Phosphorylation of the Nicotiana benthamiana WRKY8 transcription factor by MAPK functions in the defense response

Abstract

Mitogen-activated protein kinase (MAPK) cascades have pivotal roles in plant innate immunity. However, downstream signaling of plant defense-related MAPKs is not well understood. Here, we provide evidence that the Nicotiana benthamiana WRKY8 transcription factor is a physiological substrate of SIPK, NTF4, and WIPK. Clustered Pro-directed Ser residues (SP cluster), which are conserved in group I WRKY proteins, in the N-terminal region of WRKY8 were phosphorylated by these MAPKs in vitro. Antiphosphopeptide antibodies indicated that Ser residues in the SP cluster of WRKY8 are phosphorylated by SIPK, NTF4, and WIPK in vivo. The interaction of WRKY8 with MAPKs depended on its D domain, which is a MAPK-interacting motif, and this interaction was required for effective phosphorylation of WRKY8 in plants. Phosphorylation of WRKY8 increased its DNA binding activity to the cognate W-box sequence. The phospho-mimicking mutant of WRKY8 showed higher transactivation activity, and its ectopic expression induced defense-related genes, such as 3-hydroxy-3-methylglutaryl CoA reductase 2 and NADP-malic enzyme. By contrast, silencing of WRKY8 decreased the expression of defense-related genes and increased disease susceptibility to the pathogens Phytophthora infestans and Colletotrichum orbiculare. Thus, MAPK-mediated phosphorylation of WRKY8 has an important role in the defense response through activation of downstream genes.

Figure 1.

Phosphorylation of WRKY8 in Vitro and in Vivo. (A) In vitro phosphorylation of WRKY8 by MAPKs. Purified Trx and Trx-fused WRKY8 were used as substrates for active MAPKs. Proteins were separated by SDS-PAGE, were stained with Coomassie Brilliant Blue (CBB; bottom panel), and were exposed to x-ray film (kinase assay; top panel). (B) Ala scanning analysis of SIPK-mediated phosphorylation sites in WRKY8. Phosphorylation of WRKY8 variants was detected by autoradiography (middle), and the intensities of each band were quantified (top). Protein loads were monitored by CBB staining (bottom). (C) Specificity of anti-pSer79 and anti-pSer86 antibodies. Peptides of WRKY8_74-84, Ser-79–phosphorylated WRKY8_74-84 (WRKY8_74-84pSer79) and WRKY8_81-91, and Ser-86–phosphorylated WRKY8_81-91 (WRKY8_81-91pSer86) were spotted on nitrocellulose membranes. Immunoblot analyses were performed using anti-pSer79 antibody or anti-pSer86 antibody. (D) Phosphorylation of Ser-79 and Ser-86 in WRKY8 by INF1 elicitin expression. WRKY8-HA-StrepII was expressed with INF1 or GUS in N. benthamiana leaves by agroinfiltration. Total proteins were prepared 30 h after agroinfiltration. After Strep-Tactin purification, immunoblot analyses were done using anti-HA, anti-pSer79, or anti-pSer86 antibody. (E) Phosphorylation of Ser-79 and Ser-86 in WRKY8 by MEK2^DD expression. WRKY8-HA-StrepII was expressed with FLAG-MEK2^KR or FLAG-MEK2^DD in N. benthamiana leaves by agroinfiltration. Total proteins were prepared 36 h after agroinfiltration. Anti-FLAG antibody was used to detect accumulation of FLAG-MEK2^KR and FLAG-MEK2^DD. Protein loads were monitored by CBB staining of the bands corresponding to the ribulose-1,5-bisphosphate carboxylase large subunit (RBCL). Phosphorylation of Ser-79 and Ser-86 in WRKY8 was detected as described in (D). (F) Effects of infection of TRV:SIPK (S), TRV:WIPK (W), or TRV:SIPK/WIPK (S/W) on phosphorylation of Ser-79 and Ser-86 in WRKY8 in response to MEK2^DD expression. Ten micromolars of 17-β-estradiol was injected into the leaves 18 h after coinoculation with Agrobacterium strains containing pER8:FLAG-MEK2^DD and pER8:WRKY8-HA-StrepII. Total proteins were prepared 12 h after estradiol injection, and phosphorylation of Ser-79 and Ser-86 in WRKY8 was detected as described in (D). (G) Phosphorylation of Ser-79 and Ser-86 in WRKY8 by SIPK^WT or NTF4^WT expression. WRKY8-HA-StrepII was expressed with FLAG-tagged SIPK^WT, SIPK^KR, NTF4^WT, or NTF4^KR in N. benthamiana leaves by agroinfiltration. Total proteins were prepared at 48 h after agroinfiltration, and phosphorylation of Ser-79 and Ser-86 in WRKY8 was detected as described in (D). Arrowheads refer to the pertinent bands. Asterisks indicate nonspecific bands.

