Plant Physiol.
(1997) 115: 1021-1027
Histone Hyperacetylation in Maize in Response to Treatment
with HC-Toxin or lnfection by the Filamentous Fungus
-
Cochliobolus carbonum’
Richard F. Ransom and Jonathan D. Walton*
Department of Energy-Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1 31 2
HC-toxin, the host-selective toxin produced by the filamentous
fungus Cochliobolus carbonum, inhibits maize (Zea mays L.) histone
deacetylases (HDs) in vitro. Here we show that HDs are also
inhibited by HC-toxin in vivo, as demonstrated by the accumulation
of hyperacetylated forms of the core (nucleosomal) histones H3.1,
H3.2, H3.3, and H4 in both maize embryos and tissue cultures.
Hyperacetylation of H4 and all isoforms of H3 in tissue cultures of
inbred Pr (genotype hm/hm) occurred at 10 ng/mL (23 nM). l h e
effect was host-selective; acetylation of histones i n the near-
isogenic inbred P r l (genotype Hm/Hm) did not occur in tissue
cultures or embryos treated with 0.2 pg/mL or 1O pg/mL HC-toxin,
respectively. Hyperacetylation of histone H4 in embryos of P r l
began to occur at 50 j&nL HC-toxin, and 200 pg/mL HC-toxin
caused equal hyperacetylation in Pr and P r l embryos. Hyperacety-
lated core histones, especially of the isoforms of histone H3, accu-
mulated in leaves of inbred Pr, but not Prl, after infection by
toxin-producing strains of C. carbonum. Accumulation of hyper-
acetylated histones began at 24 h after inoculation, before the
development of visible disease symptoms. Hyperacetylation of H2A
or H2B histones were not detected i n any of the studies. The results
are consistent with H D being a primary site of action of HC-toxin.
The host-selective toxin HC-toxin is a critica1 determi-
nant of virulence in the interaction between the producing
fungus Cochliobolus carbonum and its host, maize (Zea mays
L.) (Walton, 1996). Maize plants that are homozygous re-
cessive at the nuclear Hm locus are susceptible to infection
by HC-toxin-producing (Tox2+) isolates of C. carbonum,
whereas plants of genotype H m / - are 100-fold less sensi-
tive to HC-toxin and develop only small, nonexpanding
lesions when inoculated with Tox2+ isolates. Isolates that
do not produce HC-toxin (Tox2T) produce only small,
nonexpanding lesions regardless of host genotype (Walton
et al., 1997).
HC-toxin is a cyclic tetrapeptide with the structure
cyclo(~-Pro-~-Ala-~-Ala-~-Aeo). HC-toxin production by
C. carbonum is under the control of a complex genetic locus,
called TOX2, that extends over more than 500 kb and
This work was supported by the U.S. Department of Energy,
Division of Energy Biosciences (grant no. DEFG02-91ER20021),
and the National Institutes of Health, Institute of General Medica1
Sciences (grant no. GM45868).
* Corresponding author; e-mail walton8pilot.msu.edu; fax 1-
517-353-91 68.
1021
consists of at least three different duplicated genes that are
unique to Tox2+ isolates (Walton et al., 1997).
The Hm gene, which confers dominant insensitivity to
HC-toxin and, hence, dominant resistance to Tox2+ isolates
of C. carbonum, encodes an NADPH-dependent carbonyl
reductase that detoxifies HC-toxin by reducing the car-
bonyl group of the side chain of Aeo (Johal and Briggs,
1992; Meeley et al., 1992).However, the demonstration that
selective detoxification is the basis of the specificity of
HC-toxin leaves open the question of the mode of action of
HC-toxin, i.e. the mechanism by which the toxin allows the
establishment of a compatible interaction between C. car-
bonum and maize of genotype hmlhm.
We previously demonstrated that HC-toxin inhibits
maize HD activity in vitro, and that it also inhibits partially
purified HD1-A, HD1-B, and HD2. The HDs of yeast Pkysa-
rum polycepkalum and chicken are also inhibited by HC-
toxin in vitro (Brosch et al., 1995). HC-toxin with a reduced
8-carbonyl group is less active against HD, consistent with
the function of Hm as a detoxifying enzyme (Johal and
Briggs, 1992; Meeley et al., 1992).
Although these in vitro results suggest that HD is the site
of action of HC-toxin, they do not establish that HD is
actually inhibited in vivo or during the disease process. We
report here that maize tissue culture cells and embryos of
sensitive (hmlhm)maize accumulate hyperacetylated forms
of histones H3 and H4, but not H2A and H2B, in response
to HC-toxin treatment, and that hyperacetylated H3 and
H4 accumulate in infected plants early in the infection
process, consistent with inhibition of HD by Tox2+ isolates
of C. carbonum during pathogenesis.
MATERIALS A N D M E T H O D S
Treatment of M a i z e (Zea mays 1.) Tissues with HC-Toxin
HC-toxin was prepared as described by Brosch et al.
(1995). The filamentous fungus Cockliobolus carbonum (also
known as Helminthosporium carbonum and Bipolaris zeicola)
lace 1 isolate SBlll (ATTC 90305) was cultured on V8 agar
plates for 7 to 9 d, and a conidiospore suspension in water
was used to inoculate HMT medium (Van Hoof et al.,
1991). After 18 to 21 d of still culture, HC-toxin was puri-
Abbreviations: Aeo, 2-amino-9,10-epoxi-8-oxodecanoicacid;
AUT, acetic acid-urea-Triton X-100; HD, histone deacetylase;
Tox2+, C. carbonum strains that make HC-toxin.
1022 Ransom and Walton
fied from the culture medium by chloroform extraction and
reverse-phase HPLC.
Maize embryos were prepared and treated with HC-
