Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae

Abstract

Background: A common feature of plant defense responses is the transcriptional regulation of a large number of genes upon pathogen infection or treatment with pathogen elicitors. A large body of evidence suggests that plant WRKY transcription factors are involved in plant defense including transcriptional regulation of plant host genes in response to pathogen infection. However, there is only limited information about the roles of specific WRKY DNA-binding transcription factors in plant defense.

Results: We analyzed the role of the WRKY25 transcription factor from Arabidopsis in plant defense against the bacterial pathogen Pseudomonas syringae. WRKY25 protein recognizes the TTGACC W-box sequences and its translational fusion with green fluorescent protein is localized to the nucleus. WRKY25 expression is responsive to general environmental stress. Analysis of stress-induced WRKY25 in the defense signaling mutants npr1, sid2, ein2 and coi1 further indicated that this gene is positively regulated by the salicylic acid (SA) signaling pathway and negatively regulated by the jasmonic acid signaling pathway. Two independent T-DNA insertion mutants for WRKY25 supported normal growth of a virulent strain of P. syringae but developed reduced disease symptoms after infection. By contrast, Arabidopsis constitutively overexpressing WRKY25 supported enhanced growth of P. syringae and displayed increased disease symptom severity as compared to wild-type plants. These WRKY25-overexpressing plants also displayed reduced expression of the SA-regulated PR1 gene after the pathogen infection, despite normal levels of free SA.

Conclusion: The nuclear localization and sequence-specific DNA-binding activity support that WRKY25 functions as a transcription factor. Based on analysis of both T-DNA insertion mutants and transgenic overexpression lines, stress-induced WRKY25 functions as a negative regulator of SA-mediated defense responses to P. syringae. This proposed role is consistent with the recent finding that WRKY25 is a substrate of Arabidopsis MAP kinase 4, a repressor of SA-dependent defense responses.

Figure 1

Sequence and DNA-binding Activity of WRKY25. A. Amino acid sequence of WRKY25. The two WRKY motifs are indicated with the highly conserved WRKYGQK sequence and the residues forming the C₂H₂ zinc fingers underlined. B. Oligonucleotides used in the electrophoretic mobility shifting assay (EMSA). The Pchn5 probe contains two direct W-box repeats, while in the mPchn5 probe, the TTGACC sequences are mutated to TTGAAC. The wild-type and mutated W-box sequences are underlined. C. EMSA to test binding of recombinant WRKY25 to the W box motif in the Pchn5 probe. Binding reactions containing WRKY25 and Pchn5 produced two major DNA/protein complexes, which are indicated by arrows. Change of the TTGACC to TTGAAC in the mPchn5 probe abolished WRKY25 binding. No retarded bands were detected in the absence of the recombinant WRKY25 protein.

Figure 2

Localization of WRKY25 in vivo. WRKY25 was fused to GFP to yield W25-GFP; this chimeric protein was localized to the nucleus of onion epidermal cells. GFP alone was detected in both the nucleus and the cytoplasm due to its small size. Bright-field image of the onion epidermal cells are shown in the top panels.

Figure 3

Expression of WRKY25. A. RNA blot analysis of WRKY25 expression in 5-week old wild-type or mutant Arabidopsis. Two fully expanded leaves were infiltrated with 10 mM MgCl₂ (mock inoculation) or Psm ES4326 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂) and harvested at the indicated times after inoculation. After separation on a gel and blotting to nylon membrane, the blot was probed with a WRKY25-specific DNA fragment. B. Induced WRKY25 expression in 5-week old wild-type Arabidopsis plants sprayed with H₂O, 2 mM SA, 0.1 mM ACC or 0.1 mM methyl JA. Leaves were harvested at the indicated times after treatment and used for preparation of total RNA and RNA blotting. Ethidium bromide stained rRNA was used as a loading control. The experiments were repeated twice with similar results.

Figure 4

Characterization of wrky25 T-DNA insertion mutants and transgenic overexpressing plants. A. Diagram of WRKY25 gene and its T-DNA insertion mutants. B. RNA blot analysis of wrky25 mutant lines. Four hours after inoculation with PsmES4326 (OD₆₀₀ = 0.0001), the inoculated leaves from two wild-type plants (Col-0) or four wrky25-1 and wrky25-2 mutant plants were harvested and total RNA was isolated. After separation and blotting to a nylon membrane, the blot was probed with a full-length WRKY25 cDNA fragment (upper panel) or a WRKY25 DNA fragment corresponding to the region downstream of the T-DNA insertion in the wrky25-2 mutant (lower panel). Ethidium bromide stained rRNA was used as a loading control. C. RNA blot analysis of WRKY25 expression in transgenic plants constitutively overexpressing WRKY25. RNA samples were prepared from leaves of two wild-type plants (Col-0) or four plants from each tansgenic 35S:WRKY25 line and probed with a WRKY25-specific DNA probe. Lines 12 and 18 expressed elevated levels of WRKY25 and contain a single T-DNA insertion based on the ratio of antibiotic-resistant progeny. F3 homozygous progeny plants of the two lines were used for further analyses.

Figure 5

Responses of the wrky25 mutant and transgenic 35S:WRKY25 plants to P. syringae. A. Bacterial titer in wild type (Col-0), wrky25 insertion mutant and F3 progeny of transgenic 35S:WRKY25 plants (line 12) at 3 days post inoculation (dpi) with PsmES4326 (OD₆₀₀ = 0.0001). The means and standard errors were calculated from six replicates. Analysis of F3 progeny from 35S:WRKY25 line 18 yielded similar results to those observed for 35S:WRKY25 line 12. B. Disease symptom development in wild type (Col-0), wrky25 mutants and transgenic 35S:WRKY25 plants inoculated with PsmES4326 (OD₆₀₀ = 0.0001). Pictures of representative inoculated leaves were taken at 4 dpi. These experiments were repeated two additional times with similar results.

Figure 6

Pathogen-induced PR1 expression and SA accumulation. A. RNA blot analysis of PR1 expression in wild type (Col-0), F3 progeny of transgenic 35S:WRKY25 plants (line 12) and wrky25 mutants following inoculation with PsmES4326 (OD₆₀₀ = 0.0001). Total RNA was isolated from inoculated leaves harvested at indicated times after inoculation and probed with a PR1 probe. Ethidium bromide-stained rRNA was used as a loading control. The experiment was repeated two additional times with similar results. B. Determination of free and SA glucoside (SAG) levels in wild type (Col-0), transgenic 35S:WRKY25 plants (line 12) and the wrky25 mutants after inoculation with PsmES4326 (OD₆₀₀ = 0.0001). Inoculated leaves were harvested at indicated times for SA and SA glucoside (SAG) determination. The means and standard errors were calculated from 2–3 replicate samples. FW, fresh weight.