Vol. 178, No. 3, 1991 August 15, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1202-1204
POTENTIAL OF ANIMAL MYELOPEROXIDASE TO PROTECT PLANTS FROM PATHOGENS T. J. Jacks, P. J. Cotty and 0. Hinojosa Southern Regional Research Center P. 0. Box 19687 New Orleans, LA 70179
Received
June
18,
1991
The effect of animal myeloperoxidase (EC 1.I 1.1.7) on the viability of a plant pathogen was determined. Lethality of hydrogen peroxide to germinating spores of Asoeraillus Singlet oxygen was present but hypochlorite flavus increased go-fold enzymically. accounted for two-thirds of the increase. The results indicate myeloperoxidase could improve microbial resistance in plants, perhaps transgenically. 0 1991 Academic Press, Inc.
Both plants and animals generate hydrogen peroxide (H,O,) in response to microbial infection, but only animals are capable of converting peroxide to a deadlier microbicide, hypochlorous acid (HOCI): H,O, + HCI -
PI
H,O + HOCI
Without myeloperoxidase (MPO) that catalyzes the conversion (1) plants lack an effective antimicrobial system that exists in animals. In anticipation of transforming plants with an animal MPO gene to provide ultimately the biosynthetic capability for hypochlorite, we needed to evaluate MPO action on the viability of a plant pathogen.
We chose
Asoeraillus flavus for nonspecific and widely spread virulence (2); it is also of interest as the principal source of the unsurpassed natural carcinogen, aflatoxin (3).
Materials and Methods Sodium hypochlorite was obtained from Aldrich Chemical Co., Milwaukee, WI, and was quantified spectrophotometrically at 292 nm using the molar absorptivity of 350 M” cm-’ (4). H,O, (30%) was from Matheson Coleman & Bell, Not-wood, OH, and was assayed at 230 nm using the molar absorptivity of 67 M” cm” (5). MPO from human polymorphonuclear leukocytes was obtained from Calbiochem Corp., La Jolla, CA. Culture media were from Campbell Soup Co., Camden, NJ, (V-8 vegetable juice) and from Difco Laboratories, Detroit, Ml. All other chemicals were reagent grade.
Abbreviations used: CPM, counts per minute; H,O,, hydrogen peroxide; hypochlorous acid; MPO, myeloperoxidase; ‘O,, singlet molecular oxygen. 0006-291X/91 Copyright All rights
$1.50
0 1991 by Academic Press, k. of reproduction in any form reserved.
1202
HOCI,
Vol.
178, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A. flavus strain 13, described previously (2), was used in all experiments. Stock cultures were maintained at 30 C on 5% V-8 vegetable juice amended with 2% agar. Spore suspensions consisting of about 550 viable spores/ml in potato dextrose broth (pH 5) were gently stirred for 5-6 hr at 27 C to initiate germination. Suspensions were then diluted with oxidant-free or oxidant-containing broths to final concentrations of 10” to 0.1 M HOCI, 1Od to 1 .O M H,O,, and about 500 spores/ml. In experiments with MPO, 0.1 unit of MPO/ml was added before dilutions. All broths contained 0.1 M NaCI. Suspensions were incubated for 1.5 hr at 30 C and surviving spores were assessed by counting colony-forming units on potato dextrose-agar (2). Dimol chemiluminescence of ‘0, and its enhancement by deuterium oxide were determined with a Packard 3255 liquid scintillation spectrometer equipped with red sensitive RCA 31OOOAN photomultiplier tubes and operating in the singlet mode (6). Reactants, initially separated in the counting chamber, were mixed 17 set after counting began. A Packard 260A ratemeter converted the pulse frequency to an analog signal for display on a chart recorder. Reactants were 7.0 to 14.0 mM H,O,, 3.0 mM HOCI and 0.1 unit of MPO/ml. All solutions were made with potato dextrose broth containing 0.1 M NaCI. In studies of chemiluminescent enhancement, deuterium oxide was substituted for water. Results and Discussion H,O,, HOCI and H,O,-MPO
mixtures had dramatic
effects on the viability of A. flavus
measured by colony formation after exposure of germinating
spores to the oxidants (Fig.
