NPR1 Promotes Its Own and Target Gene Expression in Plant Defense by Recruiting CDK8

Abstract

NPR1 (NONEXPRESSER OF PR GENES1) functions as a master regulator of the plant hormone salicylic acid (SA) signaling and plays an essential role in plant immunity. In the nucleus, NPR1 interacts with transcription factors to induce the expression of PR (PATHOGENESIS-RELATED) genes and thereby promote defense responses. However, the underlying molecular mechanism of PR gene activation is poorly understood. Furthermore, despite the importance of NPR1 in plant immunity, the regulation of NPR1 expression has not been extensively studied. Here, we show that SA promotes the interaction of NPR1 with both CDK8 (CYCLIN-DEPENDENT KINASE8) and WRKY18 (WRKY DNA-BINDING PROTEIN18) in Arabidopsis (Arabidopsis thaliana). NPR1 recruits CDK8 and WRKY18 to the NPR1 promoter, facilitating its own expression. Intriguingly, CDK8 and its associated Mediator subunits positively regulate NPR1 and PR1 expression and play a pivotal role in local and systemic immunity. Moreover, CDK8 interacts with WRKY6, WRKY18, and TGA transcription factors and brings RNA polymerase II to NPR1 and PR1 promoters and coding regions to facilitate their expression. Our studies reveal a mechanism in which NPR1 recruits CDK8, WRKY18, and TGA transcription factors along with RNA polymerase II in the presence of SA and thereby facilitates its own and target gene expression for the establishment of plant immunity.

Figure 1.

NPR1 protein facilitates its own expression and binds to its own promoter. A, NPR1 gene expression level in Col-0 wild type, npr1-1, and npr1-2. The plants were sprayed with 0.5 mm SA or water as a control for 4 h. Leaves were collected for gene expression analysis. B, NPR1 protein level in Col-0 wild type, npr1-1, and npr1-2. Plants were treated as in A. Western blots were probed with an anti-NPR1 antibody. C, npr1-2 gene expression level in Col-0 wild type, npr1-2, and NPR1-GFP npr1-2. Seedlings were treated with 0.5 mm SA or water as a control for 4 h. Samples were assayed for gene expression analysis with primers specifically recognizing npr1-2 but not NPR1. The expression levels of NPR1 and npr1-2 were normalized to UBQ5 expression. ND, Not detected. D, NPR1 associates with its own promoter. Seedlings of transgenic GFP npr1-2 and NPR1-GFP npr1-2 were treated with 0.5 mm SA for 4 h. ChIP assay was performed using GFP-Trap (ChromoTek) or control beads with the following primers: -2000bp, amplifies upstream of the NPR1 promoter at the −2,000-bp region; W box, amplifies the sequence containing the W-box at the NPR1 promoter; Actin2 was used as a negative control. All experiments were repeated at least two times with similar results. Data represent means of three independent samples with se. Asterisks indicate significant differences (Student’s t test, *, P < 0.05). Ponceau S, Ponceau staining.

Figure 2.

The interaction between NPR1 and WRKY18 is substantially enhanced by SA. A, NPR1 but not npr1-1 or npr1-2 interacts with WRKY18 in Y2H assays. Yeast strain AH109 was cotransformed with pGBKT7-WRKY6 or WRKY18 and pGADT7-NPR1, npr1-1, npr1-2, or an empty vector (EV). Cotransformed yeast selected from a double dropout (DD) plate (−Leu, −Trp) was recultured, diluted to OD_{600 nm} = 1, 0.1, or 0.01, and then 10 µL of the diluted liquid cultures was placed onto a DD plate and a TD plate (−Leu, −Trp, −His) with or without 200 μm SA. B, Co-IP between HA-NPR1 and GFP, WRKY6-GFP, or WRKY18-GFP. HA-NPR1 and GFP, WRKY6-GFP, or WRKY18-GFP proteins, expressed in N. benthamiana leaves by agroinfiltration, were immunoprecipitated with GFP-Trap for 2 h. The beads were washed, eluted with 2× loading buffer, and immunoblotted with an anti-GFP or an anti-HA antibody. C, In vitro pull-down between 6xHis-MPB-NPR1 and WRKY18-GFP. GFP or WRKY18-GFP was immunoprecipitated from N. benthamiana leaf extracts with GFP-Trap. The beads were washed and incubated with purified 6xHis-MBP-NPR1 with or without 200 μm SA. After overnight incubation, beads were washed three times with washing buffer, then eluted, and detected with anti-GFP or anti-6xHis antibody. The number beneath each blot indicates the relative strength of the band. The experiments were repeated two times with similar results.

