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. 2010 Oct 8;154(4):1805–1818. doi: 10.1104/pp.110.165563

The Arabidopsis PR-1 Promoter Contains Multiple Integration Sites for the Coactivator NPR1 and the Repressor SNI1[W]

Sebastian Pape 1, Corinna Thurow 1, Christiane Gatz 1,*
PMCID: PMC2996008  PMID: 20935179

Abstract

Systemic acquired resistance is a broad-spectrum plant immune response involving massive transcriptional reprogramming. The Arabidopsis (Arabidopsis thaliana) PATHOGENESIS-RELATED-1 (PR-1) gene has been used in numerous studies to elucidate transcriptional control mechanisms regulating systemic acquired resistance. WRKY transcription factors and basic leucine zipper proteins of the TGA family regulate the PR-1 promoter by binding to specific cis-elements. In addition, the promoter is under the control of two proteins that do not directly contact the DNA: the positive regulator NONEXPRESSOR OF PR GENES1 (NPR1), which physically interacts with TGA factors, and the repressor SUPPRESSOR OF NPR1, INDUCIBLE1 (SNI1). In this study, we analyzed the importance of the TGA-binding sites LS5 and LS7 and the WKRY box LS4 for regulation by NPR1 and SNI1. In the absence of LS5 and LS7, NPR1 activates the PR-1 promoter through a mechanism that requires LS4. Since transcriptional activation of WRKY genes is under the control of NPR1 and since LS4 is not sufficient for the activation of a truncated PR-1 promoter by the effector protein NPR1-VP16 in transient assays, it is concluded that the LS4-dependent activation of the PR-1 promoter is indirect. In the case of NPR1 acting directly through TGA factors at its target promoters, two TGA-binding sites are necessary but not sufficient for NPR1 function in transgenic plants and in the NPR-VP16-based trans-activation assay in protoplasts. SNI1 exerts its negative effect in the noninduced state by targeting unknown proteins associated with sequences between bp −816 and −573. Under induced conditions, SNI1 negatively regulates the function of WRKY transcription factors binding to WKRY boxes between bp −550 and −510.

The plant signaling hormone salicylic acid (SA) is synthesized upon pathogen infection and serves to elicit plant defense reactions (Malamy et al., 1990; Vlot et al., 2009). Increased SA levels in systemic leaves lead to the onset of systemic acquired resistance (SAR), an inducible defense program that renders the plant immune against a broad spectrum of pathogens (Ross, 1961; Durrant and Dong, 2004). Treatment of plants with SA or its analogs 2,6-dichloro-isonicotinic acid (INA) or benzothiadiazole S-methyl ester is sufficient for SAR establishment.

The signaling cascade leading to the activation of SAR has been elaborated in Arabidopsis (Arabidopsis thaliana) using SA-inducible PATHOGENESIS-RELATED (PR) genes (Cao et al., 1994; Lebel et al., 1998; Zhang et al., 2003; Rochon et al., 2006; Kesarwani et al., 2007). After performing a mutant screen designed to isolate plants that cannot activate a PR-2:GUS reporter construct in the presence of SA or INA, Cao and colleagues (Cao et al., 1994, 1997; Wang et al., 2006) identified the protein NONEXPRESSOR OF PR GENES1 (NPR1), which is essential for the regulation of 2,248 genes after benzothiadiazole S-methyl ester treatment and for SAR establishment. In the absence of SA, the majority of the NPR1 proteins reside in the cytosol as an oligomer (Mou et al., 2003). Upon pathogen attack or INA treatment, NPR1 becomes reduced and the oligomer dissociates. NPR1 monomers are translocated into the nucleus, where they interact with TGA factors (Zhang et al., 1999). TGA factors constitute a subfamily of basic Leu zipper transcription factors that recognize TGACG motifs found in NPR1-dependent (Zhang et al., 1999; Thibaud-Nissen et al., 2006) but also in NPR1-independent (Fode et al., 2008; Blanco et al., 2009) SA-inducible promoters. Mutation of the three redundant members of clade II TGA factors, TGA2, TGA5, and TGA6, leads to increased basal PR-1 expression levels that cannot be induced any longer (Zhang et al., 2003).

The negative regulatory protein SUPPRESSOR OF NPR1, INDUCIBLE1 (SNI1) was identified in a genetic screen for mutants that induce the PR-2:GUS reporter construct in the npr1 mutant background (Li et al., 1999). In sni1 npr1 plants, INA induction of the PR-1 promoter is reestablished, unraveling an NPR1-independent pathway that can activate the promoter in the absence of SNI. This observation implies that NPR1 is required to inactivate SNI1. The molecular mechanism that drives the NPR1/SNI1-independent induction pathway is not yet understood, but it requires the recombination/repair protein RAD51D (Durrant et al., 2007). In the presence of NPR1, SNI1 dampens both basal and induced expression of PR genes. Chromatin immunoprecipitation experiments indicate that histone modifications are involved in SNI1-mediated repression (Mosher et al., 2006).

Deletion and linker-scanning (LS) mutations have identified functionally important cis-regulatory sequences between positions −698 and −621 of the PR-1 promoter (Lebel et al., 1998). Interestingly, two 10-bp LS substitutions that affect the TGA-binding sites LS5 and LS7 (Fig. 1A) had different effects on promoter activity: substitution of LS7 by a linker sequence led to a complete loss of promoter activity, whereas mutation of LS5 led to enhanced basal expression levels, which could be induced to wild-type-like levels by INA. Thus, it appeared that the two TGA-binding sites have different functions. Based on the finding that TGA factors physically interact with the positive regulator NPR1 (Zhang et al., 1999; Fan and Dong, 2002), it was suggested that LS7 facilitates NPR1-mediated activation of the promoter. As TGA factors repress the promoter in the uninduced state (Zhang et al., 2003; Boyle et al., 2009), it was proposed that their binding to LS5 mediates the repression of basal levels (Kesarwani et al., 2007). Mutation of LS4, which encodes a potential target site for WRKY transcription factors, enhanced promoter activity (Lebel et al., 1998). Therefore, this site was postulated to be involved in the repressive effect of SNI1 (Kesarwani et al., 2007; for a schematic representation of the current model, see Supplemental Fig. S1). LS10, a potential binding site for DOF (DNA binding with one finger) transcription factors, is, like LS7, of crucial importance for the activation of the promoter.

Figure 1.

Figure 1.