1). According to linear portions of mortality curves in Fig. 1, 50% killing of fungal spores was obtained with about 0.3 mM HOCI and with about 0.2 mM H,O, combined
with MPO.
In marked contrast, about 18 mM H,O, was required in the absence of MPO. These results show that HOCI and H,O,-MPO were about 66fold and SO-fold more effective, respectively, than H,O, at killing germinating no microbicidal
spores of A. flavus. MPO without H,O, had
effect.
HOCI, which is generated
in H,O,-MPO
thirds of the increase in microbicidal
mixtures (Equation l), accounted for about twoactivity of H,O, brought about by MPO (Fig. 1). In
addition to HOCI, however, ‘0, might also be produced in H,O,-MPO spontaneous reaction of H,O, with newly formed HOCI (7,8): H,O, + HOCI ->
mixtures, from the
H,O + HCI + ‘0,
PI To ascertain whether ‘0, was being generated, red chemiluminescence of incubation media was measured. A burst of chemiluminescence occurred in H,O,-HOCI mixtures where reactants are consumed instantly, whereas chemiluminescence was produced steadily during the enzymically catalyzed generation of HOCI in H,O,-MPO (Fig. 2). Enhancement by deuterium oxide was about 3-fold and 2-fold for the respective mixtures. Chemiluminescence required two reactants but was absent in HOCI-MPO. These results show ‘0, was being produced in incubation media containing H,O,-MPO during germination of A. flavus spores and consequently accounted for part of the microbicidal activity besides that from enzymically generated HOCI. 1203
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 178, No. 3, 1991
2 t
01 QW
1M OXIDANT
tmolarityl
02
’ T
1
2
MIN
2
4
6
Fia. 1. Killing A. with several concentrations of HOC1 and H,Op, the latter alone and in combinationwith MPO. Symbol sizes approximate S.E. Fio. 2. Redchemiluminescenceof incubationmediaprepared in water or heavy water and containing mixtures of 14.0 mfvl H,O, with either 3.0 mM HOC1 or 0.1 unit MPO/ml: A, H,O,-HOC1 in water; 6, H,O,-HOC1 in heavy water; C, H,Oz-MPO in water; D, H,O,-MPO
in heavy water. Arrow at 17 set marks mixing of H,O, with HOCI or MPO in the spectrometer.
Concentrations of HOC1 effective in killing A. flavu8 (Fig 1) were comparable to that produced by MPO in animal
cells responding to microbial infection (4). Furthermore, amounts of H,O, required for catalysis by MPO (9) are generated in plant cells during
microbially
induced
stress (IO).
These findings suggest
generating
plant cells would produce microbicidal
microbially
infected,
H,O,-
amounts of deadlier HOC1 if an active
catalyst was available. Acquiring MPO such as by genetic transformation might greatly improve the defense capacity of plants.
and expression
References 1. Klebanoff, S. J. (1980) In Mononuclear Phagocytes: Functional Aspects (R. van Furth, Ed.), Pt. II, pp. 1105-1137. Mattinus Nijhoff PublishersBoston, MA. 2. Cotty, P. J. (1989) Phytopathology 79, 808-814. 3. Dvo%Ekov& I. (1990) Aflatoxins and Human Health, 154 pp. CRC Press, Boca Raton, FL. 4. McKenna, S. M., and Davies, K. J. A. (1988) Biochem. J. 254, 885892. 5. Maehly, A. C., and Chance, 6. (1954) Meth. Biochem. Anal. 1, 357-424. 8. Cadenas, E., and Sies, H. (1984) Meth. Enzymol. 105, 221-231. 7. Harrison, J. E., and Schultz, J. (1978) J. Biol. Chem. 251, 1371-1374. 8. Kasha, M., and Khan, A. U. (1970) Ann. N. Y. Acad. Sci. 171, 5-23. 9. Andrews, P. C., and Krinsky, N. I. (1982) J. Biol. Chem. 257, 13240-13245. 10. Apostol, I., Heinstein, P. F., and Low, P. S. (1989) Plant Physiol. 90, 109-118. 1204
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1202-1204
POTENTIAL OF ANIMAL MYELOPEROXIDASE TO PROTECT PLANTS FROM PATHOGENS T. J. Jacks, P. J. Cotty and 0. Hinojosa Southern Regional Research Center P. 0. Box 19687 New Orleans, LA 70179
Received
June
18,
1991
The effect of animal myeloperoxidase (EC 1.I 1.1.7) on the viability of a plant pathogen was determined. Lethality of hydrogen peroxide to germinating spores of Asoeraillus Singlet oxygen was present but hypochlorite flavus increased go-fold enzymically. accounted for two-thirds of the increase. The results indicate myeloperoxidase could improve microbial resistance in plants, perhaps transgenically. 0 1991 Academic Press, Inc.
