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Keywords:

  • Bipolaris sorokiniana ;
  • common wheat;
  • drought;
  • MYB transcription factor;
  • resistant response;
  • Triticum aestivum

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • In this study, we report new insights into the function of a wheat (Triticum aestivum) MYB gene TaPIMP1 through overexpression and underexpression, and its underlying mechanism in wheat.

  • Electrophoretic mobility shift and yeast-one-hybrid assays indicated that TaPIMP1 can bind to five MYB-binding sites including ACI, and activate the expression of the genes with the cis-element, confirming that TaPIMP1 is an MYB transcription activator.

  • TaPIMP1-overexpressing transgenic wheat exhibited significantly enhanced resistance to the fungal pathogen Bipolaris sorokiniana and drought stresses, whereas TaPIMP1-underexpressing transgenic wheat showed more susceptibility to the stresses compared with untransformed wheat, revealing that TaPIMP1 positively modulates host-defense responses to B. sorokiniana and drought stresses. Microarray analysis showed that a subset of defense- and stress-related genes were up-regulated by TaPIMP1. These genes, including TaPIMP1, RD22, TLP4 and PR1a, were regulated by ABA and salicylic acid (SA). TaPIMP1-underexpressing transgenic wheat showed compromised induction of these stress-responsive genes following ABA and SA treatments.

  • In summary, TaPIMP1, as a positive molecular linker, mediates resistance to B. sorokiniana and drought stresses by regulation of stress-related genes in ABA- and SA-signaling pathways in wheat. Furthermore, TaPIMP1 may provide a transgenic tool for engineering multiple-resistance wheat in breeding programs.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Wheat (Triticum aestivum) is one of the most important staple crops. Drought, a main abiotic stress, profoundly affects plant growth and productivity, and reduces yield of wheat worldwide. Common root rot is a soilborne disease in many areas of the world. The primary pathogen that causes this disease is the fungus Bipolaris sorokiniana (teleomorph Cochliobolus sativus), which has a short biotrophic phase followed by successful tissue infection in the necrotrophic growth phase (Kumar et al., 2002). To improve wheat resistance to both B. sorokiniana and drought stress, it is vital to identify resistance-related genes and to unravel mechanisms underlying the function of these genes.

Plants have evolved various mechanisms to cope with biotic and abiotic stresses (Mengiste et al., 2003; Yi et al., 2004). In model plants, the molecular and cellular responses to the stresses and underlying regulatory mechanisms have been studied. The molecular mechanisms involved in each stress have been revealed to be comparatively independent. Recent studies have shown that crosstalks exist between biotic and abiotic stresses, but understanding of the crosstalks remains rudimentary (Fujita et al., 2006). Phytohormones, such as salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and ABA, regulate primarily the protective responses of plants against both biotic and abiotic stresses via synergistic or antagonistic actions (Fujita et al., 2006). Usually, SA is associated with biotrophic pathogen resistance, whereas JA and ET are associated with necrotrophic pathogen resistance responses (Pieterse et al., 2009). ABA plays a major role in regulation of growth and development, and in defense responses to abiotic and biotic stresses (Fan et al., 2009; Lee & Luan, 2012).

Transcription factors (TFs), including the MYB family, play important roles in biotic and abiotic stress crosstalks and signaling cascades through regulation of gene expression. Since the first plant MYB gene, COLORED1 (C1) required for the synthesis of anthocyanins in the aleurone of maize (Zea mays) kernels, was isolated (Paz-Ares et al., 1987), a large number of MYB proteins have been identified in different plant species. MYB proteins can be divided into four types – MYB1R, R2R3-MYB, R1R2R3 MYB (MYB3R), and 4R MYB – with one, two, three, and four repeats of the MYB DNA-binding domains, respectively (Dubos et al., 2010). The functions of MYB proteins have been investigated in numerous plant species, such as Arabidopsis thaliana, maize, rice (Oryza sativa), grapevine (Vitis vinifera), poplar (Populus tremuloides) and apple (Malus domestica). MYB proteins have been implicated in various developmental and physiological processes, including control of the cell cycle and development, regulation of primary and secondary metabolism, participation in defense responses to biotic and abiotic stresses, and hormone synthesis and signal transduction (Dubos et al., 2010). For instance, R2R3-MYB proteins, including AtMYB30, AtMYB60, and AtMYB96, are involved in responses to drought stress and disease resistance (Dubos et al., 2010). AtMYB15 accounts for cold- and drought-stress tolerances (Agarwal et al., 2006; Ding et al., 2009). AtMYB108, which belongs to Arabidopsis MYB subgroup 20, the other members of which include AtMYB2, AtMYB62, AtMYB78, AtMYB112, and AtMYB116 (Dubos et al., 2010), is involved in biotic and abiotic stress crosstalks (Mengiste et al., 2003). Certain rice MYB proteins contribute to abiotic stresses (Vannini et al., 2004; Dai et al., 2007; Ma et al., 2009; Su et al., 2010).

Although there has been much progress in the identification and functional analyses of MYB genes in model plants, less is known about the function of the MYB family in wheat because of the huge and complex genome of wheat. Chen et al. (2005) used degenerate primers to obtain 23 MYB gene fragments and six near-complete coding sequences. Himi & Noda (2005) isolated a wheat MYB gene, TaMYB10, which controls the color development of wheat seed. Morimoto et al. (2009) cloned a wheat MYB gene orthologous to the maize rough sheath2 (RS2), and showed that it had conserved function with RS2. Zhang et al. (2011) isolated 60 unique MYB genes from wheat full-length cDNA libraries, and analyzed their expression during abiotic stresses. Using a computational pipeline, Cai et al. (2012) identified 218 potential MYB genes from wheat expressed sequence tags (ESTs), encoding MYB1R, R2R3-MYB, MYB3R, and 4RMYB transcription factors. Xue et al. (2011) showed that TaMYB13 is an MYB transcriptional activator of fructosyltransferase genes in β-2,6-linked fructan synthesis in wheat. Ectopic expression of TaMYB2A confers enhanced tolerance to multiple abiotic stresses in Arabidopsis (Mao et al., 2011). Ectopic expression of the wheat MYB genes TaMYB73 and TaMYB32 improves salinity stress tolerance in transgenic Arabidopsis (He et al., 2011; Zhang et al., 2011). However, there is no published research regarding the role of wheat MYB genes in disease resistance. We have previously described the cloning of a wheat pathogen-induced MYB gene TaPIMP1, whose ectopic expression enhanced resistance to both a pathogen, Ralstonia solanacearum, and to abiotic stresses in transgenic tobacco (Liu et al., 2011). However, functional assays of MYB genes through overexpression (gain of function) and RNA interference (RNAi, loss of function) in wheat, and molecular mechanisms underlying these functions have not been reported previously.

