Reduced Contamination by the Fusarium Mycotoxin Zearalenone in Maize Kernels through Genetic Modification with a Detoxification Gene

Maize is subject to ear rot caused by toxigenic Aspergillusand Fusarium species, resulting in contamination with aflatoxins,fumonisins, trichothecenes, and zearalenone (ZEN). The trichothecenegroup and ZEN mycotoxins are produced by the cereal pathogenFusarium graminearum. A transgenic detoxification system forthe elimination of ZEN was previously developed using an egfp::zhd101gene (gfzhd101), encoding an enhanced green fluorescent proteinfused to a ZEN-degrading enzyme. In this study, we produceda transgenic maize line expressing an intact copy of gfzhd101and examined the feasibility of transgene-mediated detoxificationin the kernels. ZEN-degrading activity has been detected intransgenic kernels during seed maturation (for a period of 6weeks after pollination). The level of detoxification activitywas unaltered after an additional storage period of 16 weeksat 6°C. When the seeds were artificially contaminated byimmersion in a ZEN solution for 48 h at 28°C, the totalamount of the mycotoxin in the transgenic seeds was uniformlyreduced to less than 1/10 of that in the wild type. The ZENin the transgenic maize kernels was also efficiently decontaminatedunder conditions of lower water activity (a_w) and temperature;e.g., 16.9 µg of ZEN was removed per gram of seed within48 h at an a_w of 0.90 at 20°C. F. graminearum infectionassays demonstrated an absence of ZEN in the transgenic maizeseeds, while the mycotoxin accumulated in wild-type kernelsunder the same conditions. Transgene-mediated detoxificationmay offer simple solutions to the problems of mycotoxin contaminationin maize.

INTRODUCTION

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Introduction

Maize is one of three major cereal crops that dominate worldagriculture. Maize kernels are processed primarily for livestockfeed (78%) and, to some extent, for human consumption (13%)(28). In substantial proportions of maize-producing areas worldwide,the crops are subject to contamination from mycotoxins (22).Although numerous toxic fungal metabolites may be found in maizekernels, studies have focused on four major mycotoxins: aflatoxinsassociated with Aspergillus ear rot and fumonisins, trichothecenes,and a myco-estrogen zearalenone (ZEN) associated with Fusarium/Gibberellaear rot (16, 27). The risk of contamination with these mycotoxinshas been recognized for decades, but preventive measures remaincostly and inadequate.

Recent advances in the study of plant pathology have openedthe way to achieving transgenic resistance to fungal pathogens,including toxigenic fungi and their toxins (2). However, transgenicstrategies have not been successful in achieving a convincingand practical level of resistance to fungi in the field (4).An alternative way to address mycotoxin contamination couldbe transgene-mediated detoxification in plants by the deploymentof anti-mycotoxin antibodies (plantibodies) (35) or mycotoxin-degradingenzymes (12). The latter idea is being tested with geneticallymodified (GM) maize engineered so that fumonisin B₁ is eliminatedthrough the expression of the fumonisin esterase and amine oxidasegenes of Exophiala spinifera (4). However, there have been nopublished data that support the practicality of mycotoxin-degradingmaize.

Fusarium graminearum is a broad-range fungal pathogen that infectsa number of important crops worldwide (7). It is a causal agentof Gibberella ear rot in maize and Fusarium head blight in wheatand barley, diseases by which grains become contaminated withtrichothecene group and ZEN mycotoxins. The fungus enters maizeears down the silk or through insect wounds to the kernels andcob. Under field conditions, the time courses of the fungalgrowth and mycotoxin level vary depending on the environmentalconditions (25, 26), but the mycotoxin appears to increase towardthe late milk stage. As to the removal of ZEN, a gene for detoxification(designated zhd101) encoding an alkaline lactonohydrolase wascloned and characterized (29). In this study, we establisheda homozygous maize line stably expressing zhd101 and conductedlaboratory scale experiments to see whether transgenic detoxificationis feasible or not for mitigation of the mycotoxin problemscaused by F. graminearum.

