Arabidopsis WRKY38 and WRKY62 transcription factors interact with histone deacetylase 19 in basal defense

Abstract

Arabidopsis thaliana WRKY38 and WRKY62, encoding two structurally similar type III WRKY transcription factors, are induced in a Nonexpressor of PR Gene1 (NPR1)-dependent manner by salicylic acid (SA) or by virulent Pseudomonas syringae. Disease resistance and SA-regulated Pathogenesis-Related1 (PR1) gene expression are enhanced in the wrky38 and wrky62 single mutants and, to a greater extent, in the double mutants. Overexpression of WRKY38 or WRKY62 reduces disease resistance and PR1 expression. Thus, WRKY38 and WRKY62 function additively as negative regulators of plant basal defense. WRKY38 and WRKY62 interact with Histone Deacetylase 19 (HDA19). Expression of HDA19 is also induced by P. syringae, and the stability of its induced transcripts depends on SA and NPR1 in infected plants. Disruption of HDA19 leads to compromised resistance, whereas its overexpression results in enhanced resistance to P. syringae. Thus, HDA19 has a role opposite from those of WRKY38 and WRKY62 in basal resistance to the bacterial pathogen. Both WRKY38 and WRKY62 are transcriptional activators in plant cells, but their activation activities are abolished by overexpressed HDA19. Interaction of WRKY38 and WRKY62 with HDA19 may act to fine-tune plant basal defense responses.

Figure 1.

Pathogen- and SA-Induced Expression of WRKY38 and WRKY62. (A) Time course of pathogen-induced expression of WRKY38 and WRKY62. Five-week-old Arabidopsis plants (Col-0) were infiltrated with 10 mM MgCl₂ or PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). The infiltrated leaves were collected at the indicated times after inoculation for RNA isolation. RNA gel blot analysis was performed with a ³²P-labeled WRKY38 probe. The blot was stripped and reprobed with the WRKY62 probe. Ethidium bromide staining of rRNA is shown for the assessment of equal loading. (B) SA and NPR1 dependence of pathogen-induced expression of WRKY38 and WRKY62. Five-week-old wild-type (Col-0), npr1-3, and sid2-3 mutant plants were infiltrated with PstDC3000. Leaf collection, RNA isolation, and RNA gel blot analysis of WRKY38 and WRKY62 expression were performed as in (A). (C) Time course of SA-induced expression of WRKY38 and WRKY62. Five-week-old Arabidopsis plants (Col-0) were sprayed with water or SA (1 mM). Leaf collection, RNA isolation, and RNA gel blot analysis of WRKY38 and WRKY62 expression were performed as in (A). (D) NPR1 dependence of SA-induced expression of WRKY38 and WRKY62. Five-week-old wild-type (Col-0) and npr1-3 mutant plants were sprayed with 1 mM SA. Leaf collection, RNA isolation, and RNA gel blot analysis of WRKY38 and WRKY62 expression were performed as in (A). These experiments were performed three times with similar results.

Figure 2.

Altered Responses of the WRKY Mutants to PstDC3000. (A) and (B) Altered bacterial growth in the WRKY mutants. Wild type, single mutants, and double mutants for WRKY38 and WRKY62 were infiltrated with a suspension of PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Samples were taken at 0 (open bars) or 3 (closed bars) DAI to determine bacterial growth. Means and se were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units do not differ at 0 DAI significantly if they are indicated with the same lowercase letter and do not differ significantly at 3 DAI if they are indicated with the same uppercase letter. (C) and (D) Altered disease symptom development in the WRKY mutants. Pathogen inoculation of wild-type and mutant plants was performed as in (A) and (B). Photographs of pairs of representative inoculated leaves were taken at 4 DAI (C) and 5 DAI (D). (E) and (F) Pathogen-induced PR1 expression. Wild type, single mutants, and double mutants for WRKY38 and WRKY62 were infiltrated with a suspension of PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Inoculated leaves were collected at the indicated DAI for RNA isolation. RNA gel blot analysis was performed with ³²P-labeled PR1. These experiments were performed four times with similar results.

Figure 3.

Characterization of WRKY38 and WRKY62 Overexpression Lines. (A) Altered bacterial growth. Wild-type, overexpression line, and npr1 mutant plants were infiltrated with a suspension of PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Samples were taken at 0 (open bars) or 3 (closed bars) DAI to determine the growth of the bacterial pathogen. Means and se were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units do not differ significantly at 0 DAI if they are indicated with the same lowercase letter and do not differ significantly at 3 DAI if they are indicated with the same uppercase letter. (B) Altered disease symptom development. Pathogen inoculation was performed as in (A). Photographs of representative inoculated leaves were taken at 3 DAI. (C) Pathogen-induced PR1 expression. Wild-type, overexpression line, and npr1 mutant plants were infiltrated with a suspension of PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Inoculated leaves were collected at the indicated DAI for RNA isolation. RNA gel blot analysis was performed with ³²P-labeled PR1. These experiments were performed four times with similar results.

Figure 4.

Roles of WRKY38 and WRKY62 in Plant SA Sensitivity for PR1 Induction. Five-week-old wild-type (Col-0), knockout mutant, and overexpression plants for WRKY38 and WRKY62 were sprayed with SA at the indicated concentrations. Leaf samples were collected at the indicated times after spraying for RNA isolation. RNA gel blot analysis was performed with ³²P-labeled PR1. These experiments were performed twice with similar results.

