Synthetic promoters consisting of defined cis-acting elements link multiple signaling pathways to probenazole-inducible system#

Synthetic promoters consisting of defined cis -acting elements link multiple signaling pathways to probenazole-inducible system # # 基于已知顺式元件的人工启动子响应性分析揭示烯丙异噻唑诱导系统可触发植物体内多条信号途径 1 3 first-author 2 2 2 2 1 4 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:bkkuai@fudan.edu.cn †E-mail:bkkuai@fudan.edu.cn bkkuai@fudan.edu.cn The Southern Modern Forestry Collaborative Innovation Center, Nanjing Forestry University, Nanjing 210037, China The Southern Modern Forestry Collaborative Innovation Center, Nanjing Forestry University, Nanjing 210037, China State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200438, China State Key Laboratory of Genetic Engineering and Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200438, China College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China Bamboo Research Institute, Nanjing Forestry University, Nanjing 210037, China Bamboo Research Institute, Nanjing Forestry University, Nanjing 210037, China Probenazole (3-allyloxy-1,2-benzisothiazole-1,1-dioxide, PBZ), the active component of Oryzemate, could induce systemic acquired resistance (SAR) in plants through the induction of salicylic acid (SA) biosynthesis. As a widely used chemical inducer, PBZ is a good prospect for establishing a new chemical-inducible system. We first designed artificially synthetic promoters with tandem copies of a single type of cis -element (SARE, JERE, GCC, GST1, HSRE, and W-box) that could mediate the expression of the β-glucuronidase ( GUS ) reporter gene in plants upon PBZ treatment. Then we combined different types of elements in order to improve inducibility in the PBZ-inducible system. On the other hand, we were surprised to find that the cis -elements, which are responsive to jasmonic acid (JA) and ethylene, also responded to PBZ, implying that SA, JA, and ethylene pathways also would play important roles in PBZ’s action. Further analysis demonstrated that PBZ also induced early events of innate immunity via a signaling pathway in which Ca 2+ influx and mitogen-activated protein kinase (MAPK) activity were involved. We constructed synthesized artificial promoters to establish a PBZ chemical-inducible system, and preliminarily explored SA, JA, ethylene, calcium, and MAPK signaling pathways via PBZ-inducible system, which could provide an insight for in-depth study. 目的 2 构建有效响应烯丙异噻唑（PBZ）诱导的人工合成启动子，了解植物体内受PBZ诱导系统触发的信号途径。 创新点 2 通过分析包含已知顺式元件的人工合成启动子对PBZ的响应性，为构建一种基于PBZ诱导系统的新型化学诱导启动子提供了可能性，并初步揭示了除水杨酸（SA）外，茉莉酸（JA）和乙烯等多条信号途径可能共同参与了PBZ诱导的植物免疫反应过程。 方法 2 利用已知的响应相关信号通路的顺式作用元件构建人工合成启动子，融合GUS报告基因后，稳定转化拟南芥。通过检测PBZ处理过程中GUS酶活性的变化，了解人工合成启动子对PBZ的响应性，分析PBZ诱导系统可能触发的信号途径。 结论 2 除了SA响应元件SARE可以有效响应PBZ诱导外，利用JA和乙烯响应元件JERE和GCC，超敏反应（HR）相关的顺式元件HSRE和GST1，以及植物抗病反应中重要顺式作用元件W-box构建的人工合成启动子也均可有效响应PBZ。另外，通过人工合成启动子响应性分析的手段，初步揭示了包括SA、JA、乙烯、钙离子和丝裂原活化蛋白激酶（MAPKs）在内的多条信号通路可能共同参与了PBZ诱导植物免疫反应的过程。 Probenazole Systemic acquired resistance (SAR) Cis -acting element Chemical-inducible system Synthetic promoter 烯丙异噻唑 人工合成启动子 顺式作用元件 化学诱导系统 Introduction 1 In eukaryotes, the promoter is the core DNA region that controls the initiation of gene expression. A gene promoter usually contains a battery of cis -acting regulatory elements, with their combined effect shaping the expression profile in response to both developmental and environmental signals (Rushton et al., 2002 2002 ). Promoters of plant hormone and several other key signaling pathway genes have been extensively studied, and a list of cis -acting elements has been isolated, which exhibit diversified responsive characteristics (Rushton and Somssich, 1998 1998 ; Rushton et al., 2002 2002 ). The GST1 box is one of the elements that were isolated early on from the promoter of the potato GST1 gene encoding a glutathione S -transferase, which is used as a reliable marker for the generation of reactive oxygen species (ROS) (Levine et al., 1994 1994 ; Strittmatter et al., 1996 1996 ); and SARE, a cis -acting element responsive to salicylic acid (SA), was then isolated from the upstream region of tobacco PR2 gene (Shah and Klessig, 1996 1996 ). Then JERE was reported to modulate the expressions of both elicitor- and jasmonate-responsive genes (Menke et