MATERIALS AND METHODS

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Materials and Methods

Plant and plasmid materials.
The Zea mays inbred line A 188 (JP no. 808) was supplied bythe National Institute of Agrobiological Biosciences (NIAS)GenBank-Plant. Plants were grown in a greenhouse under conditionsof a 12-h photoperiod (400 W metal halide lamp; Toshiba) attemperatures of 25°C in the day and 20°C in the night.

For easy monitoring of the ZHD101 enzyme's distribution in varioustissues, we previously constructed an egfp::zhd101 gene (hereafterdesignated gfzhd101) that encodes an enhanced green fluorescentprotein (EGFP) N-terminally fused to ZHD101 (30). In this study,we constructed the plasmid pEU- ${Omega}$ gfzhd101 containing gfzhd101under the control of the Act1 promoter (20) and an ${Omega}$ translationenhancer (32).

Transformation of maize.
The culture and transformation of maize were done followingthe method reported by Frame et al. (5) with modifications.Briefly, immature embryos (approximately 1.5 mm) were placedembryo axis-side down on D1.5 medium (Murashige minimal organicmedium [Invitrogen, Carlsbad, CA] supplemented with 1.5 mg/liter2,4-dichlorophenoxyacetic acid, 0.5 mg/liter nicotinic acid,0.5 mg/liter pyridoxine HCl, 700 mg/liter L-proline, 1.7 mg/litersilver nitrate, 20 g/liter sucrose, and 8 g/liter agar) so thatcalluses could form. The plates (90 by 20 mm; Eiken Co., Tokyo,Japan) containing the plant materials were sealed with surgicaltape (3M Health Care, Borken, Germany) and incubated at 28°Cin the dark.

After 4 to 7 days, embryos producing calluses were transferredto D2Man medium (Murashige minimal organic medium supplementedwith 2.0 mg/liter 2,4-dichlorophenoxyacetic acid, 30 g/litersucrose, 72.8 g/liter mannitol, and 2.5 g/liter gellun gum)with the scutellum side up. The embryos were cobombarded withplasmids pDM302 (3) and pEU ${Omega}$ -gfzhd101 (2 µg each) at 650lb/in² at a target distance of 9 cm using a PDS-1000/He biolisticsystem (Bio-Rad, Hercules, CA). After an overnight culture,the bombarded embryos were transferred to a selection medium(D1.5 medium without L-proline, supplemented with 2.5 mg/literbialaphos) and subcultured every 2 weeks.

Selected calluses showing expression of EGFP and resistanceto bialaphos were transferred to SE medium (Murashige minimalorganic medium supplemented with 0.5 mg/liter nicotinic acid,0.5 mg/liter pyridoxine HCl, 0.5 mg/liter bialaphos, 60 g/litersucrose, and 1.5 g/liter gellun gum) for somatic embryogenesisand cultured at 25°C under 24-h illumination. The somaticembryos were transferred to RE medium (1/2 Murashige minimalorganic medium supplemented with 0.5 mg/liter nicotinic acid,0.5 mg/liter pyridoxine HCl, 30 g/liter sucrose, and 8 g/literagar) for plant regeneration.

Southern and Western blot analyses.
Genomic DNA was isolated from mature leaves using a NucleonPhytoPure plant and fungal DNA extraction kit (Amersham Biosciences,Piscataway, NJ). For Southern blot analysis, a digoxigenin-labeledprobe was prepared by using a PCR digoxigenin probe synthesiskit (Roche Applied Science GmbH, Manheim, Germany). The probeconsists of the entire coding region of gfzhd101 amplified withthe following primers: 5'-ATGGTGAGCAAGGGCGAGGAGCTGTT-3' and5'-TCAAAGATGCTTCTGCGTAGTTTCCA-3'. After agarose gel electrophoresis,DNA was transferred to a Nytran N membrane (Schleicher and Schell,Dassel, Germany). Standard hybridization techniques recommendedby the manufacturer were used.