toxin, as described by Brosch et al. (1995). Maize tissue
cultures were obtained from Sheila Maddock and Joyce
Maddox (Pioneer Hi-Bred International, Johnston, IA). The
cultures were established from immature embryos of a
selfed plant of the genotype Hmlhm, which was the product
of a cross between inbreds K61 (hmlhm) and B73 (HmlHm).
From the resulting segregating cultures, one sensitive line
(genotype kmlhm) and one resistant line (genotype HmlHm
or Hmlhm) were selected and used in this study. The tissue
cultures were grown in shake culture in 250-mL flasks
containing 60 mL of Murashige and Skoog medium with
vitamins, 1 pg/mL 2,4-D, and 3% (w/v) SUCat 28OC. The
medium was removed and fresh medium was added every
3 to 4 d, then the tissue cultures were split into two por-
tions when the volume of the settled tissue exceeded
approximately 25 mL. Tissue cultures were treated with
HC-toxin immediately after a regular medium change; HC-
toxin at the appropriate concentration was included in the
fresh medium, and the tissue cultures were incubated for
15 h under normal culture conditions. After treatment, the
medium was removed, and the cells were washed twice
with distilled water, drained, frozen under liquid nitrogen,
and stored at -80°C.
Preparation of lnfected Maize Leaves
Maize plants were grown from seed in pots in the green-
house until the fifth true leaf emerged (approximately
21 d). The near-isogenic inbreds Pr (hmlkm, susceptible)
and Prl (HmlHm, resistant) were used. Plants were sprayed
with a suspension of conidia prepared from plate cultures
of C. carbonum isolate SBlll (5.0 X 105 conidia/mL in 0.1%
Tween 20) using an atomizer. After inoculation plants were
kept at high RH inside polyethylene bags for 18 h and then
grown under normal greenhouse conditions. After photo-
graphing, the central sections of the upper leaves were
collected at various time points after inoculation, frozen
under liquid nitrogen, and stored at -80°C.
Purification and Analysis of Maize Histones
Maize histones were extracted from maize tissues as
described by Waterborg (1990). Histones were isolated by
C-4 reverse-phase chromatography as described by Brosch
et al. (1995). Fractions containing core (nucleosomal) his-
tones were pooled and lyophilized, and the histones and
acetylated isoforms were separated by AUT-PAGE (Water-
borg et al., 1987; Brosch et al., 1995). Typically, 15 pg of
protein was loaded per gel lane. The gels were stained with
colloidal Coomassie brilliant blue G-250 (Neuhoff et al.,
1988). Stained gels were destained for 2 min in 25% (v/v)
methanol to remove surface stain and were stored in 25%
(w / v) ammonium sulfate before scanning. Gels were
scanned on a densitometer (model 300A, Molecular Dy-
namics, Sunnyvale, CA) using ImageQuant version 3.3
software (Molecular Dynamics). The relative amounts of
the acetylated forms of histones H3 and H4 were deter-
Plant Physiol. Vol. 11 5, 1997
mined from the densitometric scan by resolving the over-
lapping Gaussian curves for each band. The triacetylated
form of histone H3.2 and the unacetylated form of histone
3.1 co-migrated in our separations of H3 histones. To en-
sure that the resulting bias emphasized the unacetylated
forms, we made no attempt to determine the contribution
of triacetylated H3.2 to this band and assigned the entire
value to unacetylated H3.1.
RESULTS
Crude histone preparations were separated by C-4
reverse-phase HPLC into three fractions (Fig. 1).The iden-
tity of the histones in each fraction was determined by
subsequent gel electrophoresis and comparison with the
known HPLC and electrophoretic behavior of maize his-
tones (Waterborg et al., 1987, 1989, 1990; Waterborg, 1990,
1991) H2B elutes over the range of 46 to 47% acetonitrile
(fraction I), H2A and H4 elute at 47 to 48.5% acetonitrile
(fraction 11), and the three H3 isoforms elute at 52.5 to
54.5% acetonitrile (fraction 111). Histones H2A and H4 were
subsequently separated from each other by AUT-PAGE
(Waterborg et al., 1990). The migration of acetylated his-
tone forms in AUT-PAGE is retarded relative to the migra-
tion of histone forms with fewer acetyl groups, permitting
the analysis of the relative degree of acetylation of the
histones (Waterborg et al., 1987, 1989).
We determined the effect of HC-toxin on histone acety-
lation in tissue cultures and embryos, and the effect of
infection with a Tox2+ isolate of C. carbonum on histone
acetylation in maize leaves. Maize embryos and tissue
cultures were incubated in the presence of various concen-
trations of HC-toxin, whereas for infection studies the up-
per leaves of maize plants were sprayed with a suspension
of C. carbonum spores and collected at various times after
inoculation.
In treatments lasting 16 h, hyperacetylated histones (i.e.
two or more acetyl groups per molecule) began to accumu-
late in Pr embryos at 2 pg/mL HC-toxin, whereas Prl
embryos only began to accumulate hyperacetylated H4
histones at 50 p g / mL (Fig. 2). The response of H4 histones
60
50
40
RCD
30
2.
rc
20 3
CD
10
O
10 20 30 40 50 I
Elution Time, min
Figure 1. Reverse-phase HPLC separation of semipurified histones.
Solid line, A2,4;dashed line, percent acetonitrile in elution solvent.
I, 11, and 111 indicate fractions collected for histones H2B, H2A and
H4, and H3, respectively.
 Toxin and Infection-Induced Histone Hyperacetylation 1023