al., 1999 1999 ); GCC box, an ethylene-responsive element, was identified in the promoters of both ethylene signaling pathway genes and pathogen-related genes (Ohme-Takagi et al., 2000 2000 ). More recently, S box, a GCC-like element, was shown to be responsible for the activation of fungal elicitors-triggered gene expression (Kirsch et al., 2000 2000 ; Rushton et al., 2002 2002 ); HSRE, an HSR203 responsive element, was located in the promoter region of HSR203J to mediate the hypersensitive response (HR) in Nicotiana tabacum (Pontier et al., 2001 2001 ). Recently, advances in promoter construction technology allow us to specifically manipulate the transcriptional activities of target genes. Combining pathogen and/or chemical-responsive defined cis -acting elements with constitutive promoters to generate inducible synthetic promoters could not only provide a precise control of transgene expression but also modulate its expression in various contexts (Venter, 2007 2007 ; Mehrotra et al., 2011 2011 ). In addition, these inducible synthetic promoters can be introduced into plants as molecular markers, which may provide a powerful approach for studying plant-pathogen interactions and characterizing signaling pathways induced by various chemicals at the molecular level. Probenazole (PBZ) is the active component of Oryzemate, an environment-friendly rice blast controlling agrochemical since 1975 (Watanabe, 1977 1977 ; Sekizawa and Watanabe, 1981 1981 ). Previous reports revealed that many defense-related genes, including PBZ1 and RPR1 , as well as accumulation of superoxide and hydroxyl unsaturated fatty acids, are induced by PBZ in rice (Midoh and Iwata, 1996 1996 ; Sakamoto et al., 1999 1999 ; Yasuda et al., 2008 2008 ). Moreover, PBZ triggers the activation of multiple defense-related enzymes, including 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, peroxidase, phenylalanine ammonia-lyase, and lipoxygenase in rice seedlings inoculated with the blast fungi Magnaporthe grisea (Sekizawa and Watanabe, 1981 1981 ; Nikolaev and Aver'yanov, 1991 1991 ; Midoh and Iwata, 1997 1997 ; Sasaki et al., 2004 2004 ). Research carried out on Arabidopsis and tobacco revealed that, among chemicals being identified as systemic acquired resistance (SAR) activators, PBZ is the only one that can initiate the SA/NPR1 (nonexpressor of PR genes 1)-mediated defense signaling pathway (Yoshioka et al., 2001 2001 ; Nakashita et al., 2002 2002 ; Yasuda et al., 2008 2008 ). However, it is not known yet how PBZ contributes to SA accumulation. In addition, the possibility cannot be ruled out that PBZ induces other pathways as well. To address these issues, we constructed a series of synthetic promoters by combining a minimal cauliflower mosaic virus (CaMV) 35S promoter with a range of aforementioned defined defense-related cis -acting elements, either separately or in combination, with the β-glucuronidase ( GUS ) gene as a reporter. By taking an advantage of the efficient Arabidopsis stable expression system, we demonstrate that, in addition to the SA signaling pathway, both jasmonic acid (JA) and ethylene signaling pathways are also somehow involved in the PBZ-induced immune response. In addition, HR, Ca 2+ signal and kinase activity likely play roles in the upstream of signal transduction triggered by PBZ. Moreover, we illustrate that, by combining diverse PBZ inducible cis -acting elements into one synthetic promoter, promoter efficacy can be significantly improved. Materials and methods 1 Plant growth conditions 2 All Arabidopsis plants were grown in a short day condition (9 h light/15 h dark) in a growth chamber at (21±2) °C. Plasmid construction of synthetic promoters fused with GUS gene 2 Both sense and antisense strands of a minimal CaMV 35S promoter (−46, +1) were synthesized with a Pst I site at the 5' end and an Nco I site at the 3' end. After annealing, the double-stranded DNA fragment containing the Pst I/ Nco I overhangs was subcloned into the Pst I/ Nco I-digested pCAMBIA1301 vector, resulting in pCAMBIA1301-1. Promoter constructs were produced in a similar way by annealing sense and antisense oligoes, which contained an Xba I site at the 5' end and a Pst I site at the 3' end. These synthetic promoters were introduced into the Xba I and Pst I sites of the binary vector pCAMBIA1301-1 (Fig. 1a 1a ). Scheme of the synthetic promoters. Elements were inserted between the Xba I and Pst I sites in 1301-1 upstream of the −46 CaMV 35S minimal promoter. (b) GUS activity in the leaves of CaMV 35S promoter transgenic T3 lines. Min-6 and Min-11 were two independent lines of the −46 CaMV 35S minimal promoter. 