Crude protein was extracted from mature leaves (50 mg) of nontransgenicand transgenic plants by grinding in a mortar with 200 to 500µl of extraction buffer (50 mM Tris-HCl, 10 mM 2-mercaptoethanol,10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5% [vol/vol]glycerol, 2% [wt/vol] polyvinylpyrrolidone K25, and 1.5% [wt/vol]poly[vinylpolypyrrolidone], pH 8.0). Twenty micrograms of denaturedprotein was subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis using a 12.5% polyacrylamide gel and electroblottedto a polyvinylidene difluoride membrane (Bio-Rad). The membranewas first incubated with a polyclonal antibody that recognizesGFPs (living colors full-length A.v. polyclonal antibody; TakaraBIO Inc., Ohtsu, Japan) and then with a biotinylated goat anti-rabbitimmunoglobulin G (heavy plus light chains) (Bio-Rad) as a secondaryantibody. Detection was achieved using an AP conjugate substratekit (Bio-Rad).

Statistical analyses.
Observed segregation ratios were compared to the Mendelian 3:1,15:1, or 63:1 ratio using the chi-square test ( ${chi}$ ², >3.84;P, <0.05).

In vitro ZEN degradation assay using maize kernels.
The wild-type and homozygous T₃ seeds (self-pollinated progeniesderived from line N11-2-19 or N11-2-21) were harvested afterseed maturation (6 weeks after pollination) and then storedfor 16 weeks at 6°C. The moisture content was determinedby heating the grains at 80°C for 16 h. The value of thewater activity (a_w) was measured using a water activity meterPawKit (Decagon Devices Inc., Pullman, WA) calibrated with thesalt solutions.

For assaying the degradation of ZEN in vitro, seeds were collectedat 2 weeks (moisture content, 78%; a_w, 0.99), 4 weeks (moisturecontent, 45%; a_w, 0.98), and 6 weeks (moisture content, 14%;a_w, 0.82) after pollination, and some seeds collected at 6 weeksafter pollination were stored for 16 weeks at 6°C (a totalof 22 weeks after pollination). Seeds were ground in a chilledmortar and the crude protein was extracted with buffer A (50mM Tris-HCl, pH 7.0, 10 mM EDTA, and 5% glycerol). The proteinconcentration of this crude protein extract was adjusted to0.2 mg/ml with buffer A. The enzymatic reaction was initiatedby adding 5 µl of ZEN stock solution (5 mg/ml in dimethylsulfoxide) to 495 µl of crude protein extract in a siliconizedmicrotube. After 4 or 24 h of incubation at 30°C, a portionof the reaction mixture (50 µl) was mixed with methanol(450 µl) and the resulting sample was passed through a0.2-µm Millex-LG filter (Millipore, Billerica, MA). Analiquot (10 µl) was analyzed using a high-performanceliquid chromatography (HPLC) system (SCL-10A; Shimadzu) equippedwith a PEGASIL octyldecyl silane column (diameter, 4.6 mm; length,250 mm; Senshu Scientific Co., Tokyo, Japan). Samples were elutedwith 0.1% trifluoroacetic acid-acetonitrile (4:6) at a flowrate of 1.0 ml/min at 40°C. ZEN and its degradation product,1-(3,5-dihydroxyphenyl)-10'-hydroxy-1'-undecen-6'-1 (DPHU) (11,29), were monitored at 254 nm. The experiments were carriedout in triplicate for both the wild-type and T₃ seeds.

In vivo ZEN degradation assay using maize kernels.
Four-week-old wild-type and homozygous T₃ seeds (self-pollinatedprogenies of N11-2-9) were surface sterilized with 80% ethanol,rinsed with sterilized water, and placed in a siliconized polypropylenecentrifuge tube (50 ml; BD Falcon, Franklin Lakes, NJ). An equalamount of ZEN solution (50 µg/ml) was added to each tubeand the kernels were allowed to absorb ZEN at 28°C for 48h. After the incubation, the seeds were lightly tapped againstthe inner wall of the tube to remove excess liquid on the surfaceand then dissected into three parts: embryos, endosperms, andpericaps. After each tissue was ground in a chilled mortar,the sample was transferred to a 50-ml tube and mixed with 80%acetonitrile (4 to 10 times the amount of each tissue). Eachsample was homogenized using a handy micro homogenizer (Physcotron;Microtech Co., Ltd., Chiba, Japan) and ZEN was extracted bygently shaking the mixture for 2 h. The concentration of ZENwas determined by HPLC based on the fluorescence of ZEN (excitation,278 nm; emission, 460 nm).