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Influence of the cis-elements LS4, LS5, LS7, and LS10 and the trans-factors NPR1 and SNI1 on PR-11294 promoter activity. A, Mutations introduced into the PR-11294 promoter context to study the functional significance of LS4, LS5, LS7, and LS10. The W box of LS4 and conserved positions within the ideal TGA-binding site TGAC/GTCA are depicted in boldface letters; each TGAC half-site is marked by an arrow provided that 3 or 4 bp are in consensus with this motif. Vertical lines denote the centers of the palindromes. The LS mutations as originally introduced by Lebel et al. (1998) are shown in gray boxes and lowercase letters. B, Influence of LS4, LS5, LS7, and LS10 on PR-11294 promoter activity. C, Expression of PR-11294 in npr1, sni1, and sni1 npr1. For B and C, LUC activities of 2-week-old plants grown axenically on MS plates without (gray columns) and with (black columns) 30 μm INA were measured. The indicated LS elements were replaced by linker sequences within the PR-11294 promoter context. The genotypes of the analyzed plants are indicated. Values represent means of the activities of the indicated number (#) of independent lines. LUC activities are expressed as relative light units (RLU) per μg of total protein. Error bars represent the sd of two independent experiments. wt, Wild type.

Since NPR1, SNI1, and TGA transcription factors are key regulators of SAR, the elucidation of their mode of action is of major interest. Recent studies have yielded further results (Mosher et al., 2006; Rochon et al., 2006; Zhang et al., 2006; Durrant et al., 2007; Tada et al., 2008; Boyle et al., 2009; Spoel et al., 2009) that need to be integrated into the current model of their interplay on the PR-1 promoter (Kesarwani et al., 2007). Since this model is based on two different studies, expression analysis of the endogenous promoter in different genetic backgrounds (Kesarwani et al., 2007) and deletion and LS analysis of different PR-1 promoter variants in transgenic wild-type plants (Lebel et al., 1998), we challenged the postulated significance of LS4, LS5, and LS7 for the functions of NPR1, SNI1, and TGA factors in one experimental setup: we generated numerous PR-1 promoter mutants and compared their expression levels in the npr1, sni1, and sni1 npr1 genetic backgrounds. Unexpectedly, we found that simultaneous mutation of LS5 and LS7 did not disrupt NPR1-dependent expression of the PR-1 promoter. This LS5/LS7-independent promoter activity is repressed by LS5 and appears to be driven by LS4-bound WRKY transcription factors whose expression is regulated by SA and NPR1. In the case of NPR1 acting through TGA-binding sites at target promoters, two TGA-binding motifs as well as not yet identified sequences between these motifs are required for NPR1 function. Likewise, efficient repression of the promoter by TGA factors is mediated by an intact LS5/LS7 motif. In contrast to previous assumptions (Kesarwani et al., 2007), SNI1 acts independently from LS4. In the noninduced state, it targets yet unknown sequences between −816 and −573. Further WKRY boxes (W boxes) outside this region are required for hyperinduction in sni1. An updated model of the transcriptional control mechanisms operating on the PR-1 promoter is presented.

RESULTS

The 1,294-bp-Long PR-1 Promoter Fragment Is Regulated Like the Endogenous Gene in npr1, sni1, and sni1 npr1

As treatment of plants with the SA analog INA is sufficient for SAR establishment and PR-1 induction, PR-1 transcription was monitored either 3 d after spraying Murashige and Skoog (MS) plate-grown plantlets with 350 μm INA (Lebel et al., 1998) or after growing seedlings for 14 d on 30 μm INA (Kesarwani et al., 2007). Since the latter treatment had been successfully deployed for the isolation of the two SAR-regulating proteins, NPR1 (Cao et al., 1994) and SNI1 (Li et al., 1999), we decided to base our analysis on this induction procedure. As displayed in Figure 1B, this treatment basically recapitulated the results reported before (Lebel et al., 1998): induction of a luciferase (LUC) reporter gene construct under the control of a 1,294-bp-long PR-1 promoter (PR-11294) was enhanced when substituting LS4 by a linker sequence; replacement of only LS5 did not interfere with INA induction, and substitution of either LS7 or LS10 compromised promoter activity. Quantitative differences from the previous report (Lebel et al., 1998), like the less pronounced enhanced basal expression levels of PR-11294LS4mut and PR-11294LS5mut, might be the result of the different reporter gene used in this study. Because of the high stability of the GUS enzyme used by Lebel et al. (1998), background levels might have accumulated to a higher degree, thus exaggerating subtle effects on basal expression levels.

When testing the PR-11294:LUC construct in npr1, sni1, and sni1 npr1 plants, we observed that it displayed the relevant characteristics of the endogenous PR-1 promoter (Fig. 1C; Li et al., 1999). Induction was severely compromised in the npr1 mutant, whereas basal and induced levels were enhanced in the sni1 mutant, demonstrating that the sequences necessary for the regulation of PR-1 by NPR1 and SNI1 are located within 1,294 bp upstream of the transcriptional start site. The described NPR1/SNI1-independent activation mode was also functional (Fig. 1C, lane 4), although with a higher efficiency than described before for the endogenous gene: LUC activities reached the same induced values in wild-type plants as in sni1 npr1 plants, whereas the endogenous PR-1 RNA levels were less abundant in sni1 npr1 (Li et al., 1999; Supplemental Fig. S2). It might well be that the NPR1/SNI1-independent activation mode is negatively affected by sequences outside the 1,294-bp promoter fragment used in this study.

NPR1-Dependent INA-Induced Activation of the PR-1 Promoter Occurs Even in the Absence of TGA-Binding Sites

Since LS5 and LS7 are both recognized by TGA transcription factors, it had remained elusive why only mutation of LS7 interfered with INA-inducible expression. Unexpectedly, simultaneous mutation of LS5 and LS7 within PR-11294 yielded a promoter that responded to INA almost like the wild-type promoter (Fig. 2, lane 2), although expression levels were somehow higher both in the noninduced state and the induced state. Thus, mutating LS5 suppresses the negative effect of the LS7 mutation.

Figure 2.

Figure 2.

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Influence of the simultaneous mutation of the TGA-binding motifs LS5 and LS7 on PR-11294 promoter activity. LUC activities of 2-week-old plants grown axenically on MS plates without (gray columns) and with (black columns) 30 μm INA were measured. The indicated LS elements were replaced by linker sequences within the PR-11294 promoter context (Fig. 1A). The genotypes of the analyzed plants are indicated. Values are means of the activities of the indicated number (#) of independent lines. LUC activities are expressed as relative light units (RLU) per μg of total protein. Error bars represent the sd of two independent experiments. wt, Wild type.

In order to address the question of whether removal of the TGA-binding sites affects the influence of the TGA-interacting protein NPR1 and its suppressor SNI1, we analyzed the expression of the PR-11294LS5,7mut promoter in npr1 and sni1 mutant plants. Although the putative NPR1 recruitment site LS7 was mutated, NPR1 was still required for the activation (Fig. 2, lane 3). Thus, an NPR1-dependent activation mode that does not depend on the TGA-binding sites must exist.

In the sni1 mutant, basal expression levels of the PR-11294LS5,7mut promoter were higher than those of the wild-type promoter (Fig. 2, compare lanes 4 and 5) and were comparable to those of the induced wild-type promoter in the wild-type background (Fig. 2, compare lanes 1 and 4). This indicates that basal levels of the PR-1 promoter are suppressed by two independent mechanisms: by TGA factors at LS5/LS7 as well as by SNI1. In the presence of INA, the sni1 allele had no effect on expression of PR-11294LS5,7mut (Fig. 2, compare lanes 2 and 4).

