Targets of AtWRKY6 regulation during plant senescence and pathogen defense

Abstract

In Arabidopsis, WRKY factors comprise a large gene family of plant-specific transcriptional regulators controlling several types of plant stress responses. To understand the regulatory role of WRKY proteins during such processes, we identified targets of the senescence- and defense-associated WRKY6 factor. WRKY6 was found to suppress its own promoter activity as well as that of a closely related WRKY family member, indicating negative autoregulation. On the other hand, WRKY6 positively influenced the senescence- and pathogen defense-associated PR1 promoter activity, most likely involving NPR1 function. One novel identified target gene, SIRK, encodes a receptor-like protein kinase, whose developmental expression is strongly induced specifically during leaf senescence. The transcriptional activation of SIRK is dependent on WRKY6 function. Senescing leaves of wrky6 knockout mutants showed a drastic reduction, and green leaves of WRKY6 overexpression lines showed clearly elevated SIRK transcript levels. Furthermore, the SIRK gene promoter was specifically activated by WRKY6 in vivo, functioning very likely through direct W-box interactions.

Figure 1

Identification of wrky6 knockout mutants. (A) Schematic representation of the Arabidopsis WRKY6 gene. Exons (boxes) and introns (lines) are indicated. The regions of the leucine zipper (light gray) and the WRKY domain (dark gray) as well as the position of the En-1 insertion (triangle) are shown. (B) Expression analysis of WRKY6 in senescent leaves of two knockout mutants, wrky6-1 and wrky6-2, compared with wild type (WT). The 28S rRNA band of the ethidium bromide-stained gel is shown for loading control.

Figure 2

WRKY6 overexpression lines. (A) Expression of WRKY6 in nine independent T₂ transgenic plants carrying a CaMV 35S::WRKY6 construct compared with wild type (WT). The ethidium bromide-stained 28S rRNA band is shown for loading control. (B) Dosage-dependence of the mutant phenotypes. Plants of the overexpressor lines CaMV 35S::WRKY6-3, -5, and -9 showing increasing levels of WRKY6 transcript are compared with wild type. (C) Comparison of the strongest overexpressor line CaMV 35S::WRKY6-9 with wild type. Plants were grown either under short-day (SD) or long-day (LD) conditions.

Figure 3

Repressor activity of WRKY6. (A) WRKY6 promoter-driven GUS reporter gene activity (6p) was monitored in wild-type plants, in the knockout mutants wrky6-1 and wrky6-2, as well as in the overexpressor line CaMV 35S::WRKY6-9 (-9). Shown are GUS-stained roots (R), senescing leaves (SL), and mature leaves 5 h postinoculation with 10⁸-CFU bacterial solution (+Ps avrRPM1) or with 10 mM MgCl₂ (control). (B) Transient cotransfection assays with different target gene promoters and WRKY6. Presented are relative activities of WRKY6 promoter (6p), WRKY42 promoter (42p), tetramerized W₂-box (4xW₂), and mutated tetramerized W₂-box (4xmW₂) driven GUS reporter gene constructs after transfection of cell culture-derived Arabidopsis protoplasts. Transient transfections were done either with reporter constructs alone or combined with an effector construct containing a CaMV 35S-driven WRKY6 cDNA (WRKY6) or a CaMV 35S-driven fusion of the WRKY6 cDNA to the VP16 activation domain (WRKY6–VP16). Each bar represents the median of four independent transfections. Normalized GUS values were obtained using a control luciferase plasmid for standardization. Relative fold induction or repression values ⩾twofold are depicted.

Figure 4

Effect of WRKY6 overexpression on PR1 and NPR1. (A) Activation of PR1-promoter-driven reporter gene activity by WRKY6. GUS activity observed in representative leaves of transgenic plants harboring the PR1 promoter-driven reporter gene in wild type (PR1p), wrky6-1 mutant, and WRKY6-9 (-9) overexpressor. GUS-stained mature leaves are shown either 5 h postinoculation with 10⁸-CFU bacterial solution (+Ps avrRPM1) or with 10 mM MgCl₂ (control), as well as senescing leaves (SL). (B) RNA blot analysis of PR1 in mature leaves of wild-type (WT) and WRKY6 overexpression lines (-9). The 28S rRNA ethidium bromide-stained band is shown as loading control. (C) RT–PCR analysis of NPR1 in mature leaves of wild-type (WT) and WRKY6 overexpression lines (-9). For control RPL4 (ribosomal protein L4) transcript was amplified.

Figure 5

Dependence of SIRK gene expression on WRKY6. (A) Expression analysis of SIRK transcripts in different tissues, including roots (R), young leaves (YL), mature leaves (ML), senescing leaves (SL), stems (ST), flowers (F), or siliques (SI), derived from wild-type plants (WT), the wrky6-2 knockout mutant, or from the overexpressor lines -3, -5, and -9. The 28S rRNA band of the ethidium bromide-stained gel is shown as loading control. (B) Effect of the bacterial-derived elicitor flagellin 22 (Flg22) on SIRK promoter activity in transient transfection assays. Presented are relative activities of the SIRK promoter (SIRKp) driven GUS reporter gene after addition of the active (Flg22) and inactive (Flg15Δ5) forms of the elicitor. Each bar represents the median of four independent transfections. Normalized GUS values were obtained using a control luciferase plasmid.

Figure 6

Specificity of the WRKY6–SIRK promoter interaction. (A) Representative leaves of four independent biolistic-mediated transient transfection assays using a 0.9-kb SIRK promoter-driven GUS reporter gene (SIRKp). Bombardments of detached mature leaves of wrky6-2 knockout mutants and the 35S::WRKY6-9 overexpressor line were done either with SIRKp alone or in combination with a CaMV 35S-driven WRKY6 cDNA construct (WRKY6). (B) Results of bombardment assays using various SIRK-promoter deletion/mutation GUS constructs as depicted schematically on the left. The respective sizes relative to the transcription start site (bent arrow) as determined by 5′RACE (O. Noubibou, pers. comm.; GenBank accession no. AF486619) are indicated by arrows. W boxes (TGACC/T) are marked by blue, TGACA motifs by orange rectangles, and mutated elements by crosses. SIRK promoter deletion constructs, Δ1, Δ2, Δ3, and mutation constructs, Δ3m2 and Δ3m1/2/3, were bombarded in detached mature wild-type leaves either alone (control) or in combinations with the effectors (+WRKY6, +WRKY52, +PcWRKY1, +WRKY42). Activation of the SIRK promoter constructs is indicated by relative values ranging from − (no GUS staining), +/− (1–5 GUS-positive cells in total), ++ (>20 GUS-positive cells per leave), to +++ (>50 GUS-positive cells per leave). Three independent experiments were performed.