Both plants and animals generate hydrogen peroxide (H,O,) in response to microbial infection, but only animals are capable of converting peroxide to a deadlier microbicide, hypochlorous acid (HOCI): H,O, + HCI -
PI
H,O + HOCI
Without myeloperoxidase (MPO) that catalyzes the conversion (1) plants lack an effective antimicrobial system that exists in animals. In anticipation of transforming plants with an animal MPO gene to provide ultimately the biosynthetic capability for hypochlorite, we needed to evaluate MPO action on the viability of a plant pathogen.
We chose
Asoeraillus flavus for nonspecific and widely spread virulence (2); it is also of interest as the principal source of the unsurpassed natural carcinogen, aflatoxin (3).
Materials and Methods Sodium hypochlorite was obtained from Aldrich Chemical Co., Milwaukee, WI, and was quantified spectrophotometrically at 292 nm using the molar absorptivity of 350 M” cm-’ (4). H,O, (30%) was from Matheson Coleman & Bell, Not-wood, OH, and was assayed at 230 nm using the molar absorptivity of 67 M” cm” (5). MPO from human polymorphonuclear leukocytes was obtained from Calbiochem Corp., La Jolla, CA. Culture media were from Campbell Soup Co., Camden, NJ, (V-8 vegetable juice) and from Difco Laboratories, Detroit, Ml. All other chemicals were reagent grade.
Abbreviations used: CPM, counts per minute; H,O,, hydrogen peroxide; hypochlorous acid; MPO, myeloperoxidase; ‘O,, singlet molecular oxygen. 0006-291X/91 Copyright All rights
$1.50
0 1991 by Academic Press, k. of reproduction in any form reserved.
1202
HOCI,
Vol.
178, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
A. flavus strain 13, described previously (2), was used in all experiments. Stock cultures were maintained at 30 C on 5% V-8 vegetable juice amended with 2% agar. Spore suspensions consisting of about 550 viable spores/ml in potato dextrose broth (pH 5) were gently stirred for 5-6 hr at 27 C to initiate germination. Suspensions were then diluted with oxidant-free or oxidant-containing broths to final concentrations of 10” to 0.1 M HOCI, 1Od to 1 .O M H,O,, and about 500 spores/ml. In experiments with MPO, 0.1 unit of MPO/ml was added before dilutions. All broths contained 0.1 M NaCI. Suspensions were incubated for 1.5 hr at 30 C and surviving spores were assessed by counting colony-forming units on potato dextrose-agar (2). Dimol chemiluminescence of ‘0, and its enhancement by deuterium oxide were determined with a Packard 3255 liquid scintillation spectrometer equipped with red sensitive RCA 31OOOAN photomultiplier tubes and operating in the singlet mode (6). Reactants, initially separated in the counting chamber, were mixed 17 set after counting began. A Packard 260A ratemeter converted the pulse frequency to an analog signal for display on a chart recorder. Reactants were 7.0 to 14.0 mM H,O,, 3.0 mM HOCI and 0.1 unit of MPO/ml. All solutions were made with potato dextrose broth containing 0.1 M NaCI. In studies of chemiluminescent enhancement, deuterium oxide was substituted for water. Results and Discussion H,O,, HOCI and H,O,-MPO
mixtures had dramatic
effects on the viability of A. flavus
measured by colony formation after exposure of germinating
spores to the oxidants (Fig.
