Chinese Science Bulletin

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Citation: Shang Y, Xiao J, Ma L L, et al. Characterization of a PDR type ABC transporter gene from wheat (Triticum aestivum L.). Chinese Sci Bull, 2009, 54:

3249―3257, doi: 10.1007/s11434-009-0553-0

Characterization of a PDR type ABC transporter gene from wheat (Triticum aestivum L.)

SHANG Yi1,2, XIAO Jin1, MA LuLin1, WANG HaiYan1, QI ZengJun1, CHEN PeiDu1, LIU DaJun1† & WANG XiuE1† 1 State Key Laboratory of Crop Genetics and Germplasm Enhancement, Cytogenetics Institute, Nanjing Agricultural University, Nanjing 210095, China;

2 Institute of Crop Research and Nuclear Technique Utilization, Zhejiang Academy of Agricultural Science, Hangzhou 310021, China

DON, as a virulence factor, plays an important role in the infection of Fusarium graminearum in wheat. The infection ability of F. graminearum depends on its capacity of producing DON. The production of DON by F. graminearum is significantly decreased in the wheat varieties with scab resistance. In this study, GeneChip analysis indicated that an EST encoding an ATP-binding cassette (ABC) transporter was up-regulated by 45 times in a wheat landrace Wangshuibai, which is resistant to DON accumulation. A pair of EST-derived primers were designed based on the EST sequence, and a clone was then iso-lated from a wheat genomic DNA TAC library. The TAC clone was sequenced using chromosome walking and gene prediction was conducted using Softberry. A cDNA clone of this gene was subse-quently isolated from Wangshuibai induced by DON using gene-specific primers designed according to the untranslated sequence of the gene. The genome size of the gene is 7377 bp, consisting of 19 exons with coding sequences of 4308 bp. It encodes a protein with 1435 amino acid residues and the calcu-lated molecular weight is about 161 kD. BLAST analysis indicated that the gene may belong to plei-otropic drug resistance (PDR) sub-family, and hence designated as TaPDR1 (Triticum aestivum plei-otropic drug resistance). TaPDR1 was located on chromosome 5A of wheat using nullisomic-tetrasomic lines of Chinese Spring. TaPDR1 was up-regulated by induction of both DON and F. graminearum. Ex-pression patterns of TaPDR1 were different in wild-type Wangshuibai and the fast-neutron induced Wangshuibai mutant lacking FHB1, a major QTL of FHB resistance and DON resistance in chromosome arm 3BS. These results suggested that TaPDR1 might be a candidate gene responsible for DON ac-cumulation resistance. The expression profile showed that TaPDR1 expression was neither induced by hormones typically involved in biotic stress, such as JA and SA, nor by abiotic stresses, such as heat, cold, wounding and NaCl. However, TaPDR1 expression was regulated by Al3+ and [Ca2+], indicating that [Ca2+]i might mediate the signal of TaPDR1 expression.

wheat, deoxynivalenol (DON), ABC transporter, PDR (pleiotropic drug resistance)

Fusarium head blight (FHB) is a fungal disease caused by Fusarium graminearum, which can infect wheat, barley, small grain cereals and maize, and has become a destructive disease worldwide. FHB causes an enormous yield loss[1]. More seriously, it deteriorates grain quality by contaminating infected grains with trichothecene toxins, thus bringing food safety problems. Deoxyniva-lenol (DON) is one of the most important toxin mem-bers[2]. It inhibits syntheses of DNA, RNA and protein in eukaryotic cells, affects mitochondrial functions, sup-

presses cell division and changes plasma membrane permeability. In addition, DON can act as a virulence factor and plays an important role in FHB-induced plant

Received March 21, 2009; accepted June 6, 2009 doi: 10.1007/s11434-009-0553-0 † Corresponding authors (email: xiuew@njau.edu.cn; djliu@njau.edu.cn) Supported by the National Natural Science Foundation of China (Grant No. 30330380), Programme of Introducing Talents of Discipline to Universities (Grant No. B08025), Chinese High Technology Research and Development Program of China (Grant No. 2006AA10Z1F6), Natural Science Foundation of Jiangsu Province (Grant No. BK2006720), Important National Science & Technology Specific Pro-jects of Transgenic Research (Grant No. 2009Zx08002-001B) and McKnight Foun-dation CCRP Program