In this study, biochemical assays indicated that the wheat MYB protein TaPIMP1 was an MYB transcription activator. Both gain- and loss-of-function assays showed that TaPIMP1 positively modulates defense responses to B. sorokiniana and drought stress in wheat. Through microarray analysis, we identified a group of defense and stress-responsive genes activated by TaPIMP1. TaPIMP1 and these genes activated by TaPIMP1 could be induced by B. sorokiniana and drought stress, and by ABA and SA. The data suggest that TaPIMP1, as an important integrator, contributes to biotic (B. sorokiniana) and abiotic (drought) stress resistance by regulating defense- and stress-related genes in ABA–SA signal pathways in wheat.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and treatments

Wheat (Triticum aestivum L.) cv Yangmai 12 seeds were collected from the Lixiahe Agricultural Institute of Jiangsu, China.

The Yangmai 12 specimens were grown hydroponically in a glasshouse under a 16 h light : 8 h dark (22 : 12°C, 60% humidity) regime. According to Xiang et al. (2011), Hansen & Grossmann (2000), and Zhang et al. (2007), seedlings at the three-leaf stage were sprayed with 1.0 mM SA (Sigma), 0.2 mM ABA (Sigma), 0.1 mM methyl jasmonate (MeJA; Sigma), 0.1 mM paclobutrazol (PBZ, the inhibitor of benzoic acid 2-hydroxylase that converts benzoic acid to SA; Sigma; Xiang et al., 2011), and 2 mM tungastate (an inhibitor of ABA biosynthesis, Hansen & Grossmann, 2000), all of which were dissolved in 0.1% Tween-20, or 0.1% Tween-20 (mock), and treated with 0.2 mM ethephon, which releases ET in a sealed container.

Primers

All primers used for vector constructions, PCR detection for the TaPIMP1 transgene detection, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays are listed in Supporting Information, Table S1.

Cis-element binding assay in vitro

The coding sequence of TaPIMP1 was cloned into the pGEX-4T-1 vector with a glutathione S-transferase (GST) gene. The GST-TaPIMP1 recombinant protein was induced in Escherichia coli BL21 cells with 0.3 mM isopropyl-β-D-thiogalactopyranose, and purified using MicroSpin module (GE Amersham).

The primers of seven probes with cis-acting elements were synthesized as the sequences in Fig. 1(a). These cis-acting elements contained the MYB-binding site (MBS) ACI (Romero et al., 1998; Patzlaff et al., 2003), RT1 bound by OsMBY3R-2 (Ma et al., 2009), St1R bound by StMYB1R-1 (Shin et al., 2011), MBS1 (including MBS1-Bz bound by AtMYB2 (Urao et al., 1993) and MBS1-W existing in the promoters of certain defense- and stress-related genes in wheat (Table S2)), the GCC-box bound by ERF TFs (Zhang et al., 2007), or the DRE-box bound by DREB TFs (Liu et al., 1998). The promoter sequences of defense- and stress-related genes were searched from wheat Draft Genome Assembly database (http://www.cerealsdb.uk.net/CerealsDB/) and MBSs in the promoters were predicted by the website (www.dna.affrc.go.jp/PLACE/).

image

Figure 1. Cis-element binding ability and transcriptional-activation assays of TaPIMP1. (a) The oligonucleotide sequences for 5 MYB-binding sites, GCC-box and DRE-box cis-elements used as probes for the electrophoretic mobility shift assays (EMSAs). (b) The EMSA indicated that TaPIMP1 could bind to MYB-binding site ACI and not to GCC-box or DRE-box. Lanes 1–3, 1000 ng ACI probe; lane 4, 500 ng ACI probe; lane 5, 1000 ng GCC-box probe; lane 6, 1000 ng DRE-box probe. The free probe and the binding band of recombinant GST-TaPIMP1 to the ACI probe are indicated. (c) The EMSA indicated that TaPIMP1 can bind to five MYB-binding site probes, including ACI, MBS1-bz, MBS-W, RT1, and St1R. Lanes 1–7, 1000 ng probe. (d) The yeast one-hybrid assay showed that the TaPIMP1 protein had transcriptional-activation activity. Panel (1) shows yeast cells containing distinct effector and reporter constructs grown on an SD/-Trp/-Ura medium plate. 1, Y-pYTaPIMP1/pACI-LacZi; 2, Y-pYepGAP/pACI-LacZi; 3, Y-pYTaPIMP1/pLacZi; 4, Y-pYepGAP/pLacZi. Panel (2) shows that β-galactosidase (encoded by LacZ) activity was detected using X-gal staining in the lifted filter for the yeast cells shown in panel (I).

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Electrophoretic mobility shift assays (EMSAs) were used to determine whether the TaPIMP1 protein can bind to these MBS-containing probes. Following a modified protocol (Dong et al., 2010), each probe was mixed with c. 2 μg of recombinant GST-TaPIMP1 or GST in a binding buffer. Each reaction mixture was incubated on ice for 30 min and loaded onto 8% polyacrylamide gel. After electrophoresis was performed at 100 V for 30 min, the gels were stained with ethidium bromide for visualization of the DNA bands.

Transcriptional activation assay in yeast

A yeast-one-hybrid system was used to assay the transcriptional activation ability of the protein TaPIMP1. Following Zhang et al. (2007), we prepared the effector construct pYTaPIMP1, in which the TaPIMP1 coding sequence was subcloned into the yeast expression vector pYepGAP (Liu et al., 1998) under the control of the glyceraldehydes-3-phosphate dehydrogenase (GAP) promoter. The MBS ACI sequence with two copies was inserted upstream of the reporter gene LacZ in the vector pLacZi to prepare the reporter construct pACI-LacZi. The effector and reporter vectors did not contain any yeast activation domain or binding domain, except for the domains present in the TaPIMP1 protein.

According to the yeast-one-hybrid manual (Clontech, Palo Alto, CA), the effector and reporter or control constructs were transformed into competent cells of the yeast strain YM4271, resulting in the following yeast cells: Y-pYTaPIMP1/pACI-LacZi, Y-pYepGAP/pACI-LacZi, Y-pYTaPIMP1/pLacZi, and Y-pYepGAP/pLacZi. These cells were first grown on a SD/-Trp/-Ura medium. Subsequently, they were lifted to a filter paper and subjected to X-gal staining for assaying the activity of β-galactosidase (encoded by LacZ).

DNA isolation and RNA extraction

Genomic DNA for each sample was isolated from the wheat leaves using the CTAB method (Saghai-Maroof et al., 1984).

Total RNA was extracted from wheat leaves using TRIzol (Invitrogen, Carlsbad, CA), and subjected to RNase-free DNase I (Promega, Madison, WI, USA) digestion and purification.

RT-PCR and qRT-PCR

Four micrograms of purified RNA of each sample was reverse-transcribed to cDNA (Invitrogen).