1). According to linear portions of mortality curves in Fig. 1, 50% killing of fungal spores was obtained with about 0.3 mM HOCI and with about 0.2 mM H,O, combined
with MPO.
In marked contrast, about 18 mM H,O, was required in the absence of MPO. These results show that HOCI and H,O,-MPO were about 66fold and SO-fold more effective, respectively, than H,O, at killing germinating no microbicidal
spores of A. flavus. MPO without H,O, had
effect.
HOCI, which is generated
in H,O,-MPO
thirds of the increase in microbicidal
mixtures (Equation l), accounted for about twoactivity of H,O, brought about by MPO (Fig. 1). In
addition to HOCI, however, ‘0, might also be produced in H,O,-MPO spontaneous reaction of H,O, with newly formed HOCI (7,8): H,O, + HOCI ->
mixtures, from the
H,O + HCI + ‘0,
PI To ascertain whether ‘0, was being generated, red chemiluminescence of incubation media was measured. A burst of chemiluminescence occurred in H,O,-HOCI mixtures where reactants are consumed instantly, whereas chemiluminescence was produced steadily during the enzymically catalyzed generation of HOCI in H,O,-MPO (Fig. 2). Enhancement by deuterium oxide was about 3-fold and 2-fold for the respective mixtures. Chemiluminescence required two reactants but was absent in HOCI-MPO. These results show ‘0, was being produced in incubation media containing H,O,-MPO during germination of A. flavus spores and consequently accounted for part of the microbicidal activity besides that from enzymically generated HOCI. 1203
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 178, No. 3, 1991
2 t
01 QW
1M OXIDANT
tmolarityl
02
’ T
1
2
MIN
2
4
6
Fia. 1. Killing A. with several concentrations of HOC1 and H,Op, the latter alone and in combinationwith MPO. Symbol sizes approximate S.E. Fio. 2. Redchemiluminescenceof incubationmediaprepared in water or heavy water and containing mixtures of 14.0 mfvl H,O, with either 3.0 mM HOC1 or 0.1 unit MPO/ml: A, H,O,-HOC1 in water; 6, H,O,-HOC1 in heavy water; C, H,Oz-MPO in water; D, H,O,-MPO
in heavy water. Arrow at 17 set marks mixing of H,O, with HOCI or MPO in the spectrometer.
Concentrations of HOC1 effective in killing A. flavu8 (Fig 1) were comparable to that produced by MPO in animal
cells responding to microbial infection (4). Furthermore, amounts of H,O, required for catalysis by MPO (9) are generated in plant cells during
microbially
induced
stress (IO).
These findings suggest
generating
plant cells would produce microbicidal
microbially
infected,
H,O,-
amounts of deadlier HOC1 if an active
catalyst was available. Acquiring MPO such as by genetic transformation might greatly improve the defense capacity of plants.
and expression
References 1. Klebanoff, S. J. (1980) In Mononuclear Phagocytes: Functional Aspects (R. van Furth, Ed.), Pt. II, pp. 1105-1137. Mattinus Nijhoff PublishersBoston, MA. 2. Cotty, P. J. (1989) Phytopathology 79, 808-814. 3. Dvo%Ekov& I. (1990) Aflatoxins and Human Health, 154 pp. CRC Press, Boca Raton, FL. 4. McKenna, S. M., and Davies, K. J. A. (1988) Biochem. J. 254, 885892. 5. Maehly, A. C., and Chance, 6. (1954) Meth. Biochem. Anal. 1, 357-424. 8. Cadenas, E., and Sies, H. (1984) Meth. Enzymol. 105, 221-231. 7. Harrison, J. E., and Schultz, J. (1978) J. Biol. Chem. 251, 1371-1374. 8. Kasha, M., and Khan, A. U. (1970) Ann. N. Y. Acad. Sci. 171, 5-23. 9. Andrews, P. C., and Krinsky, N. I. (1982) J. Biol. Chem. 257, 13240-13245. 10. Apostol, I., Heinstein, P. F., and Low, P. S. (1989) Plant Physiol. 90, 109-118. 1204