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diseases[3]. There are two stages in the process of the infection of FHB by Fusarium graminearum, including biotrophy and necrotrophy. In the initial stage of infec-tion, F. graminearum is a biotrophic pathogen. In the conversion process from biotrophy to necrotrophy, DON and other trichothecene toxins produced and accumu-lated by F. graminearum play an important role[4,5]. The infection ability of F. graminearum depends on its ca-pacity of DON production, and there exists significant correlation between the disease level of FHB in the field and the DON content in seeds or tissues of the infected spikes. Typical FHB symptoms appear when wheat tis-sues are treated with DON only[6]. The content of DON in wheat varieties with different resistant levels was ob-viously different[7,8], indicating that the production of DON in the infected spike might be affected signifi-cantly by the resistant varieties. It is proposed that there are some factors inhibiting DON syntheses or making it degraded in the resistant varieties. At present, several genes related to DON resistance have been isolated from various plants, such as acetyltransferase in wheat[9], the ribosomal protein L3 (RPL3) gene in rice[10] and wheat[11], the DOGT1 gene of the UDP-glycosyltrans-ferases (UGTs) family in A. thaliana[12], and the PDR type ATP-binding cassette (ABC) transporter in Sac-charomyces cerevisiae[12,13]. In wheat, FHB1, a major QTL of FHB resistance and DON resistance in chromo-some arm 3BS has been identified[14–17], and it probably encodes or regulates DON detoxification proteins[18]. In order to better understand molecular mechanisms of FHB or DON resistance, it is critical to identify new genes that are expressed closely upon DON treatment, because the inheritance of FHB resistance and DON resistance is a complicated quantitative trait and mecha-nisms under FHB resistance have not yet been clarified.

Wangshuibai is one of the very few wheat varieties with stable FHB resistance. More importantly, the in-fected grains of Wangshuibai contain lower DON con-tent[19–21]. Several major QTLs related to the FHB resis-tance and low DON content, such as FHB1, have been found in Whangshuibai[16,22,23]. To better understand the molecular mechanism of low DON content of Wang-shuibai, the GeneChip method was used to clone DON resistance related genes in the present research. Accord-ing to the Genechip result, an EST coding an ABC transporter gene was up-regulated by 45 times. The ABC transporter gene was subsequently identified from Wangshuibai cDNA. The expression of the gene was

induced by DON and F. graminearum infection, indi-cating that the gene might contribute to the low DON content of Wangshuibai.

1 Materials and methods

1.1 Materials

T. aestivum cv Wangshuibai, Wangshuibai mutant iden-tified from a fast-neutron treated Wangshuibai popula-tion, showing stable susceptibility to F. graminearum, (The characterization of the mutant will be published in another paper), Alondra’s, nullisomic-tetrasomic lines of Chinese Spring (T. aestivum), and wheat genomic DNA TAC library were maintained in Cytogenetics Institute, Nanjing Agricultural University, China.

1.2 Cloning of TaPDR1

A pair of primers SH49S (5′-ACTTTGACAACAAC CGACAG-3′) and SH49A (5′-GCATTAAGCACGATT TCC-3′) was designed based on the sequence of EST BQ161629. The primers were used to screen the wheat genomic DNA TAC library to identify clones containing the full length of the ABC transporter gene. PCR (poly-

merase chain reaction) was performed in a 25-L reac-

tion volume containing 1× PCR buffer, 2 L template,

0.2 mol/L of each primer, 200 mol/L (each) of de-oxyribonucleotides, 1.5 mmol/L MgCl2 and 1 U Taq DNA polymerase (Takara Bio Inc., Japan). The PCR thermocycling program was as follows: initial denatura-

tion at 94℃ for 3 min; followed by 33 cycles of dena-

turation at 94℃ for 30 s, annealing at 55℃ for 45 s,

extension at 72℃ for 50 s; and a final extension step at

72℃ for 10 min. PCR was carried out in a PTC-220

Tetrad Thermal Cycler (MJ Research). PCR products were examined by electrophoresis with 8% non-dena-turing polyacrylamide gels.

