histone hyperacetylation maize response treatment with hc-toxin by

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 in the near- isogenic inbred Pr l (genotype Hm/Hm) did not occur in tissue cultures or embryos treated with 0.2 pg/mL or 1 O pg/mL HC-toxin, respectively. Hyperacetylation of histone H4 in embryos of Pr l began to occur at 50 j&nL HC-toxin, and 200 pg/mL HC-toxin caused equal hyperacetylation in Pr and Pr l embryos. Hyperacety- lated core histones, especially of the isoforms of histone H3, accumulated in leaves of inbred Pr, but not Prl, after infection by toxin-producing strains of C. carbonum. Accumulation of hyperacetylated histones began at 24 h after inoculation, before the development of visible disease symptoms. Hyperacetylation of H2A or H2B histones were not detected in 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 sensitive 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.

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 carbonyl 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. carbonum 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 METHODS

Treatment of Ma ize (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-oxodecanoic acid; AUT, acetic acid-urea-Triton X-100; HD, histone deacetylase; Tox2+, C. carbonum strains that make HC-toxin.

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1022 Ransom and Walton Plant Physiol. Vol. 11 5, 1997

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) SUC at 28OC. The medium was removed and fresh medium was added every 3 to 4 d, then the tissue cultures were split into two portions 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 greenhouse 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) histones 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-

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 histones (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 histone forms in AUT-PAGE is retarded relative to the migration 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 acetylation 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 concentrations of HC-toxin, whereas for infection studies the upper 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 accumulate in Pr embryos at 2 pg/mL HC-toxin, whereas Prl embryos only began to accumulate hyperacetylated H4 histones at 50 pg/ mL (Fig. 2). The response of H4 histones

10 20 30 40 50 Elution Time, min

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Toxin and Infection-Induced Histone Hyperacetylation 1023

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in Prl embryos to 200 pig/mL HC-toxin is roughly equal tothe response of Pr embryos to 50 ^g/mL (Fig. 2). Byquantitative densitometric analysis of the gel shown inFigure 2, H4 histone shows a 1.5- to 3-fold increase in thepercentage of tetra- and pentaacetylated H4 in Pr versusPrl embryos over the range of 2 to 10 jag/mL, and a smallerdifferential accumulation of triacetylated H4 over the sameconcentration range (Fig. 3A). The differential accumula-tion of tri-, tetra-, and pentaacetylated H4 histones in Prversus Prl disappears at 200 ^ig/mL, the concentration atwhich the embryos of the two genotypes accumulate equalamounts of hyperacetylated forms (Fig. 3A).

A similar analysis of H4 histone acetylation in maizetissue cultures in response to HC-toxin treatment revealeda differential accumulation of tri- and tetraacetylated H4 inPr cultures over the range of 0.01 to 0.2 ^g/mL, which areconcentrations approximately 100-fold lower than the con-centrations required to induce a differential response inembryos (Fig. 3B). In a separate experiment both Pr and Prltissue cultures accumulated equal amounts of hyperacety-lated H4 histones after treatment with HC-toxin at concen-trations of 1.0 jug/mL or greater (data not shown). Thus,histone deacetylation in tissue cultures is not only moresensitive to HC-toxin than it is in embryos, but there is alsoa greater differential between Pr and Prl in tissue cultures(particularly in the amount of triacetylated H4; Fig. 3, Aand B). However, a greater percentage of the acetylated H4histone is monoacetylated and there is less accumulation ofhyperacetylated H4 in tissue cultures compared with em-bryos (Fig. 3, A and B).

Hyperacetylated H4 histones also accumulated in Prmaize 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 beforethe first appearance of disease symptoms (Fig. 4A). Hyper-acetylated H4 histones continued to accumulate during thecourse 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 ofH4 do not change significantly during the course of the

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Figure 3. H4 histone acetylation in maize em-bryos (A) and tissue cultures (B) as a function ofHC-toxin concentration, and in Pr and Prlmaize leaves as a function of time after infectionby a Tox2+ isolate of C. carbonum (C). The datain A are derived from the gel shown in Figure 2.Histones were isolated and electrophoresed byAUT-PAGE as described in "Materials andMethods," and the portions of the gel containingthe histones were scanned and quantified. Q,Sensitive (Pr) maize; •, near-isogenic-resistant(Prl) maize. Pentaacetylated H4 in maize tissuecultures was present but not quantifiable.