Reverse transcription PCR consisted of 28–30 cycles of amplification (40 s, 94°C; 45 s, 60°C; 45 s, 72°C) with gene-specific primers. qRT-PCR was performed using SYBR Green I Master Mix in a volume of 25 μl and applied to the ABI 7300 RT-PCR system (Applied Biosystems, Foster City, California, USA). The amplification of the wheat actin gene was used as an internal control to normalize all data. The inline image method (Livak & Schmittgen, 2001) was used to evaluate the relative expression of each gene. All RT-PCR reactions were repeated three times.

Generation and molecular characterization of TaPIMP1 transgenic wheat plants

To generate the TaPIMP1-overexpressing (TaPIMP1-O) transformation vector pTaPIMP1-O, the open reading frame (ORF) of TaPIMP1 was used to replace the GUS gene of a pAHC25 vector (Christensen & Quail, 1996), and was controlled by the promoter of the maize ubiquitin gene. The pTaPIMP1-O plasmid was transformed into wheat cv Yangmai 12 by biolistic bombardment following Chen et al. (2008). The transgene TaPIMP1-O was detected by a PCR product (533 bp) specific to the transgene using specific primers (UBIP-OF locating in the ubiquitin promoter region, and MYB-OR in the TaPIMP1 coding region).

A 295 bp sequence derived from a specific sequence (585–879 nt) of the TaPIMP1 ORF was fused in antisense and sense orientations to flank the 148 bp maize alcohol dehydrogenase gene (Adh) intron, forming the TaPIMP1-RNAi fragment. The TaPIMP1-RNAi fragment was then used to replace the GUS gene in pAHC25, generating the RNAi construct pTaPIMP1-Ri. The pTaPIMP1-Ri plasmid was transformed into Yangmai 12 by bombardment following Chen et al. (2008). The TaPIMP1-RNAi transgene was detected by PCR using the TaPIMP1-RNAi transgene-specific primers (MYB-Ri-F locating in the TaPIMP1 antisense sequence region, and MYB-Ri-R in the Adh intron region).

Southern and western blottings

The integrative sites and copy numbers of the TaPIMP1-O transgene in the overexpressing wheat lines were detected by Southern blotting following Sharp et al. (1989). Genomic DNA (20 μg each) was digested with HindIII, resolved in a 0.8% agarose gel, and blotted on to a Hybond-N+ membrane (Amersham). The TaPIMP1-O transgene-specific amplified fragment (533 bp) labeled by α-32P-dCTP was used as the probe. The membrane was prehybridized for 4 h and hybridized with the probe for at least 16 h at 65°C.

The protein expression of TaPIMP1 was evaluated by western blotting. Total proteins were extracted from 0.3 g of ground leaf powders. Approx. 10 μg of total soluble proteins were separated on 12% sodium dodecyl sulfate polyacrylamide gels and transferred to a polyvinyl difluoride (PVDF) membrane. The western blots were incubated with a dilution of the polyclonal GST-TaPIMP1 antibody, which was developed from purified GST-TaPIMP1 protein in rabbit. The TaPIMP1 protein was visualized using an ECL plus Western Blotting Reagent Pack (Amersham).

The fungal pathogen B. sorokiniana and the resistance test

The fungal pathogen Bipolaris sorokiniana ACC30209 was purchased from the Agricultural Microbial Culture Collection, CAAS, Beijing, China.

At the tillering stage, at least 20 plants per line in each test were inoculated with B. sorokiniana mycelia following Dong et al. (2010). At 40 d postinoculation (dpi), common root rot symptoms on wildtype (WT) plants were clearly visible. At 60 dpi, the B. sorokiniana infection types (ITs) were categorized from 0 to 4 based on the brown lesion square on the base stem (IT 0, no necrotic lesion; IT 1, ≤ 1/4 coverage; IT 2, 1/4–1/2 coverage; IT 3, 1/2–2/3 coverage; IT 4, 2/3 to complete coverage), and the disease indexes of plants were calculated following Dong et al. (2010). The tests were repeated at least three times.

Microscopic obversation followed by trypan blue staining was performed to analyze the inhibition effect of TaPIMP1 overexpression on B. sorokiniana hyphae. At 22 dpi, the base sheath was kept in the fixative (ethanol : acetic acid = 1 : 1) overnight before staining with trypan blue solution for 6 h at room temperature, followed by washing with distilled water and observation under a light microscope (Leica Microsystems M165FC, Wetzlar, Germany).

Drought tolerance assay

Wheat plants were cultured in the soil of 20 cm pots in a glasshouse under 15 h light : 9-h dark, 25 : 16°C regime. At least 12 plants per independent line were tested in each test with three replications. The volumetric water content (VWC) of each pot was monitored every day, and plants under the same VWC were compared. At the three-leaf stage of wheat seedlings, the initial VWC of each pot was 72%. Water was withheld for 22 d, when VWC reached 2% and the RNAi plants showed severe wilting, and then plants were rewatered. The survival rate was counted after resumption of watering for 7 d.

Water loss assay

Following Dai et al. (2007) and Shin et al. (2011), the leaves of 10 plants per line at the tillering stage were detached, and incubated in the growth chamber under a regime of 23°C and 45% humidity. The FW was measured at each 30 min interval. Water loss was calculated from the decrease in FW compared with time zero. All tests were repeated three times.

Proline content assay

Afetr rewatering for 7 d, the leaves of 10 plants per line were pooled as a sample for assaying the free praline content. Free proline was extracted and quantified from the fresh leaf tissues (0.5 g) as previously described (Hu et al., 1992). Three replications were set for each line.

Microarray assay

Fresh leaves from the 20 seedlings of the stable TaPIMP1-overexpressing lines M80 and M499, or of wild type Yangmai12, treated by B. sorokiniana inoculation for 40 d or deprived of water for 18 d, were quickly cut and pooled, and used to extract the total RNA. RNA quality was examined using the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA). A cyanine 3-CTP- or cyanine 5-CTP-labeled cDNA probe was prepared from the mixed RNA of M80 + M499 or the RNA of Yangmai 12 (control) according to the manufacturer's protocol. The labeled probes, Cy3-(M80 + M499) and Cy5-Yangmai 12, were hybridized to the Agilent Wheat Gene Expression Microarray containing 43 803 probe sets following the Two-Color Microarray-Based Gene Expression Analysis Manual. The hybridized arrays were conducted with three replications. The hybridization signals were scanned with an Agilent G2505C Microarray Scanner System, and microarray data were extracted using Feature Extraction Software (v.10.7.1.1) available from Agilent using the default variables. Data files were loaded into GeneSpring GX 11.5 (Agilent Technologies). The signal values between both experimental groups (M80 + M499 mixture probe vs Yangmai 12 probe) were assessed for each gene based on the normalized probe signals, and correlation coefficients between biological replications were found to be > 0.90. Significant differentially expressed transcripts were identified by one-way ANOVA with a stringent cutoff at the false discovery rate (FDR) < 0.05 that corresponds to P < 0.01.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

TaPIMP1 is an MYB transcriptional activator

The amino acid sequence analysis indicated that the TaPIMP1 protein contains R2 and R3 DNA-binding domains, and phylogenetic analysis revealed that TaPIMP1 was clustered with R2R3-MYB proteins of different plants, and closely related to AtMYB108 and also AtMYB2 (Fig. S1; Liu et al., 2011).