A pair of primers CPDR1S (5′-CACGGATCCACAG ACAGGCACAGAAAGA-3′) and CPDR1A (5′-AAAC CCGGGTTGACAACAACCG ACAGA-3′) was designed based on the 5′- and 3′-untranslated regions of the gene predicted by Softberry. The full size cDNA was amplified from Wangshuibai cDNA using primers CPDR1S and

CPDR1A. PCR was performed in a 50-L reaction vol-

ume containing 1× GC buffer II, 5 L template, 0.4

mol/L of each primer, 400 mol/L (each) of deoxyri-bonucleotides and 2.5 U of LA Taq DNA polymerase (Takara Bio Inc., Japan). The amplification was con

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ducted with the following cycling parameters: 3 min at

94℃; 33 cycles of 94℃ for 30 s, 45 s at 58℃, 4 min at

72℃; and a final extension step at 72℃ for 10 min. The

PCR was carried out in a PTC-220 Tetrad Thermal Cy-cler (MJ Research). PCR products were examined by electrophoresis with 1% agarose gel. DNA fragments were recovered from agarose gel after electrophoresis, and then cloned into pGEM-T Easy cloning vector (Promega). Clones containing the insert of right size were randomly selected for sequencing in Shanghai Bioasia Biotechnology Co. Ltd., China.

1.3 RT-PCR

(i) Treatment of wheat spikes with DON and F. graminearum. The young spikes of wheat Wangshui-bai and Alondra’s were treated with 100 mg/L DON, and the young spikes of wild type Wangshuibai and

Wangshuibai mutants were inoculated with 50 spores/L F. graminearum using single flower inoculation for 0, 6, 12, 24, 48, and 72 h and then frozen in liquid nitro-gen.

(ii) Treatment of wheat leaves with SA, JA, heat, cold and wounding. The young leaves of Wangshuibai were

treated with 1 mmol/L SA, 0.1 mmol/L JA, heat (40℃, 1

h), cold (4℃) and mechanical wounding for different

times and then frozen in liquid nitrogen. (iii) Treatment of wheat roots with NaCl. Three-

day-old roots of Wangshuibai were treated with 300 mmol/L NaCl for different times and then frozen in liq-uid nitrogen.

(iv) Treatment of wheat roots with Al3+ and [Ca2+]. Wheat seeds were germinated in 0.5 mmol/L CaCl2 solu-tion (pH 4.5) and the solution was changed daily. After growing for 3 d, the roots were treated with 0.5 mmol/L CaCl2 solution (pH 4.5) containing 0, 10, 20, 50, 100 or

200 mol/L AlCl3 and 0.05 mmol/L CaCl2 (pH 4.5) for 6 h and then frozen in liquid nitrogen.

(v) RNA extraction and RT-PCR. Total RNA was isolated from the above treated plant tissues frozen in liq-uid nitrogen using TRIzol reagent (Invitrogen). cDNA

was synthesized from 1 g of total RNA using AMV Re-verse Transcriptase XL (TaKaRa, Japan). RT-PCR was performed to analyze the expression level of TaPDR1 in different tissues of wheat. The gene-specific primers were SH49S and SH49A. The expression level of Tubulin gene was used as the control and the primers were TubulinF (5′-CTCATCACAGGCAAGGAAGAT-3′) and TubulinR

(5′-TTAAGGTAAGTGTAGGTTGGG-3′). The PCR was performed following the similar procedure as in TAC li-brary screening except that the number of cycles was changed to 28. PCR products were examined by electro-phoresis with 1% agarose gel.

1.4 Construction of the phylogenetic tree

A NJ phylogenetic tree was constructed in MEGA4 pro-gram[24] with 1000 bootstrap replicates.