Influence of water activity (a_w) and temperature on ZEN degradation.
To evaluate the influence of environmental conditions on theZEN-degrading activity of transgenic kernels, the in vitro andin vivo ZEN degradation assays were also carried out under conditionsin which the a_w was adjusted to 1.00 (at 30°C), 0.97 (at25°C), and 0.90 (at 20°C) by adding sodium chloride.The in vitro and in vivo ZEN-degrading activities of transgenickernels were measured as described above, except that 80% methanolwas used for the ZEN extraction in the in vivo experiment (normalizedto the efficiency of 80% acetonitrile extraction for comparison).These experiments were carried out in triplicate.

Inoculation of maize kernels with F. graminearum.
Four-week-old maize seeds (moisture content, 45%; a_w, 0.98)surface-sterilized as described above were placed in a sterilized100-ml Erlenmyer flask and mixed with a mycelial plug of F.graminearum ZEA-1 (33). Each flask contained a mixture of sixwild-type and six homozygous T₃ seeds (self-pollinated progeniesof N11-2-9). The flasks were placed in a sealed plastic boxcontaining water (equilibrium relative humidity, 100%) and incubatedfor 7 to 8 days at 25°C. After the incubation, the myceliaon the surface of the kernels were lightly scrubbed with paper,and then the seeds were lightly rinsed with water. The seedswere divided into wild-type and transgenic groups, and eachgroup was separately analyzed by HPLC for the amount of ZENas described above. The experiment was independently carriedout 5 times.

RESULTS

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Results

Transformation of maize with pEU- ${Omega}$ gfzhd101.
Maize embryos cobombarded with pEU- ${Omega}$ gfzhd101 and pDM302 wereselected with bialaphos, and the calluses with EGFP fluorescencewere regenerated into plantlets as described in Materials andMethods. Seven independent T₀ lines were obtained from the approximately1,500 embryos bombarded. The genomic DNA of these lines wasdigested with XbaI, which does not cut the vector, and examinedby Southern blotting using gfzhd101 as a probe. As shown inFig. 1a, many lines carried multiple copies of the transgenein the genome, which is often the case with the biolistic method.Epifluoresence stereomicroscopic observation of mature T₀ leavesrevealed that three transgenic lines, N4, D2, and M3, whichcarry multiple copies of the transgene, did not show EGFP fluorescenceat this developmental stage, presumably due to gene silencing.Consistent with this observation, Western blot analysis alsodemonstrated that the fluorescent ZEN-degrading enzyme was producedonly in lines N11, D9, ND24, and J30 (Fig. 1b).

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FIG. 1. Analyses of T₀ transgenic lines of maize expressing gfzhd101. (a) Southern blot analysis of DNA from wild-type and transgenic lines. Genomic DNA was digested with XbaI, which does not cut the probe DNA. M, ${lambda}$ HindIII marker DNA. (b) Western blot analysis of total leaf protein extracted from wild-type and transgenic lines. The sizes of biotinylated marker proteins (M) are shown by arrows.

Inheritance of the transgene.
The EGFP-positive transgenic lines were self-crossed to obtainT₁ seeds, which were used for self-crosses in successive generationsto achieve homozygosity for the transgene. While lines N11,D9, and J30 were fertile and produced viable T₁ seeds, ND24failed to produce anther dusts and T₁ seeds were not obtained.Out of 301 and 473 T₁ seeds originating from T₀ lines D9 andJ30, 262 and 311 seeds, respectively, showed EGFP fluorescence.These expression patterns of the transgene differed significantlyfrom a single- or multiple-site integration of the transgeneas assessed with a chi-square test. As to progenies of N11,52 of 71 T₁ seeds showed EGFP fluorescence, which did not differsignificantly from a 3:1 Mendelian segregation for a singleinsertion site ( ${chi}$ ², 0.117; P, <3.84). For the subsequent characterizationof transgenic plants, we used progenies of line N11.

