shoots injected with phosphate buffer (O-O) or C. 200 pg DNA from virulent (A-A) or avirulent (O-O) E. amylouora and challenged 24 h later with lo3 cells of E. amylouora. Twelve plants observed per treatment.
large cell populations are not present to release DlNA upon cell death and lysis. Factors other than DNA may also play a role in protection by some bacteria. Pseudomonas tabaci produces a toxin [7] which may affect either E. amylovora or sites of E. herbicola produces P-glucosidase [S] which action in the host for E. amylovora. has been suggested hydrolyzes arbutin to hydroquinone and glucose. Hydroquinone to play a role in resistance of pear to fire blight [l3-1.51, and therefore release of this enzyme by the bacterium may also affect protection. Torula yeast RNA may delay symptom expression in a manner unrelated to protection with DNA, such as directly inhibiting E. amylouora or blocking possible active sites for E. amylouora. DNA contamination in the yeast RNA may have also caused this delay. DNA from virulent and avirulent E. amylovora may find practical application as a protectant against fire blight under field conditions. Concentration of DNA was directly related to protection of etiolated seedlings. More permanent protection occurred in etiolated seedlings from which the radicle was removed and the seedlings placed into solutions containing DNA, presumably because this method allowed more uptake and better distribution of DNA, or its degradation products, than did the injection procedure. This study shows that DNA from virulent and avirulent E. amylouora protects Bartlett pear against fire blight, as does DNA from several other bacteria. Mode of action for protection is unknown, but data indicate some specificity for bacterial DNA, and in particular specificity for DNA from E. amylovora. Work in other laboratories [I, 8, 17, 20-23, 351 has indicated uptake and expression of bacterial DNA by plant cells. We are presently exploring the means by which DNA from E. amylovora interacts with the host to protect against the pathogen.
Protection
of Bartlett pear against fire blight
169
The authors wish to thank Dr A. 0. Jackson, Department of Botany and Plant Pathology, Purdue University, for supplying purified virus samples and for valuable advice and assistance during the course of performing these experiments. We also wish to thank Dr Mike Byers, Biology Department, Purdue University, for performing DNA melting curves, and Cita Helkie and Cindy Mayes for technical assistance. REFERENCES 1. ANKER, P., STROUN, M., GAHAN, P., ROSSIER, A. & GUEPPIN, nucleic acids into plant cells and crown gall induction. Systemr, Ed. by L. Ledoux, pp. 187-192. North Holland 2. ARK, P. A. (1937). Variability in the fire blight organism,
H. (1971). Natural release of bacterial In Informative Molecules in Biological Publishing Co., Amsterdam. Erwinia amylovora. Phytopathologr 27,
l-28. 3. BRAKKJZ, M. K. (1971). Degradation of brome mosaic virus and tobacco mosaic virus in bentonite. Virology 46, 575-585. 4. BRAKKE, M. K. & VAN PELT, N. (1970). Linear-log sucrose gradients for estimating sedimentation coefficients of plant viruses and nucleic acids. Anabtical Biochemistry 38, 56-64. 5. CARROL, R. B. & LUKEZIC, F. L. (1972). Induced resistance in alfalfa to Corynebacterium insidiosum by prior treatment with avirulent cells. Phytopathology 62, 555-564. 6. CHATTERJEE, A. K., GIBBONS, L. N. & CARPENTER, F. A. (1968). Some observations on the physiology of Erwinia herbicola and its possible implication as a factor antagonistic to Erw+ia amylovora in the “fire blight” syndrome. Canadian3ournal of Microbiology 15, 640-642. 7. CLAYTON, E. E. (1936). Water soaking of leaves in relation to the development of the wildfire disease in tobacco. Journal of Agricultural Research 52, 239-269. 8. DOY, C. H., GRESSHOF, P. M. & ROLFE, B. G. (1973). Biological and nuclear evidence for the trangenosis of genes from bacteria into plant cells. Proceedings of the .National Academy of Scimce, U.S.A. 70, 723-726. 9. FARABEE, G. J. & LOCKWOOD, J. L. (1958). Inhibition of Erwinia amylovora by bacterium sp isolated from fire blight cankers. Phytojathology 48, 209-2 11. 10. GARDNER, J. M. & Ado, C. I. (1972). Comparative base sequence homologies of the deoxyribonucleic acids of Erwinia species and other Enterobacteriaceae. International 3ourml of Systematic Bacteriology 22, 201-209. 