Exchanging the as-1-like element of the PR-1 promoter by the as-1 element of the CaMV 35S promoter abolishes salicylic acid responsiveness and regulation by NPR1 and SNI1

Abstract

The plant defense hormone salicylic acid (SA) activates gene expression through a number of different mechanisms. In Arabidopsis thaliana, the SA-induced PATHOGENESIS RELATED (PR)-1 promoter is regulated through TGA transcription factors binding to the two TGACG motifs of the so called as-1 (activation sequence-1)-like element which is located between base pair positions -665 and -641. Activation is mediated by the transcriptional co-activator NPR1 (NON EXPRESSOR OF PR GENES1), which physically interacts with TGA factors. Moreover, the promoter is under the control of the negative regulator SNI1 (SUPPRESSOR OF NPR1, INDUCIBLE1). We have recently reported that SNI1-mediated repression of basal promoter activities and NPR1-dependent induction are maintained in a truncated PR-1 promoter that contains sequences between -816 and -573 upstream of the -68 promoter region. In this addendum, we report that the expression characteristics of this truncated PR-1 promoter is changed profoundly when its as-1-like element is replaced by the as-1 element of Cauliflower Mosaic Virus 35S promoter which also contains two TGACG motifs. The resulting chimeric promoter showed high constitutive activity that was independent from SA, NPR1 and SNI1. Thus, the configuration of two TGA binding sites within the PR-1 promoter determines whether NPR1 can induce and whether SNI1 can repress the promoter.

Introduction

Plants respond to invading pathogens by activating the synthesis of a distinct set of hormones¹ which orchestrate transcriptional activation of defense genes.² Salicylic acid (SA) is the predominant plant hormone counteracting the attack of biotrophic pathogens.³ In Arabidopsis thaliana and several other plants, the BTB/ankyrin repeat protein NPR1 (NON EXPRESSOR OF PR GENES1)⁴ has been identified as the key positive regulator of many SA-induced genes. SA-activated NPR1 translocates to the nucleus,⁵ where it interacts with members of the TGA family of basic leucine zipper (bZIP)-type transcription factors.⁶ TGA factors were originally identified because of their binding activity to the two TGACG motifs of the as-1 (activation sequence-1) element of the Cauliflower Mosaic Virus 35S promoter.⁷ Simultaneous knock-out of the three Arabidopsis clade II TGA factors (TGA2, TGA5, TGA6) led to an npr1-like phenotype, as revealed by compromised induction of the SA marker gene PATHOGENESIS RELATED-1 (PR-1). Moreover, the SA-dependent plant defense response “systemic acquired resistance (SAR)” did not develop after pathogen infection in tga256 mutant plants.⁸ Early reports have suggested that NPR1 stimulates TGA factors to bind to their cognate cis elements in SA-responsive promoters.^9,10 In contrast, more recent reports provide evidence that TGA factors bind constitutively to their target promoters thus repressing basal levels.^11,12 It is hypothesized that NPR1 acts as a transcriptional co-activator after association with DNA-bound TGA factors.¹¹ The repressor SNI1 (SUPPRESSOR OF NPR1, INDUCIBLE1)¹³ dampens both basal and induced levels of PR-1 expression by a yet unexplored mechanism that seems to involve histone modifications.¹⁴

In our recent publication¹⁵ we have provided indirect evidence that NPR1 can only associate with the PR-1 promoter when both TGA binding sites of the as-1-like element are intact. However, such a configuration of two TGA binding sites is not only found in NPR1-dependent but also in NPR1-independent promoters.^16–18 Since the as-1 element of the CaMV 35S promoter¹⁹ functions as an autonomous NPR1-independent SA-responsive element,^20–22 we have asked the question whether it would confer SA-induced but NPR1-independent expression to the PR-1 promoter. To this end, we exchanged the as-1-like element of the PR-1 promoter by the as-1 element of the CaMV 35S promoter and monitored the expression of the resulting chimeric promoter in transgenic tga256, npr1 and sni1 plants.

The as-1 Element of the CaMV 35S Promoter Confers De-regulated High Expression to the PR-1 Promoter