1024 Ransom and Walton Plant Physiol. Vol. 115, 1997

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 afterinoculation; C, 96 h after inoculation. Plants from this experimentwere 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, referredto as H3.1, H3.2, and H3.3, which are numbered accordingto their relative mobility in AUT-PAGE (Waterborg, 1991).In our AUT-PAGE separations the triacetylated form ofH3.2 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 H3.1and omitted the analysis of triacetylated H3.2. Althoughthis approximation overestimates the amount of unacety-lated H3.1 in each of the experiments, the increase inunacetylated H3.1 is not evident in the comparisons be-tween the relative amounts of H3.1 histone present in Prand Prl tissues because the amount of H3.1 is much greaterthan the amount of the other two isoforms.

HC-toxin treatment of embryos and tissue cultures re-sults in considerable differential accumulation of hyper-

acetylated forms of all three H3 isoforms (Figs. 5 and 6 ). Incontrast to the accumulation of hyperacetylated H4 in thesetissues, in which hyperacetylated forms are never morethan 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 ofthe total in Pr tissues after HC-toxin treatment. In each casethe increase in di- and triacetylated isoforms in Pr tissue isaccompanied by a corresponding decrease in the amount ofunacetylated histone, whereas the amount of monoacety-lated histone remains at approximately the same levels asin Prl tissue or decreases slightly. The relative sensitivity ofH3 hyperacetylation in tissue cultures compared withembryos 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 approximately100-fold less than are required to induce a similar accumu-lation in embryos (Fig. 5). The concentrations of HC-toxinrequired to induce both Pr and Prl tissues to accumulateequal amounts of hyperacetylated histones is also similarfor both H3 and H4 histones; hyperacetylated H3.1, H3.2,and H3.3 accumulate in equal amounts in Pr and Prlembryos 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 duringthe course of infection in Prl leaves, the three H3 isoformswere primarily unacetylated or monoacetylated, whereasin infected Pr leaves di- and triacetylated H3 forms wereincreasingly evident as the infection proceeded (Figs. 7 and8). In Pr leaves at 48 and 96 h after inoculation, whendisease symptoms are severe (Fig. 4), the majority of the H3histones were either di- or triacetylated, whereas theamount of unacetylated H3 histone decreased to less thanone-half of the amount present in uninfected leaves (Figs. 7and 8). At 108 to 120 h after inoculation, the infected Prleaves were dead and desiccated, whereas the Prl leavesremained essentially as shown in Figure 4C.

We detected no effect of HC-toxin on acetylation ofhistones H2A and H2B in maize embryos (Fig. 9), in tissuecultures (data not shown), or in leaves in response toC. carbonum infection (data not shown).

DISCUSSION

HC-toxin causes maize embryos and tissue cultures toaccumulate hyperacetylated forms of the core histones H3and H4. Histone hyperacetylation occurs in a host-selectivemanner during infection of Pr (genotype hm/hm) maize, butnot Prl maize (genotype Hm/Hm), by a Tox2+ isolate of C.carbonum. These results are consistent with earlier workshowing that in vitro maize HDs are inhibited by HC-toxin,and support the hypothesis that HD is the biologicallysignificant 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 beforethe appearance of macroscopic disease symptoms. Further-more, necrosis in the incompatible interaction, even whenmacroscopically visible because of the high inoculationdensity used, does not induce histone hyperacetylation. Itsearly induction during the pathogenesis process, as well as www.plantphysiol.orgon January 3, 2019 - Published by Downloaded from

Copyright © 1997 American Society of Plant Biologists. All rights reserved.


Toxin and Infection-lnduced Histone Hyperacetylation 1025

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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 uptake, 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 cultures compared with embryos or other tissues is unknown, but might be attributable to tissue cultures having an especially 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

A B Figure 6. H 3 histone acetylation in Pr (O) and Prl (O) maize tissue cultures in response to HC-toxin. After HC-toxin treatment, histones were extracted and separated by AUT-PAGE. A, Histone H3.1; B, histone H3.2; and C, histone H3.3. Triacetylated H3.2 is not shown because it migrates at the same position as unacetylated H3.1 (see "Materials and Methods").

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1026 Ransom and Walton Plant Physiol. Vol. 115, 1997

H3.1

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Figure 7. AUT-PAGE separation of H3 histones from Pr and Pr1maize leaves infected with Tox2+ C. carbonum 0, 12, 24, 48, or 96 hafter inoculation. The positions of the three H3 isoforms (H3.1, H3.2,and H3.3) and their acetylated forms are indicated at the right.