Figure 3.

SA promotes the interaction between NPR1 and CDK8. A, NPR1 but not npr1-1 or npr1-2 interacts with CDK8 in Y2H assays. Y2H assays were carried out using CDK8 as bait (in pGBKT7 vector with a DNA-binding domain) and NPR1, npr1-1, npr1-2, or an EV as prey (in pGADT7 vector with an activation domain). Cotransformed yeast selected from a DD plate were recultured, diluted to OD_{600 nm} = 1, 0.1, or 0.01, and then 10 µL of the diluted liquid cultures was placed onto a DD plate and a TD plate with or without 1 mm 3-AT or 200 μm SA. B, In vitro pull-down assay between GST-NPR1 and 6xHis-MBP-CDK8. Purified 6xHis-MBP-CDK8 was incubated with magnetic beads conjugated with GST or GST-NPR1 with or without 200 μm SA. After overnight incubation, beads were washed four times with washing buffer, then eluted, and detected with anti-GST or anti-6xHis antibody. C, Co-IP between HA-CDK8 and NPR1-GFP proteins in N. benthamiana NahG transgenic plants. Proteins, extracted from N. benthamiana leaves transiently expressing NPR1-GFP and HA-CDK8 or GFP and HA-CDK8 proteins by agroinfiltration, were immunoprecipitated with GFP-Trap with or without 200 μm INA overnight. The beads were washed, eluted with 2× loading buffer, and immunoblotted with an anti-GFP or an anti-HA antibody. The number beneath each blot indicates the relative strength of the band. The experiments were repeated two times with similar results.

Figure 4.

SAR and defense gene expression are compromised in the cdk8 mutants. A, CDK8 gene expression level during the time-course infection of the avirulent pathogen. Col-0 plants were infiltrated with Psm avrRpt2 (OD_{600 nm} = 0.005) or MgCl₂ buffer as a control. Leaf samples were collected at the indicated times for gene expression analysis. hpi, Hours post inoculation. B, SAR phenotypes in Col-0 wild-type, npr1-2, cdk8-4, and cdk8-1 mutant plants. Two lower leaves were infiltrated with Psm avrRpt2 (OD_{600 nm} = 0.02; SAR+) or MgCl₂ buffer (SAR−) as a control. After 2 d, two upper healthy leaves were challenged with virulent Psm (OD_{600 nm} = 0.0005). The leaf discs from the second inoculation were collected at 3 d post inoculation for counting bacterial colonies. CFU, Colony-forming unit. C, NPR1 gene expression levels in the local leaves of Col-0 wild-type, npr1-2, and cdk8-4 mutant plants infected with Psm avrRpt2 (OD_{600 nm} = 0.02) or MgCl₂ buffer as a control. Leaves were collected 4 h after infection for gene expression analysis. D, PR1 gene expression level in the local leaves of Col-0 wild-type, npr1-2, and cdk8-4 mutant plants. Leaves were infected with Psm avrRpt2 (OD_{600 nm} = 0.001) or MgCl₂ buffer as a control. After 24 h, the infected leaves were collected for gene expression analysis. E, PR1 gene expression levels in the systemic leaves of Col-0 wild-type, npr1-2, and cdk8-4 mutant plants. Two lower leaves were infected with Psm avrRpt2 (OD_{600 nm} = 0.02) or MgCl₂ buffer as a control. After 2 d, the upper uninfected leaves were collected for gene expression analysis. F, NPR1 gene expression level in Col-0 wild type, npr1-2, and cdk8-4 mutants. Col-0 and mutant plants were irrigated with 0.5 mm SA or water as a control, and then leaf samples were collected after 4 h for NPR1 gene expression analysis. G, NPR1 protein accumulation in Col-0 wild type, npr1-2, and cdk8-4 mutants during SA treatment. H to J, Col-0 wild type, npr1-2, and cdk8-4 mutants were treated with SA for 24 h, and leaf samples were collected for PR1 (H), PR2 (I), and WRKY38 (J) gene expression analysis. The expression levels of CDK8, NPR1, PR1, PR2, and WRKY38 were normalized to UBQ5 expression. Data represent means of three independent samples with se. Asterisks indicate significant differences (Student’s t test, *, P < 0.05 and **, P < 0.01). The experiments were repeated three times with similar results.