LS substitutions in only one of the two TGA-binding sites, as in PR-11294LS5mut and PR-11294LS7mut, yielded high basal expression levels in sni1, indicating that one binding site is not sufficient for efficient repression (Fig. 2, lanes 6 and 7). In contrast, hyperinduction of PR-11294 in sni1 was severely compromised when mutating LS7 and to a lesser degree when mutating LS5 (Fig. 2, compare lane 5 with lanes 6 and 7). It is concluded that LS5 and LS7 are both needed for efficient hyperactivation in sni1.

The LS5/LS7-Independent Pathway Requires the W box LS4

The NPR1 dependency of the PR-11294LS5,7mut promoter (Fig. 2, lane 3) implied that it might be indirectly regulated through WRKY genes that are directly up-regulated by NPR1 (Wang et al., 2006). Although LS4 (a W box located upstream of LS5) dampens promoter activity (Lebel et al., 1998; Fig. 1B, lane 2), we considered this motif as a candidate sequence to be responsible for the activation of PR-1 lacking the LS5/LS7 motif. Indeed, induction of a promoter carrying linker substitutions of LS4, LS5, and LS7 (PR-11294LS4,5,7mut) was severely compromised (Fig. 3, lane 3). It is concluded that LS4 is required for the activation of the promoter in the absence of LS5 and LS7. In the presence of these two motifs, LS4 is dispensable for induction (Fig. 3, lane 4). The promoter constructs PR-11294LS4,5mut and PR-11294LS4,7mut did not show any activities, indicating that neither LS5 nor LS7 is sufficient for activation of the promoter in the absence of LS4 (Fig. 3, lanes 5 and 6).

Figure 3.

Figure 3.

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Influence of LS4 on PR-11294 promoter activity. LUC activities of 18-d-old plants grown axenically on MS plates without (gray columns) and with (black columns) 30 μm INA were measured. The indicated LS elements were exchanged by linker sequences within the PR-11294 promoter context. The genotypes of the analyzed plants are indicated. Values are means of the activities of the indicated number (#) of independent lines. LUC activities are expressed as relative light units (RLU) per μg of total protein. Error bars represent the sd of two independent experiments. wt, Wild type.

The LS4-Independent Pathway Requires Two TGA-Binding Sites

The results displayed in Figure 3 suggest that two alternative activation pathways exist: one that is driven by LS4, and a second that requires LS5 and LS7. Since chromatin immunoprecipitation analysis has demonstrated an association of NPR1 with the PR-1 promoter (Rochon et al., 2006) and since NPR1 directly interacts with TGA factors (Zhang et al., 1999), we assume that the second pathway works through the direct association of NPR1 with TGA factors bound to the LS5/LS7 motif. As we were unable to obtain a suitable anti-NPR1 antiserum for chromatin immunoprecipitation assays, we developed a transient assay that would support our idea that activation through LS4 is not mediated through a mechanism that recruits NPR1 whereas the combined action of LS5 and LS7 would be able to do so. To this end, truncated PR-1 promoter derivatives were constructed that contain the sequences from −816 to −573, including LS4, LS5, and LS7 upstream of the PR-1 core promoter (−68 to +1; Fig. 4A). When fused to the LUC gene, this PR-1816–573 promoter yields low background activity when transfected into SA-treated npr1 protoplasts (Fig. 4B). Coexpression of NPR1 or NPR1 fused to the activation domain of herpes simplex virus protein 16 (VP16) led to a 5- and 20-fold enhancement of promoter activity, respectively (Supplemental Fig. S3). Because of the higher activation capacity of NPR1-VP16, subsequent studies were performed with this construct. This activation was enhanced by SA (Supplemental Fig. S4) and was abolished after mutation of LS5 and LS7 (Fig. 4B, lane 2). Apparently, the LS4-dependent pathway is not functional in this system. Probable explanations are that the WRKY factors driving this activation are not induced in time upon expression of NPR1-VP16 or, alternatively, that this activation mode requires additional sequences outside bp −813 to −573. Only residual activation was observed for PR-1816–573LS5mut (Fig. 4B, lane 3) and to a lesser extent for PR-1816–573LS7mut (Fig. 4B, lane 4). Although some activation through LS7 is detected, this experiment demonstrates that NPR1 can only efficiently activate the PR-1816–573 promoter if both TGA-binding sites are present. Based on the previous data that NPR1 interacts with TGA factors (Zhang et al., 1999), we conclude that the activation observed in the transient assays is due to the direct association of NPR1 with TGA factors bound to LS5 and LS7 and that such a mechanism cannot operate on LS4.

Figure 4.

Figure 4.

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Importance of the cis-elements LS5 and LS7 for NPR1-VP16-mediated activation of the PR-1816–573 promoter in npr1 protoplasts. A, Schematic presentation of the PR-1816–573 promoter. Sequences between bp −573 and −68 and upstream of bp −816 were removed. The black box depicts the relative position of the LS5/LS7 motif. Mutations in LS5 and LS7 are as indicated in Figure 1. B, PR-1816–573 activities in mesophyll protoplasts isolated from npr1 plants. PR-1816–573 promoter derivatives with mutations in LS5, LS7, or both elements were fused to the firefly LUC gene. Expression was analyzed in the presence of effector plasmids encoding NPR1 fused to the VP16 activation domain (black bars) under the control of the 35SC4PPDK promoter (Sheen et al., 1995). After transfection, protoplasts were incubated for 14 h in the presence of 5 μm SA. Relative LUC activities are expressed in arbitrary luminescence units, normalized to Renilla LUC activity. The PR-1816–573 promoter activity in the absence of effectors was set to 1. Values are means of three replicates ± sd.

Two TGA-Binding Sites Are Not Sufficient for NPR1-Mediated Activation

Two TGA-binding sites are not only found in NPR1-dependent, but also in NPR1-independent, SA-inducible promoters (Fode et al., 2008; Blanco et al., 2009). These promoters are inactive in the tga256 mutant, indicating that the same clade of TGA factors is required for NPR1-dependent and NPR1-independent promoters. When fusing the promoter regions of two NPR1-independent (GSTF8, IEGT38) and two NPR1-dependent (WRKY38, WRKY70) genes upstream of the PR-1 core promoter (−68 to +1), we observed trans-activation by NPR1-VP16 only in the case of WRKY38 and WRKY70 (Fig. 5B). In contrast, all four promoters were activated by VP16-TGA2 in tga256 mutant protoplasts (Fig. 5C). This analysis shows that only promoters that are under the control of NPR1 respond to NPR1-VP16 in our transient system. Based on our hypothesis that the activation through NPR1-VP16 is due to recruitment of the protein to TGA factors at the promoter region, we conclude that two TGA-binding sites are not sufficient for this type of activation.