35S-12 and 35S-22 were two independent lines of the full-length CaMV 35S promoter. 4-MU: 4-methylumbelliferone. Error bars indicate standard deviations ( n =6) Arabidopsis transformation 2 The resulting constructs expressing the GUS reporter gene under respective synthetic promoters were introduced into the Agrobacterium tumefaciens strain GV3101. Plant transformation was performed as previously described (Clough and Bent, 1998 1998 ) and transgenic plants were identified on Murashige and Skoog (MS) agar medium supplemented with 40 μg/ml hygromycin. T3 homozygotes were used for the GUS activity assay. PBZ, salicylic acid, La 3+ , K252a, and apigenin treatments of plants 2 Four-week-old transgenic Arabidopsis plants were treated with 0.5 mmol/L PBZ or 1 mmol/L SA by root drenching and/or leaf spraying. Transgenic lines containing 4× HSRE were pretreated with 4 μmol/L K252a, 0.4 mmol/L La 3+ , and 0.25 mmol/L apigenin prior to PBZ treatment, and then leaves were taken and frozen in liquid nitrogen for analysis. RNA extraction and qRT-PCR analysis 2 Total RNA was isolated from 100 mg leaves by TRIzol (Invitrogen, Carlsbad, CA, USA). The extracted RNA was dissolved in 20 μl diethyl pyrocarbonate (DEPC)-treated water. The SuperScript® First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA, USA) was used for reverse transcription. The generated complementary DNA (cDNA) was subsequently used as a template for the quantitative real-time polymerase chain reaction (qRT-PCR) analysis. The qRT-PCR was performed using the SYBR® Green I PCR Kit (Toyobo, Osaka, Japan) on a Bio-Rad iCycler according to the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). AtACT2 was used as a reference gene. Analysis of GUS activity 2 Measurement of GUS activity was conducted according to Jefferson et al. ( 1987 1987 ). In brief, 0.1 g of frozen leaf samples were homogenized in 0.2 ml of pre-cooled lysis buffer (0.1 mol/L sodium phosphate pH 7.5, 10% glycerol, and 1 mmol/L ethylenediaminetetraacetic acid (EDTA)) and then centrifuged at 4000 g for 10 min at 4 °C. The supernatant was transferred to a new tube and used for measuring GUS activity. GUS activity was calculated as described by Jefferson et al. ( 1987 1987 ). Microarray analysis 2 Arabidopsis plants overexpressing NPR1 were grown under a short day condition (9 h light/15 h dark) for about four weeks. Total RNAs of PBZ-pretreated plants and control were extracted by the RNeasy Mini Kit (Qiagen) and repeated isopropanol precipitations. The extracted total RNA was subject to cDNA and complementary RNA (cRNA) syntheses with the Affymetrix GeneChip One-Cycle Target Labeling Kit. Microarray was performed in Shanghai Biotechnology Corporation (China) according to the standard Affymetrix manual. Differential gene expression was calculated by their relative expression of a PBZ-treated sample as compared with that of a mock sample. Results 1 Minimal 35S CaMV promoter does not respond to PBZ treatment 2 In this study, a variant pCAMBIA1301 binary expression vector, harboring GUS gene driven by the minimal CaMV 35S promoter, was used as a basic cloning vector, and a series of hormone-inducible constructs was constructed by placing defined elements to the 5' of the minimal CaMV 35S promoter, as shown in Fig. 1a 1a . To clarify whether the minimal 35S promoter responds to PBZ treatment, the cloning vector harboring the minimal 35S promoter as well as the original pCAMBIA1301 harboring the full-length 35S promoter was introduced into Arabidopsis plants separately. By examining the GUS activity of T3 transgenic lines after 0.5 mmol/L PBZ application, we showed that neither the minimal CaMV 35S promoter nor the full-length CaMV 35S promoter responded to PBZ treatment (Fig. 1b 1b ). Moreover, the transgenic plants harboring the 35S minimal promoter showed only a trace level of background GUS activity. These findings