Electrophoretic mobility shift assays were used to investigate if TaPIMP1 can bind five MBS elements, which were bound by known functional R2R3-MYB, StMYB1R-1 and OsMYB3R-2 TFs or present in the promoters of certain wheat defense- and stress-related genes (Fig. 1a, Table S2). The results showed that the recombinant GST-TaPIMP1 protein bound to the tested MBS probes, including ACI, MBS1-Bz, MBS1-W, RT1, and St1R (Fig. 1b–c), but not to the GCC-box or to the DRE-box (Fig. 1b), whereas GST failed to bind to the cis-element ACI (Fig. 1a), indicating that TaPIMP1 can bind to the five MBS elements.

To explore if TaPIMP1 possesses transcriptional-activation activity, we cotransformed the TaPIMP1 effector vector pYTaPIMP1 and the reporter vector pACI-LacZ into yeast cells. The yeast-one-hybrid and β-galactosidase activity assays showed that the yeast cells with the pYTaPIMP1/pACI-LacZ combination exhibited strong β-galactosidase activity (Fig. 1d), whereas the other yeast cells, harboring pYepGAP/pLacZi, pYepGAP/pACI-LacZ, or pYTaPIMP1/pLacZi combinations, did not show visible β-galactosidase activity (Fig. 1d). These results prove that TaPIMP1 is an R2R3-MYB transcriptional activator that can activate transcription of genes after binding with the MBS cis-element in their promoter.

Molecular characteristics and phenotypes of TaPIMP1 transgenic wheat

To dissect the function of TaPIMP1 in wheat by gain- and loss-of-function approaches, we generated transgenic wheat plants with an overexpressing or underexpressing (RNAi)- TaPIMP1 gene. Eight TaPIMP1-overexpressing transgenic wheat lines (M80, M240, M443, M460, M492, M499, M535, and M556) and four TaPIMP1-RNAi transgenic lines (Z70, Z99, Z107, and Z110) were generated and identified by specific amplications (Fig. 2a–b). Using the probe derived from the TaPIMP1-O transgene-specific fragment, Southern blot results showed that the TaPIMP1-overexpressing transgene was integrated with one to two copies by diverse patterns into the genomes of the eight TaPIMP1-overexpressing lines (Fig. 2c), confirming that these transgenic lines were derived from independent transformation events. RT-PCR and qRT-PCR assays showed that, compared with WT Yangmai 12 (host), the transcript abundance of TaPIMP1 in the RNAi lines was significantly reduced (Figs 2d, 5, 6), whereas the transcript abundance of TaPIMP1 in the overexpressing lines had markedly increased (Figs 2e, 5, 6). The western blotting assay indicated that the TaPIMP1 protein levels in the TaPIMP1-overexpressing lines were markedly increased compared with the WT Yangmai 12 (Fig. 2f).

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Figure 2. Molecular characterization of TaPIMP1 transgenic wheat (Triticum aestivum) plants. (a) PCR analysis of eight TaPIMP1-overexpressing transgenic lines (M80, M240, M443, M460, M492, M499, M535, and M556) and the wildtype wheat Yangmai 12 (WT) using the TaPIMP1-overexpressing transgene-specific primers. P, the transformation vector pTaPIMP1-O as positive control. (b) PCR analysis of four TaPIMP1-RNAi transgenic lines (Z70, Z99, Z107, and Z111) and WT Yangmai 12 using the TaPIMP1-RNAi transgene-specific primers. P, the transformation vector pTaPIMP1-Ri as positive control. (c) Southern blot assay of the eight TaPIMP1-overexpressing transgenic lines. Genomic DNA isolated from the WT or transgenic plants was digested with HindIII, and then hybridized with a probe of the amplified fragment specific for the TaPIMP1-overexpressing transgene. (d) Reverse transcription polymerase chain reaction (RT-PCR) (top) and quantitative RT-PCR (qRT-PCR) analyses (above) of the expression of TaPIMP1 in four RNAi transgenic lines. Three biological triplicates per line were averaged and statistically treated using Student's t-test (**, < 0.01). (e) qRT-PCR analysis of the expression of TaPIMP1 in eight overexpressing transgenic lines. Three biological replicates per line were averaged and statistically treated (t-test,**, < 0.01). (f) Western blot analysis of eight overexpressing transgenic lines, WT, and positive control GST-TaPIMP1 with the TaPIMP1 antibody. Bars indicate + SE of the mean.

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The seed germination, growth, and major agronomic traits (including plant height, spike length, spikelet number, and 1000-grain weight) were not significantly different between the TaPIMP1-overexpressing lines and the WT Yangmai 12 (Fig. S2, Table S3). These results indicate that TaPIMP1 overexpression did not obviously affect the growth and other agronomic traits in wheat.

TaPIMP1 contributes positively resistance to B. sorokiniana in wheat

As TaPIMP1 was induced after B. sorokiniana infection (Fig. 3a), its role in the defense in wheat was explored. Following inoculation with B. sorokiniana, compared with the WT wheat Yangmai 12, the TaPIMP1-overexpressing lines exhibited significantly enhanced resistance to B. sorokiniana, and the TaPIMP1-RNAi plants displayed greater susceptibility to the pathogen (Fig. 3b, Table 1). Most TaPIMP1-overexpressing plants displayed resistance with ITs 0–1 (Fig. 3b), whereas most RNAi plants exhibited greater susceptibility to B. sorokiniana with ITs 3–4 (Fig. 3b). The disease indexes of the TaPIMP1-overexpressing lines were 23.18–29.43%, whereas those of the RNAi lines were 73.21–77.41% and that of WT Yangmai 12 was 55.90% (Table 1). Moreover, microscopic observation revealed fewer hyphae of B. sorokiniana at the base sheaths of TaPIMP1-overexpressing transgenics than on WT Yangmai 12 (Fig. 3c,d), providing supporting evidence that TaPIMP1-overexpressing transgenics were more resistant to B. sorokiniana. These results indicated that TaPIMP1 contributed to wheat's resistance response to B. sorokiniana infection.