Figure 5.

Figure 5.

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Trans-activation of SA-induced promoters by NPR1-VP16 in npr1 protoplasts. A, Schematic presentation of the two TGA-binding sites within the four promoters (GSTF8, IEGT38, WRKY38, WRKY70) and their relative positions within their promoter contexts. Conserved positions with respect to the ideal TGA-binding site TGAC/GTCA are depicted in boldface letters; each TGAC half-site is marked by an arrow provided that 3 or 4 bp are in consensus with this motif. Vertical lines denote the centers of the palindromes. B, Activities of chimeric promoters in mesophyll protoplasts isolated from npr1 plants. Upstream sequences of the promoters of GSTF8, IEGT38, WRKY38, and WRKY70 were amplified and fused to the −68 PR-1 promoter fragment so that the TGA-binding sites were in the same relative position to the TATA box as in PR-1816–573. Promoter derivatives were fused to the firefly LUC gene. Expression was analyzed in the presence of effector plasmids encoding NPR1 fused to the VP16 activation domain (black bars) under the control of the 35SC4PPDK promoter (Sheen et al., 1995). After transfection, protoplasts were incubated for 14 h in the presence of 5 μm SA. C, The same promoters as in B were analyzed in tga256 protoplasts in the presence and absence of TGA2 fused to the VP16 activation domain (black bars) under the control of the cauliflower mosaic virus 35S promoter. Relative LUC activities are expressed in arbitrary luminescence units, normalized to Renilla LUC activity. The PR-1816–573 promoter activities in the absence of effectors were set to 1. Values are means of three replicates ± sd.

Comparison of basal expression levels of the five promoters in the absence of the effector proteins revealed that their relative activities differed depending on the genotype of the protoplasts. The three NPR1-dependent promoters showed lower activities than the NPR1-independent promoters in the npr1 mutant, whereas their activities were higher in the tga256 mutant (Fig. 5, B and C). Thus, only those promoters that are targets of NPR1 can be subject to the repressive effect of TGA factors.

Comparison of the sequence covering the two TGA-binding sites in NPR1-dependent and NPR1-independent promoters has suggested that the number of base pairs between the palindromic centers might be a relevant feature: in NPR1-independent promoters, the spacing is conserved (12 bp; Krawczyk et al., 2002) and differs from the number of base pairs between the palindromic centers of the respective sites within the PR-1 (17 bp), WRKY38 (14 bp), and WRKY70 (15 bp) promoters. Therefore, we tested whether changing the sequence between the palindromic centers of LS5 and LS7 to 12 bp would interfere with NPR1 recruitment. The sequence CTATTTTAC between LS5 and LS7, which can be exchanged by linker sequences without affecting promoter activity (Lebel et al., 1998), was replaced by AAAA (Fig. 6A). In the transient assay system, the PR-1816–573LS54xaLS7 promoter was activated less efficiently by NPR1-VP16 than the corresponding promoter with the wild-type LS5/LS7 motif (Fig. 6B). In order to investigate whether the spacing or the sequence between the two TGA-binding sites is the discriminating factor, we constructed PR-1816–573LS59xaLS7, thus restoring the original spacing found in the PR-1 promoter. However, this promoter was as inefficiently activated by NPR1-VP16 as PR-1816–573LS54xaLS7 (Fig. 6B, lane 3), indicating that the spacing between the two TGA-binding sites is not the only determining factor and that certain sequence requirements between the TGA-binding sites are relevant for NPR1 function.

Figure 6.

Figure 6.

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Influence of the number of base pairs between LS5 and LS7 on NPR1-VP16-mediated activation of the PR-1816–573 and PR-11294 promoter activities. A, Mutations introduced into the PR-11294 promoter context to study the relevance of the number of base pairs between the two TGA-binding sites. The sequence CTATTTTAC between LS5 and LS7 was replaced by four and nine A residues, respectively. Conserved positions compared with the ideal palindrome TGAC/GTCA are depicted in boldface letters, and vertical lines denote the centers of the two palindromes. Each TGAC half-site is marked by an arrow provided that 3 or 4 bp are in consensus with this motif. B, PR-1 promoter activities in mesophyll protoplasts isolated from npr1 plants. Cells were transfected with the indicated plasmids. PR-1 promoter derivatives that contain the sequences from −816 to −573 upstream of the PR-1 core promoter (−68 to +1) fused to the firefly LUC gene were used as reporter constructs. Mutated cis-elements are indicated on the left. Expression was analyzed in the presence of effector plasmids encoding NPR1 fused to the VP16 activation domain (black bars) under the control of the 35SC4PPDK promoter (Sheen et al., 1995). After transfection, protoplasts were incubated for 14 h in the presence of 5 μm SA. Relative LUC activities are expressed in arbitrary luminescence units, normalized to Renilla LUC activity. The PR-1816–573 promoter activity in the absence of effectors was set to 1. Values are means of three replicates ± sd. C, LUC activities of 2-week-old plants grown axenically on MS plates without (gray columns) and with (black columns) 30 μm INA were measured. The genotypes of the analyzed plants are indicated. Values are means of the activities of the indicated number (#) of independent lines. LUC activities are expressed as relative light units (RLU) per μg of total protein. Error bars represent the sd of two independent experiments. wt, Wild type.

Comparison of the expression data of PR-11294LS54xaLS7 and PR-11294LS5,7mut in transgenic plants reveals that mutating the distance between the TGA recognition sites has a different effect than mutating both elements: PR-11294LS5,7mut shows a wild-type-like expression pattern (Fig. 2, lane 2), whereas PR-11294LS54xaLS7 is severely compromised (Fig. 6C, lane 2). Obviously, the LS4-dependent pathway cannot be activated in PR-11294LS54xaLS7. Assuming that the activity of the LS4-dependent pathway is repressed by LS5, we can also explain why the activity of PR-11294LS7mut is compromised: in this promoter, the LS4-dependent pathway is repressed by LS5, and the LS5/LS7-dependent pathway cannot operate because NPR1 functions through an intact LS5/LS7 motif rather than through LS7 alone.

In sni1, PR-11294LS54xaLS7 did not reach the induced expression levels of PR-11294 (Fig. 6C, lanes 3 and 4), thus resembling PR-11294LS5,7mut (Fig. 6C, lane 5). The reduced expression levels are most likely due to the fact that NPR1 cannot function through TGA factors at these promoters.

The PR-1 Promoter Contains at Least Two Different Integration Sites for SNI1-Mediated Suppression

Next, we addressed the question of which promoter sequences are important for SNI1 to act as a repressor. Previously, it has been speculated that SNI1 negatively affects the PR-1 promoter by targeting LS4 (Kesarwani et al., 2007). This hypothesis was based on the findings that basal and induced levels of the PR-1 promoter are elevated in sni1 (Li et al., 1999), a phenomenon that has also been observed for a promoter lacking LS4 (Lebel et al., 1998). Analysis of PR-11294LS4mut in wild-type and sni1 plants indicated that SNI1 still influences PR-1 in the absence of LS4 (Fig. 7A).

