Inhibition of the K+-stimulated ATPase of maize root microsomes by Helminthosporium maydis Race T pathotoxin

BIOCHEMICAL

Vol. 51, No. 3,1973

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

INHIBITION OF THE K+-STIMULATEDATPase OF FAIZE ROOT MICROSOMES BY HELMINTHOSPORIUM MAYDIS RACET PATHOTOXIN*

Carl L. Tipton,

Mohammad H. Mondal and John Uhlig

Department of Biochemistry and Biophysics Iowa State University,

Ames, Iowa 50010

Received February 12,1973 SUMMARY : The microsomes obtained by centrifugation between 12,000 g and 80,000 g of an homogenate of maize root tips contain K+-stimulated ATPase Pre-incubation of the microsomes from roots of cytoplasmic male activity. sterile (cmsT) plants with partially urified pathotoxin from Helminthosporium maydis Race T strongly inhibits the KP stimulation; the sametreatment of microsomes from normal roots has no effect. We suggest this inhibition is closely related to the mode of action of H. maydis Race T pathotoxin. -

INTRODUCTION The southern leaf blight

of corn, which reached epiphytotic

proportions

in the United States in 1970, is caused by a fungus, Helminthosporium maydis Nisikado and Miyaka Race T, which selectively cytoplasmic gene cmsT for male sterility H. maydis race T, produce pathotoxins symptoms (2). identical

(1).

specificity

others.

(2), Periconia circinata

pathotoxin

Miller

pathotoxins

changes, resulting produced by -H.

of the pathotoxins with plant cells

has

and Koeppe (7) observed that the H, maydis race T

caused swelling and other changes in mitochondria

although mitochondria from normal plants were unaffected,

from cmsT plants,

but this cannot

*Supported by USDA, CSRSSpecial Grant, Project 117-15-06. Journal Paper No. J-7517 of the Iowa Agriculture and HomeEconomics Experiment Stat&on. Project No. 1914.

Copyright 0 1973 by Academic Press, Inc. All rights of reproduction in any form reserwd

is

(4) and H. maydis race T (5,6) and perhaps

The nature of the interaction

not been determined.

producing disease

of the pathotoxin

Membranepermeability

in ion leakage, are caused by the host-specific victoriae

Several plant pathogens, including

that damageplant cells,

In somecases the host-plant

to that of the pathogen (3).

attacks maize plants bearing the

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

account for the rapid change in membranepermeability This communication is to report

caused by the pathotoxin.

that the -H. maydis Race T pathotoxin

inhibits

the K+-dependent ATPase from roots of Tcms maize plants but has no effect

on

the enzyme from normal plants. MATERIALSANDMETHODS Ethyl acetate-soluble (1,6) and purified

pathotoxin

further

was produced as described previously

by thin-layer

gel GF254

chromatography on silica

(E. Merck, Darmstadt, Germany) with a solvent composedof chloroform: 9:l v/v. acetate, ethyl

The portion yielding

of the plates from Rf 0 to 0.4 was eluted with ethyl

a partially

acetate solution.

redissolved

purified

pathotoxin

which was stored at -20" in

Before use, the solvent was evaporated and the residue

in water to a volume corresponding to the original

from which that aliquot was stimulation

of ion

maize variety

methanol

was derived. leakage

from

The pathotoxin roots

assay used in purification

of cmsT plants

B37, with normal (N) or male-sterile

culture volume

(6).

Seed of the

(T) cytoplasm was obtained

from Dr. Peter A. Peterson, Department of Agronomy, Iowa State University. The seeds were germinated and cell essentially

fractions

from root tips were prepared

as described by Leonard and Hansen (8) except that Na2-EDTAwas

used in the solution

in which the roots were ground.

Assay for ATPase activity

was conducted as described by Leonard and Hansen (8) with minor modification. RESULTSANDDISCUSSION K+-stimulated ATPase activity microsomes) from maize root tips.

is found in the 80,000 pellet, The corresponding fraction

(designated

from oat roots

has been shown to contain fragments of plasma membraneand the Kf-stimulated ATPase is strongly

implicated

the microsomal preparation

in K+ transport

from maize roots strongly

completely abolished, K'-stimulated inhibition

is actually

tion is indicated

(9).