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Figure 3. Wheat (Triticum aestivum) TaPIMP1 responding to Bipolaris sorokiniana infection. (a) Transcriptional profiles of TaPIMP1 in WT Yangmai 12 after B. sorokiniana inoculation by qRT-PCR with three replications. The relative transcript abundances of TaPIMP1 in plants treated were compared with that at 0 h, and the statistically significant difference was calculated based on the results of three replications (t-test: **, < 0.01). Bars indicate SE of the mean. (b) Typical infection phenotypes of a resistant TaPIMP1-overexpressing line M80, susceptible WT Yangmai 12 and TaPIMP1-RNAi line Z99 after B. sorokiniana inoculation for c. 60 d. IT, infection type. (c) Microscopic observation of the B. sorokiniana hyphae on the base leaf sheath of the TaPIMP1-overexpressing transgenic plant M80. (d) Microscopic observation of the B. sorokiniana hyphae on the base leaf sheath of the WT Yangmai 12 plant.

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Table 1. Bipolaris sorokiniana responses of TaPIMP1 transgenic and wildtype (WT) wheat (Triticum aestivum) plants
LinesInfection types to B. sorokinianaAverage disease IndexaResponse
01234
  1. a

    Ox, overexpressing lines; Ri, RNAi lines; R, resistance; MS, moderately susceptible; S, susceptible.

    **Significant difference between the transgenic lines and the host WT Yangmai 12 at < 0.01 (t-test).

M80 (Ox)143680022.63**R
M240 (Ox)113850022.22**R
M443 (Ox)103770023.44**R
M460 (Ox)93851023.45**R
M492 (Ox)143640020.53**R
M499 (Ox)103650022.18**R
M535 (Ox)153530019.78**R
M556 (Ox)152840020.26**R
Yangmai 12 (WT)172716454.46MS
Z70 (Ri)00716573.21**S
Z99 (Ri)00420777.41**S
Z107 (Ri)00617574.10**S
Z110 (Ri)00520675.81**S

TaPIMP1 positively modulates drought tolerance in wheat

As transcript analysis of TaPIMP1 indicated that it is involved in response to water deficit (Fig. 4a), the drought tolerance of the seedlings of TaPIMP1-overexpressing and RNAi wheat lines was assessed. After a 19 d water-withholding period, most TaPIMP1-overexpressing plants grew normally, whereas the WT Yangmai 12 plants showed slight wilting. On the 22nd day (just before rewatering), most WT and RNAi plants displayed severe wilting, whereas some TaPIMP1-overexpressing transgenic plants showed wilting (Fig. 4b). After resumption of watering for 7 d, the survival rates of the eight TaPIMP1-overexpressing lines were 51.52–55.22%; whereas those of the RNAi lines were 7.69–10.52%, and 14.97% of the WT Yangmai 12 plants had recovered (Fig. 4c, Table 2). These results indicate that TaPIMP1 overexpression significantly enhanced drought tolerance in wheat, TaPIMP1 underexpression significantly decreased drought tolerance, and thus TaPIMP1 mediated wheat adaptive response to drought stress.

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Figure 4. TaPIMP1 responding to drought stress treatments in wheat (Triticum aestivum). (a) Transcript abundances of TaPIMP1 were up-regulated after dehydration treatments in WT Yangmai 12 by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The relative expressions of TaPIMP1 in dehydrated plants were relative to that at 0 h, and statistically treated based on three replicates (t-test: *, < 0.05). (b) Tolerance responses of the TaPIMP1-overexpressing (M80 and M499) and TaPIMP1-RNAi transgenic (Z99 and Z107) lines to drought stress. D18d, withholding water for 18 d; D22d, withholding water for 22 d; R7d, resumption of water for 7 d after withholding water for 22 d. (c) Survival rates of TaPIMP1-overexpressing (M80 and M499) and TaPIMP1-RNAi (Z99 and Z107) transgenics, and WT plants on day 7 after resuming water following the withholding of water for 22 d based on three replications. At least 20 plants were counted and averaged for each line in each test. (d) The water losses in detached leaves from WT, TaPIMP1-overexpressing (M80 and M499) and TaPIMP1-RNAi transgenic (Z99 and Z107) plants. Twenty plants for each line were tested. (e) Measurement of proline contents in Yangmai 12 (WT), TaPIMP1-overexpressing (M80 and M499) and TaPIMP1-RNAi transgenic line (Z99 and Z107) plants. Statistically significant differences of transgenic lines from WT were determined based on three replications (t-test, **, < 0.01). Bars indicate SE.

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Table 2. Drought tolerance of TaPIMP1 transgenic and widetype (WT) wheat (Triticum aestivum) plants
LinesTotal plantsSurvived plantsSurvival rate (%)a
  1. a

    Ox, overexpressing lines; Ri, RNAi lines.

    **Significant difference between the transgenic lines and the host WT Yangmai 12 at < 0.01. Values are presented as ± SE.

M80 (Ox)683652.9 ± 3.4**
M240 (Ox)693753.6 ± 4.9**
M443 (Ox)733953.4 ± 4.2**
M460 (Ox)663451.5 ± 3.1**
M492 (Ox)754154.7 ± 2.1**
M499 (Ox)723852.8 ± 2.9**
M535 (Ox)673755.2 ± 4.9**
M556 (Ox)673552.2 ± 3.1**
Yangmai 12 (WT)1672515.0 ± 3.3
Z70 (Ri)38410.5 ± 3.1**
Z99 (Ri)3837.8 ± 4.9**
Z107 (Ri)39410.3 ± 4.2**
Z110 (Ri)3937.7 ± 2.9**

As the rate of detached leaf water loss is an essential parameter of water status in plants and has been proposed as an important indicator of water status measurement (Dhanda & Sethi, 1998), water losses of the WT Yangmai 12 and transgenic plants were assayed at 12 time points over 6 h (Fig. 4d). From 1.5 to 6 h, the water loss of the TaPIMP1-overexpressing lines (M80 and M499) was significantly slower (< 0.05) than that of WT Yangmai 12, whereas that of the RNAi lines (Z99 and Z107) was quicker than that of Yangmai 12. At 6 h treatment, the water losses of TaPIMP1-overexpressing (M80 and M499) leaves were 31.74 and 34.96%, whereas those of the RNAi transgenics (Z99 and Z107) were 50.60 and 48.45%, and that of the WT leaves was 45.12%. These results indicate that TaPIMP1 contributes to wheat drought tolerance by enhancing its water retention capability. To explore if the difference in water loss from these leaves is related to stomatal closing rate, we observed the effect of TaPIMP1 overexpression on stomatal apertures in leaves under a microscope. The results showed that, following drought stress treatment, overexpression of TaPIMP1 resulted in an earlier closure of leaves’ stomata than in the WT (Fig. S3a,b), which therefore reduced water loss and led to improved drought tolerance among the TaPIMP1-overexpressing plants.

Furthermore, it is well documented that free proline is the most widely distributed multifunctional osmoprotectant, playing an important role in enhancing osmotic stress tolerance (Szabados & Savour, 2009). Free proline content in TaPIMP1-overexpressing transgenics was significantly higher than in the WT plants, whereas that in RNAi plants was lower (Fig. 4e), suggesting that TaPIMP1 overexpression improved the proline synthesis, thus resulting in enhanced drought tolerance in wheat.