Figure 7.

Figure 7.

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Identification of promoter regions containing SNI1 integration sites. A, Influence of LS4 on the expression in sni1. B, Expression of PR-1816–573 in npr1, sni1, and sni1 npr1. LUC activities of 2-week-old plants grown axenically on MS plates without (gray columns) and with (black columns) 30 μm INA were measured. The indicated LS elements were replaced by linker sequences within the PR-11294 or the PR-1816–573 promoter context. The genotypes of the analyzed plants are indicated. Values represent means of the activities of the indicated number (#) of independent lines. LUC activities are expressed as relative light units (RLU) per μg of total protein. Error bars represent the sd of two independent experiments. wt, Wild type.

In order to address the question of which sequences might be relevant for the repressive effect of SNI1, we analyzed PR-1816–573, which contains 244 bp, including the sequences LS4, LS5, and LS7 upstream of the −68 core promoter region (Fig. 4A), in npr1, sni1, and sni1 npr1 mutant plants. The PR-1816–573 promoter still mediated INA-inducible expression in the wild-type background, although the expression levels were lower than those of PR-11294 (Fig. 7B, lane 2). As observed for the PR-11294 promoter (Fig. 1C), expression of PR-1816–573 required NPR1 (Fig. 7B, lane 3). As NPR1 is only necessary in the presence of SNI1 (Li et al., 1999), it is concluded that PR-1816–573 contains either direct or indirect target sites for SNI1. This notion is supported by the observation that basal expression levels of PR-1816–573 were enhanced in sni1 (Fig. 7B, lane 4). However, whereas the PR-11294 promoter was induced to higher levels in sni1 than in wild-type plants (Fig. 1C), the PR-1816–573 promoter did not show this hyperinduction (Fig. 7B, compare lanes 2 and 4). Thus, sequences outside the −816 to −573 region are important for hyperinduced expression of the promoter in sni1. Whereas the PR-11294 promoter is still inducible in the sni1 npr1 mutant background (Fig. 1C, lane 4), this is not the case for the PR-1816–573 construct (Fig. 7B, lane 5). Apparently, this promoter fragment lacks sequence information that allows induction of the promoter in the absence of NPR1 and SNI1.

In summary, the analysis displayed in Figure 7 has several implications: first, regulatory sequences between −816 and −573 are sufficient for INA induction in wild-type plants and increased background expression in the absence of SNI1; second, regulatory sequences outside the −816 to −573 region are necessary for full promoter activity in wild-type plants, hyperinduction in sni1, and INA-induced expression in the sni1 npr1 mutant background.

W Boxes Are Required for Full Promoter Activity in Wild-Type Plants and Hyperinduced Expression in the sni1 Mutant Background

When searching for putative positive regulatory cis-elements outside the −816 to −573 region, we noticed two W boxes [(T)TGAC(T)] and one W box-like element [(C)TGAC(T)] spaced by approximately 20 bp between positions −550 and −480 (Fig. 8A). As transcription of many genes encoding WRKY factors is SA inducible (Dong et al., 2003; Wang et al., 2006), we hypothesized that these boxes might contribute to PR-1 promoter activity. Indeed, deletion of box W1 within the PR-11294 promoter context reduced promoter activity (Fig. 8B, lane 2). Simultaneous deletion of W1, W2, and W3 (Fig. 8B, lane 3) had the same effect as deletion of W1 alone, indicating that W2 and W3 have no function in the absence of W1.

Figure 8.

Figure 8.

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Influence of W boxes W1, W2, and W3 on expression of the PR-1 promoter. A, Schematic presentation of the “W box cluster” within the PR-1 promoter. The small black box within the −816 to −573 region depicts the relative position of the LS5/LS7 motif. The sequence between −550 and −480 containing the three W boxes is indicated. PR-11294ΔW1 and PR-11294ΔW1,2,3 contain deletions between bp −547 to −537 and −573 to −483, respectively. For constructing PR-1816–573+W1,2, bp −572 to −509 were added to the PR-1816–573 promoter. B, GUS activities of 2-week-old plants grown axenically on MS plates without (gray columns) and with (black columns) 30 μm INA were measured. Distinct mutations in the W box cluster were introduced into the PR-11294 promoter context. The genotypes of the analyzed plants are indicated. Values represent mean GUS activities (pmol methylumbelliferyl glucuronide mg−1 protein min−1) of the indicated number (#) of independent lines. Error bars represent the sd of two independent experiments. C, LUC activities of 2-week-old plants grown axenically on MS plates without (gray columns) and with (black columns) 30 μm INA were measured. Sequences encoding two W boxes were added to PR-1816–573. The genotypes of the analyzed plants are indicated. Values are means of the activities of the indicated number (#) of independent lines. LUC activities are expressed as relative light units (RLU) per μg of total protein. Error bars represent the sd of two independent experiments. wt, Wild type.

Next, we added sequences from –572 to –509, which contained the two canonical W boxes W1 and W2, downstream of the PR-1816–573 construct and investigated the activity of the resulting promoter PR-1816–573+W1,2 in the wild-type and the sni1 background. Whereas the added W boxes did not alter the general expression levels of PR-1816–573 under inducing conditions in wild-type plants (Fig. 8C, lanes 2 and 4), the PR-1816–573+W1,2 construct showed hyperinduced expression in the sni1 mutant background (Fig. 8C, lanes 4 and 5). Thus, hyperinduction in sni1 does not only require NPR1 (Fig. 1C) and a functional LS5/LS7 motif (Figs. 2 and 6C) but also W boxes downstream of −573.

DISCUSSION

Since expression of the PR-1 gene correlates with the onset of the long-lasting plant immune response SAR, it has been used as a paradigm to analyze transcriptional control mechanisms that are operational in SAR leaves (Cao et al., 1994, 1997; Lebel et al., 1998; Zhang et al., 2003; Rochon et al., 2006; Kesarwani et al., 2007). The strong difference between basal and induced transcript levels is not merely due to the induced nuclear translocation of the coactivator NPR1 but is supported by the partially redundant negative functions of SNI1 and TGA factors in the noninduced state (Kesarwani et al., 2007). When trying to integrate data on the expression characteristics of the PR-1 promoter in npr1, sni1, and tga2 sni1 mutants with results obtained from previous promoter analysis (Lebel et al., 1998), the following model of the interplay of these regulatory proteins on the promoter had been proposed (Kesarwani et al., 2007; Supplemental Fig. S1). The W box LS4 functions as a target site of SNI1, LS5 is the target site of the negative function of TGA2, and TGA factors at LS7 function in concert with the coactivator NPR1. In this study, we specifically addressed the question of which sequences are important for the regulation of the PR-1 promoter by negatively and positively acting TGA factors, SNI1 and NPR1. To this end, we analyzed PR-1 promoter derivatives encoding different combinations of mutated LS4, LS5, and LS7 elements in sni1 and npr1. The updated working model is illustrated in Figure 9.