A similar analysis of H4 histone acetylation in maize

»**«•**•**
0 2 10 50 200 0 2 10
PM Pr
Figure 2. AUT-PACE of H4 histones from Pr and Pr1 embryos treated
for 16 h with HC-toxin at 0, 2, 10, 50, or 200 ng/mL. Degree of
acetylation is indicated on the right (0, unacetylated; 1, monoacety-
lated; 2, diacetylated; 3, triacetylated; 4, tetraacetylated; and 5,
pentaacetylated).
in Prl embryos to 200 pig/mL HC-toxin is roughly equal to
the response of Pr embryos to 50 ^g/mL (Fig. 2). By
quantitative densitometric analysis of the gel shown in
Figure 2, H4 histone shows a 1.5- to 3-fold increase in the
percentage of tetra- and pentaacetylated H4 in Pr versus
Prl embryos over the range of 2 to 10 jag/mL, and a smaller
differential accumulation of triacetylated H4 over the same
concentration range (Fig. 3A). The differential accumula-
tion of tri-, tetra-, and pentaacetylated H4 histones in Pr
versus Prl disappears at 200 ^ig/mL, the concentration at
which the embryos of the two genotypes accumulate equal
amounts of hyperacetylated forms (Fig. 3A).
45 tissue cultures in response to HC-toxin treatment revealed
44 a differential accumulation of tri- and tetraacetylated H4 in
43 Pr cultures over the range of 0.01 to 0.2 ^g/mL, which are
concentrations approximately 100-fold lower than the con-
41 centrations required to induce a differential response in
40 embryos (Fig. 3B). In a separate experiment both Pr and Prl
tissue cultures accumulated equal amounts of hyperacety-
lated H4 histones after treatment with HC-toxin at concen-
50 200 trations of 1.0 jug/mL or greater (data not shown). Thus,
histone deacetylation in tissue cultures is not only more
sensitive to HC-toxin than it is in embryos, but there is also
a greater differential between Pr and Prl in tissue cultures
(particularly in the amount of triacetylated H4; Fig. 3, A
and B). However, a greater percentage of the acetylated H4
histone is monoacetylated and there is less accumulation of
hyperacetylated H4 in tissue cultures compared with em-
bryos (Fig. 3, A and B).
Hyperacetylated H4 histones also accumulated in Pr
maize leaves infected with a Tox2+ strain of C. carbonum.
Tri-, tetra-, and pentaacetylated H4 histones began to ac-
cumulate 24 h after inoculation (Fig. 3C), which is before
the first appearance of disease symptoms (Fig. 4A). Hyper-
acetylated H4 histones continued to accumulate during the
course of the infection, whereas the percentage of diacety-
lated H4 increased slightly and the amount of monoacety-
lated H4 declined (Fig. 3C). The accumulation of hyper-
acetylated H4 is host selective; hyperacetylated forms of
H4 do not change significantly during the course of the

