Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression

Abstract

The Arabidopsis NPR1 gene is a positive regulator of inducible plant disease resistance. Expression of NPR1 is induced by pathogen infection or treatment with defense-inducing compounds such as salicylic acid (SA). Transgenic plants overexpressing NPR1 exhibit enhanced resistance to a broad spectrum of microbial pathogens, whereas plants underexpressing the gene are more susceptible to pathogen infection. These results suggest that regulation of NPR1 gene expression is important for the activation of plant defense responses. In the present study, we report the identification of W-box sequences in the promoter region of the NPR1 gene that are recognized specifically by SA-induced WRKY DNA binding proteins from Arabidopsis. Mutations in these W-box sequences abolished their recognition by WRKY DNA binding proteins, rendered the promoter unable to activate a downstream reporter gene, and compromised the ability of NPR1 to complement npr1 mutants for SA-induced defense gene expression and disease resistance. These results provide strong evidence that certain WRKY genes act upstream of NPR1 and positively regulate its expression during the activation of plant defense responses. Consistent with this model, we found that SA-induced expression of a number of WRKY genes was independent of NPR1.

Figure 1.

Scheme of The Arabidopsis NPR1 Gene Promoter.

Figure 2.

Sequence-Specific Recognition of the W-Box Elements in the NPR1 Gene Promoter by W-Box Binding Proteins. (A) Sequences of the PN1 probe (the −99 to −132 region of the NPR1 gene promoter) and the mPN1 probe with the TTGAC sequences mutated into TTGAA. Underlining signifies W-box sequences. Asterisks represent the mutated bases in the W-box elements. (B) Sequence-specific binding of PN1 by the AtWRKY18 protein (lane 2). Change of the TTGAC sequences to TTGAA in the mPN1 probe drastically decreased the intensities of retarded bands (lane 4). No retarded bands were detected in the absence of proteins (lanes 1 and 3). (C) The PN1 probe recognized DNA binding activities present at low levels in untreated plants (lane 1) and induced in plants treated with SA (2 mM for 6 hr) (lane 2). The mPN1 probe failed to recognize these DNA binding activities from untreated (lane 3) or SA-treated plants (lane 4).

Figure 3.

Antibodies against the Conserved WRKYGQK Sequence of WRKY Proteins Inhibit Binding of AtWRKY18 and SA-Induced W-Box Binding Activities to the PN1 Probe. (A) Sequence-specific binding of PN1 by the recombinant AtWRKY18 protein without added antibodies (lane 1) or with added preimmune antiserum IgG (lane 2) or affinity-purified WRKY antibodies (lane 3). (B) Sequence-specific binding of PN1 by SA-induced W-box binding activities in the nuclear extracts isolated from SA-treated Arabidopsis plants without added antibodies (lane 1) or with added preimmune antiserum IgG (lane 2) or affinity-purified WRKY antibodies (lane 3).

Figure 4.

Importance of the W-Box Elements for NPR1 Gene Promoter Activity. (A) Constructs of NPR1-GUS (the +1 to −2419 promoter sequence of the NPR1 gene fused to the GUS reporter gene), mNPR1-GUS (the +1 to −2419 promoter sequence with mutated TTGAA sequences fused to the GUS reporter gene), and −GUS (the GUS reporter gene with no upstream promoter). Underlining signifies W-box sequences. Asterisks represent the mutated bases in the W-box elements. (B) RNA gel blotting of GUS transcripts in transgenic Arabidopsis plants harboring the three promoter constructs shown in (A) before SA treatment (−SA) or 24 hr after SA treatment (+SA). Ethidium bromide staining of rRNA is shown to demonstrate equal loading of RNA in each lane. (C) GUS activities in transgenic Arabidopsis plants harboring the three promoter constructs shown in (A) before SA treatment (−SA) or 24 hr after SA treatment (+SA). MU, 4-methylumbelliferone. Error bars indicate ±se.

Figure 5.

Transcription of the GUS Transgene under the Control of the Wild-Type or Mutant NPR1 Gene Promoter. ³²P-CTP–labeled transcripts were prepared from nuclear run-on assays of nuclei isolated from transgenic plants transformed with the NPR1-GUS or mNPR1-GUS construct before (−) and after (+) SA spraying (2 mM for 12 hr). The filters contained immobilized, linearized plasmids containing sequences for GUS and AphII genes. The AphII gene under the control of the nos promoter conferred kanamycin resistance in the transgenic plants, and its transcription was used as an internal control.

Figure 6.

Complementation for PR1 Gene Expression. (A) Structures of the NPR1, mNPR1, and mNPR1a genes used for complementation. mNPR1 contains a mutant NPR1 gene promoter with the three TTGAC sequences mutated into TTGAA. mNPR1a contains a mutant NPR1 gene promoter with the two canonical W-box sequences mutated from TTGAC to TTGAA. Underlining signifies W-box sequences. Asterisks represent the mutated bases in the W-box elements. (B) RNA gel blotting of PR1 gene expression in the wild type (WT), npr1-3, and npr1-3 transformants with NPR1 or mNPR1. RNA samples were prepared from 4-week-old plants 24 hr after treatment with 2 mM SA. Ethidium bromide staining of rRNA is shown to demonstrate equal loading of RNA in each lane. (C) RNA gel blotting of PR1 gene expression 24 hr after SA treatment (2 mM) in the wild type (WT), npr1-3, and npr1-3 transformants with NPR1 or mNPR1a.

Figure 7.

Complementation for Response to P. syringae. Wild type (WT), npr1-3, and npr1-3 transformants with NPR1, mNPR1, or mNPR1a were inoculated with P. syringae pv tomato DC3000 (OD₆₀₀ = 0.001), and samples were taken 3 days after infection. cfu, colony-forming units. Error bars indicate ±se.

Figure 8.

Induction of Arabidopsis WRKY Genes by SA. Arabidopsis wild-type (WT) and npr1-3 mutant plants were sprayed with 2 mM SA, and leaves were harvested at the indicated times after treatment for preparation of total RNA. Five separate blots were prepared from the same preparation of total RNA and hybridized with the first set of five separate WRKY gene probes. The blots were subsequently stripped and rehybridized with the second set of five WRKY gene probes. Ethidium bromide staining of rRNA for one of the five identical blots is shown to demonstrate equal loading of RNA in each lane.