HC-toxin (Meeley and Walton, 1991; Meeley et al., 1992).Alternatively, the increased sensitivity of tissue culturesmight be attributable to more efficient uptake of the toxinby 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 observedin embryos treated with high (50-200 jug/mL) concentra-tions of HC-toxin. At these toxin concentrations, Prl (HmlHm) embryos accumulate both hyperacetylated H3 and H4histones, but no accumulation of hyperacetylated histoneswas observed in Prl leaves even after prolonged infection.Two possible explanations for this are: (a) the quantities oftoxin present in the incubation medium are sufficient tosaturate 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 Prlleaves compared with Pr leaves results in much loweramounts 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 thataccumulate 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 H3histone decreases with increasing toxin concentration or

H2B

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time of infection, and there is consistently a large decrease(>50%) in the amount of unacetylated H3. The decrease inmonoacetylated H4 is smaller (<10%), and there is nodetectable decrease in the amount of unacetylated H4 (datanot shown), although the relative amount of unacetylatedH4 is never less than 75% of the total in any of the analysesand, therefore, small decreases in the amount of the unac-etylated form may not be detectable. There is no observableeffect on acetylation of H2A and H2B in embryos, tissuecultures, or during infection. The differential response ofthe different histones could be because of differential sen-sitivities of the different HDs responsible for deacetylationof 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 toHC-toxin (Brosch et al., 1995), we favor the hypothesis thatthe differential effects of HC-toxin on H3 and H4 hyper-acetylation are attributable to different rates of turnover ofthe 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, anHC-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) andPr1 (•) maize leaves in response to infection byan HC-toxin-producing isolate of C. carbonum.Histones were extracted from infected leaves atthe indicated times after inoculation and sepa-rated by AUT-PAGE. The portions of the gelcontaining the histones were scanned and quan-tified. A, Histone H3.1; B, histone H3.2; and C,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-lated isoforms. Triacetylated H3.2 is not shownbecause it migrates at the same position as un-acetylated H3.1 (see "Materials and Methods").

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Toxin and Infection-lnduced Histone Hyperacetylation 1027

strongly than H3 i n mouse a n d human cells. Furthermore, the effect of trapoxin on H 4 acetylation i n mammalian cells is more drastic than the effect of HC-toxin on H 4 acetylation in plant cells, because i n treated mammalian cells tetraacetylated H 4 becomes the predominant form. Tricho- statin, a n HD inhibitor that is chemically unrelated to HC-toxin and trapoxin, also causes preferential hyperacetylation of H 4 over H3 i n mammalian cells (Yoshida et al., 1990). The dramatic differences i n the relative amounts of acetylated H3 and H 4 histones that accumulate in plants versus animal cells after treatment with these compounds may be attributable to the different chemistries of trapoxin a n d HC-toxin, resulting i n differential inhibition of the HDs that deacetylate H3 versus H4. Alternatively, the acetyl groups of H3 and H 4 might have different turnover rates in plants versus animal cells. ,

In both mammalian and plant tissues trapoxin and HC- toxin have little effect on acetylation of H2A a n d H2B (Yoshida et al., 1990; Kijima et al., 1993). This is probably not because H2A and H2B are deacetylated by a different, HC-toxin-insensitive HD, because all known forms of maize H D are sensitive to HC-toxin (Brosch e t al., 1995; Lusser e t al., 1997).

The evidence presented here provides further support for the hypothesis that HC-toxin acts to promote infection of maize of genotype hmlhm by Tox2+ isolates of C. carbonum by inhibiting HD and thereby causing the accumulation of hyperacetylated core (nucleosomal) histones. Al- though histones are critica1 for chromatin structure, interference with the process of reversible histone acetylation has surprisingly moderate effects on overall gene expression (Rundlett e t al., 1996). The expression of fewer than 2% of the 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 to the role of HC-toxin i n allowing the development of a compatible disease interaction, it is plausible that, by inhibiting HD, HC-toxin interferes with the proper expression of a subset of genes necessary for maize to 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 acetylated histone purification and analysis.

Received April 4, 1997; accepted July 29, 1997. Copyright Clearance Center: 0032-0889/97/ 115/ 1021/07.

LITERATURE ClTED

Brosch G, Ransom R, Lechner T, Walton JD, Loidl P (1995) Inhibition of maize histone deacetylases by HC toxin, the host- selective toxin of Cochliobolus carbonum. Plant Cell 7: 1941-1950

Darkin-Rattray SJ, Gurnett AM, Myers RW, Dulski PM, Crum- ley TM, Allocco JJ, Cannova C, Meinke PT, Colletti SL, Bednarek MA, and others (1996) Apicidin: a nove1 antiproto- zoa1 agent that inhibits parasite histone deacetylase. Proc Natl Acad Sci USA 93: 13143-13147

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