Figure 3. H4 histone acetylation in maize em-

100
B bryos (A) and tissue cultures (B) as a function of
100r 100,
monoacetylated monoacetylated monoacetylated HC-toxin concentration, and in Pr and Prl
80 801
60
maize leaves as a function of time after infection
60
by a Tox2 + isolate of C. carbonum (C). The data
40 40
in A are derived from the gel shown in Figure 2.
20 20 Histones were isolated and electrophoresed by
<D AUT-PAGE as described in "Materials and
diacetylated c diacetylated diacetylated
30 o 30 Methods," and the portions of the gel containing
.'a 20 the histones were scanned and quantified. Q,
O 20 O 2Q
c Sensitive (Pr) maize; •, near-isogenic-resistant
O I 10
M 10 tn 10 (Prl) maize. Pentaacetylated H4 in maize tissue
± cultures was present but not quantifiable.
triacetylated _ triacetylated
X triacetylated
.£>
T3 10 8 •a
•2 4 V
£•
0
5 S
o ,o 0)
o

.~s
tetraacetylated tetraacetylated tetraacetylated
"5 1.5
1

0.5#
0
pentaacetylated 0 0.05 0.1 0.15 02 pentaacetylated
HC-Toxin [ug/ml]

~So i 3 o T 5 o 200 20 40 60 80 100

HC-Toxin [ug/ml] Time After Infection, h
1024
Figure 4. Time course of infection of Pr (susceptible, right) and Prl
(near-isogenic-resistant, left) maize leaves by a Tox2* isolate of C.
carbonum. A, Twenty-four hours after inoculation; B, 48 h after
inoculation; C, 96 h after inoculation. Plants from this experiment
were used for isolation of histones (see Figs. 3, 7, and 8).
incompatible response of Prl maize leaves to C. carbonum
(Fig. 3C).
Maize has three distinct isoforms of histone H3, referred
to as H3.1, H3.2, and H3.3, which are numbered according
to their relative mobility in AUT-PAGE (Waterborg, 1991).
In our AUT-PAGE separations the triacetylated form of
H3.2 co-migrates with the unacetylated form of H3.1. Be-
cause we were unable to separate this band into its com-
ponents, we assigned its entire value to unacetylated H3.1
and omitted the analysis of triacetylated H3.2. Although
this approximation overestimates the amount of unacety-
lated H3.1 in each of the experiments, the increase in
unacetylated H3.1 is not evident in the comparisons be-
tween the relative amounts of H3.1 histone present in Pr
and Prl tissues because the amount of H3.1 is much greater
than the amount of the other two isoforms.
HC-toxin treatment of embryos and tissue cultures re-
sults in considerable differential accumulation of hyper-
Ransom and Walton Plant Physiol. Vol. 115, 1997
acetylated forms of all three H3 isoforms (Figs. 5 and 6 ). In
contrast to the accumulation of hyperacetylated H4 in these
tissues, in which hyperacetylated forms are never more
than a small (<5%) proportion of the total H4 histone (Fig.
3), di- and triacetylated H3 forms increase from approxi-
mately 20% of the total of each isoform to 50% or more of
the total in Pr tissues after HC-toxin treatment. In each case
the increase in di- and triacetylated isoforms in Pr tissue is
accompanied by a corresponding decrease in the amount of
unacetylated histone, whereas the amount of monoacety-
lated histone remains at approximately the same levels as
in Prl tissue or decreases slightly. The relative sensitivity of
H3 hyperacetylation in tissue cultures compared with
embryos is similar to that seen in the analysis of H4 hy-
peracetylation, i.e. tissue cultures accumulate di- and tri-
acetylated H3 at HC-toxin concentrations approximately
100-fold less than are required to induce a similar accumu-
lation in embryos (Fig. 5). The concentrations of HC-toxin
required to induce both Pr and Prl tissues to accumulate
equal amounts of hyperacetylated histones is also similar
for both H3 and H4 histones; hyperacetylated H3.1, H3.2,
and H3.3 accumulate in equal amounts in Pr and Prl
embryos at 200 jag/mL HC-toxin and in tissue cultures at 1
(xg/mL (data not shown).
