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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.
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- Materials and Methods
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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 B. sorokiniana 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 B. sorokiniana infection. B. sorokiniana is a hemibiotrophic pathogen (Kumar et al., 2002). In the B. sorokiniana 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 B. sorokiniana. 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 B. sorokiniana 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.