NPR1 modulates cross-talk between salicylate- and jasmonate-dependent defense pathways through a novel function in the cytosol

Abstract

Plant defenses against pathogens and insects are regulated differentially by cross-communicating signal transduction pathways in which salicylic acid (SA) and jasmonic acid (JA) play key roles. In this study, we investigated the molecular mechanism of the antagonistic effect of SA on JA signaling. Arabidopsis plants unable to accumulate SA produced 25-fold higher levels of JA and showed enhanced expression of the JA-responsive genes LOX2, PDF1.2, and VSP in response to infection by Pseudomonas syringae pv tomato DC3000, indicating that in wild-type plants, pathogen-induced SA accumulation is associated with the suppression of JA signaling. Analysis of the Arabidopsis mutant npr1, which is impaired in SA signal transduction, revealed that the antagonistic effect of SA on JA signaling requires the regulatory protein NPR1. Nuclear localization of NPR1, which is essential for SA-mediated defense gene expression, is not required for the suppression of JA signaling, indicating that cross-talk between SA and JA is modulated through a novel function of NPR1 in the cytosol.

Figure 1.

Enhanced JA Accumulation and JA-Responsive Gene Expression in Pseudomonas DC3000–Infected Arabidopsis NahG Plants. (A) Endogenous levels of free and conjugated SA in wild-type Col-0 (open squares) and SA-degrading NahG (open circles) plants after inoculation with the bacterial pathogen Pseudomonas DC3000. Error bars represent se (n = 5). (B) JA levels in Pseudomonas DC3000–infected Col-0 (open squares) and NahG (open circles) plants. The experiment was performed three times with similar results. (C) RNA gel blot analysis of SA-responsive (PR-1) and JA-responsive (LOX2, VSP, and PDF1.2) genes during pathogen infection. Plants were inoculated with virulent Pseudomonas DC3000 by dipping the leaves in a bacterial suspension containing 2.5 × 10⁷ colony-forming units/mL. At different days after inoculation (dpi), leaves were harvested for SA, JA, and RNA extraction. To check for equal loading, RNA gel blots were stripped and hybridized with a gene-specific probe for β-tubulin (TUB). Transcript levels of the constitutively expressed TUB gene decreased during the course of the infection process as a result of progressing cell death. FW, fresh weight.

Figure 2.

Effect of the Cosuppression of LOX2 on Pathogen-Induced JA Production in Arabidopsis S-12 Plants. JA levels in wild-type Col-0 and LOX2-deprived S-12 plants at 2 days after inoculation with virulent Pseudomonas DC3000 or avirulent Pseudomonas DC3000/avrRpt2. Plants were inoculated by pressure-infiltrating the leaves with a bacterial suspension containing 10⁷ colony-forming units/mL. FW, fresh weight; n.i., not inoculated.

Figure 3.

Effect of the npr1 Mutation on the SA-Mediated Suppression of JA-Responsive Gene Expression. RNA gel blot analysis of SA-responsive (PR-1) and JA-responsive (LOX2, VSP, and PDF1.2) genes after exogenous application of MeJA, SA, or a combination of both in Arabidopsis wild-type Col-0 and mutant npr1 plants. Five-week-old plants were induced by dipping the leaves in a 0.015% (v/v) Silwet L-77 solution containing 1 mM SA, 0.1 mM MeJA, or a combination of both. Two days later, leaves were harvested for RNA extraction. Equal loading of RNA samples was checked using a probe for the constitutively expressed β-tubulin (TUB) gene.

Figure 4.

In Vitro Binding of TGA2 to the TGACG Motif in the Promoter of the Arabidopsis PDF1.2 Gene. (A) Oligonucleotides used in the gel mobility shift assay. The as-1 probe contains two inverted TGACG motifs and represents the SA-responsive as-1 element in the promoter of the Arabidopsis PR-1 gene. The pdf probe resembles the wild-type sequence surrounding the TGACG motif in the JA-responsive PDF1.2 gene. The mpdf probe is similar to the pdf probe but contains three point mutations in the TGACG motif. (B) Gel mobility shift assay to test the binding of TGA2 to the TGACG motif in the PDF1.2 promoter. Binding reactions contained 7 × 10⁴ cpm of ³²P-labeled probe incubated with 1 μg of either a control protein preparation (lanes 1, 3, and 14) or partly purified TGA2 (lanes 2, 4 to 13, and 15). The specificity of the binding of TGA2 to the TGACG motif in the PDF1.2 promoter was tested by the addition of 100×, 50×, 10×, 1×, or 0.5× molar excess amounts of unlabeled pdf (lanes 5 to 8) or mpdf (lanes 9 to 13) competitor probes. The double asterisks indicate specific binding of TGA2 to the oligonucleotide probe, and the single asterisk indicates specific TGA2 binding and dimerization as a result of the presence of two TGACG motifs in the oligonucleotide probe. FP, free probe.

Figure 5.

The TGACG Motif Is Not Required for SA-Mediated Suppression of the MeJA-Induced Activation of the Arabidopsis PDF1.2 Promoter. (A) Scheme of the 5′ deletions of the PDF1.2 promoter fused to the uidA gene. Red bars represent the TGACG motif located at positions −445 to −441 upstream of the predicted translational start of the uidA reporter gene. (B) Histochemical staining of GUS activity in seedlings of transgenic Arabidopsis lines PG15, which constitutively expresses the uidA gene, and P1 to P6, which contain 5′ deletions of the Arabidopsis PDF1.2 promoter fused to the uidA reporter gene. Twelve-day-old seedlings grown on Murashige and Skoog (1962) medium were transferred to fresh medium containing 0.5 mM SA, 0.02 mM MeJA, or a combination of both and stained for GUS activity 2 days later.

Figure 6.

SA-Mediated Suppression of MeJA-Induced PDF1.2 Expression through a Function of NPR1 in the Cytosol. RNA gel blot analysis of the SA-responsive PR-1 gene and the MeJA-inducible PDF1.2 gene in wild-type Col-0, mutant npr1, and the NPR1-overexpressing transformants 35S:NPR1 (in npr1) and 35S:NPR1-HBD (in npr1). Seedlings were grown for 12 days on Murashige and Skoog (1962) medium with or without DEX (5 μM). Subsequently, the seedlings were transferred to fresh medium with (+) or without (−) 0.5 mM SA and 0.02 mM MeJA. Two days after induction, the seedlings were harvested for RNA gel blot analysis. Equal loading of RNA samples was checked by staining rRNA bands with ethidium bromide.

Figure 7.

Proposed Model for Cytosolic NPR1 as a Modulator of Cross-Talk between SA- and JA-Dependent Plant Defense Responses. In wild-type Col-0 plants, SA accumulates after pathogen infection, resulting in the activation of NPR1 (asterisk). Activated NPR1 then is localized to the nucleus, where it interacts with TGA transcription factors, ultimately leading to the activation of SA-responsive PR genes. In the cytosol, activated NPR1 negatively regulates JA-responsive gene expression, possibly by inhibiting positive regulators of JA-responsive genes or by facilitating the delivery of negative regulators of JA-responsive genes to the nucleus. The suppression of JA-responsive genes that encode enzymes from the octadecanoid pathways, such as LOX2, ultimately results in the inhibition of JA formation.