Before infection in Pr leaves, and both before and during
the course of infection in Prl leaves, the three H3 isoforms
were primarily unacetylated or monoacetylated, whereas
in infected Pr leaves di- and triacetylated H3 forms were
increasingly evident as the infection proceeded (Figs. 7 and
8). In Pr leaves at 48 and 96 h after inoculation, when
disease symptoms are severe (Fig. 4), the majority of the H3
histones were either di- or triacetylated, whereas the
amount of unacetylated H3 histone decreased to less than
one-half of the amount present in uninfected leaves (Figs. 7
and 8). At 108 to 120 h after inoculation, the infected Pr
leaves were dead and desiccated, whereas the Prl leaves
remained essentially as shown in Figure 4C.
We detected no effect of HC-toxin on acetylation of
histones H2A and H2B in maize embryos (Fig. 9), in tissue
cultures (data not shown), or in leaves in response to
C. carbonum infection (data not shown).
DISCUSSION
HC-toxin causes maize embryos and tissue cultures to
accumulate hyperacetylated forms of the core histones H3
and H4. Histone hyperacetylation occurs in a host-selective
manner during infection of Pr (genotype hm/hm) maize, but
not Prl maize (genotype Hm/Hm), by a Tox2+ isolate of C.
carbonum. These results are consistent with earlier work
showing that in vitro maize HDs are inhibited by HC-toxin,
and support the hypothesis that HD is the biologically
significant site of action of HC-toxin (Brosch et al., 1995).
In planta accumulation of hyperacetylated histones be-
gins approximately 24 h after inoculation, which is before
the appearance of macroscopic disease symptoms. Further-
more, necrosis in the incompatible interaction, even when
macroscopically visible because of the high inoculation
density used, does not induce histone hyperacetylation. Its
early induction during the pathogenesis process, as well as
 Toxin and Infection-lnduced Histone Hyperacetylation 1025

A B C Figure 5. H3 histone acetylation in Pr (O)and

60 80 Prl (O) maize embryos in response to HC-toxin.
A, Histone H3.1; B, histone H3.2; and C, his-
60
40 tone H3.3. Triacetylated H3.2 is not shown be-
40 cause it migrates.at the same position as unac-
20 20
a, 2o
etylated H3.1 (see "Materials and Methods").
C unacetylated
c
.- I monoacetylated I
I

=-xm
0 40
C 40
3
$ 20 20
c
-7 '?
P
2 s

40H
9 diacetylated

20 s 420 0 ! 4 4
I I
O 2 4 6 8 1 0
HC-toxin [pg/ml]
4 4 I
20

'
0 2 4 6 8 1 0
HC-toxin [pglml] HC-toxin [pglml]

its lack of correlation with tissue necrosis, argues that
histone hyperaccumulation is not a secondary effect of
pathogenesis.
Hyperacetylation in embryos of inbred Pr occurs at an
HC-toxin concentration of approximately 0.5 to 2 p g / mL,
which is similar to toxin concentrations that affect other
physiological processes, such as root growth, nitrate up-
take, chlorophyll synthesis, and chloroplast redistribution
in protoplasts (Yoder and Scheffer, 1973; Walton et al.,
1982; Rasmussen and Scheffer, 1988; Wolf and Earle, 1991).
However, hyperacetylation of H4 and a11 three isoforms of
H3 in tissue cultures is much more sensitive to HC-toxin,
being affected at concentrations as low as 10 ng/mL. To the
best of our knowledge, this is the most sensitive plant
response to HC-toxin yet described. (Mammalian cells,
parasitic protozoa, and their HDs are sensitive to HC-toxin
at 4 to 30 ng/mL [Walton et al., 1985; Darkiri-Rattray et al.,
19961.) The reason for the greater sensitivity of tissue cul-
tures compared with embryos or other tissues is unknown,
but might be attributable to tissue cultures having an es-
pecially low background leve1 of carbonyl reductases other
than HC-toxin reductase that are capable of reducing HC-
toxin. Earlier we showed that Pr (hmlhm) maize seedlings
have a low but detectable ability to reductively detoxify