Figure 9.

Figure 9.

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Mechanistic models showing the interplay of different regulatory factors at the PR-1 promoter under noninduced (–INA) and induced (+INA) conditions. A, In the absence of INA, basal expression levels of the wild-type promoter are under the control of two partially redundant repression mechanisms. We assume that an activator at a yet unknown site between –816 and –573 is repressed by the LS5/LS7 motif through the TGA2/NPR3/4 complex and by SNI1. A negative influence at LS4 by negatively acting WRKY transcription factors (R) is favored by TGA factors at the LS5 site. Under noninduced conditions, transcription of genes coding for activating WRKY transcription factors is low. Average LUC activities of the construct in the wild type (wt) and sni1 are given in the right corner. B, In the presence of INA, NPR1 acts as a transcriptional coactivator by binding to the TGA factors at the LS5/LS7 motif. The function of SNI1 and its target is inactivated. LS4 remains occupied by negatively acting WRKY transcription factors. Positively acting WRKY factors can only be recruited in the absence of LS5, allowing the LS5/LS7-independent activation mode. Activating WRKY transcription factors, which are under the control of the SNI1/NPR1 regulon, contribute to high induction of the promoter by binding to WRKY boxes W1 to W3. NPR1 synergistically interacts with WRKY transcription factors at W1, W2, and W3. Average LUC activities of the construct in the wild type and sni1 are given in the right corner.

Direct NPR1-Mediated Activation Requires an Intact LS5/LS7 Motif

Numerous studies have suggested that the key activator of PR-1 expression, NPR1, functions through TGA factors at the cis-element LS7 of the PR-1 promoter (Lebel et al., 1998; Johnson et al., 2003; Rochon et al., 2006). Here, we provide evidence that NPR1 can activate the promoter either through a functional LS5/LS7 unit or, in the absence of LS5/LS7, through LS4. Based on previous results that NPR1 activates WRKY promoters (Wang et al., 2006) and as it directly interacts with TGA factors (Zhang et al., 1999), we postulate that NPR1 activates the LS4-dependent pathway through activated WRKY factors, whereas the LS5/LS7-dependent pathway exploits the interaction between NPR1 and TGA factors at the promoter. In contrast to previous assumptions, LS7 is not sufficient for the latter activation mechanism. This concept is supported by transient assays with a truncated promoter construct that showed that LS4 alone, or LS4 in combination with one TGA-binding site or two binding sites with an inappropriate intervening sequence, is not sufficient for efficient NPR1-VP16-mediated promoter activation (Figs. 4B and 6B). Simultaneous mutation of LS4 and LS5, which have been previously characterized as negative elements, wipes out promoter activity in transgenic plants, indicating that LS7 alone cannot support transcription (Fig. 3). Analysis of the PR-1 promoter derivatives in sni1 led to the same conclusions: PR-11294 activity was higher in sni than in sni1 npr1 (Fig. 1C), indicating that NPR1 does not only interfere with the function of SNI1 but that it also functions as a transcriptional coactivator in the absence of SNI1. Full NPR1-dependent hyperactivation, which exceeds the expression levels of the induced promoter in wild-type plants, is not observed with promoter derivatives that either contain only one TGA-binding site (Fig. 2) or an inappropriate sequence between both sites (Fig. 6C).

Consistently, 21 out of 48 promoters that were candidates to be directly targeted by NPR1 (Wang et al., 2006) contain two TGA-binding sites (Supplemental Fig. S5). When cloning the respective sequences of two selected promoters (WRKY38, WRKY70) upstream of the PR-1 core promoter, the resulting chimeric promoters were activated by NPR1-VP16 in transiently transformed protoplasts (Fig. 5B). Equivalent promoter constructs containing two TGA-binding sites from SA-inducible NPR1-independent promoters (GSTF8, IEGT38; Blanco et al., 2005) were not activated by NPR1-VP16. Comparison of the sequence around the TGA-binding sites in the NPR1-dependent promoters did not reveal any obvious pattern. Our transient assay system might allow the definition of the sequence requirements that are important for NPR1 function.

LS5 Interferes with LS4-Dependent Activation of the PR-1 Promoter

The importance of LS5 for the direct activation of the PR-1 promoter by NPR1 was previously occluded by the observation that mutation of LS5 did not compromise promoter activity (Lebel et al., 1998). The severely compromised activity of the PR-11294LS4,5mut promoter indicates that activation of PR-11294LS5mut is, like the activation of PR-11294LS5,7mut, due to an activation pathway that depends on LS4 (Figs. 2 and 3). In turn, the severely compromised activities of PR-11294LS7mut and PR-11294LS54xa7 indicate that the LS4-dependent pathway is repressed by LS5 (Figs. 1B and 6C). A possible scenario is that TGA factors at LS5 recruit repressive WRKY factors to LS4, which in turn would interfere with the access of positively acting WRKY factors (Fig. 9). Due to the complexity and redundancy of the WRKY transcription factor family, it seems almost impossible to define which WRKY transcription factors are responsible for the function of LS4 (Xu et al., 2006; Eulgem and Somssich, 2007).

Other NPR1-dependent PR genes like PR-2 and PR-5 do not contain TGA-binding sites and thus are likely to be regulated indirectly through a mechanism that relies on the transcriptional activation of WRKY factors. Interestingly, these promoters can be activated by ectopic expression of WRKY70, whereas only an aberrantly long and faint PR-1 mRNA is detected in these plants (Li et al., 2004). Likewise, ectopic expression of WRKY18 is not sufficient for the activation of PR-1, unless leaves get older (Chen and Chen, 2002). Thus, it might well be that LS5 prevents the activation of PR-1 by ectopically expressed WRKY factors and that such a mechanism is not operational in other PR promoters.

Repression of Basal PR-1 Promoter Activity by TGA Factors Requires an Intact LS5/LS7 Motif

It has been shown that TGA factors and SNI1 are partially redundant in repressing the PR-1 promoter (Kesarwani et al., 2007). Thus, the importance of the cis-elements required for TGA-dependent repression can be best monitored in sni1. In sni1, mutation of LS5 or LS7 or both sites led to high basal expression levels (Fig. 2). As the two NPR1-related proteins NPR3 and NPR4 interact with TGA factors and as the npr3 npr4 mutant shows increased basal levels of PR-1 expression (Zhang et al., 2006), we speculate that repression through the LS5/LS7 motif is mediated through these proteins (Fig. 9A). Like NPR1, NPR3 and NPR4 seem to require the intact LS5/LS7 motif to be efficiently recruited. This notion is consistent with the observation that the two NPR1-dependent promoters of WRKY genes WRKY38 and WRKY70 have lower basal expression levels in the npr1 mutant than the two NPR1-independent promoters GSTF8 and IEGT38, whereas their expression levels are higher in the tga256 mutant, which cannot recruit NPR proteins to promoter regions (Fig. 5, B and C). Thus, basal levels of WRKY38 and WRKY70 promoters might be repressed through NPR3 and NPR4 by the same mechanism as the PR-1 promoter.