ATPase activity

due to the pathotoxin

by the specificity,

Addition inhibited, (Table I).

of pathotoxin

to

and sometimes That the

and not to impurities

in the prepara-

since ATPase from N plants is unaffected.

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BIOCHEMICAL

Vol.51,No.3,1973

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Effect maize

on k-stimulated

of F. maydis Race T pathotoxin root microsomes.

ATPase

activity

of

___K+-stimulated ATPase activity Pi, p moles/hr/g fresh ----tissue Cytoplasm

Pathotoxin

N N

+

T T

Experiment Number II III ----- IV

I

+

V

2.60

2.41

2.41

2.60

2.30

2.28

3.08

2.00

2.43

2.41

2.50

0.00

0.73

0.27

0.39

0.00

-~I

Reaction mixtures contained 0.75 u moles ATP (Tris salt, pH 6.8); 0.875 u moles MgC12, when added; 12.5 u moles KCl, when added; from 4.0 to 9.0 u moles 90 ug protein, in a final Tris-EC1 buffer, pH 6.8 and enzyme, approximately Incubation was at 30C for 30 min. volume of 1.5 ml. In addition, assay)

toxin

by being

--in vacua

dissolved

results

and inactivates plants

the cells

are

which

unable

in water,

to maintain

leakage

toxin

work ATPase

activity

then

evaporated

on the

ATPase

from

ensue.

of this

located

in

or something is

ion

leakage

to dryness activity.

consistent

after

exposure

is

that

either

inhibition

of the toxin

with

the rapidity

to toxin

(6).

cytoplasmic the

gene

K"-

it.

the differences

Use of the

727

the

membrane, with

As a result, K+ concentration

is

associated

purification

sterility.

intracellular

finding

to determine

N and T plants.

male

This

the plasma

closely

in progress

to facilitate

for

can be detected

implication

ATPase

gene

the necessary

alterations

cmsT has a gene product

dependent

v/v,

has no effect

cmsT cytoplasmic

metabolic

An interesting

Further

(as shown by the root

in CHC13:AcOH 98:2

the

electrolyte

stimulated

inactivated

suggest that the -H. maydis race T pathotoxin combines with + a K transport system in the plasma membrane of root cells from

having

and extensive with

has been

and redissolved

These

maize

that

also

between

the K"-

as an assay is being

for

investigated.

Vol. 51, No. 3, 1973

8lOCHEMfCAL

AND BIOPHYSKAL

RESEARCH COMMUNICATIONS

ACKNOWLEDGEMENI: Wewish to thank Dr. C. A. Martinson for the H. maydis cultures from which the pathotoxin

was extracted

and for helpful

discussions.

REFERENCES

(1) (2)

(3 (4) (5) (6) (7) (8)

(9)

Turner, M. T. and Martinson, C. A., Plant Dis. Rep. 56, 29 (1972). Owens, L. D., Science 165, 18 (1969).Scheffer, R. P. and Samaddar, K. R., Recent Advances g Phytochemistty 2, 123 (1970). Gardner, J. M., Mansour, I. S. and Scheffer, R. P., Physiol. -Plant Pathol. 2, 197 (1972). Gracen, V. E., Grogan, C. 0. and Forster, M. J., ---Can. J. Bot. 50, 2167 (1972). Halloin, J. M., Comstock, J. C., Martinson, C. A. and Tipton, C. L., Phytopathology in press (1973). Miller, R. .I. Lnd Koeppe, D. E., Science 173, 67 (1971). Leonard, R. T. and Hansen, J. B., Plant Physiol. 49-, 436 (1972). Hodges, T. K., Leonard, R. T., Bracker, C. E. and Keenan, T. W., Proc. Nat. Acad. Sci. U.S.A. 69 3307 (1972). ---e-s

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