Figure 5.

CDK8-associated Mediators are involved in SAR and defense gene expression. A, SAR phenotypes in Col-0 wild-type, npr1-2, cycCab, med12, and med13 mutant plants. Two lower leaves were infiltrated with Psm avrRpt2 (OD_{600 nm} = 0.02; SAR+) or MgCl₂ buffer (SAR−) as a control. After 2 d, two upper healthy leaves were challenged with virulent Psm (OD_{600 nm} = 0.0005). The leaf discs from the second inoculation were collected at 3 d post infection for counting bacterial colonies. CFU, Colony-forming units. B, PR1 gene expression levels in the systemic leaves of Col-0 wild-type, npr1-2, cycCab, med12, and med13 mutant plants. Two lower leaves were infected with Psm avrRpt2 (OD_{600 nm} = 0.02) or MgCl₂ buffer as a control. After 2 d, the upper uninfected leaves were collected for PR1 gene expression analysis. C and D, NPR1 and PR1 gene expression levels in Col-0 wild-type, npr1-2, cycCab, med12, and med13 mutant plants treated with SA. Col-0 and mutant plants were irrigated with 0.5 mm SA, and then leaf samples were collected after 4 h for NPR1 gene expression analysis (C). After 24 h, leaves were collected for PR1 gene expression analysis (D). The expression levels of NPR1 and PR1 were normalized to UBQ5 expression. Data represent means of three independent samples with se. Asterisks indicate significant differences (Student’s t test, *, P < 0.05). Columns with different letters indicate significant differences determined by Duncan’s multiple range test. The experiments were repeated three times with similar results.

Figure 6.

CDK8 connects WRKY6 and WRKY18 with RNA polymerase II to facilitate NPR1 gene expression. A, Y2H assays using CDK8 as bait and WRKY6, WRKY18, WRKY36, or an EV as prey. After cotransformation, yeast was selected on DD medium lacking Leu and Trp. The yeast was then recultured, diluted to OD_{600 nm} = 1, 0.1, or 0.01, and 10 µL of these diluted cultures was placed onto a DD plate and a TD plate. B, Co-IP of HA-CDK8 with WRKY6-GFP, WRKY18-GFP, and WRKY36-GFP in N. benthamiana. WRKY6-GFP, WRKY18-GFP, or WRKY36-GFP was coexpressed with HA-CDK8 proteins in N. benthamiana by agroinfiltration and then immunoprecipitated with magnetic GFP-Trap for 2 h. After washing four times, magnetic beads containing protein complexes associated with GFP or WRKY-GFP were eluted with 2× Laemmli sample buffer and immunoblotted with an anti-GFP antibody. The coimmunoprecipitated proteins were immunoblotted with an anti-HA antibody. Fifty micrograms of the proteins was used as the input and detected with an anti-HA antibody. C, The CDK8 protein is associated with the NPR1 promoter in ChIP assays. Col-0 and CDK8-MYC cdk8-1 transgenic plants were treated with 0.5 mm SA for 4 h. Chromatin was extracted, then immunoprecipitated with an anti-MYC antibody. D, CDK8 is required for RNA polymerase II (Pol II) association with the NPR1 promoter and coding region in ChIP assays. Col-0 and cdk8-4 plants were treated with 0.5 mm SA for 16 h. Chromatin was extracted, then immunoprecipitated with anti-RNA polymerase II antibody or IgG as a negative control. Primers used in the quantitative PCR (qPCR; C and D) amplify the following amplicons: -2000bp, amplifies upstream of the NPR1 promoter at the −2,000-bp region; W-box, amplifies the sequence containing the W-box at the NPR1 promoter; Actin2 was used as a negative control. Data represent means of three independent samples with se. Asterisks indicate significant differences (Student’s t test, *, P < 0.05). The experiments were repeated at least two times with similar results.

