JXB Advance Access published online on August 28, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm151
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
A novel ERF transcription activator in wheat and its induction kinetics after pathogen and hormone treatments
1The National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 10008, PR China
2The National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Biotechnology, Chinese Academy of Agricultural Sciences, Beijing 100081, PR China
* To whom correspondence should be addressed. E-mail: zyzh-68{at}163.com; zhangzy{at}mail.caas.net.cn
Received 18 April 2007; Revised 3 June 2007 Accepted 5 June 2007
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Abstract |
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In this study, a pathogen-inducible ERF (ethylene-response factor) gene in wheat, designated TaERF3, was isolated and characterized in detail. The sequence of the TaERF3 protein possesses all of the traits commonly associated with ERFs, but its entire sequence shares low identity with other ERFs of transcription factor families. The results of assays on subcelluar localization, GCC box-binding ability, and transactivation activity indicated that TaERF3 is a nuclear targeting protein and functions as a GCC box-binding transcriptional activator. Following infection with Blumeria graminis, the induction peak of TaERF3 expression occurring at 12 h in the resistant line was about six times higher than that in its susceptible parent. Following infections with Fusarium graminearum or Rhizoctonia cerealis, the TaERF3 maximum inductions in the susceptible line occurring at 12 h were about three or six times higher than those in the resistant lines, whereas after 24 h or 48 h, the transcript inductions in the resistant lines were much higher than that in the susceptible line. Furthermore, the TaERF3 transcript peak induced by salicylic acid (SA) treatment occurred at 4 h, whereas the peaks induced by exogenous ethylene and methyl jasmonate (MeJA) occurred at 24 h, all of which were earlier than those induced by pathogens in the resistant lines. These results suggested that TaERF3 might be mainly involved in the active defence response to B. graminis at an earlier stage through SA signalling, and to F. graminearum and R. cerealis at a later stage through the ethylene/jasmonic acid signalling pathways.
Key words: Defence response, ethylene-response factor (ERF), induction kinetics, transcription activator, wheat, Triticum aestivum
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Introduction |
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Plants cope with pathogen attack through complex adaptive responses. Depending on the severity of the responses, a plant may succumb to the pathogen (compatible interactions), or prevent the pathogen from colonizing it (incompatible interactions), in which the latter shows resistance to the pathogen (Glazebrook, 2005). Appropriate regulation of the defence response is important for plant fitness (Glazebrook, 2005). Many members of ERF, WRKY, bZIP, and MYB transcription factor families are involved in regulating the defence response (Singh et al., 2002). ERF is also named the ethylene-responsive element binding protein (EREBP), containing a highly conversed ERF/AP2 domain (Hao et al., 1998). The ERF family is unique to plants and some ERFs have been identified from some plant species (Ohme-Takagi and Shinshi, 1995; Zhou et al., 1997; Solano et al., 1998; Fujimoto et al., 2000; Park et al., 2001; Mazarel et al., 2002; Tournier et al., 2003; Guo et al., 2004; Zhang et al., 2004; Cao et al., 2006a). ERF proteins can specifically bind the GCC box cis-acting element (core sequence AGCCGCC) that is present in promoters of pathogenesis-related (PR) genes (Ohme-Takagi and Shinshi, 1995; Park et al., 2001). Some ERFs can activate transcripts of the PR genes, contributing to the plant's ability to overcome disease through the ethylene, jasmonic acid (JA), and salicylic acid (SA) signalling pathways (Park et al., 2001; Berrocal-Lobo et al., 2002; Gu et al., 2002; Berrocal-Lobo and Molina, 2004; Guo et al., 2004; Zhang et al., 2004; McGrath et al., 2005; Cao et al., 2006b). Interestingly, consistent with their defensive function, the mRNA expression of the ERF genes mentioned above are up-regulated by pathogen infection, and ethylene, JA/methyl jasmonate (MeJA), and SA (Gu et al., 2000; Park et al., 2001; O
ate-Sánchez and Singh, 2002; Lorenzo et al., 2003; Gutterson and Reuber, 2004; Zhang et al., 2004; McGrath et al., 2005). Based on these characteristics, McGrath et al. (2005) put forward a two-step strategy to identify transcription factors (TFs) with defence roles: firstly to identify TF genes that show altered transcript levels during the early stage of the defence response; and, secondly, to perform functional analysis of these candidate genes. Some TFs with a defence function in Arabidopsis were successfully identified using this strategy (McGrath et al., 2005). Wheat (Triticum aestivum) is an important source for the human diet. The production of wheat in China has been seriously affected by various diseases, such as powdery mildew caused by Blumeria graminis f.sp. tritici, stripe rust caused by Puccinia striiformis f.sp. tritici, scab caused by Fusarium graminearum, and sharp eyespot caused by Rhizoctonia cerealis. According to the lifestyles of the pathogens, B. graminis and P. striiformis belong to the biotrophic fungi, whereas F. graminearum and R. cerealis belong to the necrotrophic fungi. However, none of the ERF proteins in wheat with a defence function has yet been studied in detail, although two wheat ERFs, TaERF1 and TaERF2, were found in the GenBank database. To understand the defence mechanisms of wheat further, it is necessary to isolate, characterize in detail, and functionally to analyse the wheat ERFs related to defence responses.