This model is consistent with the finding that the tga2 sni1 double mutant shows higher PR-1 expression levels in the absence of INA as compared with the respective single mutants (Kesarwani et al., 2007). We postulate that NPR3/4 is not recruited to the promoter if TGA2 is missing. However, a recent report showed that TGA2 forms oligomers in vitro and that these aggregates are resolved by NPR1 (Boyle et al., 2009). It was concluded that TGA2 oligomers repress the promoter. In our view, the association of NPR3/4 with TGA factors at an intact LS5/LS7 motif has to be considered as an alternative explanation.

SNI1 Target Sequences for Repression of Basal Levels Are Located between bp −816 and −573

Based on the finding that the W box LS4 acts as a negative element, it has been suggested that SNI1 exerts its negative effect either directly or indirectly at this site (Kesarwani et al., 2007). As PR1294LS4mut is derepressed in the sni1 mutant background (Fig. 7A), we suggest that SNI1 represses basal expression levels through an LS4-independent mechanism. The finding that basal levels of the PR-1816–573 promoter are repressed by SNI1 (Fig. 7B) points at a functionally relevant SNI1 integration site within these 244 bp. As this promoter does not show any hyperinduction in sni1 (Fig. 7B), we postulate that SNI1 influences a different mechanism in the induced state.

Negative Regulation of Induced PR-1 Expression by SNI1 Requires W Boxes Downstream of Position −573

In contrast to the PR-11294 promoter, PR-1816–573 did not show different induced expression levels in sni1 (Figs. 1C and 7B). As extension of the PR-1816–573 promoter by W boxes W1 and W2 reestablished hyperinduction in sni1 (Fig. 8C), we conclude that SNI1 exerts a negative effect through these sites. This repression might be mediated either directly or indirectly by regulating the expression of cognate WRKY transcription factors. As hyperinduced expression in sni1 depends on NPR1 (Fig. 1C) and an intact LS5/LS7 motif (Figs. 2 and 6C), we postulate that hyperinduction in sni1 is mediated by the synergistic action of these WRKY transcription factors with NPR1 (Fig. 9B).

Sequences Downstream of bp −573 Are Necessary for NPR1/SNI1-Independent Regulation of the PR-1 Promoter

Apart from not showing a hyperinduction in sni1, PR-1816–573 is also not induced in the sni1 npr1 mutant (Fig. 7B). As W boxes W1 and W2 restore the hyperinduction in sni1 (Fig. 8C), they might play a role in the induction process in sni1 npr1. This conclusion is supported by functional analysis of these W boxes in the PR-11294LS4,5mut construct. This promoter is not a target of NPR1 because it lacks LS4 and an intact LS5/LS7 motif, which mimics the situation in npr1. However, it is inducible in sni1 (Supplemental Fig. S6), thus resembling processes in the sni1 npr1 mutant. Its induction is abolished upon removal of the three W boxes downstream of −573 (Supplemental Fig. S6), which is consistent with the hypothesis that these W boxes are likely important for induction in sni1 npr1. This finding has implications for unraveling the SNI1/NPR1-independent induction pathway that depends on RAD51D (Durrant et al., 2007).

CONCLUSION

The updated working model (Fig. 9) for the regulation of the PR-1 promoter is based on an experimental approach that, to our knowledge, has not been taken to this extent before: multiple combinations of mutations in previously identified regulatory cis-elements have been tested in different mutant backgrounds. Previous results of single promoter mutants in the wild-type background have been misleading, because the regulation of the PR-1 promoter is complicated by the fact that it is not only directly regulated by NPR1 and SNI1 but also indirectly by WRKY transcription factors that are subject to a similar regulatory node.

We conclude from our results that the promoter is repressed by SNI1 at yet unidentified target sites between positions −816 and −573 (Fig. 9A). This target site is redundantly regulated by TGA factors at the LS5/LS7 motif, possibly through the action of the redundant proteins NPR3 and NPR4. LS4 works as a negative element, potentially due to the recruitment of negative WRKY factors by TGA factors residing at LS5. W boxes downstream of bp −573 do not play a major role due to low expression of the cognate WRKY transcription factors in the noninduced state. Under inducing conditions, NPR1 functions as an inhibitor of SNI1, displaces NPR3/4, and acts as a transcriptional coactivator. As soon as the promoter is activated, the SNI1-regulated positive activity does not play a role any longer. NPR1 acts synergistically with WRKY transcription factors at W boxes downstream of bp −573. The activity of these factors is under the negative control of SNI1.

This study has yielded novel insights into the complex regulation of the PR-1 promoter and provides a framework for the integration of future results. The finding that two TGA-binding sites are required for the recruitment of NPR1 to the promoter and for repression in the absence of SNI1 has implications for further efforts to advance our understanding of the mechanism of action of NPR1 as a transcriptional coactivator and of the mechanism of action of NPR3 and NPR4 as negative regulators of PR-1 expression. Analysis of PR-1816–573 promoter derivatives in wild-type and sni1 tga2 plants might yield the cis-elements that are essential for high basal expression levels. This might be instrumental for unraveling the mechanism of action of SNI1. To elucidate the influence of the sni1 allele under induced conditions, the expression of WRKY transcription factors in INA-induced sni1 and sni1 npr1 plants has to be studied. WRKY genes that are under the control of an NPR1-independent induction mode in sni1 would explain the SNI1/NPR1-independent induction of the PR-1 promoter and might serve as future targets to study the function of RAD51D (Durrant et al., 2007).

MATERIALS AND METHODS

Vector Construction

The PR-11294 promoter, which covers the sequence from −1,294 to +34 relative to the transcriptional start site, was amplified with primers P1 and P2 (for primer sequences, see Supplemental Table S1) from genomic DNA isolated from Arabidopsis (Arabidopsis thaliana) accession Columbia (Col-0) plants using the iProof high-fidelity PCR kit (Bio-Rad). The resulting PCR fragment was digested with SalI and XbaI and cloned into pUC18-Entry2, which contains a multiple cloning site flanked by AttL recombination sites. Substitution and deletion constructs were obtained by overlap extension PCR. To this end, P1 and P2 were utilized separately in two iProof PCRs in combination with primers carrying the mutations of interest (P3–P22; Supplemental Table S1). The two different fragments yielded by the first PCRs were purified after separation on a 1% agarose gel and diluted 1:200. The two diluted fragments were used as templates in a second PCR, again employing the primers P1 and P2. The resulting PCR fragment contained the desired DNA modification and was digested with Eco91I (−816) and Mph1103I (−573; for modifications of LS motifs) or Mph1103I and BpiI (−370; for deletions of W boxes) before ligation into pUC18-Entry2-PR-1, cut with the corresponding enzymes.