Figure 2 traces the integration patterns of gfzhd101 in self-pollinatedprogenies of line N11. When DNA was digested with ClaI, whichdoes not cut the probe, one intense and two faint bands weredetected from the T₀ and T₁ progenies, except for the line N11-3T₁ progenies (Fig. 2a and b). Lines N11-5 and N11-6 failed toproduce seeds, and lines N11-3 and N11-4 were heterozygous,as suggested by the segregation pattern of the EGFP-fluorescencein the self-pollinated T₂ generation. Among these T₁ progenies,only line N11-2 produced 100% fluorescent T₂ seeds expressinggfzhd101. The T₂ plants N11-2-9 and N11-2-20 lost the two faintbands that were observed on the Southern blot of the parentN11-2, and only a single ClaI fragment was detected (Fig. 2c).When the T₃ progenies derived from N11-2-9 were analyzed bySouthern blotting, they were found to have similarly inheritedthis single banding pattern (Fig. 2d). These T₃ progenies andtheir T₂ parent N11-2-9 revealed a single band with anotherno-cut enzyme, BglII, and double bands with a single-cut enzyme,Bsp1047I (data not shown), suggesting that they are homozygouscarrying a single copy of the transgene. These self-pollinatedprogenies of N11-2-9 were used for in vivo ZEN detoxificationand Fusarium infection assays.

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FIG. 2. Inheritance of gfzhd101 in progenies of line N11. The extended names joined by hyphens represent the self-crossed next generation derived from the original parent (e.g., "N11-2-9-z" represents T₃ progeny "z" descended from the T₂ parent "N11-2-9"). The arrows with asterisks indicate faint ClaI bands that segregated from the main single band in the two progenies in the T₂ generation. M, ${lambda}$ HindIII marker DNA.

In vitro ZEN-degrading activities of transgenic seeds.
To evaluate the ZEN-degrading activities of the transgenic maizekernels, crude protein extracts were prepared from the T₃ seedsand used for the in vitro detoxification assay. Compared toa blank control (data not shown), no decontamination was observedwith the protein extract prepared from the wild-type kernelsduring seed maturation (Fig. 3). In contrast, the amount ofZEN decreased significantly in the transgenic reaction mixturecompared to the amount in the control reaction mixture withheat-denatured transgenic seed extract (data not shown) or theamount in the wild-type seed extract. With 4 h of incubation,the detoxifying activity was most evident in developing seedsat 2 weeks after self-pollination. The enzyme activities weredecreased in seeds at 4 weeks, 6 weeks, and 22 weeks (6 weekson the cob with an additional 16 weeks of storage at 6°C).When the incubation time was increased to 24 h, nearly all theZEN in the reaction mixture was eliminated by the transgenicprotein at all stages of seed maturation. The cleavage productof ZEN, DPHU, was also detected in the reaction mixture of thetransgenic seeds by the HPLC analysis (data not shown). Theseresults indicate that the EGFP-tagged ZHD101 enzyme is producedin an active form in the transgenic maize kernels.

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FIG. 3. Degradation of ZEN by crude proteins extracted from T₃ seeds at 2, 4, 6, and 22 weeks (developing on the cob for 6 weeks and then harvested and stored for 16 weeks at 6°C) after pollination. The enzymatic reaction was carried out for 4 h or 24 h at 30°C. The vertical axis represents the concentration of ZEN in the reaction mixture.

In vivo ZEN-degrading activities of transgenic seeds.
T₃ homozygous N11-2-9-z seeds (4 weeks after self-pollination)were immersed in a 50-µg/ml ZEN solution and allowed toabsorb the mycotoxin for 48 h. After removal of the ZEN solutionon the surface, the ZEN-soaked seeds were dissected into embryos,endosperms, and pericarps and then observed under an epifluorescencemicroscope (MZFLIII; Leica Microsystems GmbH, Wetzlar, Germany).The EGFP-tagged enzyme was found mostly in the embryos and endosperms,and to a lesser extent in the pericarp fraction (Fig. 4a). Thisdemonstrates that the enzyme can be expressed at a certain levelin maize kernels and distributed throughout the kernel tissues.

