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JXB Advance Access published online on February 4, 2008
Journal of Experimental Botany, doi:10.1093/jxb/erm353
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© 2008 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

Abscisic acid regulates TSRF1-mediated resistance to Ralstonia solanacearum by modifying the expression of GCC box-containing genes in tobacco

Jinxin Zhou1,2 * {dagger}, Hongbo Zhang1,4 *, Yuhong Yang3, Zhijin Zhang1, Haiwen Zhang1, Xinwen Hu2, Jia Chen4, Xue-Chen Wang4 and Rongfeng Huang1,{ddagger}

1Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
2College of Agriculture, South China University of Tropical Agriculture, Danzhou 571737, Hainan, China
3Vegetable and Flower Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China
4National Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China

{ddagger} To whom correspondence should be addressed. E-mail: rongfeng{at}public3.bta.net.cn

Received 31 October 2007; Revised 13 December 2007 Accepted 14 December 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Although recent studies have established a significant regulatory role for abscisic acid (ABA) and ethylene response factor (ERF) proteins in plant pathogen resistance, it is not clear whether and how ABA performs this role. Previously, it was reported that an ERF protein, TSRF1, activates the expression of GCC box-containing genes and significantly enhances the resistance to Ralstonia solanacearum in both tobacco and tomato plants. Here, it is reported that TSRF1-regulated pathogen resistance is modified by ABA application. TSRF1 activates the expression of ABA biosynthesis-related genes, resulting in the increase of ABA biosynthesis, which further stimulates ethylene production. More interestingly, ABA application decreases, while the inhibitor of ABA biosynthesis fluridone increases, the TSRF1-enhanced resistance to R. solanacearum. This observation is further supported by the finding that ABA and fluridone reversibly modify the ability of TSRF1 to bind the ethylene-responsive GCC box, consequently altering the expression of element-controlled genes. These results therefore establish that TSRF1-regulated resistance to R. solanacearum can be modified in tobacco by ABA.

Key words: Abscisic acid, ERF protein TSRF1, GCC box-containing genes, Ralstonia solanacearum, tobacco


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Interaction of the plant hormones, abscisic acid (ABA) and ethylene, plays important roles in plant development. These include root elongation, organ senescence, the initiation of adaptive responses to various environmental conditions, and resistance to pathogens via a complex network of synergistic and antagonistic interactions (Beaudoin et al., 2000; Anderson et al., 2004; Guo and Ecker, 2004). Genetic studies on the components of ethylene and ABA pathways have suggested the antagonistic interaction between these two pathways in plant biotic and abiotic stress responses. For example, mutants of ethylene signalling (etr1, ein2, ein3), and mutants of the ABA pathway (aba1, aba2, abi1, abi2) are reported to antagonize the expression of defence and stress-responsive genes, thereby modulating plant stress responses (Beaudoin et al., 2000; Ghassemian et al., 2000; Anderson et al., 2004). More interestingly, there is increasing evidence for the involvement of ABA in the regulation of plant responses to pathogen attack. For instance, exogenous application of ABA prior to inoculation increases the susceptibility to various pathogens (Ward et al., 1989; Audenaert et al., 2002). By contrast, the ABA-deficient tomato mutants flacca and sitiens, which are both impaired in the conversion of ABA-aldehyde to ABA, display a reduction in susceptibility to Botrytis cinerea (Kettner and Dorffling, 1995; Audenaert et al., 2002). However, recent researches have shown that ABA increases the resistance of plants to pathogens by affecting callose deposition (Ton and Mauch-Mani, 2004) and jasmonic acid biosynthesis (Adie et al., 2007), suggesting the possible synergistic regulation of ethylene/jasmonic acid and ABA pathways in plant pathogen resistance. Thus, understanding the complex molecular mechanism of the interaction between the ethylene and ABA pathways in the plant's response to pathogens should be of benefit to breeders developing pathogen-resistant crops.