The objectives of this study are (i) to isolate a pathogen-inducible gene encoding a novel member of the ERF proteins; (ii) to characterize in detail the properties of the predicted ERF protein, including its subcellular localization, GCC box-binding ability, and transactivation activity; and (iii) to investigate whether the ERF gene is involved in defence responses by analysing the gene expression patterns in incompatible and compatible interactions between different wheat lines and different pathogens.
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Plant material and treatments
Seeds of the wheat lines/cultivars were germinated and grown in growth chambers for 12/12 h light/dark at 25/20 °C. Seedlings at the two-leaf stage were treated by the following pathogens or hormones. Inoculation with B. graminis was carried out by spraying with a conidial suspension of 106 spores ml–1 on to wet leaf surfaces of a powdery mildew-resistant line ‘Pm97034’ possessing Pm21 and its susceptible parent ‘Wan7107’. Inoculation with F. graminearum was performed by rubbing fresh mycelium onto wet leaf surfaces of a scab-resistant cv. ‘Sumai-3’ (supplied by Professor Weizhong Lu, Jiangsu Academy of Agricultural Sciences), and a susceptible line ‘Wenmai6’. Inoculation with R. cerealis was performed by rubbing fresh mycelium onto wet leaf surfaces of a sharp eyespot-resistant line ‘Shannong0431’ (provided by Professor Sishen Li, Agricultural University of Shandong), and a susceptible line ‘Wenmai6’. The hormone treatments on the seedlings of ‘Sumai-3’ or ‘Pm97034’, including 1 mM salicylic acid (SA), 0.1 mM methyl jasmonate (MeJA), and ethylene released from 0.2 mM ethephon, were carried out following the protocol described by Zhang et al. (2004). In addition, the resistant ‘Pm97034’ was inoculated with B. graminis on seedlings treated for 48 h with 1 mM SA or water (CK) using the method of benzothiadiazole (BTH) with Magnaporthe grisea treatments as described by Cao et al. (2006b). Leaves from the treatments mentioned above were harvested at various time intervals, then quickly frozen in liquid nitrogen and stored at –80 °C until use.
RNA extraction and the first-strand cDNA synthesis
Total RNA of each wheat sample was extracted using the TRIZOL reagent according to the manufacturer's protocol (Invitrogen, USA), and then subjected to RNase-free DNase I (Promega, USA) digestion and purification. Five micrograms (µg) of RNA per sample was used to synthesize the first-strand cDNA using the Superscript II First-Strand Synthesis Kit for RT-PCR (Invitrogen).
