A Model Transgenic Cereal Plant with Detoxification Activity for the Estrogenic Mycotoxin...

8/7/2019 A Model Transgenic Cereal Plant with Detoxification Activity for the Estrogenic Mycotoxin Zearalenone

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Short communication

A model transgenic cereal plant with detoxication activity for the estrogenicmycotoxin zearalenone

Arisa Higa-Nishiyama 1 , Naoko Takahashi-Ando 1 , Tsutomu Shimizu 2 , Toshiaki Kudo 3 ,Isamu Yamaguchi 1,4 & Makoto Kimura 1,3, *1Laboratory for Remediation Research, Plant Science Center, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama,Kanagawa 230-0045, Japan2Life Science Research Institute, Kumiai Chemical Industry Co., Ltd., Tamari 276, Kakegawa, Shizuoka 436-0011,Japan3Environmental Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan4Laboratory for Adaptation and Resistance, Plant Science Center, RIKEN, 1-7-22 Suehiro, Tsurumi, Yokohama,Kanagawa 230-0045, Japan

Received 7 January 2005; accepted 28 April 2005

Key words: detoxifying gene, Fusarium head blight of wheat, barely, and maize, genetically modied (GM)cereals, lactonohydrolase, mycotoxin decontamination

Abstract

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the necrotrophic cereal pathogen Fusarium

graminearum . This mycotoxin is detoxied by ZHD101, a lactonohydrolase from Clonostachys rosea , orEGFP:ZHD101, its fusion to the C-terminus of an enhanced green uorescence protein. We previouslyshowed that egfp:zhd101 is efficiently expressed in T 0 leaves of rice. In this study, we assessed the feasibilityof in planta detoxication of the mycotoxin using progeny. When protein extract from T 1 leaves wasincubated with ZEN, the amount of the toxin decreased signicantly as measured by HPLC. ZEN deg-radation activity was also detected in vivo in transgenic T 2 seeds. These results suggest that zhd101 can beexploited as an efficient and cost-effective system for protection of important cereals that are more sus-ceptible to the pathogen (e.g., wheat and maize) from contamination with the estrogenic mycotoxin.

Introduction

Fusarium head blight (FHB) is a signicant diseaseof small grain cereals (e.g., wheat, barley andmaize) caused by Fusarium species such asF. culmorum and F. graminearum . In addition tothe yield loss, Fusarium -infected grains are oftencontaminated with the mycotoxins trichothecenes(e.g ., deoxynivalenol) and zearalenone (ZEN).Although several fungicides reduce the diseaseseverity of FHB, mycotoxin contamination is not

necessarily correlated with the visible diseasesymptoms (Edwards et al., 2001). For example, a

number of eld and greenhouse studies demon-strated that application of the strobilurin fungicideazoxystrobin increases the amount of deoxynivale-nol in wheat grains (Simpson et al., 2001). Inaddition, sublethal concentrations of certain fungi-cides were reported to increase trichothecene pro-duction by Fusarium species (Magan et al., 2002).

To reduce the risks of mycotoxin contamina-tion, transgenic inactivation using a detoxicationgene is an alternative strategy (Karlovsky, 1999).Previously, we have isolated zhd101 , a ZEN detox-ifying gene, from a fungal strain of Clonostachys

* Author for correspondenceE-mail : [email protected]

Transgenic Research (2005) 14:713717 Springer 2005DOI 10.1007/s11248-005-6633-2


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rosea (Kakeya et al., 2002; Takahashi-Ando et al.,2002). Although the molar activity of ZHD101 was

quite low and its optimal pH inadequate for livingorganisms (i.e., pH = 10.5) (Takahashi-Ando etal., 2004), the genetically modied (GM) microbescompletely converted 2 l g/mL of ZEN to a cleavedproduct that is devoid of estrogenic activity(Takahashi-Ando et al., 2005). This result suggeststhe feasibility of the transgenic strategy for efficientdecontamination of ZEN in important cereal crops.

The success of the detoxication approach formycotoxins depends in part on theextent to whichtheGM plant-produced detoxication enzyme reachesits substrate mycotoxin. We previously showed that

egfp:zhd101 , a ZEN detoxication gene fused to theC-terminus of an enhancedgreenuorescence proteingene ( egfp), is efficiently expressed in T 0 leaves of rice.In this study, the ZEN degradation activity of theGM crops was characterized in grains using the seedsof the T 2 generation.