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FIG. 4. Degradation of ZEN in vivo by mature T₃ seeds. (a) Dissection of maize kernels into three parts: embryos (A), endosperms (B), and pericarps (C). Photographs of dissected parts are shown in the left panel. An epifluorescent stereomicroscopic image of transgenic seeds is shown in the right panel (the bar represents 5 mm). (b) Reduced amounts of ZEN in transgenic seeds soaked in the mycotoxin solution (50 µg/ml of ZEN) at 28°C. W and T represent experiments with wild-type and transgenic seeds, respectively, which were independently carried out three times (distinguished by a number after a hyphen). The stacked bars represent the amount of ZEN (µg) detected per gram of seed in each experiment. (c) Reduced amounts of ZEN in transgenic embryos, endosperms, and pericarps soaked with the mycotoxin solution (50 µg/ml of ZEN) at 28°C. The bars represent the amount of ZEN (µg) per gram of each tissue; the error bars represent standard deviations (n = 3).

The amount of ZEN in the supernatant and each of the above threefractions was quantified by HPLC. As shown in Fig. 4b, the wild-typeseeds contained a total of 24.6 ± 1.7 µg of ZENper gram of seed, which is far above the usual levels of ZENfound in the field (1/2,000 to 1/500 the level of contaminationcompared to the level found in this experiment). The concentrationof ZEN in the supernatant solution was reduced to 0.83 µg/ml,suggesting that most of the mycotoxin in the solution was absorbedby the seeds. Although slightly less ZEN tended to accumulatein the endosperms than in the embryos and pericarps, the distributionpatterns of this mycotoxin were not remarkably different inthe wild-type seeds (Fig. 4c).

In spite of the assay conditions that result in such high accumulationlevels of ZEN, the transgenic seeds contained only 1.6 ±0.4 µg of ZEN per gram of seed, and the amount of ZENin the supernatant solution was reduced to 0.16 µg/ml.The reduction in the amount of ZEN in the seeds is attributedto degradation by the enzyme, since a large fluorescent peakof DPHU was observed in the HPLC chromatogram of the reactionmixture (data not shown). Again, there were no remarkable differencesin the residual amounts of ZEN in the embryos, endosperms, andpericarps of the transgenic seeds (Fig. 4c). Therefore, theZEN-degrading enzyme and ZEN are similarly distributed withinthe maize kernels, suggesting the feasibility of the transgene-mediateddetoxification strategy.

Degradation of ZEN in transgenic maize kernels under conditions of lower water activity and temperature.
To assess the ability of the recombinant enzyme to decontaminatethe mycotoxin under different conditions that are less preferablefor enzymes in general, we examined the influence of water activityand temperature on ZEN degradation. Since the toxigenic Fusariumno longer produces ZEN when the a_w is reduced to 0.90 (21),the effect of the water activity on ZEN-degrading activity wasexamined at an a_w above this value. Experiments with purifiedrecombinant ZHD101 (30) showed that the enzyme activity whenthe a_w was 0.90 was at least 64% of that when the a_w was 1.00when compared at the same temperature (in the range of 15 to30°C) and that, at an a_w of 0.90 at 15°C, the enzymestill retained 33% of its activity compared to that when thea_w was 1.00 at 30°C (data not shown).

The ZEN-degrading activities of transgenic kernels were assayedby an in vitro experiment with an incubation period of 24 h.Consistent with the relative insensitivity of purified ZHD101to lower water activity and temperature, the crude cell extractof the transgenic seeds could remove 84% of ZEN in the reactionmixture at an a_w of 0.90 at 20°C (Fig. 5a). This in vitroexperiment also demonstrated that as much as 95% of ZEN in thereaction mixture was degraded when the a_w was 0.97 at 25°C,one of the conditions under which the fungus is known to produceits highest yield of ZEN (10, 21). The activity under theseconditions was substantially equal to that at an a_w of 1.00at 30°C.

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FIG. 5. Degradation of ZEN under conditions of different water activities and temperatures (a_w of 0.90 at 20°C; a_w of 0.97 at 25°C; and a_w of 1.00 at 30°C). (a) Degradation of ZEN by crude proteins extracted from T₃ seeds at 4 weeks after pollination. The enzymatic reaction was carried out for 24 h. The vertical axis represents concentrations of ZEN in the reaction mixture. (b) Degradation of ZEN in vivo by mature T₃ seeds at 4 weeks after pollination. Maize kernels artificially contaminated with ZEN as described in the Fig. 4 legend (placed under the conditions for 48 h) were analyzed as described in Materials and Methods. The vertical axis represents the amount of ZEN (µg) per gram of seed.