Isolation of the TaERF3 gene
The cDNA of ‘Sumai-3’ at 12 h post-infection with F. graminearum was used as template for isolating the cDNA sequence of the wheat ERF gene. The sequence of Arabidopsis AtERF1 (O80337
[GenBank]
) was used as a query sequence for a BLAST search on GenBank. The search returned rice ERF, OsBIERF3 (AAV98702
[GenBank]
), which is homologous to AtERF1. Then two wheat ESTs (expression sequence tags), CN012725 and CN010562, encoding the partial sequence of a putative ERF protein with ERF/AP2 domain, were found to be homologous to OsBiERF3. Based on these sequences, a pair of primers (WE-F1: 5'-ACGGCGAGGATGCTGCTGAAC-3’ and WE-R1: 5'-CGATCTCGGAGCGGATGCGGA-3’) were designed, synthesized, and used to amplify the partial sequence of a putative ERF gene from wheat. Then, the end sequences of the ERF gene were obtained by RACE (rapid amplification of cDNA end) method according to the manufacturer's instructions (Invitrogen). The full-length cDNA sequence of the resulting ERF gene with 1479 bp length was obtained by analysing the overlaid sequences. Based on the full-length cDNA sequence, a pair of gene-specific primers (WE-F1 and WE-CR: 5'-TTGATCCACTTGGGCGAGTTG-3’) were designed, synthesized, and used to amplify the reconstituted cDNA sequences of TaERF3, containing the complete ORF (open reading frame) with 909 bp length, from cDNAs of ‘Sumai-3’ and ‘Pm97034’ infected by B. graminis. The purified PCR products were cloned into the pMD18-T vector (Takara, Japan) and transformed into E. coli competence cells. More than five positive clones respectively from ‘Sumai-3’ and ‘Pm97034’ were sequenced using the ABI 3730 sequencer (Applied Biosystems, USA) by SinoGenoMax Co. LD (China). The confirmed ERF gene of wheat was designated TaERF3. Its sequence has been deposited in the National Center for Biotechnology Information (NCBI) database under the accession number EF570122.
In order to analyse the genomic sequence of the TaERF3 gene, genomic DNA of ‘Sumai-3’ as the template was amplified using the gene specific primers (WE-F1 and WE-CR). The sequence of TaERF3 derived from the genomic DNA was compared with that from cDNA.
Sequence analysis
Sequence analysis was performed using DNAMAN software (Lynnon Biosoft, USA), DNASTAR software (http://www.dnastar.com/web/index.php), and BLAST online (http://www.ncbi.nlm.gov/blast). Phylogenetic analysis on TaERF3 and the other published ERF-related proteins was performed using the MegAlign program of DNASTAR software. The phylogenetic tree was drawn after the sequences entered were aligned by ClustalW. Homology modelling of TaERF3 was performed using program facilities at http://expasy.chs. TaERF3 modelling was performed based on AtERF1 (PDB accession number 1GCC/2GCC/3GCC).
Construction of TaERF3 expression vector and GCC box-binding assay in vitro
The partial sequence of TaERF3 with 147 amino acids containing the ERF/AP2 domain was amplified by the primers (W3-APF: 5'-ATGGATCCCCGTCCTGCTTCGGTTTC-3', the underlined sequence is the BamHI site, and W3-APR: 5'-ATGTCGACTTCCTCCTTTTCGGG GAC-3', the underlined sequence is the SalI site) and then fused in-frame to the BamHI/SalI sites at the 3'-terminus of the coding region of glutathione S-transferase (GST) in the pGEX-4T-1 vector (Amersham). After sequencing confirmation, the resulting GST-TaERF3 and the control GST proteins were overexpressed in E. coli strain BL21 cells after being induced with 0.4 mM isopropyl-ß-D-thinogalactopyranoside (IPTG) for 4 h at 30 °C and purified using MicroSpin GST Purification Module (Amersham).
The DNA binding ability of the TaERF's ERF/AP2 domain in vitro was analysed by the electrophoretic mobility-shift assay (EMSA). Both the wild-type GCC probe containing two copies of the GCC box sequence (GCC: 5'-AATTCATAAGAGCCGCCACT-3’) and the mutant mGCC probe containing two copies of the mutated GCC box sequence (mGCC: 5'-AATTCATAAGATCCTCCACT-3’) were synthesized and prepared following protocols by Ohme-Takagi and Shinshi (1995) and Mazarel et al. (2002). The GCC box and mGCC box probes were labelled by 
-32P-dATP with the Klenow end-fill reaction following the prime--gene labelling kit (Promega; Mazarel et al., 2002). The binding reaction was performed on ice for 30 min in binding buffer [10 mM TRIS–HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 0.5 mM Na2EDTA, 0.5 mM DTT, 4% glycerol, 0.05 mg l–1 poly (dI-dC)] with approximately 3 ng probe and 1.5 µg purified protein in a total volume of 10 µl. The reaction mixture was subjected to 8% polyacrylamide gel in 0.5x TRIS-borate-EDTA (TBE) buffer and autoradiographed using a phosphoimaging system.