Materials, results and discussion

Transgene integration and inheritance

We previously obtained six T 0 rice plants express-ing egfp:zhd101 on the basis of the bright-greenEGFP uorescence of regenerated shoots (Higaet al., 2003), the intensity of which is proportionalto the ZEN detoxication activity (Takahashi-Andoet al., 2004). The transgenic rice plants owerednormally and produced viable seeds. In this com-munication, we focus on description of molecularand genetic analyses of one promising line, Z54.

The genomic DNA of T 0 line Z54 and itsuorescent T 1 progenies were examined by South-ern blot analysis. The DNA (10 l g) was digestedwith Pst I, whose recognition sequences are present

in the polylinker upstream of the Act1 promotercassette and in the coding region of zhd101(between the Bsr GI and Nco I sites) in pAct1-egfp:zhd101 (Higa et al., 2003), separated onan agarose gel, and transferred to a Nytran Nmembrane (Schleicher and Schell, Dassel,Germany). As a probe, the entire coding regionof egfp:zhd101 was labeled with digoxigenin (DIG)using the PCR DIG Probe Synthesis kit(Roche Applied Science GmbH, Manheim,Germany) with primers 5-egfp:zhd101 (5 -ATG-GTGAGCAAGGGCGAGGAGCTGTT-3 ) and

3-egfp: zhd101 (5 -TCAAAGATGCTTC TGCG-TAGTTTCCA-3 ). Probe-target hybrids were

detected using the DIG Nucleic Acid Detection kit(Roche). The Southern blot of the T 0 line and its T 1progenies revealed the same hybridization patternscomprising of ve bands (see Figure 1a), suggesting amultiple-copy integration of the transgene at a singlelocus in line Z54. The transgene showed a stablepattern of integration through the three generationsinvestigated in this study (data not shown).

Expression of egfp:zhd101 was visually exam-ined by monitoring the bright-green EGFP uo-rescence under a Leica MZFLIII uorescencestereomicroscope. For the observation of shoots,

roots, and owers, lter sets GFP-Plus (460 500 nm excitation and >510 nm barrier lter)were used. Expression of egfp:zhd101 in thetransgenic lines could easily be conrmed by thepresence of bright-green EGFP uorescence,which was clearly visible in the pistil and stameninside the owering spikelets (Figure 1b).

Figure 1 . Analyses of T 0 and T 1 rice plants transformed withegfp:zhd101 . (a) Southern blot analysis of Pst I-digested geno-mic DNA from wild-type and transgenic rice plants. Z54 isthe name of the T 0 plant, and the extended names joined byhyphens represent T 1 progenies derived from the T 0 parents(e.g., Z54-8 implies a T 1 progeny 8 from the T 0 parentZ54). (b) Expression of egfp:zhd101 in owering spikelets.The owers of wild-type and T 1 line Z54-9 were dissected andobserved under an epiuorescence stereomicroscope.

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As demonstrated with the T 0 leaves in ourprevious report (Higa et al., 2003), individual uo-

rescent T 1 lines showedbands for EGFP:ZHD101onthe western blots (data not shown). T 1 line Z54-9 andtheir T 2 seeds Z54-9- n (n = 1, 2, 3, . . .) were used forthe in vitro and in planta ZEN detoxication assay,respectively (see below).

Degradation of ZEN by crude enzyme fractionfrom leaves

To evaluate the ZEN degradation activity of thetransgenic rice plants, crude protein extracts wereprepared from the leaves of T 1 line Z54-9 and used

for the in vitro detoxication assay. Rice leaves(about 0.1 g FW) ground in liquid nitrogen werevigorously shaken in extraction buffer (50 mMTrisHCl, pH7.5, 10 mM EDTA, 0.1 % TritonX-100 and 0.1 % -mercaptoethanol) and the super-natant enzyme fraction was recovered. The assaywas initiated by adding 89 l L of this crude proteinextract (protein concentration adjusted to 1 l g/ l L)to 11 l L of substrate solution (1 l g of ZEN in0.1 M TrisHCl, pH9.5) at 37 C. The enzymereaction was terminated by adding 10 l L of 1 NHCl. The samples were diluted 2-fold with meth-