First, we replaced the as-1-like element of the PR-1 promoter by the CaMV 35S-derived as-1 element within the context of a 1294 bp long fragment yielding the promoter PR-1₁₂₉₄as-1 (Fig. 1A). The construct was fused to the luciferase reporter gene and transformed into Arabidopsis plants (for experimental details see Pape et al. 2010).¹⁵ In the absence of the SA analog INA (2,6-dichloro-isonicotinic acid), the resulting promoter (PR-1₁₂₉₄as-1) showed 400-fold higher activities than the wild-type PR-1₁₂₉₄ promoter (Fig. 1B and lane 2). This high basal activity was further enhanced by a factor of two in plants grown on INA. Induced expression levels were about 40-fold higher than those delivered by the activated wild-type PR-1₁₂₉₄ promoter. PR-1₁₂₉₄as-1 promoter activity was completely abolished in the tga256 mutant (Fig. 1B and lane 3), indicating that these TGA factors are essential components of the activation process. As the activity of the element alone is very weak,²² strong synergistic interactions with other transcriptional activators on the PR-1 promoter have to be postulated. A similar observation has been described before: When fusing a multimer of the GT-box of the light-regulated Ribulose-1,5-bisphosphate Small Subunit promoter to the CaMV 35S core promoter, no promoter activity was observed.²³ However, after insertion of the as-1 element, a light-regulated promoter was generated. It seems that the as-1 element has a high capacity to cooperate with other cis elements and that it synergistically interacts with factors on the PR-1 promoter even in the absence of INA.

Figure 1.

Influence of the as-1 element of the CamV 35S promoter on PR-1₁₂₉₄ activity in wild-type and tga256 mutant plants. (A) Sequences of the as-1-like element of the PR-1 promoter and the as-1 element of the CamV 35S promoter. Conserved positions within the TGA binding site TGAC/GTCA (gray box) are depicted in boldface letters; each TGAC half site is marked by an arrow provided that 3 or 4 bps are in consensus with this motif. Vertical lines denote the centers of the palindromes. Bases that are different in the CamV 35S-derived as-1 element as compared to the PR-1 as-1-like element are represented as lowercase letters. (B) Luciferase activities of 2-week-old plants grown axenically on plates without (gray columns) and with 30 µM INA (black columns) were measured. The genotypes of the analyzed plants are indicated. Values are the means of the activities of the indicated number (#) of independent lines. Luciferase activities are expressed as relative light units (RLU) per µg of total protein. Error bars represent the standard deviation of two independent experiments. Experimental conditions are as previously reported in reference ¹⁵. wt, wild-type.

The as-1 Element of the CaMV 35S Promoter Renders a Truncated PR-1 Promoter Independent from NPR1 and SNI1

The activity of the PR-1₁₂₉₄ promoter is supported by WRKY factors whose transcription is induced by SA-activated NPR1 and potentially repressed by SNI1. In order to analyze the influence of NPR1 and SNI1 on PR-1(as-1) in the absence of this indirect activation mode, we deleted W-box containing sequences between base pair positions −573 and −68 and sequences upstream of −816 (Fig. 2A). This PR-_1816-573 promoter is inactive in the npr1 mutant and reveals enhanced basal levels in the sni1 mutant.¹⁵ However, exchanging the as-1-like element by the as-1 element of the CaMV 35S promoter generated an INA-independent constitutive promoter whose activity exceeded the induced levels of the original PR-1_816-573 promoter by a factor of 25 (Fig. 2B). Obviously, the as-1 element cannot confer INA responsiveness in this promoter context. This constitutive activity was independent from NPR1 most likely because NPR1 cannot recognize TGA factors at the CaMV 35S-derived as-1 element, even though it is embedded in the PR-1 promoter context. Moreover, the promoter was as strong in wild-type plants as in sni1. As SNI1 can repress a PR-1₁₂₉₄ promoter derivative lacking the as-1-like element, we tentatively conclude that the repressive effect of SNI1 that is observed for the PR-1_816-573 promoter is overridden by the strong activity of the as-1 element.

Figure 2.

Influence of the as-1 element of the CamV 35S promoter on PR-_1816-573 activity in wild-type, npr1 and sni1 plants. (A) Schematic presentation of the PR-_1816-573 promoter construct. Sequences between bp positions −573 and −68 and upstream of bp position −816 were removed. the black box depicts the relative position of the as-1-like element. (B) Luciferase activities of 2 week-old plants grown axenically on plates without (gray columns) and with 30 µM INA (black columns) were measured. The genotypes of the analyzed plants are indicated. Values are the means of the activities of the indicated number (#) of independent lines. Luciferase activities are expressed as relative light units (RLU) per µg of total protein. Error bars represent the standard deviation of two independent experiments. Experimental conditions are as previously reported in reference ¹⁵. wt, wild-type.

In conclusion, we have shown that changing the configuration of two TGA binding sites within the PR-1 promoter disconnects the promoter from is regulation by SNI1 and INA-activated NPR1, although its expression still depends on clade II TGA factors. It remains to be determined, whether other strong transcriptional activators at the as-1 element of the CaMV 35S promoter compete with the access of the NPR1 protein and/or whether NPR1 requires a certain topology of TGA factors bound to the DNA to be able to recognize them as a target.

Addendum to: Pape S, Thurow C, Gatz C. The Arabidopsis thaliana PR-1 promoter contains multiple integration sites for the co-activator NPR1 and the repressor SNI1. Plant Physiol. 2010;154:1805–1818. doi: 10.1104/pp.110.165563.