Transactivation assay of TaERF3 in yeast
To prepare the effector construct pYepGAP::TaERF3 that can be expressed in yeast cells, the full ORF of TaERF3 was cloned downstream of the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene (GAP) in the yeast expression vector pYepGAP (provided by Drs Qiang Liu and Jun Zhao). Reporter vectors containing the wild-type GCC or mutated GCC, 3x GCC-lacZ and 3x mGCC-lacZ, were prepared and integrated into the genome of the yeast strain YM4271, in which three copies of the GCC box sequence (3x GCC) or the mGCC box sequence (3x mGCC) were, respectively, inserted upstream of the minimal promoter element (MP) and the reporter gene lacZ existed in the vector placZi (Clontech). The effector vector was transformed into the yeast competence cells carrying the 3x GCC-lacZ or 3x mGCC-lacZ reporter vector following the manufacturer's instructions for the yeast one-hybrid protocol (Clontech). The ß-galactosidase (encoded by lacZ) filter-lifted assay was performed according to the manufacturer's protocol (Clontech). The relative quantification of ß-galactosidase activity was carried out using O-nitrophenyl-ß-D-galactopyranoside (ONPG, Sigma) as a substrate following the method of Chen et al. (2003). The data for ß-galactosidase relative activity were averaged from triplicate samples and three independent experiments.
Subcellular localization of TaERF3
The termination codon of TaERF3 was removed by the PCR method. The modified ORF of TaERF3 was fused in-frame to the 5'-terminus of the coding region of the green fluorescent protein (GFP) and driven by a CaMV 35S promoter. The resulting 35S::TaERF3-GFP fusion construct and 35S::GFP vector as a control were respectively bombarded into 20 living onion epidermal segments (about 0.5 cM2) and then used for transient expression assay referring to the protocol of Guo et al. (2004). Expression of the genes introduced into the onion epidermal cells was observed using a Confocal Laser Scanning Microscopy (Leica TCS-SP2, Germany).
Expression analysis of TaERF3 by quantitative RT-PCR
Expression patterns of TaERF3 in response to pathogen and hormone treatments were analysed using the real-time quantitative RT-PCR (Q-RT-PCR) method. Q-RT-PCR analyses were conducted using the ABI Prism 7000 system (Applied Biosystems, USA) according to the modified protocol of Livak and Schmittgen (2001). The actin gene, expressed constitutively in wheat (Okubara et al., 2002), was used as an internal reference using the gene-specific primers (ActA: 5'-CACTGGAATGGTCAAGGCTG-3’ and ActB: 5'-CTCCATGTCATCCCAGTTG-3’). The transcript analysis of the TaERF3 gene was performed using the TaERF3 gene-specific primers (WE-U2: 5'-GCAATCAGGCAAAGCAACC-3’ and WE-L2: 5'-CGACTCAGAAGGAACCACGA-3’), which were located in the 3'-untranslated region of the gene. Validation experiments were performed to demonstrate that amplification efficiency of the TaERF3 specific primers were approximately equal to the amplification efficiency of the endogenous reference primers. The PCR reaction was performed in 25 µl of reaction mixture containing 1x SYB Premix Ex Taq (Takara, Japan), 0.2 µM of each primer, 1x Rox Reference Dye, and about 10 ng cDNA per sample. The program used was as follows: 1 min at 94 °C; followed by 40 cycles of 5 s at 94 °C and 15 s at 60 °C. Quantification of the target gene expression was carried out with comparative CT method (Livak and Schmittgen, 2001). Average CT values for the target gene from at least three PCRs were normalized to average CT values for actin from the same cDNA preparations and analysed using Microsoft Excel. The relative expression of TaERF3 indicated the increasing fold of the gene expression over the control (0 h before treatment).
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Identification and sequencing analysis of TaERF3
Using RT-PCR and RACE methods, a full-length cDNA of the ERF gene of wheat with 1479 bp length was obtained from ‘Sumai-3’ infected by F. graminearum (Fig. 1). In order to study the character and function of the gene, the reconstituted cDNA sequences of the ERF gene with 1258 bp, including the complete ORF with 909 bp, 5'-untranslated region (UTR) with 9 bp, and 3’ UTR with 340 bp, were amplified, cloned, and sequenced, respectively, from ‘Sumai-3’ infected with F. graminearum and ‘Pm97034’ infected with B. graminis using the gene-specific primer pairs of WE-F1 and WE-CR (Fig. 1). The ORF sequences from the different wheat accessions, i.e. ‘Pm97034’ and ‘Sumai-3’, are identical. This wheat ERF gene is designated as TaERF3.