Ethylene response factor (ERF) proteins have been presumed to modulate multiple responses, including regulation of the plant pathogen response (Ohme-Takagi and Shinshi, 1995) and plant development (Chakravarthy et al., 2003). In such processes, ERF proteins are regulated by ETR1, CTR1, EIN2, and EIN3 (Alonso and Stepanova, 2004; Anderson et al., 2004; Guo and Ecker, 2004). Most of the identified ERF proteins, such as tomato Pti4/5/6 (Gu et al., 2002), Arabidopsis ERF1 (Solano et al., 1998), AtERFs (Fujimoto et al., 2000), bind to the GCC box and modulate the expression of pathogenesis related (PR) genes that function in plant pathogen resistance. Some ERF proteins, such as CBF1 (Cook et al., 2004), interact with a dehydration-responsive element (DRE) that is involved in drought, salt, and cold stress (Yamaguchi-Shinozaki and Shinozaki, 2005). Interestingly, the ABA responsive element, coupled element 1 (CE1), is important in determining the specific expression of ABA responsive genes (Shen and Ho, 1995). Most importantly, the ERF proteins Tsi1 (Park et al., 2001) and TERF1 (Huang et al., 2004; Zhang et al., 2005) regulate the expression of GCC box- and DRE-containing genes. Subsequently, plants have enhanced tolerance to biotic and abiotic stresses, suggesting that ERF proteins are important regulators in pathogen responses mediated by the ethylene and ABA pathways. What is not clear is how ABA affects ERF protein-mediated plant pathogen resistance.

It has been proved that some of ERF proteins might be the downstream component of EIN3 (Solano et al., 1998), which is further regulated by upstream regulators in ethylene pathways. This cascade regulation ultimately results in the increase of expression of PR genes (Alonso and Stepanova, 2004; Anderson et al., 2004; Guo and Ecker, 2004). A more recent report demonstrates that components in the ethylene pathway, such as MAPK kinase 2, which is inducible upon pathogen infection, modulate jasmonic acid and salicylic acid levels (Brader et al., 2007). More importantly, by affecting jasmonic acid biosynthesis ABA modulates the expression of defence genes (Adie et al., 2007). Previously, it was demonstrated that an ERF protein, TSRF1, significantly enhanced resistance to Ralstonia solanacearum, Pseudomonas syringae, and B. cinerea in both tobacco and tomato, by activating the expression of GCC box-containing genes (Zhang et al., 2004, 2007). In the present study, the focus is on the ABA-regulated pathogen response to R. solanacearum. Using the expressing TSRF1 tobacco, the relationship between ABA and ERF protein-mediated plant pathogen resistance was investigated.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Plant materials and growth conditions
Tobacco (Nicotiana tabacum cv. Gexin1) plants were grown in a growth chamber at 25 °C with a 16/16 h light/dark cycle (except where mentioned in the text). For detecting the expression of TSRF1-downstream genes in tobacco plants, leaves from normally growing 4-week-old plants were used after treatment with 50 µM ABA (Dingguo, Beijing), or 50 µM fluridone (Abel Industries, Canada) for 4 h. Both ABA and fluridone were separately dissolved in ethanol, and were diluted to the above final concentration, containing less than 0.005% Tween-20 and 0.2% ethanol. The control was treated with 0.005% Tween-20 and 0.2% ethanol, which did not have a significant effect on the expression of the target genes in these assays. The transgenic tobacco plants expressing TSRF1 were generated as described by Zhang et al. (2004). T3 tobacco plants were used in this paper. Wild-type tobacco and TSRF1-expressing tobacco are indicated as WTB and OTB, respectively.