Figure 7.

CDK8 interacts with TGAs and is associated with the PR1 promoter. A, CDK8 interacts with TGA5 and TGA7, but not TGA1, TGA2, TGA3, TGA4, or TGA6, in Y2H assays. Yeast strains Y187 and AH109 were transformed with pGBKT7-CDK8 and pGADT7-TGA1-7 or an EV, respectively. Selected yeast diploids from a DD plate were recultured, diluted to OD_{600 nm} = 1, 0.1, or 0.01, and placed onto a DD plate and a TD plate. B, Co-IP assay between HA-CDK8 and TGA5-GFP or TGA7-GFP in N. benthamiana. Proteins extracted from N. benthamiana leaves transiently expressing GFP, TGA5-GFP, or TGA7-GFP and HA-CDK8 by agroinfiltration were immunoprecipitated with an anti-GFP magnetic trap and immunoblotted with an anti-GFP or an anti-HA antibody. C, CDK8 is associated with the PR1 promoter in ChIP assays. Col-0 and CDK8-MYC cdk8-1 transgenic plants were treated with 0.5 mm SA for 4 h. Chromatin was extracted, then immunoprecipitated with an anti-MYC antibody. D, CDK8 is required for the association of RNA polymerase II (Pol II) with the PR1 gene promoter and coding region. After Col-0 and cdk8-4 mutant plants were treated with 0.5 mm SA for 16 h, chromatin was extracted, then immunoprecipitated with an anti-RNA polymerase II antibody. Primers used in the qPCR (C and D) amplify the following amplicons: -2000bp, amplifies upstream of the PR1 promoter at the −2,000-bp region; as-1, amplifies the sequence containing the as-1 region at the PR1 promoter; Actin2 was used as a negative control. The results are representative data from three independent experiments. Data represent means of three independent samples with se. Asterisks indicate significant differences (Student’s t test, *, P < 0.05). The experiments were repeated at least two times with similar results.

Figure 8.

Schematic model of the roles of NPR1 and CDK8 in the transcriptional regulation of NPR1 and PR1 genes. Left, Activation of NPR1 gene expression by NPR1 and CDK8. Pathogen infection induces the expression of the CDK8 gene. CDK8 interacts with the WRKY6 and WRKY18 TFs, which bind to the NPR1 gene promoter at the W-box motif. SA promotes the interactions between NPR1 and CDK8 and between NPR1 and WRKY18. CDK8 and other proteins in the kinase module of the Mediator complex, including MED12 and MED13, bring RNA polymerase II (Pol II) to the NPR1 gene promoter and coding region to promote its transcription. NPR1 mRNA is then exported to the cytosol for NPR1 protein synthesis. Right, CDK8 facilitates the expression of the PR1 gene. After CDK8 and NPR1 induce the transcription of the NPR1 gene, NPR1 protein is synthesized and reduced from oligomers to monomers upon pathogen infection, then NPR1 monomers enter the nucleus. In the nucleus, NPR1 forms a protein complex with CDK8, TGA5, and TGA7, which bind to the PR1 promoter at the as-1 sequence. RNA polymerase II is then recruited to the promoter and coding region of the PR1 gene by CDK8, MED12, and MED13 to facilitate PR1 gene expression to activate plant defense.