1.5 Chromosome location of the TaPDR1 in wheat

A set of nullisomic-tetrasomic lines of Chinese Spring were used to determine the chromosome location of the TaPDR1 orthologs in wheat using the primer pair SH49S and SH49A. PCR procedure was the same as described in the TAC library screening.

2 Results

2.1 Cloning of the TaPDR1

GeneChip analysis using cDNA of DON-induced spikes of Wangshuibai (date not shown) revealed that an EST of the ABC transporter was up-regulated by 45 times. A positive TAC clone was identified using the primer pair SH49S and SH49A. The TAC clone was sequenced us-ing a chromosome walking method and the gene was predicted using Softberry software (http://www. soft-berry.com). Based on the genomic sequence, the cDNA of the gene was obtained by amplification of cDNAs from DON-induced Wangshuibai using the full-length gene specific primers CPDR1S and CPDR1A. The ge-nome size of the gene is 7377 bp, containing 19 exons with the coding sequence of 4308 bp, which encodes a 1435 aa protein with a calculated molecular weight of about 161 kD (Figure 1). Protein database search (http:// www. ncbi.nlm.nhi.gov) of the gene product revealed high similarity to amino acid sequences of PDR (plei-otropic drug resistance) sub-family (Figure 2). The PDR family is characterized by a configuration in which the nucleotide binding domain is closer to the N-terminus of the protein than the transmembrane domain. Thus, the gene was named TaPDR1 (Triticum aestivum pleiotropic drug resistance). The accession numbers of the TaPDR1 cDNA and genomic sequences in GenBank are FJ185035 and FJ858380, respectively.

2.2 Homology analysis

Plant PDR genes have been arbitrarily divided into five

clusters (I－V)[25]. TaPDR1 belongs to cluster IV (Fig-

ure 3). The closest TaPDR1 homologs are OsPDR3,

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Figure 1 The genomic organization of TaPDR1.

Figure 2 Conserved domains of TaPDR1 protein.

Figure 3 Phylogenetic analysis of plant PDR ABC transporters. The neighbor-joining method was used to construct a phylogenetic tree in MEGA4 with 1000 bootstrap replicates. All genes belonging to subfamily IV were taken into account; for other subfamilies, representative genes from different species were used. The accession numbers are as follows: GmPDR12, CAK03587; TaPDR1, ACL8065; other accession numbers refer to ref.[25]. Blue triangles mean the protein which has been best characterized at the functional level. The red circle indicates the protein reported in the present research.

OsPDR4, and OsPDR5 in rice, with which it shares 79%, 71% and 68% amino acid identity, respectively. TaPDR1 also shares 61% identity with both AtPDR6 and AtPDR11 in Arabidopsis. To date, the functions of these Arabidopsis and rice homologs are unknown. To-bacco NpPDR2, which shares 61% identity with TaPDR1, is the only one that has been characterized at the functional level, and NpPDR2 is in cluster IV[26]. Unlike other PDR genes, the expression of NpPDR2 was

not induced in leaf tissues by any hormone which is typically related with biotic and abiotic stresses. Fur-thermore, RNA interference of the expression of NpPDR2 showed no obviously altered phenotypes. The in vivo function of this PDR gene cluster in plants re-mained elusive.

2.3 Chromosome location

Nulli-tetrasomic analysis indicated that three orthologs of TaPDR1 were present in wheat genome. They were

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located in the chromosomes 4A, 5A and 5D, respec-tively (Figure 4). A PCR product of 269 bp was ampli-fied from the TAC clone using the primer pair SH49S and SH49A, which had similar size with that amplified from chromosome 5A. Thus, TaPDR1 was located in chromosome 5A. Wheat is an allohexaploid. Many genes have the same copy numbers in three homoeolo-gous genomes. As shown in Figure 4, TaPDR1 had three copies. However, only the 269 bp band could be ampli-fied from cDNA of the DON-induced Wangshuibai spike, while the other two bands could not. It was specu-lated that the other two copies of the gene were silenced through the genetic diploidization in the process of poly-ploidization, or these genes differentiated following al-lopolyploidization and became no longer involved in DON resistance.