RNA isolation and gene transcript detections
For semi-quantitative RT-PCR analysis, total RNA from tobacco was isolated using the Trizol agent (Invitrogen, Carlsbad, CA) and subjected to DNase (Promega) treatment to digest the DNA in RNA samples. 1 µg of total RNA was used to produce cDNA for RT-PCR amplifications. PCR products detected with ethidium bromide staining were recorded with an FX Imager (Bio-Rad) and then analysed using Quantity One (Bio-Rad) software for the quantification of the target gene. The transcript abundance for each gene is relative to the actin transcript level measured in the same sample. The primers for the GCC box-containing genes are the same as described by Zhang et al. (2004), and the primers for ACO1 and NtSDR genes are given as follows: 5'-CCACACAGGTGTGATGGTTG-3' and 5'-CACGTCGCACTTCATGATCG-3' for actin, 5'-GATTACACAAACAGACGGGACT-3' and 5'-TTGATTCCACCACACACAATAC-3' for ACO1, 5'- TGCGACAG TGGTCTCTGG-3' and 5'-CCCACTCATTTTGAAAAAATCA-3' for NtSDR.

Measurement of ethylene and ABA
To determine ethylene production, 6-week-old tobacco seedlings were weighed and sealed in a 100 ml vial. After 60 min, 1 ml of gas from each vial was used to analyse the ethylene content using a gas chromatograph (Hitatch, Japan). For ABA measurements, tissues (100 mg per sample) were ground in liquid nitrogen to extract ABA with 80% (v/v) ethanol for 4 h. ELISA, using monoclonal anti-ABA antibody, was used to quantify ABA content (Banowetz, 1992).

Plant infection with R. solanacearum strain BJ1057
The pathogen inoculation of tobacco leaves followed our previous report (Zhang et al., 2004). The inoculated plants were maintained at 28 °C with a 16/16 h light/dark cycle. Four days post-inoculation (DPI), the bacterial growth was measured by macerating five leaf discs of 1 cm2 from the inoculated tissue of each sample in 10 mM MgCl2, plating the serial dilutions on nutrient agar plates, and counting the colony-forming units (cfu). For ABA or fluridone treatment, 4–5-week-old wild-type and TSRF1-expressing tobacco plants were sprayed with 100 µM ABA or 100 µM fluridone containing 0.1% ethanol and 0.005% (v/v) Tween-20 16 h before infection with R. solanacearum strain BJ1057. Controls were treated with 0.1% ethanol and 0.005% (v/v) Tween-20.

β-glucuronidase transient assay in vivo
To construct the reporter vector, the four times-repeated GCC box sequences were inserted upstream of the minimal TATA box (–46 to +10) to replace the cauliflower mosaic virus (CaMV) 35S promoter in pBI121 (Clontech, Palo Alto, CA). The effector was constructed by replacing the β-glucuronidase (GUS) gene in pBI121 with the full encoding region of TSRF1. Transient assays were performed on the 10-d-old wild-type tobacco seedlings as described (Zhang et al., 2004).

Accession numbers
The GenBank accession numbers for the sequences used as materials in this article are AF494201 (TSRF1), X63603 [GenBank] (actin), AY426756 (ACO1), AJ223177 (NtSDR), EF434388 (NtSDR promoter), X66942 (PRB1B), M60402 (GLA), X51599 (CHN50), and X95308 (Osmotin).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Expressing TSRF1 in tobacco increases ABA and ethylene production
In view of the inducible nature of TSRF1 in response to pathogens (Zhang et al., 2004) and ABA (our unpublished data), the question was asked whether the regulation of TSRF1 in pathogen resistance is associated with the action of ABA. To address this hypothesis, the ABA content of WTB and OTB plants were compared first. As shown in Fig. 1A, OTB plants possess more than 3-fold the ABA levels of controls. Further studies showed that OTB plants also have greatly enhanced expression levels of NtSDR (tobacco short-chain dehydrogenase/reductase) (Fig. 1B), which is reported to be associated with ABA biosynthesis in tobacco (Wu et al., 2007).


Figure 1
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  		Fig. 1. Expressing TSRF1 in tobacco enhances the production of ABA. (A) ABA contents in WTB and OTB. (B) OTB displays increased expression of tobacco NtSDR under normal growth conditions. The expression levels are relative to the WTB control (as 100). The data are the average of three independent experiments. Error bars indicate ±SE.