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To find out whether the TaERF3 gene has any intron, the primers WE-F1 and WE-CR were used to amplify the gene sequence from the genomic DNA of ‘Sumai-3’. The results showed that the size of the PCR product derived from the genomic DNA was the same as that from the cDNA sequence obtained (data not shown). Sequencing analysis confirmed that the gene sequence was identical to the cDNA sequence, proving that the genomic transcription unit of TaERF3 probably has no intron, at least in the region flanked by these two primers.
The deduced TaERF3 protein contains 303 amino acids with a predicted molecular weight of 31.7 kDa. The TaERF3 sequence possesses the molecular characteristics commonly associated with ERFs (Fig. 1). TaERF3 has an ERF/AP2 domain, in which there were the 14th A (ala) and the 19th D (asp) residues, two key residues contributing a functional GCC box-binding activity in many ERFs (Sakuma et al., 2002). In addition to the ERF/AP2 domain, the TaERF3 protein includes an acidic region shown in other species to act as a transcription activation domain (Fujimoto et al., 2000), a putative nuclear localization signal (PKRRKR), and a serine-rich region.
The results of the BLAST-Protein (BLASTP) online (http://www.ncbi.nlm.gov/blast) showed that the TaERF3 protein shares a highly conserved ERF/AP2 domain with other ERF proteins, including rice OsBIERF3, Arabidopsis AtERF1, AtERF13, and AtERF2, tomato Pti4 and LeERF1, and tobacco NtERF1 and NsERF2 (Fig. 2A). This domain is divided into two conserved segments of YRG and RAYG, in which three ß-sheets and 1 -helix are predicted, all of which are important for DNA binding (Allen et al., 1998; Mazarel et al., 2002). The ERF/AP2 domain of TaERF3 is predicted to have such structures (Fig. 2A). However, there is an obvious difference in that the 57th residue is asp (D) in the ERF/AP2 domain of TaERF3, but asn (N) in those of other ERFs (Fig. 2A). The molecular modelling and multiple 3D alignments showed that the unique 57th residue D in the ERF/AP2 domain is not located in the key conformation regions. Comparing the complete protein sequences, the TaERF3 protein shares a low identity (24
62.5%) with the ERF proteins described above. Sequence analysis also revealed that TaERF3 shares very low homology (<20%) with the other ERF proteins of wheat searched from public databases, including TaERF1, TaERF2, TaEREBP, and TaEREBP-c. Based on the views of Gutterson and Reuber (2004) and our reconstructed phylogenetic tree (Fig. 2B), TaERF3 is most closely related to OsBIERF3 and belongs to the previously described B3 subgroup of the ERF family, whereas TaERF1, TaERF2, TaEREBP, and TaEREBP-c are closely related to CaPF1 in hot pepper and JERF3 in tomato, belonging to the B2 subgroup of the ERF family. These results suggested that TaERF3 encodes a novel member of the ERF family.
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TaERF3 protein interacts with the GCC box in vitro
Since the ERF/AP2 domain of TaERF3 contained the key amino acids to bind the GCC box, the recombinant GST–TaERF3 protein containing the ERF/AP2 domain of TaERF3 was constructed and overexpressed in E. coli, purified, and used to examine the DNA binding ability. The purified GST–TaERF3 protein was mixed, respectively, with the labelled wild-type GCC probe or the mutated GCC probe in the binding reaction. The results of EMSA showed that the gel mobility shift was specific to the GST–TaERF3 protein with the labelled GCC probe (lane 1 in Fig. 3). As expected, there were no shifted bands in the combination of GST–TaERF3 plus the mutated GCC (mGCC) probe (lane 4 in Fig. 3) and in the negative controls, including GST with the labelled GCC probe (lane 2) or mGCC probe (lane 5), and only the labelled GCC probe (lane 3) or mGCC probe (lane 6) (Fig. 3). The results demonstrated that the ERF/AP2 domain of TaERF3 was able to bind to the GCC box cis-acting element, but not to the mGCC box.