11. GOODMAN, R. N. (1967). The protection of apple stem tissue against Erwinia amylovora infection by avirulent strains and three other bacterial species. Phytojathologr 57, 22-24. 12. GOODMAN. R. N. & SHAFPER. W. H. (1971). nrocedure for evaluating the efficacv ~ , An inoculation 1 of toxicants against Erwinia amylovora. Proceedings Second Workshop Fire Blight Research, Michigan State University. 13. HILDEBRAND, D. C. (1970). Fire blight resistance in Pyrus: hydroquinone formation as related to antibiotic activity. Canadian Journal of Botany 48, 177-181. 14. HILDEBRAND, D. C. & SCHROTH, M. N. (1964). Antibiotic activity of pear leaves against Erwinia amylovora and its relation to 8-glucosidase. Phytopathologv 54, 59-63. 15. HILDEBRAND, D. C. & SCHROTH, M. N. (1964). Arbutin-hydroquinone complex in pear as a factor in fire blight development. Phytofiathology 54, 640-645. 16. JACKSON, B., JACKSON, A. 0. & BRAKKE, M. K. (1973). Pipetting calculations for linear log gradients for Spinco swinging bucket rotors. Special Publication Plant Virology Laboratory, Department of Plant Pathology, University of Nebraska, Lincoln. 17. JOHNSON, C. B., GRIERSON, D. & SMITH, H. (1973). The expression of A plac 5 DNA within cultured cells of a higher plant. Nature, New Biolopv 244, 105-107. 18. KIRBY, K. S. (1965). Isolation and characterization of ribosomal ribonucleic acid. Biochemical 3ourd 96, 266-269. 19. LANGLEY, R. A. & KADO, C. I. (1972). Studies on Agrobacterium tumfaciem: condition for mutogenesis by methyl-N’-nitro-N-nitrosoguanidine and relationships of A. tumefacienr mutants to crown gall tumour induction. Mutation Research 14, 277-286, 20. LEDOUX, L. & HUART, R. (1968). Integration and replication of DNA of M. lysodeikticus in DNA of germinating barley. Nature 218, 1256-1259. 2 1. LEDOUX, L. & HUART, R. ( 1969). Fate of exogenous bacterial DNA in barley seedlings. 3ournal of Molecular Biology 43, 243-262. 22. LEDOUX, L., HUART, R. & JACOB, M. (1971). Fate of exogenous DNA in Arabidopsis thaliani. I. Translocation and integration. European Journal of Biochemistry 23, 96108.
170
J. L. McIntyre,
23. LEDOUX, 24. 25.
26. 27. 28. 29.
30. 31. 32. 33.
34.
35.
J. Kui:
and
E. 6. Williams
L., HUART, R. &JACOB, M. (1971). Fate of rx~~enous DNA in Arabidopsis thaiiani. II. Evidence for replication and preliminary results at the biological level. In Informative Molecules In BiologicalSystems, Ed. by L. Ledoux, pp. 159-175. North Holland Publishing Co., Amsterdam. MARMUR, J. (1961). A procedure for the isolation of deoxyribonucleic acid from microorganisms. Journal of‘ Molecular Biology 3: 208-2 18. MCINTYRE, J. L. (1974). Protection of pear against fire blight by bacteria, bacterial sonicates and bacterial deoxyribonucleic acid. Ph.D. Thesis, Purdue University, Lafayette. MCINTYRE, J. L. & WILLIAB~S, E. B. (1972). Protection of Bartlett pear by avirulent Ew,&io spp. and Pseudomonas tabaci. P&ytopatholog)i 62, 777. MCINTYRE, J. L., KuC, J. & WILLIAMS, E. B. (1973). Protection of pear against fireblight by bacteria and bacterial sonicates. Phytofiathology 63, 872.-877. MILLER, J, H. (1972). Preparation and plaque assay of a phage stock. In Ex@&ments in Molecular Genetics, pp. 37-46. Cold Spring Harbor Laboratory, New York. PARISH, J. H. (1972). Principles and Practice of Experiments zvith Nucleic Acids. Longman Group Ltd, London. ROE, J. H. (1955). The determination of sugar in blood and spinal fluid with anthrone reagent. Journal of Biological Chemistry 212, 335-343. SCHILDKRAUT, C. L., MARMUR, J. & DOTY, P. (1962). Determination of the base composition of deoxyribonucleic acid from its bouyant density in CsCI. Journal of Molecular Biology 4; 430-443. SLEESMAN, H. C., PERLEY, J. E. & HOITINK, H. A. J, (1970). Susceptible and hypersensitive reactions in tobacco and their prevention by cell-free extracts of Pseudomonas tabaci and Pseudomonas glycinea. Phytojathology 60, 13 14. STARR, M. P. & MANDEL, M. (1969). DKA base composition and taxonomy of phytopathogenic bacteria and other Enterobacteria. Journal of General MicrobioloD 56, 113-l 23. STERN, H. (1968). Isolation and purification of plant nucleic acids from whole tissues and from isolated nuclei. In Methods in Enzymology, Ed. by L. Grossman & K. Moldave. XII-B 108: 111. STRAUN, M., ANKER, P., GAHAS, P., ROSSIER, 4. 8i GREPPIX, H. J. (1971). Agrobacterium tumefaciens ribonucleic acid synthesis in tomato cells and crown gall induction. 3oumal of Bacteriology 106,
634-639.