Figure 2.

D Domain–Dependent Interactions Required for Phosphorylation of WRKY8 by SIPK. (A) In vitro interaction assays between WRKY8 and MAPKs. GST, GST-SIPK, GST-NTF4, GST-WIPK, and GST-NTF6 purified proteins were incubated with WRKY8-His₆ as indicated. Pulled-down fractions were analyzed by immunoblotting using anti-His₆ antibody (top). Input proteins were monitored by Coomassie blue staining (bottom). (B) Subcellular localization of WRKY8, SIPK, NTF4, WIPK, and NTF6 in N. benthamiana epidermal cells. N. benthaniana leaves were transformed with GFP-NLS-GUS, GFP-WRKY8, GFP-SIPK, GFP-NTF4, GFP-WIPK, and GFP-NTF6 by agroinfiltration. DAPI, 4′,6–diamidino–2–phenylindole; DIC, differential interference contrast. (C) BiFC visualization of WRKY8-SIPK, WRKY8-NTF4, and WRKY8-WIPK interactions. N. benthaniana leaves were cotransformed with the C-terminal part of YFP-fused WRKY8 (cYFP-WRKY8) and the N-terminal part of YFP-fused NLS-GUS or MAPKs (nYFP-SIPK, nYFP-NTF4, nYFP-WIPK, and nYFP-NTF6) by agroinfiltration. (D) Deduced D domain found in WRKY8. Lys and Leu conserved in the D domain are indicated in pink and orange, respectively. The numbers indicate position of amino acids of WRKY8 protein. Key interacting residues were substituted with Ala (mD). (E) In vitro interaction assays between D domain–mutated WRKY8 and SIPK. (F) BiFC visualization of the interaction between D domain–mutated WRKY8 and SIPK. (G) In vitro analysis of D domain–mutated WRKY8 phosphorylation by SIPK. Purified Trx-fused WRKY8^WT and WRKY8mD were phosphorylated by active SIPK for the times indicated above each lane. Proteins were separated by SDS-PAGE and were exposed to x-ray film. (H) In vivo phosphorylation analysis of D domain–mutated WRKY8 by MEK2^DD or MEK2^KR. These experiments were repeated at least three times with similar results. Bars = 50 μm.

Figure 3.