Figure 6. H 3 histone acetylation in Pr (O)

and
A B C Prl (O) maize tissue cultures in response to
80 80
HC-toxin. After HC-toxin treatment, histones
60 60 were extracted and separated by AUT-PAGE. A,
40 40 Histone H3.1; B, histone H3.2; and C, histone
20 20 H3.3. Triacetylated H3.2 is not shown because
c it migrates at the same position as unacetylated
O
H3.1 (see "Materials and Methods").
60 60
a,
2
3 40
C
O 40
I
u) .-
v)
i 20 I 20
v
m
r
60 60
m
6ol diacetylated
I
c
8
8
40

20

triacetylated 2
40L4
20

- O0
HC-Toxin [pg/ml] 60

20

oO 0.05 0.1 0.15

HC-Toxin [pg/ml]
0.2
zow
OO 0.05 0.1 0.15

HC-Toxin [pg/ml]
0.2
1026 Ransom and Walton Plant Physiol. Vol. 115, 1997

H3.1 H2B

1 H3.2

Pr
H3.3
H2A
III i
'WMWM iii
ii
0 2 10 50 200 0 2 10 50 200
Figure 7. AUT-PAGE separation of H3 histones from Pr and Pr1
maize leaves infected with Tox2 + C. carbonum 0, 12, 24, 48, or 96 h Pr1 Pr
after inoculation. The positions of the three H3 isoforms (H3.1, H3.2, Figure 9. AUT-PAGE separation of histones H2B (top) and H2A
and H3.3) and their acetylated forms are indicated at the right. (bottom) from Pr and Pr1 embryos treated for 16 h with HC-toxin at
0, 2, 10, 50, or 200 jig/ml.

HC-toxin (Meeley and Walton, 1991; Meeley et al., 1992).

Alternatively, the increased sensitivity of tissue cultures
might be attributable to more efficient uptake of the toxin
by cells compared with intact, complex tissues.
Hyperacetylated H3 and H4 histones accumulate in sus-
ceptible maize leaves infected with a Tox2+ isolate of C.
carbonum at levels equal to or greater than those observed
in embryos treated with high (50-200 jug/mL) concentra-
tions of HC-toxin. At these toxin concentrations, Prl (Hml
Hm) embryos accumulate both hyperacetylated H3 and H4
histones, but no accumulation of hyperacetylated histones
was observed in Prl leaves even after prolonged infection.
Two possible explanations for this are: (a) the quantities of
toxin present in the incubation medium are sufficient to
saturate the detoxification capacity of the Hm gene product
(HC-toxin reductase) in Prl embryos but not in Prl leaves,
and (b) the limited development of C. carbonum in Prl
leaves compared with Pr leaves results in much lower
amounts of HC-toxin production in Prl than in Pr leaves.
In toxin-treated embryos and tissue cultures and in in-
fected leaves the acetylated forms of the core histones that
accumulate have at least two acetylations per histone mol-
ecule (which we refer to throughout the text as hyperacety-
lated). In most instances the amount of monoacetylated H3
histone decreases with increasing toxin concentration or
time of infection, and there is consistently a large decrease
(>50%) in the amount of unacetylated H3. The decrease in
monoacetylated H4 is smaller (<10%), and there is no
detectable decrease in the amount of unacetylated H4 (data
not shown), although the relative amount of unacetylated
H4 is never less than 75% of the total in any of the analyses
and, therefore, small decreases in the amount of the unac-
etylated form may not be detectable. There is no observable
effect on acetylation of H2A and H2B in embryos, tissue
cultures, or during infection. The differential response of
the different histones could be because of differential sen-
sitivities of the different HDs responsible for deacetylation
of the different histone classes or to different rates of turn-
over of the acetyl groups. However, because all three iso-
forms of maize HD are approximately equally sensitive to
HC-toxin (Brosch et al., 1995), we favor the hypothesis that
the differential effects of HC-toxin on H3 and H4 hyper-
acetylation are attributable to different rates of turnover of
the acetyl groups on these two histone classes.
In contrast to our findings that hyperacetylation sensi-
tivity progresses in the order H2A = H2B « H4 < H3.1 =
H3.2 = H3.2, Kijima et al. (1993) found that trapoxin, an
HC-toxin analog with the structure cyclo(L-Phe-L-Phe-o-
pipecolyl-L-Aeo), induces hyperacetylation of H4 more