TaPIMP1 regulates the expression of defense- and stress-related genes

To explore whether the change in resistance in TaPIMP1 transgenic plants was correlated with changes in the expression of stress-responsive genes, leaves of TaPIMP1-overexpressing transgenics (M80 + M499 pool) or of the WT Yangmai 12 plants after B. sorokiniana or drought stresses were sampled and used to isolate total RNAs. The labeled RNAs served as probes for microarray analysis using the Agilent Wheat GeneChip array. The raw microarray data were shown in Table S4. Relative change was calculated by comparing the data for the TaPIMP1-overexpressing plants with those for the WT plants. We used a stringent cutoff at FDR < 0.05, corresponding to P < 0.01, and identified 203 transcript sets that were up-regulated twofold or more in the overexpressing plants compared with the WT (Table S5). Based on the annotation, 55% corresponded to defense- or stress-related or signal transduction genes, such as genes coding pathogenesis-related (PR) proteins including PR1a and PR2; thaumatin-like proteins including TLP4; dehydration-responsive proteins including RD22 and dehydrin 6; glutathione S-transferases including GST22; germin-like proteins (GLPs) including GLP4; phenylalanine ammonia-lyases (PALs) including PAL5; protein kinases; transcriptional factors (i.e. TaPIMP1 and other MYB-like proteins, bZIP, CBF, and zinc finger protein); heat-shock proteins; and ABA-inducing gene (ABAI). To validate the results of the microarray analysis and to investigate whether some defense- and stress-related genes identified were regulated by drought stress or B. sorokiniana infection or by TaPIMP1, we used qRT-PCR to analyze the transcription of 10 defense- and stress-related genes (including TaPIMP1, RD22, PR1a, PR2, TLP4, GST22, GLP4, dehydrin 6, ABAI, and PAL5) in the TaPIMP1 transgenic and WT wheat plants. These tested genes are known marker genes in biotic and abiotic stress responses in plants. Moreover, the partial promoter sequences of PR1a, RD22, TLP4, GST22, and ABAI contain the MBS1 cis-element, and PAL5 contains both ACI and MBS1 core sequences (Table S2), implying that TaPIMP1 may activate the expression of these genes following binding of the cis-elements.

The transcript profiles of the 10 genes characterized by qRT-PCR assays were consistent with those in microarray analysis (Figs 5, 6), indicating that most of the microarray data were reliable. The TaPIMP1-overexpressing plants accumulated significantly more transcripts of these defense- and stress-related genes than did the WT plants, whereas TaPIMP1-RNAi plants accumulated fewer transcripts of the stress-related genes than did the WT plants (Figs 5, 6). After B. sorokiniana inoculation and drought stress, the expression of the defense- and stress-related genes increased; the induced expression levels of the defense- and stress-related genes are significantly higher in TaPIMP1-overexpressing lines than in WT plants (Figs 5, 6), whereas they were lower in the TaPIMP1-RNAi lines than in WT plants. These results indicate that these genes were activated by TaPIMP1 and involved in the TaPIMP1-mediated response to B. sorokiniana and drought stress, and that some genes responsive to B. sorokiniana and drought stress were overlapping.

image

Figure 5. Real-time quantitative PCR (qPCR) analysis on the transcription of TaPIMP1 and nine stress-related genes regulated by TaPIMP1 in TaPIMP1-overexpressing and RNAi transgenic and Yangmai 12 (WT) wheat (Triticum aestivum) plants before (0 h) and after Bipolaris sorokiniana inoculation for 40 d (40 d). The overexpressing lines (10 plants from M80 and M499) and RNAi lines (10 plants from Z99 and Z107) were pooled as the Ox and RNAi samples, respectively. The relative transcript abundances of the tested gene in the transgenics were relative to that in the WT plants at 0 h. Statistically significant differences of Ox and RNAi transgenic lines were compared with that of WT at the same time point based on three replications (t-test: *, < 0.05; **, < 0.01). Bars indicate + SE.

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image

Figure 6. Real-time quantitative PCR (qPCR) analysis on the transcript patterns of TaPIMP1 and nine stress-related genes regulated in TaPIMP1-overexpressing and RNAi transgenic and Yangmai 12 (WT) wheat (Triticum aestivum) plants before (0 h) and after withholding water for 18 d (D18d) as well as on day 7 after resumption of water following the withholding of water for 22 d (R7d). The leaves of overexpressing lines (10 plants from M80 and M499) and of RNAi lines (10 plants from Z99 and Z107) were pooled as Ox and RNAi samples, respectively. The transcript abundances of the tested gene in the transgenics were relative to that in the WT plants at 0 h. Statistically significant differences of Ox and RNAi transgenic lines were compared with that of WT at the same time point based on three replications (t-test: *, < 0.05; **, < 0.01). Bars indicate SE.

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Expression of TaPIMP1 and the stress-responsive genes is induced by ABA and SA

Phytohormones play important roles in plant responses to biotic and abiotic stresses. To determine if the transcript of TaPIMP1 is induced by phytohormones, we investigated the transcriptional patterns of TaPIMP1 in the WT Yangmai 12 after treatment for 1, 3, 6, and 12 h with exogenous hormones ABA, SA, jasmonic acid (JA), and ET. The expression of TaPIMP1 is significantly induced by ABA or SA, with greater effect for ABA than SA during the time course (Figs 7, S4). TaPIMP1 expression reaches its peak at 6 h after ABA and SA treatments. Moreover, after treatment with the SA inhibitor PBZ or the ABA inhibitor tungstate for 1, 6, or 12 h, the expression of TaPIMP1 (Fig. 7) was considerably decreased and was reduced more by tungstate than by PBZ. These results suggest that TaPIMP1 was a component of the ABA and SA signaling pathways with a greater effect from ABA than from SA.

image

Figure 7. Real-time quantitative PCR (qPCR) analysis on the transcript patterns of TaPIMP1 in Yangmai 12 after treatments by exogenous hormones or hormone inhibitors for 1, 6 and 12 h. Ten wheat (Triticum aestivum) seedlings were pooled as one sample. The transcript abundances with different letters or letter combinations are significantly different from each other based on statistical comparisons at the same time point (t-test: *, < 0.05). Bars indicate SE. The data shown are representative of three independent experiments. ET, ethylene; JA, jasmonic acid; SA, salicylic acid; PBZ, paclobutrazol.