The in vivo ZEN degradation assay also demonstrated that thetransgenic kernels could degrade significant amount of ZEN underthe above conditions. As shown in Fig. 5b, the residual amountsof ZEN in the artificially contaminated transgenic seeds weresignificantly reduced compared to the amount in the wild-typecontrol during an incubation period of 48 h. In terms of thedecontamination efficiency, 16.9 and 24.0 µg of ZEN wereremoved per gram of seed at an a_w of 0.90 at 20°C and atan a_w of 0.97 at 25°C, respectively (cf. 27.6 µg removedper gram of seed at an a_w of 1.00 at 30°C).

Detoxification of ZEN in Fusarium-infected transgenic kernels.
After the surfaces of the Fusarium-infected seeds were washed,the amounts of ZEN were quantified. The ZEN-producing conditionsfor F. graminearum in the wild-type and transgenic maize kernelsshould be equal in each experiment since the fungal inoculationof both types of kernels was carried out in a single Erlenmyerflask. The wild-type and transgenic seeds were easily distinguishedby the EGFP-tag attached to the detoxification enzyme. As shownin Fig. 6, the wild type contained 15.4 ± 3.7 ng of ZENper gram of seed. This level of contamination is comparableto the concentration of ZEN reported to occur naturally in infectedmaize kernels (e.g., 4 to 388 ng per gram of seed in the resultsreported in reference 13 and 14 to 169 ng per gram of seed inthe results reported in reference 17). In contrast, noninoculatedseeds (data not shown) and the T₃ transgenic seeds inoculatedwith the fungus contained no ZEN.

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FIG. 6. Detoxification of transgenic seeds infected with F. graminearum. The mature T₃ seeds, with wild-type (W) and transgenic (T) seeds mixed, were inoculated with the fungus and incubated for 7 to 8 days at 25°C under humid conditions (equilibrium relative humidity, 100%). After removal of the mycelia on the surface of the infected grain, the wild-type and transgenic seeds were separated and examined under the epifluorescence stereomicroscope. ZEN was extracted from the seeds and quantified by HPLC. The experiment (Exp) was carried out independently five times.

DISCUSSION

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Discussion

Although reducing fungal infections is the most desirable methodof eliminating mycotoxins from maize, cultural practices andgenetic approaches have achieved limited success in diseasecontrol in terms of effectiveness and cost. In addition, effortsto produce GM crops with enhanced disease resistance, such asthe introduction of insecticidal genes (22), overexpressionof antifungal proteins (1), engineering of novel secondary metabolites(8), and transgene-mediated enhancement of plant defense pathways(18), have not been successful on a practical level. As an alternativesolution, transgene-mediated detoxification of mycotoxins hasbeen proposed (12). A prerequisite for this is the availabilityof genes encoding enzymes with detoxification activities, andwith regard to ZEN, we have previously isolated zhd101 (29).This detoxification gene was useful for transgenic decontaminationof ZEN when expressed in baker's yeasts after the codon optimization(31); in addition, the active enzyme proved to be expressedin the grains of model cereal rice plants transformed with gfzhd101(9). Since ZEN causes a major problem in maize intended foruse in stockbreeding, we have established a homozygous transgenicmaize line stably expressing gfzhd101. Promisingly, the transgenicmaize kernels showed sufficient ZEN-degrading activity throughoutseed maturation and after storage as assessed by the in vitroenzyme assay. These results warrant the availability of activeZHD101 enzyme when and where it is needed in transgenic maize.