 
Furthermore, by measuring ethylene production, it was found that OTB plants display higher ethylene production than is found in WTB (Fig. 2A), suggesting that TSRF1 stimulates ethylene production as well. In order to clarify how TSRF1 regulates ethylene production, the expression of ACC-oxidase (ACO) genes was checked. These genes encode the key enzymes in ethylene biosynthesis (Chae and Kieber, 2005) and expression in WTB and OTB was measured using semi-quantitative RT-PCR amplifications. For these ethylene biosynthetic genes, the expression of ACO1 was c. 2.5-fold higher in OTB than in WTB (Fig. 2B), while transcripts of other ethylene biosynthesis-related genes were not significantly changed (data not shown). It is important to note that ethylene production and the expression of ACO1 was strengthened by ABA application (Fig. 2), indicating that ABA mediating the expression of TSRF1 affects ethylene biosynthesis.


Figure 2
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  		Fig. 2. Expressing TSRF1 in tobacco increases the production of ethylene. (A) OTB has higher ethylene production, which is further promoted by ABA application. (B) OTB shows the increased expression of tobacco ACO1. The expression levels are relative to the WTB control (as 100). Control indicates tobacco without ABA treatment; ABA indicates tobacco treated with 50 µM ABA for 2 d. The data are derived from triplicate samples and three independent experiments. Error bars indicate ±SE.

 
ABA reduces and fluridone increases the response to R. solanacearum challenge in TSRF1-expressing tobacco
It is known that expression of TSRF1 in tobacco and tomato significantly increases resistance to R. solanacearum (Zhang et al., 2004), and ABA stimulates the expression of ACO1 and ethylene production (Fig. 2). Therefore, the following assays were conducted by testing the resistance of WTB and OTB tobacco plants to R. solanacearum strain BJ1057, combined with pretreatment with ABA or fluridone (a carotenoid inhibitor of ABA synthesis; Kowalczyk-Schroder and Sandmann, 1992). In this pathogen inoculation system, four concentrations (107–104 cfu ml–1) of R. solanacearum were used, continuously infiltrated into leaves as previously described (Zhang et al., 2004). Under normal growth conditions (control), WTB plants developed water-soaked lesions and a chlorotic edge around the infected sites at 4 DPI at inoculation concentrations of 107~105 cfu ml–1 and no symptoms at low inoculation concentration of 104 cfu ml–1. By contrast, OTB plants showed no development of a chlorotic edge around the necrotic lesions at inoculation concentrations of 107 and 106 cfu ml–1 and almost no symptoms at 105 and 104 cfu ml–1 (Fig. 3), consistent with our previous observations (Zhang et al., 2004). The application of ABA decreased resistance to the pathogen, with WTB showing heavier water-soaked lesions around the infected sites, at all inoculation concentrations of 107~104 cfu ml–1. In ABA-treated OTB plants, water-soaked lesions and a chlorotic edge occurred around the infected sites at inoculation concentrations of 107 and 106 cfu ml–1; at inoculation concentration of 105 cfu ml–1, a resistant response was seen after ABA treatment and no symptoms at 104 cfu ml–1. Although it was observed that ABA obviously reduced the resistance of WTB and OTB to R. solanacearum, the disease symptoms in OTB were less serious than in WTB. In contrast to this observation, the ABA biosynthesis inhibitor fluridone significantly reduced disease symptoms in OTB and WTB, and the disease symptoms in OTB were much less serious than in WTB (Fig. 3). Distinctive pathogen symptoms are therefore seen after ABA and fluridone treatment depending on different inoculation concentrations of the pathogen.