Figure 8. H3 histone acetylation in Pr (D) and

Pr1 (•) maize leaves in response to infection by
an HC-toxin-producing isolate of C. carbonum. unacetylated unacetylated
Histones were extracted from infected leaves at
the indicated times after inoculation and sepa-
rated by AUT-PAGE. The portions of the gel
containing the histones were scanned and quan-
tified. A, Histone H3.1; B, histone H3.2; and C, monoacetylated monoacetylated
histone H3.3. The percentage of un-, mono-, di-,
or triacetylated isoforms is plotted as a percent-
±
age of the total of all acetylated and unacety- co
lated isoforms. Triacetylated H3.2 is not shown co
diacetylated diacetylated
because it migrates at the same position as un-
"ro
acetylated H3.1 (see "Materials and Methods").

triacetylated "0 20 40 60 80 100 triacetylated

Time After Infection, h

0 20 40 60 80 100 0 20 40 60 80 100

Time After Infection, h Time After Infection, h

 Toxin and Infection-lnduced Histone Hyperacetylation
strongly t h a n H3 i n m o u s e a n d human cells. Furthermore,
t h e effect of trapoxin on H 4 acetylation i n m a m m a l i a n cells
is m o r e drastic t h a n t h e effect of HC-toxin on H 4 acetyla-
tion in plant cells, because i n treated m a m m a l i a n cells
tetraacetylated H 4 becomes t h e predominant form. Tricho-
statin, a n HD inhibitor that is chemically unrelated t o
HC-toxin a n d trapoxin, also causes preferential hyper-
acetylation of H 4 over H3 i n mammalian cells (Yoshida e t
al., 1990). T h e dramatic differences i n the relative a m o u n t s
of acetylated H3 a n d H 4 histones that accumulate in plants
versus animal cells after treatment w i t h these c o m p o u n d s
m a y be attributable t o t h e different chemistries of trapoxin
a n d HC-toxin, resulting i n differential inhibition of t h e
HDs that deacetylate H3 versus H4. Alternatively, t h e
acetyl g r o u p s of H3 a n d H 4 m i g h t h a v e different turnover
rates in plants versus animal cells. ,
In b o t h mammalian a n d plant tissues trapoxin and HC-
toxin h a v e little effect on acetylation of H 2 A a n d H2B
(Yoshida e t al., 1990; Kijima e t al., 1993). This is probably
n o t because H 2 A and H2B a r e deacetylated b y a different,
HC-toxin-insensitive HD, because all known forms of
maize H D a r e sensitive t o HC-toxin (Brosch e t al., 1995;
Lusser e t al., 1997).
The evidence presented h e r e provides further s u p p o r t
for the hypothesis that HC-toxin acts t o promote infection
of maize of genotype hmlhm by Tox2+ isolates of C. carbo-
num by inhibiting HD a n d thereby causing t h e accumula-
tion of hyperacetylated core (nucleosomal) histones. Al-
t h o u g h histones are critica1 for chromatin structure,
interference w i t h t h e process of reversible histone acetyla-
tion h a s surprisingly moderate effects on overall gene ex-
pression (Rundlett e t al., 1996). The expression of fewer
t h a n 2% of t h e genes in h u m a n lymphoid cells is affected
by inhibition of HD (Van Lint e t al., 1996). w i t h regard t o
the role of HC-toxin i n allowing t h e development of a
compatible disease interaction, it is plausible that, by in-
hibiting HD, HC-toxin interferes w i t h t h e proper expres-
sion of a subset of genes necessary for maize t o mount a n
effective defense against C. cavbonum (Brosch e t al., 1995).
ACKNOWLEDCMENTS
We thank Sheila Maddock and Joyce Maddox (Pioneer Hi-Bred
International, Johnston, IA) for the generous gift of the maize
tissue cultures, Peter Loidl (University of Innsbruck, Austria) for
scientific discussions, and Jakob Waterborg (University of
Missouri-Kansas City) for invaluable technical advice on acety-
lated histone purification and analysis.
Received April 4, 1997; accepted July 29, 1997.
Copyright Clearance Center: 0032-0889/97/ 115/ 1021/07.
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