indicate that the cloning vector, named as pCAMBIA1301-1, is an ideal basic vector for generating PBZ-inducible constructs for the assessment of the responsiveness of diverse defined cis -elements to PBZ treatment. SARE, an salicylic acid responsive element, responds to PBZ treatment 2 Previous studies demonstrated that PBZ induces SAR through the enhancement of SA accumulation (Yoshioka et al., 2001 2001 ; Yu et al., 2010b 2010b ). To validate our PBZ-inducible system, we analyzed an SA responsive element, SARE, isolated from the upstream region of tobacco PR2 gene in SA and PBZ treatments (Fig. 2a 2a ). Tandem repeats of four SAREs (4× SARE) were used to generate a synthetic promoter in order to improve its regulatory efficiency (Rushton et al., 2002 2002 ). After introducing into Arabidopsis plants, the tandem repeats of four SAREs showed significant responses to both PBZ and SA (Fig. 2b 2b ). This indicates that our way of constructing synthetic promoters is valid and our system of assessing the responsiveness of these promoters is effective and efficient. Sequence of the SARE element. Core sequences are shown in boldface. (b) GUS activity in the leaves of 4× SARE transgenic T3 lines. 4-MU: 4-methylumbelliferone. Error bars indicate standard deviations ( n =6) JA and ethylene signaling pathways may be involved in the immune response triggered by PBZ 2 To explore whether the JA and ethylene signaling pathways also play a role in the immune responses induced by PBZ, 4× JERE element (AGACCGCC) and 4× GCC box (AGCCGCC), responsive to jasmonate and ethylene respectively, were employed to construct two inducible constructs and their transgenic lines were then generated. Leaves of T3 transgenic lines were subject to GUS activity assay 0, 1, and 3 d after soil drenching treatment with 0.5 mmol/L PBZ. Both constructs were found to significantly respond to PBZ. The GUS activity in transgenic plants harboring 4× JERE increased continuously and reached a 9-fold increment by Day 3 after treatment (Fig. 3a 3a ). The GUS activity in the transgenic plants harboring 4× GCC increased by 4-fold in a similar dynamic pattern 3 d after treatment (Fig. 3b 3b ). The highly inducible increments are partly attributable to their relatively low background expressions in the absence of PBZ. The fact that both the JA and ethylene signaling pathways respond to PBZ treatment suggests that, apart from SA signaling, both the JA signaling and ethylene signaling are also possibly involved in the immune response triggered by PBZ. (a) GUS activity in the leaves of 4× JERE transgenic T3 lines. (b) GUS activity in the leaves of 4× GCC transgenic T3 lines. 4-MU: 4-methylumbelliferone. Error bars indicate standard deviations ( n =6) To further explore the involvement of JA and ethylene signaling pathways in the PBZ-induced system, we compared our microarray data on PBZ treatment with those published data on JA and ethylene treatment (Nemhauser et al., 2006 2006 ). As listed in Tables S1 S1 and S2 S2 , 94 genes were up-regulated by both methyl jasmonate (MeJA) and PBZ, and 27 genes were induced by both ACC and PBZ (Fig. 4a 4a ). In addition, qRT-PCR analysis further revealed that PBZ can induce the immune response by stimulating the expression of PDF1.2 (Fig. 4b 4b ), a gene that is specifically induced by JA and ethylene in Col-0 (Penninckx et al., 1996 1996 ). (a) Venn diagrams showing the numbers of genes induced by PBZ, MeJA, and ACC, respectively. Details about the shared up-regulated genes were listed in Tables S1 S1 and S2 S2 . (b) The induction of PDF1.2 expression in Col-0 upon PBZ treatment. The transcript levels were quantified by qRT-PCR with ACTIN2 ( ACT2 ) as an internal control. Error bars indicate standard deviations ( n =4) Hypersensitive response is involved in the immune response triggered by PBZ 2 HR is an early event that contributes to plant resistance by restricting invading pathogens at the infection site, which may play an important role in stimulating a systemic signal for SAR (Heath, 2000 2000 ). We therefore hypothesized that PBZ might stimulate the immune response via HR. To verify this, 4× HSRE and 4× GST1, which are associated with HR, were employed to construct inducible constructs (Fig. 5a 5a ). We firstly analyzed