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TaERF3 protein activates transcription in yeast
To investigate the transcription activation activity of TaERF3 in yeast cells, the TaERF3 effector vector pYepGAP::TaERF3 and the reporter vectors of 3xGCC-lacZ and 3xmGCC-lacZ, in which the vectors did not contain any yeast activation-domain and binding-domain except domains present in TaERF3, were constructed. The results of the ß-galactosidase activity assays by lifted-filter (Fig. 4B) and relative activity quantification (Fig. 4C) showed that heterogenous expression of the TaERF3 protein effectively activated transcription of the lacZ reporter gene in the wild type GCC box yeast, but not in the mutated GCC box yeast. The ß-galactosidase relative activity of the combination of the 3xGCC-lacZ reporter with pYepGAP::TaERF3 was more than 10 times higher than those of the combination of the control vector placZi with the pYepGAP::TaERF3 effector and of the 3xmGCC-lacZ reporter with the effector (Fig. 5C). The results proved that the TaERF3 protein was a transcription activator to regulate positively downstream genes through binding the GCC box cis-acting element in promoters.
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Targeting of TaERF3 protein to the nuclei
To examine the subcellular localization of the TaERF3 protein in vivo, the fused expression vector 35S::TaERF3-GFP was constructed and used to perform a transient expression assay in onion epidermal cells. Twenty onion epidermal segments bombarded with 35S::TaERF3-GFP and 35S::GFP vectors were analysed. Evidently, the TaERF3–GFP fusion protein was exclusively localized in the nuclei of 16 cells recovered with TaERF3–GFP expression, whereas the control GFP was distributed throughout the cells (Fig. 5). The results indicated that the TaERF3 protein was targeted to the nuclei.
Expression patterns of TaERF3 after pathogen and hormone treatments
To investigate whether TaERF3 is involved in the wheat defence response, the expression patterns of TaERF3 in interactions between different wheat lines and pathogens were analysed using the Q-RT-PCR method. Following a biotrophic pathogen B. graminis infection, TaERF3 transcripts in a resistant line ‘Pm97034’ were induced at 6 h after inoculation (hai), peaked (about 7-fold increase over 0 h) at 12 hai, then decreased at 24 hai but increased slightly at 48 hai (Fig. 6A).The induced levels in the resistant line ‘Pm97034’ were much higher than in the susceptible line ‘Wan7107’, for example, the induction at 12 hai was about six times higher in the resistant line than in the susceptible line (Fig. 6A). After challenge by a necrotrophic pathogen F. graminearum, TaERF3 transcripts in a resistant cv. ‘Sumai-3’ and a susceptible line ‘Wenmai6’ were induced from 6 hai to 48 hai (Fig. 6B). Nevertheless, the induction in the susceptible cv. ‘Wenmai6’ at 12 hai with F. graminearum (the peak) was about three times higher than that in resistant cv. ‘Sumai-3’. By contrast, from 24 hai to 48 hai, the inductions in resistant ‘Sumai-3’ were higher than in the susceptible ‘Wenmai6’ (Fig. 6B). In response to another necrotrophic pathogen R. cerealis infection, TaERF3 transcripts in the susceptible line ‘Wenmai6’ were induced at 6 hai with a maximum induction (about 12-fold increase over 0 h) occurring at 12 hai and then decreased abruptly after 24 hai; the induction at 12 hai being about 6 times higher in ‘Wenmai6’ than in a resistant line ‘Shannong0431’; by contrast, at 48 hai with R. cerealis, the induction in the resistant ‘Shannong0431’ peaked and was about nine times higher than that in the susceptible ‘Wenmai6’ (Fig. 6C). The results suggested that TaERF3 might be involved in defence responses to the pathogens at different stages in their lifestyles, mainly being involved in an active defence response to B. graminis at an earlier stage (6–12 hai), and to F. graminearum and R. cerealis at a later stage (24–48 hai).