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We investigated the effects of ABA, SA, PBZ, and tungstate treatments for 6 h on the expression of the defense- and stress-related genes activated by TaPIMP1, including RD22, PR1a, TLP4, GST22, GLP4, dehydrin 6, ABAI, PR2, and PAL5, in the leaves of the WT Yangmai 12. As shown in Fig. 8, the transcript abundances of the nine stress-related genes were significantly elevated following exogenous ABA or SA treatment, but markedly decreased by PBZ or tungstate. Of these, the transcript abundances of RD22, dehydrin 6, TLP4, GLP4, and ABAI were enhanced to a greater extent by ABA than by SA, and were reduced more by tungstate than by PBZ, indicating that these genes were regulated mainly by ABA. The transcript abundances of PR1a, PR2, GST22, and PAL5 were increased more by exogenous SA than by ABA, and were decreased more by PBZ than by tungstate, suggesting that their transcription was regulated mainly by SA and to a lesser extent by ABA.

image

Figure 8. Real-time quantitative PCR (qPCR) analysis on the transcription of TaPIMP1 and the nine stress-related genes in Yangmai 12 after treatments with exogenous ABA and salicylic acid (SA) or their inhibitors for 6 h. Ten wheat (Triticum aestivum) seedlings were mixed as one sample. The transcript abundances of the genes in the treated plants were relative to those in mock plants, the significant differences were statistically analyzed based on three replications (t-test: *, < 0.05; **, < 0.01). Bars indicate SE. PBZ, paclobutrazol.

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Furthermore, following treatments with exogenous ABA or SA for 6 h, the transcript abundances of TaPIMP1 and the nine stress-related genes were increased to a significantly greater extent in the TaPIMP1-overexpressing plants (M80 and M499) than in the WT plants, whereas those in the RNAi transgenic wheat lines (Z70 and Z99) showed compromised induction compared with the WT plants (Fig. 9), suggesting that TaPIMP1 may act as a mediator to regulate defense- and stress-related genes in the ABA and SA signal pathways.

image

Figure 9. Real-time quantitative PCR (qPCR) analysis on the transcription of TaPIMP1 and nine stress-related genes in Yangmai 12 (WT), TaPIMP1-overexpressing and RNAi wheat (Triticum aestivum) plants following exogenous ABA and salicylic acid (SA) treatment for 6 h. The leaves of overexpressing (10 plants from M80 and M499) and RNAi lines (10 plants from Z99 and Z107) were pooled as the Ox and RNAi samples, respectively. The transcript abundances of the tested gene in the transgenics were relative to that in the WT plants at 0 h. Statistically significant differences of Ox and RNAi transgenic lines were compared with that of WT under the same treatment based on three replications (t-test: *, < 0.05; **, < 0.01). Bars indicate SE.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Wheat is one of the major food crops worldwide. However, the mechanisms and functional roles of wheat MYB genes in transgenic wheat have not been reported as yet, because generation of stably expressing transgenic wheat lines is difficult and time-consuming. Here, we reported the generation and functional characterization of stably transformed wheat lines overexpressing or underexpressing TaPIMP1. Assays on the phenotypes, microscopy and physiological traits showed that TaPIMP1 overexpression confers enhanced resistance to both B. sorokiniana and drought stress, whereas TaPIMP1-underexpressing transgenics showed greater susceptibility to the biotic and abiotic stresses than the WT wheat. Overexpression of TaPIMP1 inhibited B. sorokiniana mycelial growth, and accounted for the increased resistance. Overexpression of TaPIMP1 resulted in earlier closure of stomata, increased the proline content and reduced water loss, leading to improved drought tolerance of the TaPIMP1-overexpressing transgenic wheat. The degree of resistance to the biotic and abiotic stresses is correlated with TaPIMP1 expression levels. The data clearly reveal that the TaPIMP1 is an importantly positive mediator in wheat defense responses to Bsorokiniana and drought stress. In Arabidopsis, two MYB proteins, BOS1 (AtMYB108) and AtMYB96, mediate biotic and abiotic stress responses, respectively (Mengiste et al., 2003; Seo et al., 2009; Seo & Park, 2010). The discovery of new molecules in wheat biotic and abiotic stresses should be pursued to broaden insights into biotic and abiotic stress signaling pathways in various plant species.

Sequence and phylogenetic analyses showed that the TaPIMP1 protein should be an R2R3-MYB transcription factor. Our previous subcellular localization assay showed that TaPIMP1 is localized to the nucleus (Liu et al., 2011). Here, EMSA and transcriptional-activation activity assays indicated that TaPIMP1 possesses the transcriptional-activation activity, and could bind to five MBS cis-elements tested, suggesting that TaPIMP1 may bind widely to various MBS cis-elements, although the binding to ACI appeared strongest. These results reveal that the TaPIMP1 is, indeed, an R2R3-MYB transcriptional activator, consistent with the TaPIMP1 sequence analysis (Liu et al., 2011). Transcriptional activators can activate the expression of defense- and stress-related genes after binding of specific cis-elements in the promoters, thereby contributing to disease resistance and abiotic stress tolerance. For instance, a rice MYB transcriptional activator, OsMYB3R-2, contributes to rice cold tolerance by alteration of the cell cycle and ectopic expression of stress genes (Ma et al., 2009).

To explore which genes are regulated by TaPIMP1 and to gain an insight into the mechanism of regulation, we combined the experimental approaches of microarray and qRT-PCR assays. Microarray assays revealed that overexpression of TaPIMP1 in wheat induced 112 transcript sets, which are involved in defense- and stress-related and signal transduction genes, including RD22, dehydrin 6, ABAI, GLP4, GST22, PAL5, PR1a, PR2, and TLP4. In Arabidopsis, RD22 is a dehydration-responsive gene induced by exogenous ABA (Yamaguchi-Shinozaki & Shinozaki, 1993), and activated by an MYB transcriptional activator, AtMYB2, and by a bHLH transcriptional factor, AtMYC2 (Abe et al., 1997, 2003). Dehydrin genes are involved in plant drought-stress tolerance and are often induced by drought. PR1, PR2, and TLP genes are well-known marker genes for plant pathogenesis, play primary roles in disease resistance response (Seo & Park, 2010), and are also involved in responses to abiotic stresses (Seo et al., 2008). Expression of GLPs was significantly induced by salt and drought stresses and various pathogens, and the expressed GER3 and GER4 subfamilies of GLPs contributed to resistance against powdery mildew in barley and wheat, and blast fungus in rice (Breen & Bellgard, 2010). GSTs play an important role in defense response and may be involved in abiotic stress response (Anderson & Davis, 2004). PAL is an important enzyme involved in the biosynthesis of antimicrobial compounds during plant–pathogen interaction. In our study, qRT-PCR results in relation to the transcript profiles of these stress-related genes proved that the microarray data were reliable, and TaPIMP1 up-regulated the transcript of the defense- and stress-related genes. We hypothesized that the expression of defense- and stress-related genes up-regulated by TaPIMP1 led to the enhanced resistance to the biotic and drought stresses. After searching the promoter sequences and analyzing MBS cis-elements, we found that the partial promoter sequences of RD22, PR1a, TLP4, ABAI, GST22, and PAL5 contain MBS cis-elements, which could be bound by TaPIMP1 in our EMSA, suggesting that TaPIMP1 may activate the transcription of RD22, PR1a, TLP4, ABAI, GST22, and PAL5 followed by binding of the cis-elements in the promoters. TaPIMP1-mediated elevation of other stress-related genes may occur via an alternate mechanism.