Figure 4 The chromosome location of TaDPR1 with a set of nul-lisomic-tetrasomic lines of Chinese Spring. The arrow shows where the TaPDR1 gene was located.

2.4 Expression profile of TaPDR1

(i) TaPDR1 is induced by both DON and F. graminea-rum. RT-PCR was conducted to analyze the expres-sion of TaPDR1 induced by DON in resistant and sus-ceptible varieties, and different expression patterns were observed (Figure 5). The expression level reached the peak after DON inoculation for 6 h to 12 h in Wang-shuibai and thereafter declined slowly. While in the susceptible variety Alondra’s, the expression level did not reach the peak until inoculation for 12 h.

Figure 5 TaPDR1 expression is regulated by DON in the spikes of the resistant and susceptible cultivar. In the semiquantitative RT-PCR analysis of the expression level of TaPDR1 following treatment with

DON (100 mg/L), Tubulin was used as a control. 0―72 h were the

various time points of induction by DON. (a) Wangshuibai; (b) Alon- dra’s.

TaPDR1 was also induced by F. graminearum. The relative amount of the transcript of TaPDR1 increased at 48 h after inoculation in Wangshuibai (Figure 6). It was reported that DON can be detected 36 h after F. graminearum inoculation[27]. We speculate that the function of TaPDR1 in FHB resistance might be DON- related.

Figure 6 TaPDR1 expression is regulated by the inoculation of F. graminearum in spikes. The semiquantitative RT-PCR analysis of the expression level of TaPDR1 following treatment with F. graminearum

(50 spores/L), Tubulin was used as a control. 0―72 h were the

various time points of induction by F. graminearum. (a) Wangshuibai wildtype; (b) Wangshuibai mutant.

A major QTL of FHB resistance and DON resistance has been repeatedly located in chromosome arm 3BS in Wangshuibai, and the resistant locus was named FHB1. A susceptible mutant, which is a deletion of FL0.78~ 0.87 of 3BS, has been identified in our group (the result will be published seperately) and used to determine the relation of TaPDR1 and FHB1. The expression level of TaPDR1 after F. graminearum inoculation in the wild type and mutant plants indicated that the expression pat-terns of TaPDR1 were different. As shown in Figure 6, the expression level culminated 48 h after F. graminea-rum inoculation in Wangshuibai. By contrast, the ex-pression level of TaPDR1 was very low throughout the whole inducing period in the mutant. These results sug-gested that TaPDR1 might be a candidate gene respon-sible for DON accumulation resistance. Recently, a PDR type ABC transporter which shared 46% amino acid identity with TaPDR1 was reported. This gene conferred durable resistance to multiple fungal pathogens in wheat, such as Lr34, Yr18, Pm38 and Ltn1[28], further demon-strating the importance of wheat PDR genes in fungal disease resistance.

(ii) TaPDR1 is not involved in JA, SA pathway and other stress tolerances. PDR genes are widely involved in response to biotic and abiotic stresses in plants[25]. However, the expression profile showed that TaPDR1 expression was induced by neither hormones typically involved in biotic stresses, such as JA and SA, nor some abiotic stresses, such as cold, wounding and NaCl (data not shown). The result was the same as the expression

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profile of NpPDR2[26]. The expression patterns of the Arabidopsis PDR genes showed that AtPDR6 expression was only observed in roots, and was induced by epi-brassinolide (EBR), dichlorophenoxyacetic acid (2,4-D),

NaCl, and CdCl, at 11℃/low light and 4℃/darkness, but

not by cycloheximide (CHX) and abscisic acid (ABA) in seedling. Moreover, AtPDR11 expression was not found in roots, stems, leaves, inflorescences and seedlings, and was not induced by the above treatments in seedling[29]. These results suggested that transporters in cluster IV might have different functions in vivo.

(iii) TaPDR1 is induced by Al3+ and [Ca2+]. TaMD- R1, an Al-induced gene from wheat roots[30], was found to be up-regulated in our GeneChip result, which indi-cated that TaMDR1 may be involved in DON resistance. In order to determine the response of TaPDR1 to Al in-duction, the expression pattern of TaPDR1 in wheat roots treated with Al3+ and [Ca2+] was analyzed. It was found that the expression of TaPDR1 was induced as the Al3+ and [Ca2+] decreased (Figure 7). These results in-dicated that TaPDR1 was a functional homolog of TaMDR1. Likewise, yeast PDR5 was considered a func-tional homolog of human MDR1[31].