their responsive patterns 0, 1, and 3 d after soil-drenched treatment with 0.5 mmol/L PBZ. The results demonstrated that both of the elements responded to PBZ induction (Figs. 5b and 5c 5b and 5c ). We then became interested in whether they were also responsive to SA. Intriguingly, while GST1 element still showed a response to SA, the HSRE element did not show a significant response (Figs. 5b and 5c 5b and 5c ). This indicates that HSRE is likely responsive to the signal(s) in the PBZ-triggered signal pathway(s) upstream of SA accumulation. This finding suggests that the HSRE element can be exploited as an ideal responsive marker to further analyze the early signal transduction triggered by PBZ. (a) Sequences of the GST1 and HSRE elements. (b) GUS activity in the leaves of 4× GST1 transgenic T3 lines. (c) GUS activity in the leaves of 4× HSRE transgenic T3 lines. 4-MU: 4-methylumbelliferone. Error bars indicate standard deviations ( n =6) Both Ca 2+ influx and kinase activity are involved in the early signal transduction triggered by PBZ 2 The Ca 2+ signal and kinase activity as well as reactive oxygen intermediates (ROIs) play important roles in plant HR (Alvarez et al., 1998 1998 ; Torres et al., 2006 2006 ; Ma and Berkowitz, 2007 2007 ). To determine the relationship between Ca 2+ influx and kinase activity and response of the HSRE element to PBZ induction, we performed analyses by using a calcium signaling blocker and kinase activity inhibitors. Pre-treatment of Arabidopsis leaves with the calcium channel blocker lanthanum III acetylacetonate hydrate (La 3+ ) totally suppressed the response of the HSRE to PBZ induction. In addition, treatments with a broad-range protein kinase antagonist, K252a, and a specific mitogen-activated protein kinase (MAPK) inhibitor, apigenin, also abolished the responsiveness of the HSRE elements to PBZ induction (Fig. 6 6 ). These results collectively suggest that both Ca 2+ influx and kinase activity, especially MAPK activity, were involved in the early signal transduction triggered by PBZ. La 3+ , K252a, and apigenin were used to pretreat Arabidopsis leaves before 0.5 mmol/L PBZ or water treatment. 4-MU: 4-methylumbelliferone. Error bars indicate standard deviations ( n =6) WRKY protein-binding box is capable of responding to PBZ induction in Arabidopsis 2 Extensive studies have shown that WRKY proteins have regulatory functions in plant immune response (Rushton et al., 2010 2010 ). Our previous study showed that two of their three binding sites, W-box (TTGAC), in the promoter of AtNPR1 are essential for the full responsiveness of AtNPR1 to PBZ treatment (Yu et al., 2010a 2010a ). Moreover, microarray analysis showed that about 15 WRKY family transcription factors were induced in Col-0 after PBZ treatment (Yu et al., 2010a 2010a ). To study the PBZ-triggered signal transduction in SAR, we further examined the PBZ responsive gene expression in PBZ-treated NPR1 overexpressing transgenic plants in a new microarray experiment. More WRKY family transcription factors (39 members) were found to be up-regulated with at least a two-fold increase of their transcripts 8 d after PBZ treatment (Table 1 1 ). This finding strongly suggests that WRKY proteins are extensively involved in PBZ-triggered signaling pathways. To further elucidate the importance of WRKY proteins in the responsiveness to PBZ induction, we generated an inducible construct with tandem repeats of two W-boxes (2× W-box) and subsequently analyzed its transgenic lines (Fig. 7a 7a ). Although a slight background in GUS staining was detected in their leaves (Fig. 7b 7b ), a significant responsiveness in both GUS gene transcription and GUS activity to PBZ treatment was observed in the stable transgenic lines (Figs. 7c and 7d 7c and 7d ). These observations indicate that W-box(es) alone is capable of responding to PBZ induction. A list of PBZ-induced WRKY genes that were identified in the microarray experiment. RNA samples from NPR1 -overexpressing Arabidopsis plants treated with PBZ for 8 d were compared with RNA samples from NPR1 -overexpressing Arabidopsis plants treated with water for 8 d. Note that the fold change values are log base 2 transformed. The twofold difference in the expression level between the PBZ-8 d and H 2 