Increased DNA Binding and Transactivation Activities by MAPK-Mediated Phosphorylation of WRKY8. (A) Binding of recombinant WRKY8 to a W-box (W) sequence, but not to a mutated W-box (mW) sequence, using EMSA. Unlabeled W-box fragments and mutated W-box fragments were used as competitor DNAs. (B) Increased binding activity of WRKY8 to the W-box sequence by MAPK-mediated phosphorylation. After incubation with WRKY8 and SIPK^KR (lane 3), SIPK^WT (lane 4), NTF4^KR (lane 5), NTF4^WT (lane 6), WIPK^KR (lane 7), WIPK^WT (lane 8), or no recombinant MAPK (lane 2), EMSA was done using a ³²P-labeled W-box probe. Lane 1 shows mobility of free probe. (C) Schematic diagram of effector, reporter, and reference plasmids used in transient assays. (D) High transactivation activity of GAL4DBD-VP16. Data are means ± sd from three independent experiments (E) Transactivation of the GUS gene by GAL4DBD-WRKY8 variants in N. benthamiana leaves. Total proteins were extracted from leaves coinfiltrated with Agrobacterium-containing reporter plasmids, effector plasmids, or reference plasmids. GUS activity was normalized to LUC activity. AAAAA and DDDDD indicate that WRKY8_1-170 mutants mimic the nonphosphorylated form and phosphorylated form, respectively. Data are means ± sd from at least three independent experiments, *P < 0.01 versus the GAL4DBD-WRKY8^WT_1-170 alone by the two-tailed Student’s t test. WT, wild type. (F) Protein gel blot analysis of GAL4DBD, GAL4DBD-VP16, and GAL4DBD-WRKY8_1-170 variants. Anti-GAL4 antibody was used to detect accumulation of GAL4DBD and GAL4DBD-fused proteins. Protein loads were monitored by Coomassie Brilliant Blue (CBB) staining of the bands corresponding to ribulose-1,5-bisphosphate carboxylase large subunit (RBCL). Asterisks refer to the pertinent bands.

Figure 4.

Increased Disease Susceptibility to a Virulent Strain of P. infestans by Silencing of WRKY8. (A) Specific gene silencing of WRKY8 in TRV:WRKY8-infected plants. Silencing of WRKY8 was monitored by qRT-PCR using specific primers. WRKY7, which shows high homology to WRKY8 (see Supplemental Figure 1B online), was used as a silencing control. Data are means ± sd from three independent experiments. (B) Susceptibility to P. infestans in silenced plants. Photographs were taken 5 d after the inoculation. (C) Effects of infection of TRV:WRKY8 (8) or TRV:SIPK/WIPK (S/W) on P. infestans infection. Biomasses were determined by qPCR 5 d after inoculation. Data are means ± sd from three independent experiments. (D) Susceptibility to C. orbiculare in silenced plants. Photographs were taken 5 d after the inoculation. (E) Effects of infection of TRV:8 or TRV:S/W on C. orbiculare infection. The number of disease spots in the leaves was counted 5 d after the inoculation. Data are means ± sd from four independent experiments. *P < 0.05 and **P < 0.01; two-tailed Student’s t test.

Figure 5.

Induction of Target Gene Expression by a Phosphorylation-Mimicking Mutant of WRKY8. (A) Expression levels of NADP-ME (top) and HMGR2 (bottom) induced by MEK2^DD (left) or P. infestans (right) were compromised by TRV:WRKY8 or TRV:SIPK/WIPK. Data are means ± sd from three independent experiments. (B) Expression of NADP-ME and HMGR2 in response to WRKY8 variants. Total RNAs were extracted from N. benthamiana leaves 48 h after agroinfiltration and were used for qRT-PCR. Data are means ± sd from at least three independent experiments. Anti-HA antibody was used to detect accumulation of WRKY8-HA variants. Protein loads were monitored by CBB staining of the bands corresponding to RBCL. WT, wild type. *P < 0.05 and **P < 0.01; two-tailed Student’s t test. [See online article for color version of this figure.]

Figure 6.

Model of the Regulatory Mechanism of WRKY8 by MAPK-Dependent Phosphorylation. Pathogen recognition leads to activation of MEK2 by unidentified MAP3K(s). Active MEK2 phosphorylates and activates SIPK, NTF4, and WIPK. Active SIPK interacts with WRKY8 in a D domain–dependent manner and phosphorylates WRKY8. Active NTF4 and WIPK may act in a way similar to SIPK. Phosphorylated and activated WRKY8 binds to W-box sites within its target genes and upregulates the expression of these genes.