Unlike the situation in vitro, the transgenic detoxificationof mycotoxins in maize kernels was postulated to be problematicwith regard to (i) the distribution of the enzyme in relationto the mycotoxin, and (ii) the activity of the enzyme underphysiological conditions in maize tissue (4). In the case offumonisin B₁, the establishment of a system of transgene-mediateddetoxification has not yet been reported, presumably due tosuch problems. Therefore, laboratory-directed enzyme evolutionwas conducted to improve transgenic detoxification (34). Incontrast, detoxification of ZEN in vivo was successful regardlessof the conditions listed above. Indeed, the EGFP-tagged enzymewas present in all three fractions of the seeds, embryos, endosperms,and pericarps (Fig. 4a), and the amount of ZEN in the transgenickernels was markedly reduced in each tissue compared to thatin the nontransgenic control (Fig. 4b and c). ZEN's occurrenceseems not to be confined to (i) specific tissues or (ii) extracellularspaces as postulated for fumonisin B₁, because (i) exogenouslyabsorbed ZEN was detected equally in the above three fractionsof the wild-type kernels and (ii) the secretion signal was notnecessary for the in vivo detoxification. Therefore, the enzyme'sdistribution and stability appear not to be technical problemsthat need to be resolved in the case of ZEN-related decontamination.

The fungus produces ZEN under various conditions, includingthose of lower water activities and temperatures which are lessfavorable for the function of enzymes. We therefore evaluatedthe ZEN-detoxifying activities of transgenic maize under theconditions in which F. graminearum produces the highest yieldof ZEN (an a_w of 0.97 at 25°C) and in which it can no longerafford to produce this secondary metabolite (an a_w of 0.90 at20°C). Consistent with the relative insensitivity of theZHD101 enzyme to decreases in water activity and temperature,the transgenic maize kernels showed significant ZEN-detoxifyingactivity under these conditions; e.g., the in vivo experimentsdemonstrated that 16.9 µg of ZEN was removed per gramof seed within 48 h when the a_w was 0.90 at 20°C (Fig. 5b).In this way, transgenic detoxification of ZEN in maize kernelswas indicated to be useful to solve the problem of ZEN contaminationunder conditions similar to field situations.

Aside from the efficiency of ZEN's degradation to DPHU, itsfurther metabolism in maize plants (e.g., conjugation and degradation)must be examined under various abiotic and biotic stress conditionsin the field. As to the potential toxicity of DPHU, in vitroassays with human MCF-7 breast cancer cells demonstrated thatit shows no estrogenicity (11). The toxicity of DPHU other thanestrogenicity, if any, also needs to be evaluated by intensivein vivo studies, including large-scale ingestion experimentswith several different animal systems. In addition, the potentiallynegative impacts of mycotoxin-degrading GM crops, such as effectson nutritional properties and the risk from allergens, alsoneed to be assessed in the future.

F. graminearum concomitantly produces structurally unrelatedmycotoxin trichothecenes, e.g., deoxynivalenol (DON), in additionto ZEN in infected grains. Therefore, DON detoxification shouldalso be considered together with the transgenic strategies ofZEN decontamination. Although genes for irreversible detoxificationof trichothecenes are not currently available, numerous effortsare being made to solve the problems of DON contamination (6,14, 15, 19, 23, 24). In combination with such progress in biotechnologicaldetoxification of DON, the transgenic system of detoxificationof ZEN demonstrated in this paper will contribute significantlyto solving the mycotoxin problems in maize and other cereals.

ACKNOWLEDGMENTS

We thank K. Kadokura for technical assistance in the transformationof maize. We also thank T. Nakajima and M. Yoshida for the giftof F. graminearum strains.

This work was supported by the Program for Promotion of BasicResearch Activities for Innovative Biosciences (PROBRAIN).

FOOTNOTES

* Corresponding author. Mailing address: Plant & Microbial Metabolic Engineering Research Unit, Discovery Research Institute (DRI), RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Phone: 81-48-467-9796. Fax: 81-48-462-4394. E-mail: mkimura{at}riken.jp.

Published ahead of print on 5 January 2007.

T. Igawa and N. Takahashi-Ando should be considered as jointfirst authors.

Present address: Plant Protein Biology, Department of BiologicalSciences, Nara Institute of Science and Technology, 8916-5 Takayama,Ikoma-shi, Nara 630-0101, Japan.

Present address: Shibata Distinguished Senior Scientist Laboratory,Discovery Research Institute (DRI), RIKEN, 2-1 Hirosawa, Wako,Saitama 351-0198, Japan.

^¶ Present address: Presidential Office, Agricultural ChemicalsInspection Station (ACIS), 2-772 Suzuki-cho, Kodaira, Tokyo187-0011, Japan.

REFERENCES

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