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It has been reported that SA signalling primarily regulates resistance to biotrophic pathogens, while the JA and ethylene pathways are more effective in plant resistance responses to necrotrophic and soil-borne fungal pathogens (Glazebrook, 2005). Based on ‘Pm97034’ with high resistance to a biotrophic pathogen B. graminis and ‘Sumai-3’ with good resistance to a necrotrophic pathogen F. graminearum, the expression patterns of TaERF3 in ‘Pm97034’ were analysed with an exogenous application of SA, and ‘Sumai-3’ was analysed with exogenous ethylene and MeJA treatments. Following SA treatment, the transcripts of TaERF3 in ‘Pm97034’ had increased as early as 1 h, peaked at 4 h (4-fold increase over 0 h), but decreased abruptly at 24 h (Fig. 7A). ‘Sumai-3’showed a similar pattern, but with a weaker induced peak (2-fold increase over 0 h; data not shown). To elucidate the possible involvement in SA-induced defence responses, TaERF3 transcripts were examined after inoculation with B. graminis on the ‘Pm97034’ seedlings treated for 48 h by 1 M SA or water. The TaERF3 transcript abundance, induced by B. graminis infection following SA treatment, was dramatic and peaked (about 9-fold increase over the control) at 6 hai, around three times higher than that by B. graminis following water treatment (CK) (Fig. 7D), suggesting that the SA treatment enhanced TaERF3 expression earlier in response to B. graminis infection. After ethylene treatment, the transcript abundances of TaERF3 in ‘Sumai-3’ increased as early as 1 h, reached 2-fold from 2 h to 12 h, and peaked (12-fold increase over 0 h) at 24 h (Fig. 7B). In responding to the MeJA treatment, TaERF3 transcripts in ‘Sumai-3’ was induced slightly within 4 h, accumulated to about 4-fold at 12 h, and peaked (increasing 20-fold over 0 h) at 24 h (Fig. 7C). All the induction peaks by SA, ethylene, and MeJA treatments were earlier than those induced by the pathogens in the resistant lines, suggesting that TaERF3 might be involved in an active defence response to the pathogens through signalling pathways that are triggered by SA, ethylene, and JA at different times.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Isolation of pathogen-induced ERF genes from wheat is of importance as it is a starting point for a better understanding of the defence mechanisms of wheat in response to pathogen attack. In this study, a novel pathogen-inducible ERF gene, TaERF3, was isolated from wheat, exploiting the RT-PCR and RACE strategies. The deduced protein TaERF3 contains the typical ERF/AP2 domain of the ERF family, but the complete TaERF3 sequence shares a low identity with other ERF proteins aligned and with four ERF proteins of wheat searched from public databases. The ERF/AP2 domain of TaERF3 possesses the normal conformation in ß-sheets and
-helix that are essential for DNA binding (Allen et al., 1998). The obvious difference in ERF/AP2 domain is that the 57th is asp (D) for TaERF3 but asn (N) for other ERFs. It was suggested that the 57th D of the ERF/AP2 domain should not affect the DNA binding of TaERF3 since the alterative amino acid was not located in the key conformation regions by the model analysis. Results of the EMSA and transient expressions in yeast cells further demonstrated that the changed amino acid does not affect the DNA binding of the ERF/AP2 domain. Based on the deduced amino acid sequence, TaERF3 includes an acidic region, shown in other species to act as a transcription activation domain (Fujimoto et al., 2000; Tournier et al., 2003; Zhang et al., 2004), and is expected to encode a member of ERF transcriptional activators. Results of transcription activation assays in yeast cells demonstrated that TaERF3 encodes an ERF transcription activator, which has GCC box-binding ability and transactivation activity. The subcellular localization assay demonstrated that the TaERF3 is targeted to the nuclei. The results proved that the biochemical properties of TaERF3 coincide with its sequence traits, and TaERF3 indeed functions as a GCC box-binding ERF transcription activator.