O-8 d samples was set as the threshold for considering a gene to be PBZ-inducible Gene ID Annotation Fold change (log 2 ) PBZ-8 d vs. H 2 O-8 d AT5G56270 WRKY2 1.0408 AT1G62300 WRKY6 4.4347 AT5G46350 WRKY8 3.2967 AT4G39410 WRKY13 1.4983 AT1G30650 WRKY14 1.3503 AT2G23320 WRKY15 2.4708 AT2G24570 WRKY17 1.9240 AT4G31800 WRKY18 3.4418 AT4G01250 WRKY22 1.5641 AT2G30250 WRKY25 2.7352 AT5G07100 WRKY26 5.0472 AT5G52830 WRKY27 2.4086 AT5G24110 WRKY30 5.9014 AT4G22070 WRKY31 3.3596 AT2G38470 WRKY33 1.1953 AT1G69810 WRKY36 3.5674 AT5G22570 WRKY38 4.0561 AT1G80840 WRKY40 1.7052 AT4G11070 WRKY41 6.1246 AT3G01970 WRKY45 2.1999 AT2G46400 WRKY46 3.8088 AT4G01720 WRKY47 4.1209 AT5G49520 WRKY48 3.8653 AT5G26170 WRKY50 4.7801 AT5G64810 WRKY51 5.0924 AT4G23810 WRKY53 4.6245 AT2G40740 WRKY55 5.0885 AT1G69310 WRKY57 1.3964 AT3G01080 WRKY58 1.4476 AT2G21900 WRKY59 2.4599 AT2G25000 WRKY60 3.9039 AT1G18860 WRKY61 3.1380 AT5G01900 WRKY62 4.0745 AT1G66600 WRKY63 2.6046 AT1G29280 WRKY65 4.1438 AT1G80590 WRKY66 2.3205 AT1G29860 WRKY71 2.1442 AT5G15130 WRKY72 2.4972 AT5G13080 WRKY75 9.1676 (a) Sequence of the 2× W-box. Core sequences are shown in boldface. (b, c) GUS gene expression in the leaves of 2× W-box transgenic T3 lines 4 d after treatment with PBZ or water. GUS staining was used to detect the expression. The transcript levels were quantified by qRT-PCR with ACTIN2 ( ACT2 ) as an internal control. Error bars indicate standard deviations ( n =4). (d) GUS activity in the leaves of 2× W-box transgenic T3 lines. 4-MU: 4-methylumbelliferone. Error bars indicate standard deviations ( n =6) A synthetic promoter containing combinations of different cis -elements for PBZ-inducible system 2 To develop an effective strategy for creating inducible promoters with high responsiveness to PBZ induction, we attempted to combine distinctive elements, which have been previously shown to significantly respond to PBZ induction, including GST1, W-box, and HSRE. An inducible construct was generated with 2× GST1, 6× W-box, and 2× HSRE (named as GWH) (Fig. 8a 8a ) and its transgenic plants were then analyzed. A significantly higher GUS activity was detected in the leaves of GWH transgenic lines than in those of transgenic plants carrying single W-box or HSRE cis -elements after 4 d PBZ treatment (Figs. 8b and 8c 8b and 8c ). This result suggests that a more responsive synthetic promoter can be constructed by selectively combining distinctive cis -elements. (a) Sequence of the synthetic promoter. The elements are shown in gray shadow. (b) GUS gene expression in the leaves of GWH transgenic T3 lines 4 d after treatment with PBZ or water. GUS staining was used to detect the expression. (c) GUS activity in the leaves of GWH, 2× W-box, and 4× HSRE transgenic T3 lines. 4-MU: 4-methylumbelliferone. Error bars indicate standard deviations ( n =6) Discussion 1 In this study, by using the efficient stable expression system of Arabidopsis , we clearly demonstrated that a list of individual pathogen-responsive cis -acting elements is capable of responding to PBZ induction, suggesting that the respective transcription factors are sufficient to confer a responsiveness to PBZ. These results thus revealed important regulatory nodes on the signal transduction pathways triggered by PBZ, which may in turn help to further uncover the underlying mechanism of PBZ-induced immune response. First, we provided direct evidence that both JA and ethylene signaling pathways are likely involved in the PBZ-induced SAR. An early study showed that, while PBZ induced a resistance in rice at 4-leaf stage to M. grisea , no SA accumulation was observed during this process. Moreover, exogenously applying SA had no detectable effect in terms of the M. grisea resistance (Iwai et al., 2007 2007 ). These data indicate that the immune response triggered by PBZ in rice is not likely related to the accumulation of SA and some other mechanisms may be responsible for the process. This is consistent with our observations that JERE, GCC, and HSRE all respond to PBZ, indicating that, besides SA, JA and ethylene may also play important roles in PBZ-induced systemic immune responses in plants. These data collectively suggest that multiple hormone signaling pathways may act together in the immune response