The PR-1816–573 promoter construct was generated by first amplifying PR-1 promoter sequences between positions −1,294 and +1 by using primers P1 and P21. The PCR fragment was cut with SalI and XbaI and cloned into vector pUC18-Entry2 as described above. Subsequently, the region upstream of the Eco91I site at −816 was exchanged against bp +86 to +586 of the Tn5-encoded Chloramphenicol Acetyl Transferase (CAT) gene, with an additional arbitrary 24 bp inserted between the CAT gene and the 5′ end of the promoter. For PCR-mediated amplification of the CAT gene, the primer combination P23 and P25 was used on the template plasmid pTAX-CAT, which is a derivative of pGA582 (An, 1987). The deletion between −569 and −68 was created by ligation of the vector fragment pUC18-Entry2-CAT-PR-1 cut with Eco91I (−816) and HpaI (−68) and the promoter fragments from Eco91I (−816) to NdeI(fill in) (−573). As the fill-in reaction of the NdeI overhanging ends had destroyed the Mph1103I restriction site, an overlap extension PCR was performed. Primers carrying the Mph1103I restriction sites were P26 and P27, and flanking primers were P1 and P23. The resulting plasmid pUC18-Entry2-PR-1816–573 was cut with Mph1103I to insert an oligonucleotide (P28/P29) that extends the promoter from −573 to −510, thus adding the W boxes W1 and W2. Plasmids containing modifications of the LS motifs within the PR-1816–573 promoter context were generated by exchanging the wild-type Eco91I/Mph1103I fragment of PR-1816–573 against the corresponding fragment generated in PR-11294. Cloning of the promoter regions of Arabidopsis genes GSTF8, IEGT38, WRKY38, and WRKY70 was done by amplifying the corresponding sequences using primers P30/P31 (IEGT38), P32/P33, (GSTF8), P34/P35 (WRKY70), and P36/P37 (WRKY38) with attached Eco91I/Mph1103I sites and exchanging the PR-1 sequences in pUC18-Entry2-PR-1816–573. Introduced mutations were confirmed by sequencing.

Using the Gateway cloning system from Invitrogen, PR-1 promoter derivatives were recombined into the binary destination vectors pBGWL7 and pBGWFS7 (Karimi et al., 2005) that contain the Gateway recombination sites upstream of the firefly LUC and GUS reporter genes, respectively. The backbone of the NPR1-VP16 effector construct for transient assays is described by Krawczyk et al. (2002): the coding region of TGA2.2 was replaced by the coding region of NPR1. In the empty vector control construct, the coding region was deleted. The VP16-TGA2 effector construct was generated using Gateway technology by cloning the coding region of TGA2 into the pDONR201 vector (Invitrogen) and subsequent recombination into the destination vector pAlligator1 (Bensmihen et al., 2004). pAlligator1 served as an empty vector control.

Plant Material and Plant Transformation

Arabidopsis Col-0 was used. Mutant tga256 (Zhang et al., 2003) was obtained from Y. Zhang (University of British Columbia, Vancouver), sni1-1 and sni1 npr1 were from X. Dong (Department of Biology, Duke University), and npr1-1 was from the Norwich Arabidopsis Stock Centre. For measuring reporter gene activity of transgenic plants, approximately 200 F2 seeds of reporter gene lines were surface sterilized and sown on sterile MS plates containing 0.5 g L−1 MES. Plates contained 30 μm INA dissolved in dimethyl sulfoxide; control plants contained 15 μL L−1 dimethyl sulfoxide. Plants were grown for 18 d at 22°C under long-day conditions (14 h of light [70 μmol photons m−2 s−1] and 10 h of dark) and 60% humidity. For generation of transgenic plants, binary plasmids were electroporated into Agrobacterium tumefaciens strain GV3101 (pMP90). The resulting agrobacteria were used to transform Col-0, npr1-1, sni1-1, sni1 npr1, and tga256 mutant plants using the floral dipping method (Clough and Bent, 1998). For determining the recruitment of NPR1-VP16 or VP16-TGA2, 5 μg of reporter plasmids (pBGWL7 carrying different PR-1816–573 derivatives) and 15 μg of the effector plasmids were transformed in Arabidopsis npr1-1 and tga256 protoplasts (Yoo et al., 2007). Protoplasts were incubated in washing and incubation solution (Yoo et al., 2007) supplemented with 5 μm SA for 16 h before harvest. To normalize for the experimental variability, 1 μg of the plasmid p70SRuc containing the Renilla LUC gene under the control of the cauliflower mosaic virus 35S promoter was added to each sample.

LUC and GUS Assays

To determine LUC activities of transgenic lines, protein extracts from transgenic plants were prepared by application of 700 μL of 1× cell culture lysis reagent (Promega) to approximately 300 to 500 mg of ground frozen plant material. The samples were shaken in an Eppendorf mixer until the plant powder was completely dissolved. After centrifugation (10 min, 12,000 rpm, 4°C), 10 μL of protein extract was added to each well of a precooled Greiner Lumitrac 200 plate. The plate was subsequently incubated for 20 min in the dark at 4°C to avoid background illumination during the LUC activity measurement. The LUC measurements were started 5 to 10 min after insertion of the plate in the FLUOstar Ultima luminescence plate reader (BMG Labtech) by adding 50 μL of LUC substrate buffer (Promega) to each well. All measurements were performed under the same conditions, using a detection interval of 10 s and a relative gain value of 3,600. After detection of the emitted light, the values were normalized to the protein concentrations as determined with the BCA protein assay kit (Thermo Scientific). Quantitative GUS assays using 4-methyl-umbelliferyl-β-d-glucuronide (Sigma-Aldrich) as a substrate (Jefferson, 1989) were done on microtiter plates. The released fluorescence was measured with a Cyto Fluor Series 4000 plate reader (Perspective Biosystems). The total amount of protein was determined using the Bradford reagent (Carl Roth). Eighteen to 22 independent lines per construct were analyzed in one experiment. Supplemental Figure S7 documents exemplarily the variation between the lines, which is due to “position effects.” For calculating the means, the highest and the lowest expressing lines were excluded. As described before (Lebel et al., 1998), averages of the mean values from two independent experiments are presented. LUC activities of transfected protoplasts were determined with the dual LUC reporter assay system from Promega using the TD20/20 luminometer from TurnerBiosystems.

Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: PR-1 (At2g14610), NPR1 (At1g64280), GSTF8 (At2g47730), IEGT38 (At4g34138), WRKY38 (At5g22570), and WRKY70 (At3g56400).

Supplemental Data

The following materials can be found in the online version of this article.

Supplementary Material

[Supplemental Data]
pp.110.165563_index.html (807B, html)

Acknowledgments

We thank Anna Hermann and Larissa Kunze for excellent technical assistance, Y. Zhang for the tga256 mutant, and X. Dong for sni1-1 and sni1 npr1 seeds.

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