Figure 3
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  		Fig. 3. ABA affects the resistance of TSRF1-expressing tobacco to R. solanacearum strain BJ1057. Inoculation concentrations of R. solanacearum strain BJ1057 were 107 to 104, oriented from bottom to top, in separate regions between the lateral veins. Symptoms for WTB and OTB leaves with the pathogen were photographed at 4 DPI. The shown images represent three independent assays, which gave similar results. Control: seedlings were grown under normal growth conditions; ABA, ABA treatment; Fluridone, fluridone treatment.

 
As the visible symptoms were the result of bacterial multiplication and spread, the leaf bacterial population was further determined 4 DPI with 106 cfu ml–1 R. solanacearum for each treatment. In controls, the bacterial number in OTB was 99–123-fold less than in WTB. After ABA treatment, the bacterial numbers in WTB and OTB plants were increased by 2–27-fold compared with the WTB and OTB of the control, respectively, but showing 11–13-fold less in OTB than that in WTB. By contrast, after fluridone treatment, bacterial numbers decreased by 7-fold in WTB, and 646–861-fold in OTB, compared with the WTB of the control, but 6~7-fold less in OTB than that in OTB of the control (Table 1). Student's t tests indicate that the above data of bacterial numbers are significantly different at 95% probability. Therefore, these results show ABA to be an important negative regulator in the response of TSRF1-expressing tobacco plants to R. solanacearum.


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  		Table 1. Bacterial growth in tobacco leaves 4DPI with 106 cfu ml–1 R. solanacearum

 
ABA modifies the expression of TSRF1-regulated GCC box-containing PR genes
Many PR genes, such as PR1, PR2, PR3, and PR5, have been characterized as having the GCC box in their promoters (Ohme-Takagi and Shinshi, 1995; Park et al., 2001). Expressing TSRF1 in tobacco and tomato activates, while RNA interference of TSRF1 in tomato deactivates, the expression of GCC box-containing genes (Zhang et al., 2004, 2007). To determine whether ABA and fluridone could influence the expression of GCC box-containing genes in plants, the expression level of PRB1B, GLA, CHN50, and Osmotin in transgenic tobacco was tested using semi-quantitative RT-PCR amplifications. Because of the ectopic expression of TSRF1 in tobacco, there is no or little expression of PR genes in WTB after ABA or fluridone treatment. However, with the exception of Osmotin, the expression of GCC box-containing genes was obviously decreased after ABA application in OTB plants. But when ABA biosynthesis was inhibited by fluridone there was a significant increase in the expression of GCC box-containing genes (Fig. 4). Student's t tests indicate that the above data for gene transcripts are significantly different at 95% probability. Thus these results suggest that ABA application alters the expression of GCC box-containing genes.


Figure 4
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  		Fig. 4. ABA modifies the expression of ethylene-responsive GCC box-containing genes. Actin was used as loading control. The expression levels were relative to the WTB control (as 1). Error bars based on three independent experiments indicate ±SD. Control indicates tobacco seedlings without ABA treatment; ABA and fluridone indicate tobacco seedlings treated with ABA and fluridone, respectively.

 
In order to clarify whether the altered expression of GCC box-containing genes in vivo results from the transcriptional activity of TSRF1 based on GCC box after ABA application, the interaction of TSRF1 was investigated further using transient promoter-GUS expression in tobacco seedlings. As shown in Fig. 5, the transcriptional activity of TSRF1 based on GCC box was changed after ABA and fluridone treatments. In addition, GUS activity driven by the GCC box was about 25% of the control (GCC) after ABA and 182% after fluridone treatment. This supports the idea that ABA eventually deactivates the binding of TSRF1 to the GCC box, thereby decreasing the expression of GCC box-containing genes, resulting in changes to the pathogen response.