The expression of defense- and stress-inducible genes, including MYB TFs, affects disease resistance and stress tolerance in plants (Dong et al., 2010; Shin et al., 2011). In this study, the transcript of TaPIMP1 in wheat was clearly induced by both B. sorokiniana and drought stress. The defense- and stress-related genes up-regulated by TaPIMP1, including RD22, PR1a, TLP4, GST22, GLP4, dehydrin 6, ABAI, PR2, and PAL5, were also induced by both B. sorokiniana and drought stress, suggesting that B. sorokiniana and drought stress responses in wheat were partially overlapping. After B. sorokiniana inoculation and drought stress, the induced expression levels of the defense- and stress-related genes are significantly higher in TaPIMP1-overexpressing lines than in WT plants, whereas they were lower in the TaPIMP1-RNAi lines than in WT plants, suggesting that these defense- and stress-related genes were activated by TaPIMP1, and that TaPIMP1 positively regulated the responses to B. sorokiniana and drought stress through activation of defense- and stress-responsive genes.

Abscisic acid is involved in both abiotic and biotic stress signaling in plants (Fujita et al., 2006; Fan et al., 2009; Lee & Luan, 2012). Many drought- or pathogen-inducible genes, including MYB TFs, are also activated by ABA (Abe et al., 2003; Ding et al., 2009). SA is a defense signal molecule associated with resistance to biotrophic and hemibiotrophic pathogens (Pieterse et al., 2009). Many defense-related genes are activated by SA. Although ABA is generally considered to be a negative regulator of SA-mediated disease resistance against biotrophic pathogens (Fujita et al., 2006; Fan et al., 2009), recent studies imply that positive interactions between the ABA signaling pathway and the biotic signaling networks involving SA, JA and ET enhance a tolerance response to abiotic and biotic stresses (Seo & Park, 2010). Some TFs are involved in the signaling networks. For instance, the Arabidopsis BOS1 gene controls both JA- and ABA-inducible genes. A loss-of-function bos1 mutant is susceptible to both necrotrophic pathogens, and osmotic and oxidative stresses (Mengiste et al., 2003). AtMYB96-mediated ABA signaling promotes drought tolerance and resistance to the pathogen Pseudomonas syringae pv. tomato DC3000 infection by inducing SA biosynthesis (Seo et al., 2009; Seo & Park, 2010). The ABA-mediated MYB96 regulation of SA biosynthesis might be another route for balancing plant responses to pathogen infection and abiotic stresses (Seo & Park, 2010). Our study suggested that TaPIMP1 might be involved in the ABA and SA signaling pathways, and that the defense- and stress-related genes activated by TaPIMP1 might be in the ABA and SA signaling pathways. Moreover, following ABA or SA treatments, the inductions of these defense- and stress-related genes were increased to a significantly greater extent in the TaPIMP1-overexpressing transgenic wheat than in the WT plants, whereas those in the TaPIMP1-RNAi transgenic lines showed compromised induction compared with the WT plants, suggesting that TaPIMP1 may act as an integrator to regulate the defense- and stress-related genes in the ABA and SA signal pathways. TaPIMP1 positively regulates wheat resistance responses to drought stress and Bsorokiniana infection. Bsorokiniana is a hemibiotrophic pathogen (Kumar et al., 2002). In the Bsorokiniana tolerant 1 (bst1) barley mutant, the transcript abundances of SA-activated PR1a, PR2, and PR5 were obviously elevated (Persson et al., 2009), implying that SA signaling may be involved in the defense response to Bsorokiniana. When subjected to drought conditions, plants often produce and accumulate more ABA, which induces stomata closure, thus conserving water and inhibiting pathogen invasion (Lee & Luan, 2012). It would be of interest to study further how TaPIMP1 mediates the crosstalk between the ABA- and SA-signaling pathways and if the mechanism of wheat TaPIMP1-mediated signaling is similar to AtMYB96. Measurement of the ABA and SA concentrations in the TaPIMP1-overexpressing and RNAi wheat plants after pathogen and drought stress may help to address the issue.

In summary, upon Bsorokiniana infection and drought stress, TaPIMP1 expression was up-regulated, which could activate the defense- and stress-related genes in the ABA- and SA-signaling pathways, leading to enhanced resistance to both biotic and abiotic stresses in wheat. TaPIMP1 will provide a transgenic tool for improving multiple resistance in wheat and other cereal crops. This study provides novel insights into the function of the MYB family in wheat, and into the defense mechanisms of wheat in relation to B. sorokiniana and drought stress.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The authors are grateful to Dr Xin Deng (Institute of Botany, Chinese Academy of Sciences, China) for technical assistance with stomatal assays, and Dr Cunjin Zhang (Durham University, UK) for revision. This study was supported by the NSFC program (30871523), the ‘863’ programs (2012AA10A309 and 2012AA101105), and a National ‘Key Sci-Tech’ project (2013ZX08002001).

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office.

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nph4353-sup-0001-FiguresS1-S4.pdfapplication/PDF201K

Fig. S1 Phylogeny of TaPIMP1 was constructed based on peptide sequences using a neighbor–joining method of MEGA 4 software after these MYB protein sequences were aligned using ClustalW.

Fig. S2 The germination and growth of the stable TaPIMP1-overexpressing transgenic wheat lines (M80 and M499 in T3 generation) and wildtype Yangmai 12 (WT) plants as well as segregants lacking the TaPIMP1 transgene (Null).

Fig. S3 Microscopy images of leaf stomatal apertures of the wildtype and TaPIMP1-overexpressing transgenic plants withholding water for 0, 5, and 16 d.

Fig. S4 The transcript profiles of TaPIMP1 in the wildtype wheat Yangmai 12 were analyzed by qRT-PCR after treatment with 0.1 mM ABA, 1 mM SA, 0.1 mM JA, and 0.2 mM ET for 1, 3, 6, and 12 h.

nph4353-sup-0002-TableS1-S3.docWord document56K

Table S1 Sequences of primers used in this study

Table S2 Promoter sequence analysis of stress-related genes up-regulated by TaPIMP1

Table S3 Major agronomic traits in TaPIMP1 transgenic and wildtype wheat plants

nph4353-sup-0003-TableS4.xlsapplication/msexcel2489K

Table S4 Raw data of microarray analyses in the TaPIMP1-overexpressing and wildtype wheat lines

nph4353-sup-0004-TableS5.docWord document352K

Table S5 Genes up-regulated by TaPIMP1 through microarray analysis