The transcriptional activation cascades involving ERF proteins may be important for plant defence to pathogen attack (Oate-Sánchez and Singh, 2002). Several ERF proteins in the B3 subgroup were shown to confer enhanced disease resistance when overexpressed in plants, including ERF1, AtERF1, AtERF2, Pti4, TSRF1, OPBP1, and OsBIERF3 (Berrocal-Lobo et al., 2002; Gu et al., 2002; Berrocal-Lobo and Molina, 2004; Guo et al., 2004; Zhang et al., 2004; McGrath et al., 2005; Cao et al., 2006a). Interestingly, the transcripts of the ERF genes mentioned above were also induced by pathogen attack. For example, expression of Arabidopsis ERF genes was induced by avirulent and virulent strains of P. syringae pv. tomato, but there was a delay in the ERF mRNA accumulation after infection with the virulent strain when compared with the avirulent strain. As TaERF3 represents a member of the B3 subgroup of the ERF family based on the phylogenetic analysis, TaERF3 was predicted to play a regulatory role in the wheat defence response to pathogen infections. Therefore, in this study, the transcript profiles of TaERF3 were investigated in different interactions between different wheat lines and pathogens using the Q-RT-PCR method. The Q-RT-PCR method is known to be more sensitive than hybridization-based methods for the quantification of low-abundance transcripts (McGrath et al., 2005), and has been used to analyse low-abundance expression of TF genes and to identify TFs showing a significant change in expression. TaERF3 transcripts in all the resistant and susceptible lines could be induced to different extents by infection with B. graminis, F. graminearum, and R. cerealis. Nevertheless, the gene transcript-induced patterns (induction kinetics) in different resistant and susceptible lines were distinct, although the ORF sequences from different wheat lines, such as ‘Pm97034’ and ‘Sumai-3’, were identical. After infection with B. graminis, a biotrophic pathogen, TaERF3 transcript inductions in the resistant line ‘Pm97034’ at earlier stage (6–12 hai) were dramatic and 3–6 times higher than those in the susceptible parent ‘Wan7107’. However, after infection with necrotrophic pathogens F. graminearum and R. cerealis, within 12 hai the inductions were 3–6 times higher in the susceptible cv. ‘Wenmai6’ than in the resistant lines. By contrast, at 24 hai or 48 hai with F. graminearum or R. cerealis, the TaERF3 transcript inductions were 3–9 times higher in the resistant than in the susceptible lines. The induction kinetics of TaERF3 in the above interactions suggested that TaERF3 might be involved in defence responses to biotrophic pathogens and to necrotrophic pathogens, primarily in the active defence response to biotrophic pathogens at an earlier stage, but to the necrotrophic pathogens at a later stage.
The Arabidopsis ERF1 has been shown to be an integrator of the ethylene and JA signalling pathways, and its transcripts could be induced by the exogenous application of ethylene and JA (Lorenzo et al., 2003). Pti4, Tsi1, TSRF1, and OsBIERF3 could be induced by exogenous ethylene and SA treatments (Park et al., 2001; Zhang et al., 2004; Cao et al., 2006b). AtERF1 could be induced by ethylene, JA, and SA (Oate-Sánchez and Singh, 2002). These results showed that TaERF3 transcripts were induced to different extents following exogenous SA, ethylene, and MeJA treatments. The TaERF3 transcript-induced peak (increasing 4-fold over 0 h) by SA treatment occurred at 4 h, whereas the transcript peaks induced by ethylene and MeJA appeared at 24 h, increasing, respectively, 12-fold and 23-fold over 0 h. All the induction peaks by exogenous hormones were earlier than those by the pathogen infections in the different resistant lines, suggesting that TaERF3 is probably involved in the defence responses by signalling pathways that are activated by SA, ethylene, and JA at different times. The other experiment showed that SA could induce a defence response in wheat against B. graminis (L Yu, private communication). Most interestingly, TaERF3 transcript abundance induced by B. graminis infection following SA treatment was dramatic (about a 9-fold increase over the control) at 6 hai, which was about three times higher than that by B. graminis following water treatment (CK) (Fig. 7D), suggesting that TaERF3 may be involved in the defence response to B. graminis through signalling pathways that are triggered by SA. Cao et al. (2006b) once investigated the expression patterns of OsBIERF genes in the rice seedlings treated by both BTH and Magnaporthe grisea, and suggested that the OsBIERF proteins may play regulatory roles in the rice defence response to Magnaporthe grisea through signalling pathways triggered by BTH, based on the expressions induced by treatment with both the pathogen and BTH. The experiments used here, and the corresponding results regarding pathogen and hormone treatments in this study and in the paper by Cao et al. (2006b), can be used to elucidate the possible involvement of the hormone in the defence responses to pathogens in plant species without the hormone-related mutants.
To our knowledge, this is the first report on the identification and characterization in detail of a wheat ERF gene related to defence responses, and its induction kinetics after different pathogen infections and hormone treatments. The results in this study extend current knowledge of defence mechanisms in wheat against pathogen attack.
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The authors thank Professor Robert A McIntosh (Plant Breeding Institute, University of Sydney, Australia) and Professor Francesco Salamini (Max-Planck Institute for Plant Breeding Research, Germany) for kindly revising and advising on the manuscript. This study was supported by the National Natural Science Foundation of China (grant No. 30671292).
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