anol, ltered through a 0.2 l m Millex

-LG lter(Millipore, Billerica, MA), and quantied by high-performance liquid chromatography (HPLC) asdescribed previously (Takahashi-Ando et al., 2004).As shown in Figure 2a, ZEN degradation was notobserved with the protein extract prepared from thewild-type leaves. In contrast, the amount of ZEN inthe reaction mixture of the transgenic proteinextract decreased signicantly compared to thecontrol reaction with the wild-type. Approximatelytwo-thirds of the ZEN was removed from thereaction mixture of Z54-9. The cleavage product of ZEN was also detected from the reaction mixtureof the transgenic lines by the HPLC analysis (datanot shown). These results suggest that ZEN detox-ication activity can be conferred to cereal crops byexpression of zhd101 and that this activity can betransmitted to the subsequent generation.

Degradation of exogenously absorbed ZEN in mature seeds

While decreased yield and mycotoxin contamina-tion are the major problems associated with FHB

in wheat, barely, and maize (Goswami and Kistler,2004), infections of FHB pathogens are quite rare

in rice. In addition, ZEN was reported not toaccumulate in Fusarium -infected rice grains (Sugi-ura et al., 1993; Yoshida et al., 2004). In agreementwith these reports, we were not able to detect anyZEN from the rice grains infected with F. grami-nearum . Thus, to evaluate the usefulness of theGM grains, we exogenously added ZEN solutionto mature seeds and quantied the residualamount of the mycotoxin.

T 2 rice seeds (Z54-9- n) were immersed in a5 l g/mL ZEN solution containing 0.1 % Tween-20and allowed to absorb the mycotoxin for 2 days.

All progenies from Z54-9 (i.e., 60 T 2 seeds) showedbright-green EGFP uorescence (examples areshown in Figure 2b insert), suggesting that theT 1 line is homozygous for the transgene.

The contaminated seeds were lightly tapped toremove excess ZEN solution from the surface,disrupted with a commercial coffee mill, and thenground to a ne powder in a mortar in liquidnitrogen. Four ml of acetonitrilewater (21:4) wasadded to the powdered samples (1 g) and themixtures were vigorously shaken for 30 min. TheZEN contents in the supernatants were quantiedusing a RIDASCREEN FAST Zearalenon kit(R-Biopharm AG, Darmstadt, Germany). Asshown in Figure 2b, the wild-type seeds containedapproximately 30 l g of ZEN per gram of seeds. Incontrast, the T 2 seeds contained a reduced amountof the mycotoxin, 16 l g ZEN per gram of seeds.This implies that 14 l g of ZEN was decontami-nated in the GM seeds. Considering the previouslyreported levels of mycotoxin contamination,namely 0.0412 l g of ZEN per gram of cerealgrains (Luo et al., 1990; Kim et al., 1993; Parket al., 1996), a signicantly greater amount of themycotoxin initially existed in the rice grains in our

experiment. It may thus be possible to reduce thepractical level of contaminated ZEN in the trans-genic seeds regardless of the low catalytic efficiencyand high optimal pH of the detoxication enzyme(Takahashi-Ando et al., 2004).

Conclusion and perspective

Using model cereal rice plants, we have demon-strated that zhd101 can be exploited to developan efficient and cost-effective system for reducingthe amount of ZEN in GM grains. Transgene

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mediated detoxication of the mycotoxin couldprovide an efficient means of protecting othercereals, especially maize, which are actually ex-posed to serious threats of ZEN contamination.

Acknowledgements

We thank K. Mimori for excellent technicalassistance. This research was supported in partby a grant for the Integrated Research Programfor Functionality and Safety of Food Toward anEstablishment of Healthy Diet to M.K. fromthe Ministry of Agriculture, Fishery, and For-estry of Japan (MAFF).

References

Edwards SG, Pirgozliev SR, Hare MC and Jenkinson P (2001)Quantication of trichothecene-producing Fusarium speciesin harvested grain by competitive PCR to determine efficaciesof fungicides against Fusarium head blight of winter wheat.Appl Environ Microbiol 67 : 15751580.

Goswami RS and Kistler HC (2004) Heading for disaster:Fusarium graminearum on cereal crops. Mol Plant Pathol 5:515525.