triggered by PBZ and a complex mechanism is responsible for the regulation of cross-talks among these distinct signaling pathways. Increasing evidence also supports that both Ca 2+ elevation and kinase activity play important roles in response to both pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI)-associated HR (Hammond-Kosack and Parker, 2003 2003 ; Ma and Berkowitz, 2007 2007 ). In addition, MAPK signaling cascades downstream of Ca 2+ elevation activate the expression of genes related to pathogen defense, including some HR-related ones (Asai et al., 2002 2002 ; Zhang et al., 2006 2006 ). Although it remains unclear how MAPK cascades are activated by cytosolic Ca 2+ elevation, the initial phosphorylation in a Ca 2+ -dependent manner indicates a role of calcium-dependent protein kinases (CDPKs) in such a process. With HSRE, a cis -acting element specifically responsive to HR, we further explored the relationship between HR and PBZ-triggered immune response. We observed that pretreatment with a calcium channel blocker La 3+ , a broad-range protein kinase antagonist K252a or a MAPK-specific inhibitor apigenin abolished the effect of PBZ induction. Interestingly, in other work, we also observed that many CDPK and MAPK family members were up-regulated after PBZ treatment. Moreover, up-regulations of some earliest activated genes following pathogen-associated molecular pattern PAMP perception or R gene interactions, such as EDS1 , PAD4 and SAG101 , have also been observed (data not shown). Based on these analyses, we hypothesize that the immune response activated by PBZ is similar to that induced by PAMP perception or R gene interactions, in contrast to that triggered by other widely used plant immune response activators (i.e. benzothiadiazole (BTH), 2,6-dichloroisonicotinic acid (INA), and β-aminobutyric acid (BABA)) (Serrano and Guzman, 2004 2004 ; Ton and Mauch-Mani, 2004 2004 ). Given the above findings, the PBZ-inducible expression system may be exploited to further explore the cross-talks among SA, JA, and ethylene signal transduction pathways, and to reveal the precise roles of Ca 2+ elevation and kinase activity involved in the microbe-plant interaction and the plant systemic immune response, which would provide profound insights into the mechanisms of the plant immune response. Pathogens of plants are a significant and growing threat to crop production worldwide. Genetic engineering has been developed as a potential approach to tackle this formidable problem (Hammond-Kosack and Parker, 2003 2003 ). However, how to properly express diversified resistance genes has been and still is a challenging problem (Gurr and Rushton, 2005 2005 ). Recent progresses in the elucidation of mechanisms of pathogen-triggered immune response and chemical-triggered immune response have made it possible to construct a novel type of transgene expression system, i.e. an immune response activator inducible transgene expression system, which can provide dual protection mechanisms, one by driving target gene expression and the other via triggering the endogenous immune system (Roslan et al., 2001 2001 ; Rushton et al., 2002 2002 ; Camargo et al., 2007 2007 ). As a cheap, environmentally friendly, and widely used SAR activator for more than three decades, PBZ is obviously an ideal activator in this regard (Gozzo, 2003 2003 ; Iwata et al., 2004 2004 ). In this study, we attempted to improve promoter efficacy by combining HSRE, W-box(es), and GST1 into one synthetic promoter. A significant increase in responsiveness to PBZ was indeed observed by combining these three cis -acting elements together. 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Compliance with ethics guidelines Zheng ZHU, Jiong GAO, Jin-xiao YANG, Xiao-yan WANG, Guo-dong REN, Yu-long DING, and Ben-ke KUAI declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1400203 Project supported by the National Key Project of Transgenic Variety Development of China (Nos. 2011ZX08009-004 and 2013ZX08009-004), Shanghai Key Laboratory of Bio-Energy Crops, and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), China 2014-07-19 2015-03-18 2014-11-06 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2015 2015 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 16