Figure 7 TaPDR1 expression is responsive to Al3+ and [Ca2+]. The semiquantitative RT-PCR analysis of the expression level of TaPDR1 and TaMDR1 following treatment: 3-day-old wheat roots germinated in 0.5 mmol/L CaCl2 solution (pH 4.5) and in the solution changed daily were treated with: I, 0.5 mmol/L CaCl2 solution (pH

4.5); II, 0.05 mmol/L CaCl2 solution (pH 4.5); 1－6, 0.5 mmol/L

CaCl2 solution (pH 4.5) containing 0, 10, 20, 50, 100 or 200 mol/L AlCl3, respectively for 6 h. Tubulin was used as a control.

Ca2+ is an intracelluar second messenger, involved in

signal transduction of cell response to external biotic and abiotic stresses. [Ca2+]i was increased by exogenous JA and SA, as well as abiotic stresses such as heat, cold, wounding and NaCl[32–37]. Oppositely, [Ca2+]i was de-creased by Al stress, for Al is a calcium channel inhibi-tor. The decrease of the extracellular [Ca2+] also resulted in the decrease of the [Ca2+]i. In animals, it has been found that DON inhibited the calcium channel of myo-

cardial cells[38,39], but there is no report about the rela-tionship of DON and the calcium channel in plants. The DON treatment might cause the decrease of [Ca2+]i, and induce the expression of TaPDR1. According to these results, we speculate that [Ca2+]i might mediate the sig-nal of TaPDR1 expression.

3 Discussion

The ABC transporters are a kind of membrane-spanning transportation proteins composing the largest family with most extensive functions. They share a common basic structure containing an ATP-binding cassette, and most of them use energy from ATP hydrolysis to pump substrates across membranes, including peptides, sugars, lipids, heavy metal chelates, amyloses, alkaloids, ster-oids, inorganic ions and glutathione conjugates. Now ABC transporters are attracting wide attention for they are closely correlated with some important physiological processes in prokaryotes and eukaryotes, such as bacte-rial resistance, cystic fibrosis and medicinal resistance of tumor cells[25,29,40,41]. The PDR family is special in that its members have a reverse domain organization (NBD-TMD)2 and are found only in fungi and plants[25].

In plants, PDR genes in cluster I are the best charac-terized at the functional level. SpTUR2 from the aquatic plant, S. polyrrhiza, is the first identified plant PDR transporter gene, and its expression is induced by ab-scisic acid as well as cold and salt stresses, suggesting that it plays a certain role in response to abiotic stress conditions[42,43] Two other PDRs participating in the abiotic stress responses are OsPDR9 in rice[44] and AtPDR12 in A. thaliana[45]. Expression of OsPDR9 was strongly induced in roots by osmotic, salt and hypoxic stresses and also by heavy metals (Cd, Zn) and redox perturbations[44]. AtPDR12 was strongly induced by lead, and the knock-out and overexpression of AtPDR12 fur-ther proved its function in Pb resistance in plants[45]. NpPDR1 was isolated from a N. plumbaginifolia culture cell line treated with sclareol, a diterpene with antim-icrobial properties found on the leaf surface of Nicotiana species[46]. The induced expression of NpPDR1 in N. plumbaginifolia suspension cells decreased accumula-tion of a radioactively labeled sclareol analog within the cells[46]. NpPDR1 is expressed in leaf trichomes and its expression is regulated by the plant defense signaling molecules, JA and SA. These data suggest that NpPDR1 is actively involved in plant defense by extruding

