The transcriptional activator NPR1 and the repressor SNI1 are two key regulatory proteins for PR-1 induction. While it is assumed that NPR1 positively influences expression via interaction with members of the TGA factor family (Rochon et al. 2006), not much is known about the recruitment of SNI1 to promoter sequences to mediate repression. Since SNI1 does not contain a DNA binding domain a direct recruitment to regulatory sequences is unlikely (Mosher et al. 2006). However, at least one cis-element within the PR-1 promoter which contributes to SNI1-mediated repression has to be postulated. Repression can either be realized by recruitment of SNI1 to the promoter through a sequence specific DNA binding domain, or, alternatively, by recruitment of a transcriptional activator, whose expression is negatively regulated by SNI1. The critical sequence information for SNI1-dependent regulation is encoded in a 1294 bp PR-1 promoter fragment as revealed by its expression pattern after INA induction in distinct mutants, which corresponds to endogenous PR-1 regulation (figure 4.1A). (1) The reporter gene shows an increased basal expression in sni1-1 accompanied by a hyperinduction when INA is applied. (2) The 1294 promoter shows no sensitivity to INA in npr1-1 and (3) this lack of induction can be partially rescued in the sni1 npr1 double mutant. (4) The sni1 npr1 mutant displays an increased background expression level similar to sni1-1 but the hyperinduction fails likely due to the absence of the transcriptional co-activator NPR1. In conclusion, these experiments show that the SNI1/NPR1 independent pathway of PR-1 induction is operational on this promoter fragment.
Figure 4.1: Average LUC activities of the PR-11294 (A) and the PR-1816-573 (B) promoter fragment in wt and mutant plants.
Luciferase activities of 18-day-old plants grown on MS-plates supplemented without (white columns) or with (black columns) 30 µM INA were measured. The numbers of single transgenic lines investigated for each genotype is displayed on the left (n=). Error bars represent the standard deviation of at least two independent experiments. The promoter derivative illustrations show PR-1 promoter sequences fused to a LUC reporter gene. The blue boxes display the gateway® cloning sites, grey shaded regions illustrate PR-1 cis-regulatory region and the white box shows sequence substitution against coding sequence derived from the CAT gene. The orange LS-labeled region in the center of the two constructs displays the location of the LS-region. The gap in PR-1816-573 illustrates sequence deletion and the arrows show relative base pair positions.
Previous promoter deletion and linker scan analysis has shown that the region between -706 to -579 (called LS-region for linker scan from here on) encodes different
elements that positively or negatively influence PR-1 promoter activity (Lebel et al.
1998). Therefore, the LS-region has been hypothesized to be the target for NPR1 and SNI1 activity (Kesarwani et al. 2007). One important cis-element within this region is the as-1-like element which consists of two TGACG motifs designated as LS5 and LS7.
Substitution of LS5 by a linker sequence leads to slightly activated promoter activities both in the uninduced- and induced state, whereas linker scan mutagenesis of LS7 completely abolishes promoter activity, indicating that the two TGA motifs have distinct function. Because of the positive impact of LS7 on PR-1 induction it was hypothesized that it recruits NPR1 via interaction with TGA transcription factors.
In order to gain direct evidence for the critical function of this promoter sequence with regard to NPR1- and SNI1-dependent regulation the region between -816 and -573 was fused to the basal PR-1 promoter (-68bp to -1bp). The PR-1816-573 construct was transformed in the different plant genotypes (wt, npr1-1, sni1-1 and npr1 sni1) to validate the promoter activity in comparison to the PR-11294 promoter (figure 4.1 B). In the wildtype background, the modified promoter construct was still inducible by INA indicating that central regulatory elements within this region are not only necessary but also sufficient for induction. The induced expression levels of PR-1816-573 only reach approximately one third of the intensity measured in PR-11294 control plants suggesting additional positive cis-elements outside the LS-region. Interestingly, the PR-1816-573
construct shows slightly increased basal expression levels when transformed in sni1-1 while the hyperinduction displayed by the PR-11294 construct is missing. These results suggest that SNI1 affects PR-1 expression in two ways. On the one hand, SNI1 suppresses basal levels of the PR-1 promoter via a regulatory sequence present in the PR-1816-573 derivate. On the other hand, in contrast to the INA-induced transcription of PR-11294, INA induced transcription of PR-1816-573 is not influenced by SNI1. This illustrates that PR-1816-573 misses cis-elements that can serve as target sites for positive regulators that are up-regulated in the sni1-1 mutant. Though the influence of SNI1 on this modified promoter is altered, the induction of the construct is still strictly dependent on NPR1, illustrated by a complete lack of INA sensitivity in npr1-1. This confirms the notion that NPR1 may acts on the promoter through one or both of the TGACG motifs within the LS-region. The induction of PR-1816-573 in the sni1 npr1double mutant is abolished, likely due to the absence of sequence information that confers induction when no functional NPR1 protein is present. In conclusion, the analysis of the PR-11294- and the deleted PR-1816-573 promoter in the different mutant backgrounds revealed that other positive acting regulatory sequences exist outside the LS-region. Moreover these
cis-regulatory sequences contribute to hyperinduction in sni1-1 illustrating that the corresponding transcriptional activators are exclusively active in sni1-1 mutant background.