Figure 5
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  		Fig. 5. ABA and fluridone affect the interaction of TSRF1 with GCC box in vivo, determined by transient assay in wild-type tobacco leaves. The data for GUS activity quantification are averages of triplicate samples from three independent experiments, compared to the Min control (as 100). Min indicates the minimal TATA promoter fused with GUS; GCC indicates that the GCC box is inserted upstream of Min as a positive reporters; TSRF1 as a positive effector indicates that the full-length TSRF1 gene was driven by the 35S promoter. Error bars indicate ±SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
ERF proteins are important plant-specific transcription factors in pathogen resistance (Park et al., 2001; Chakravarthy et al., 2003; Alonso and Stepanova, 2004). In the present paper, evidence is provided that over-expressing TSRF1 increases ABA and ethylene levels as a result of increased expression of the NtSDR1 and the NtACO1 genes; further, that ABA is responsible for the elevated production of ethylene in this transgenic line. Our previous research demonstrated that the expression of TSRF1 enhances resistance to R. solanacearum as a result of the constitutive expression of GCC-box genes (Zhang et al., 2004). Further researches in the present paper indicate that ABA decreases resistance and the expression of the GCC-box genes. By contrast, the inhibitor of ABA biosynthesis, fluridone, enhances resistance and expression of the same genes. This both extends our knowledge of the ability of ABA to influence responses to pathogens, and is consistent with a body of literature (Ward et al., 1989; Audenaert et al., 2002; Anderson et al., 2004; Mohr and Cahill, 2007).

Recently, ERF proteins have been identified as regulating plant ABA-related responses (Zhang et al., 2005). In line with this, mutation of the transcriptional repressor ERF protein, ABR1, greatly enhances the ABA response (Pandey et al., 2005). Furthermore, gathering evidence demonstrates that ABA is involved in regulating the plant response to pathogen attack (Melotto et al., 2006; Hernandez-Blanco et al., 2007), although the detailed regulatory mechanisms are not well explored and even controversial (Mauch-Mani and Mauch, 2005). According to the current literature, the functions of ABA in plant pathogen resistance can be classified into structural and transcriptional regulation. Upon bacterial pathogen challenge, ABA is important in closing stomata to set up the barrier against bacterial challenge (Melotto et al., 2006). Mutation of cellulose synthases for secondary cell wall formation results in enhanced resistance to bacterium R. solanacearum and fungus Plectosphaerella cucumerina (Hernandez-Blanco et al., 2007). In addition, ABA can suppress the accumulation of lignin in Arabidopsis infected with P. syringae pv. tomato (Mohr and Cahill, 2007). ABA-deficient mutants are more susceptible to P. syringae infection. However, it has also been shown that ABA increases susceptibility by counteracting salicylic acid-dependent defences, and ABA-dependent priming of callose biosynthesis promotes enhanced resistance to some pathogens (Ton and Mauch-Mani, 2004).

Besides these structural regulations, one of the significant roles of ABA is to regulate the transcription of pathogen resistance genes. For instance, ABA mutants (abi1-1, abi2-1, and aba1-6) display an increased susceptibility to R. solanacearum, but ABA treatment causes increased expression of pathogen-induced genes and pathogen resistance (Adie et al., 2007; Hernandez-Blanco et al., 2007). However, there are accumulating reports that ABA may actually negatively regulate the expression of some pathogen resistance genes. For instance, exogenous application of ABA prior to inoculation increases plant susceptibility to pathogens (Ward et al., 1989; Audenaert et al., 2002; Anderson et al., 2004), by repressing the expression of defence-related genes (Anderson et al., 2004; Mohr and Cahill, 2007). In the present report, it is demonstrated that ABA reduces, while the ABA biosynthetic inhibitor fluridone enhances, the expression of PR genes in expressing TSRF1 tobacco plants. This is consistent with several reports that ABA negatively regulates pathogen resistance and PR gene expression (Ward et al., 1989; Audenaert et al., 2002; Anderson et al., 2004; Mohr and Cahill, 2007), but is contrary to the finding that ABA positively regulates resistance to R. solanacearum (Hernandez-Blanco et al., 2007). One explanation for these differences could be the specific interaction of the plant target with effectors secreted by R. solanacearum (van Wees and Glazebrook, 2003; Torres-Zabala et al., 2007). If this is the case then ABA levels could provide the key for fine-tuning plant defences against particular pathogens (Pierik et al, 2006).