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sclareo[47]. Sclareol also induced the expression of SpTUR2 and AtPDR12[25,48]. Now mounting data support involvement of these NpPDR1 orthologs in biotic stress response and plant defense. NpPDR2 was isolated from a N. plumbaginifolia and belongs to PDR cluster IV. NpPDR2 transcripts were found in the roots and mature flowers, and localized around the conductive tissues. In the latter, NpPDR2 expression was restricted to the style and only after pollination. NpPDR2 expression was in-duced in leaf tissues neither by hormones typically in-volved in biotic and abiotic stresses nor by Botrytis cinerea. No phenotype alteration was observed when NpPDR2 expression was decreased by RNA interfer-ence[26]. TaPDR1 expression was not induced by the hormones typically involved in biotic stress JA and SA, and some abiotic stresses, but its expression was regu-lated by DON and F. graminearum. These data suggest that PDR cluster IV was actively involved in biotic stresses and plant defenses. Like sclareol, a diterpenoid, could be a substrate of cluster I transporters, DON, a sesquiterpenoid, may be a substrate of cluster IV trans-porters, and could play an important role in functional characterization of cluster IV transporters.

The PDR transporter was first characterized in the yeast (S. cerevisiae). It conferred resistance to antifungal drugs as a pump export fungal mycotoxin from the cy-toplasm and prevented the cytotoxicity, and thus was one of the important disease resistance proteins. Yeast pdr5 mutants were hypersensitive to the mycotoxin DON compared with the wild type, indicating the func-tion of PDR transporter in DON resistance[12]. Wheat TaPDR1 gene expression was induced by DON and F. graminearum. The induced expression of TaPDR1 in the resistant variety was earlier than in the susceptible cul-tivar and the expression level was higher. These results indicated that theTaPDR1 transporter could export fun-gal mycotoxin DON, or detoxification products of DON from cells, because all of the characterized PDR proteins were plasma membrane localized and TaPDR1 was also located at the plasma membrane by software prediction. Other transporters may be responsible for vacuole ori-entated transportation, as some other ABC transporters, such as TaMDR1, were also found upregulated in our Genechip result.

Except for inducing by DON, TaPDR1 expression was

induced by Al3+ and free [Ca2+]. Two hypotheses were proposed: Firstly, TaPDR1 was not involved in Al toxic-ity. The explanation for the induction of TaPDR1 ex-pression by Al was that Al inhibited the calcium channel, making [Ca2+]i decreased and hence inducing the signal of TaPDR1 expression; secondly, TaPDR1 was involved in DON resistance and also responded to Al toxicity. It is worthwhile to further study these hypotheses in order to clarify the function of TaPDR1 in DON resistance.

DON inhibited the calcium channel of myocardial cells in animals and caused the decrease of [Ca2+]i

[38,39], indicating that the toxicity of DON was initially shown as its inhibition of the calcium channel of the plasma membrane. DON has important physiological signifi-cance in the infection process of F. graminearum. Ca2+, an intracelluar second messenger, is widely involved in signal transduction of cell disease resistance. [Ca2+]i could increase by both exogenous JA and SA. Calmodulin and calcium-dependent kinases were acti-vated for signal transduction of disease resistance by Ca2+. However, DON inhibited the increase of [Ca2+]i, and disturbed the signal transduction of disease resis-tance. In addition, Ca2+ was an important component of the cell membrane and the cell wall. Calcium deficiency caused disintegration of the cell membrane and the cell wall, which was beneficial to the invasion of hyphal of F. graminearum. Perception and recognition for F. graminearum by wheat were obviously different be-tween the resistant and susceptible varieties. Similar difference was also found in the interaction of DON and wheat. Ca2+ is involved in signal transduction of DON resistance, which is probably the signal transduction of FHB resistance. The results are important in elucidating the mechanism of FHB resistance.

In wheat, FHB1, a major QTL of FHB resistance and DON resistance in chromosome arm 3BS, was proposed to encode or regulate the UGTs and thus contribute to DON resistance [18]. However, until now the major QTL FHB1 has not been identified. Here, we report the TaPDR1 gene, the expression of which is regulated by the major QTL FHB1, suggesting that the major QTL FHB1 is likely a regulatory gene.

Acknowledgement The authors would like to thank Professor SUN WenXian of Chinese Agricultural University for his critical reading of the manuscript.

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