Figure 5.

BiFC Analysis of WRKY Protein Interactions with HDA19. Fluorescence was observed from complementation of the N-terminal part of the YFP fused with WRKY38 (WRKY38-N-YFP) or WRKY62 (WRKY62-N-YFP) with the C-terminal part of the YFP fused with HDA19 (HDA19-C-YFP) and colocalized with DAPI stains in the nuclear compartment of tobacco leaf epidermal cells. No fluorescence was observed when WRKY38-N-YFP or WRKY62-N-YFP was coexpressed with unfused C-YFP or when unfused N-YFP was coexpressed with HDA19-C-YFP. These experiments were performed three times with similar results.

Figure 6.

Expression of HDA19. (A) Time course of pathogen-induced expression of HDA19. Five-week-old Arabidopsis plants (Col-0) were infiltrated with 10 mM MgCl₂ or PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). The infiltrated leaves were collected at the indicated times after inoculation for RNA isolation. RNA gel blot analysis was performed with a ³²P-labeled HDA19 fragment. (B) Time course of induced expression of HDA19 by SA, 1-aminocyclopropane-1-carboxylic acid (ACC), and MeJA. Five-week-old Arabidopsis plants (Col-0) were sprayed with SA (1 mM), ACC (0.1 mM), and MeJA (0.1 mM). Leaf collection, RNA isolation, and RNA gel blot analysis of HDA19 expression were performed as in (A). (C) Pathogen-induced expression of HDA19 in defense signaling mutants. Five-week-old wild-type (Col-0), sid2-3, npr1-3, coi1-1, and ein2-1 mutant plants were infiltrated with a suspension of PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Leaf collection, RNA isolation, and RNA gel blot analysis of HDA19 expression were performed as in (A). The experiments were performed three times with similar results.

Figure 7.

Altered Responses to P. syringae by the Mutant and Overexpression Plants for HDA19. (A) and (B) Altered bacterial growth. The wild type (Col-0), hda19 mutants (A), and the overexpression lines (B) were infiltrated with a suspension of PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Samples were taken at 0 (open bars) or 3 (closed bars) DAI to determine the growth of the bacterial pathogen. Means and se were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units do not differ significantly at 0 DAI if they are indicated with the same lowercase letter and do not differ significantly at 3 DAI if they are indicated with the same uppercase letter. (C) and (D) Altered disease symptom development. Pathogen inoculation was performed as in (A) and (B). Photographs of representative inoculated leaves to determine altered disease responses of the hda19 mutants (C) were taken at 3 DAI. Photographs for the overexpression line (D) were taken at 4 DAI. (E) and (F) Pathogen-induced PR1 expression. Wild-type, hda19, and HDA19-overexpressing plants were infiltrated with a suspension of PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Inoculated leaves were collected at the indicated times after inoculation for RNA isolation. RNA gel blot analysis was performed with ³²P-labeled PR1. These experiments were performed four times with similar results.

Figure 8.

Antagonism of the Transcriptional Activation Activity of WRKY38 and WRKY62 by HDA19. Effects of overexpressed HDA19 and HDA19m on the transcriptional activation activity of WRKY38 and WRKY62. The HDA19- or HDA19m-overexpressing line was crossed to lines harboring both the GUS reporter and one of the five tested effectors. A transgenic line containing an empty vector was also crossed to the same GUS/effector double transformants as controls. The ratios of GUS activities were calculated from the GUS activities determined in the leaves harvested 18 h after DEX treatment (+) over those determined prior to DEX treatment (−).

Figure 9.

Altered Responses to P. syringae by Overexpression of WRKY62 in the Wild-Type and hda19 Mutant Backgrounds. (A) Altered bacterial growth. Wild-type, 35S:WRKY62-L1, hda19-3, and hda19-3/35S:WRKY62 plants were infiltrated with a suspension of PstDC3000 (OD₆₀₀ = 0.0001 in 10 mM MgCl₂). Samples were taken at 0 (open bars) or 3 (closed bars) DAI to determine the growth of the bacterial pathogen. Means and se were calculated from 10 plants for each treatment. According to Duncan's multiple range test (P = 0.05), means of colony-forming units at 0 DAI do not differ significantly if they are indicated with the same lowercase letter, and means of colony-forming units at 3 DAI do not differ significantly if they are indicated with the same uppercase letter. (B) Altered disease symptom development. Pathogen inoculation was performed as in (A). Photographs of representative inoculated leaves were taken at 3 DAI.

Figure 10.

A Model for the Functional Interactions of WRKY38 and WRKY62 with HDA19 during Plant Defense Responses. Infection by P. syringae leads to the accumulation of SA, which induces the expression of WRKY38 and WRKY62 in an NPR1-dependent manner. WRKY38 and WRKY62, as transcriptional activators, activate the expression of unknown regulatory genes that, in turn, repress the expression of defense genes (e.g., PR1) and basal disease resistance. Infection by P. syringae also induces HDA19, whose transcripts are stabilized by SA- and NPR1-mediated signaling. HDA19 represses the transcriptional activation activity of WRKY38 and WRKY62 and, as a result, reduces the activation of negative regulatory genes of plant basal defense by the two WRKY transcription factors.