A significant observation in the present work was that overexpression of TSRF1 in tobacco enhances both ABA and ethylene levels. Confirmation that this is mediated by activation of ABA and ethylene biosynthesis genes would indicate a role for TSRF1 as a positive regulator in ABA and ethylene biosynthesis, revealing the importance of hormone metabolites in defence. This result is consistent with the report that ABA is an essential signal for defence against Pythium irregulare via an effect on jasmonic acid biosynthesis and the pathogen-induced genes that are associated with ABA biosynthesis (Adie et al., 2007). Other investigations have shown that the ABA signalling pathway (Torres-Zabala et al, 2007), and salicylic acid catabolism (van Wees and Glazebrook, 2003; Jagadeeswaran et al., 2007) are major targets for effectors secreted by P. syringae. Tobacco NtSDR, a putative SDR family protein, is reported to associate with tobacco ABA production, possibly through transcriptional regulation of ERF protein JERF1 (Wu et al., 2007). Consistent with this report, it is shown in this paper that expression of TSRF1 in tobacco increases the expression of NtSDR and ABA production. More importantly, the inhibitor of ABA biosynthesis, fluridone, enhances pathogen resistance by affecting the expression of ethylene-responsive GCC box genes, thereby indicating that ABA biosynthesis is associated with pathogen resistance. Further, it was observed that higher endogenous ABA levels found in the overexpressing TSRF1 plants did not have any suppressive effects on the endogenous GCC box-containing gene transcripts whereas exogenous ABA treatment did suppress the expression of these genes. It is possible that TSRF1 predominantly activates the expression of those GCC box-containing genes that are responsive to increased ethylene levels under low ABA levels, consequently increasing disease resistance in the overexpressing plants. However, when plants were treated with exogenous ABA, it was found to inhibit transcript levels of these GCC box-containing genes, suggesting that the effect of ABA on the expression of PR genes and disease resistance might be dose-dependent.

Based on results in the present paper and our previous reports (Zhang et al., 2004, 2007), a model is proposed in which ABA mediates the regulation of TSRF1, thereby affecting resistance to the pathogen, R. solanacearum (Fig. 6). Expression of TSRF1 promotes ABA production in tobacco, which further promotes ethylene biosynthesis. In our scheme, production of these hormones could antagonistically affect the interaction of TSRF1 with the GCC box. Because of the low levels of ABA under normal growth conditions, endogenous ABA would not be sufficient to suppress the ability of TSRF1 to activate the expression of GCC-containing genes, consequently resulting in increased resistance to the pathogen. However, after ABA application, ABA dominantly represses the binding of TSRF1 to the GCC box, significantly reducing the expression of GCC box-containing genes, subsequently decreasing the resistance to the pathogen. In the case of suppression of ABA biosynthesis by fluridone, the resulting imbalance in the interaction of ethylene and ABA would allow TSRF1 to enhance the expression of GCC box-containing genes and enhance resistance to the pathogen (Fig. 6). The data in this paper provide a clue to understanding the regulation of ABA in ERF protein-mediated pathogen resistance.


Figure 6
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  		Fig. 6. A model for the way in which ABA mediates the regulation of TSRF1 in response to the pathogen, R. solanacearum. The line with an arrowhead indicates activation, the bar indicates suppression, and the continuous line with an arrowhead indicates where the function is unclear.

 


   Acknowledgements
 
We greatly appreciate Professor Clive Lloyd at the John Innes Centre, UK, for his help in improving the English of the text. This work was supported by the National Science Foundation of China (Grant nos 30525034 and 30471047) and the National Basic Research Program of China (2006CB100102).


   Footnotes
 
* These authors contributed equally to this work. Back

{dagger} Present address: Botanic Garden of Wuhan, Chinese Academy of Sciences, Wuhan 430074, China. Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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