Higa A, Kimura M, Mimori K, Ochiai-Fukuda T, Tokai T,Takahashi-Ando N, Nishiuchi T, Igawa T, Fujimura M,Hamamoto H, Usami R and Yamaguchi I (2003) Expressionin cereal plants of genes that inactivate Fusarium mycotoxins.Biosci Biotechnol Biochem 67 : 914918.

Kakeya H, Takahashi-Ando N, Kimura M, Onose R, Yam-aguchi I and Osada H (2002) Biotransformation of the my-cotoxin, zearalenone, to a non-estrogenic compound by afungal strain of Clonostachys sp. Biosci Biotechnol Biochem66 : 27232726.

Karlovsky P (1999) Biological detoxication of fungal toxinsand its use in plant breeding, feed and food production. NatToxins 7: 123.

Kim JC, Kang HJ, Lee DH, Lee YW and Yoshizawa T (1993)Natural occurrence of Fusarium mycotoxins (trichothecenesand zearalenone) in barley and corn in Korea. Appl EnvironMicrobiol 59 : 37983802.

Luo Y, Yoshizawa T and Katayama T (1990) Comparativestudy on the natural occurrence of Fusarium mycotoxins(trichothecenes and zearalenone) in corn and wheat fromhigh- and low-risk areas for human esophageal cancer inChina. Appl Environ Microbiol 56 : 37233726.

Magan N, Hope R, Colleate A and Baxter ES (2002) Rela-tionship between growth and mycotoxin production byFusarium species, biocides and environment. Eur J PlantPathol 108 : 685690.

Park JJ, Smalley EB and Chu FS (1996) Natural occurrence of Fusarium mycotoxins in eld samples from the 1992 Wis-

consin corn crop. Appl Environ Microbiol 62 : 16421648.Simpson DR, Weston GE, Turner JA, Jennings P and Nich-olson P (2001) Differential control of head blight pathogensof wheat by fungicides and consequences for mycotoxincontamination of grain. Eur J Plant Pathol 107 : 421431.

Sugiura Y, Fukasaku K, Tanaka T, Matsui Y and Ueno Y(1993) Fusarium poae and Fusarium crookwellense , fungiresponsible for the natural occurrence of nivalenol in Hok-kaido. Appl Environ Microbiol 59 : 33343338.

Takahashi-Ando N, Kimura M, Kakeya H, Osada H andYamaguchi I (2002) A novel lactonohydrolase responsible forthe detoxication of zearalenone: enzyme purication andgene cloning. Biochem J 365 : 16.

Takahashi-Ando N, Ohsato S, Shibata T, Hamamoto H,Yamaguchi I and Kimura M (2004) Metabolism of

Figure 2 . Detoxication of ZEN by GM cereal crops. (a) Degradation of ZEN by protein extracts from transgenic rice leaves. Pro-

tein extract was prepared from T 1 line Z54-9 as described in Materials and methods. Time course of the change in the ZEN con-centration ( l g/mL) in the reaction mixture was quantied by HPLC. (b) In planta detoxication of ZEN. Wild-type and T 2 riceseeds Z54-9- n (n = 1, 2, 3 . . .) were immersed in ZEN solution (5 l g/mL) for 2 days to absorb ZEN inside the grains. The seeds of wild-type and T 2 lines were observed under an epiuorescence stereomicroscope. The amount of ZEN in the rice seeds was quanti-ed using the RIDA SCREEN FAST Zearalenon kit. Statistical signicance between wild-type and transgenic lines were analyzedby Students t-test: * p = 0.0151.

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zearalenone by genetically modied organisms expressing thedetoxication gene from Clonostachys rosea . Appl Environ

Microbiol 70 : 32393245.Takahashi-Ando N, Tokai T, Hamamoto H, Yamaguchi I andKimura M (2005) Efficient decontaminationof zearalenone, themycotoxin of cereal pathogen, by transgenic yeasts through the

expression of a synthetic lactonohydrolase gene. Appl Micro-biol Biotechnol: DOI: 10.1007/s00253-004-1816-y in press.

Yoshida M, Nakajima T, Arai M and Suzuki F (2004) Myco-toxin productivity of Fusarium graminearum distributed inWestern part of Japan and pathogenicity of the nivalenol-producing strains. Jpn J Phytopathol 70 : 27.

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