Activation of salicylic acid-induced protein kinase, a mitogen-activated protein kinase, induces multiple defense responses in tobacco

Abstract

The activation of mitogen-activated protein kinases (MAPKs) is one of the earliest responses in plants challenged by avirulent pathogens or cells treated with pathogen-derived elicitors. Expression of a constitutively active MAPK kinase, NtMEK2(DD), in tobacco induces the expression of defense genes and hypersensitive response-like cell death, which are preceded by the activation of two endogenous MAPKs, salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK). However, the roles that SIPK and WIPK each play in the process are unknown. Here we report that SIPK alone is sufficient to activate these defense responses. In tobacco leaves transiently transformed with SIPK under the control of a steroid-inducible promoter, the induction of SIPK expression after the application of dexamethasone, a steroid, leads to an increase of SIPK activity. The increase of SIPK activity is dependent on the phosphorylation of newly synthesized SIPK by its endogenous upstream kinase. In contrast, the expression of WIPK under the same conditions fails to increase its activity, even though the protein accumulates to a similar level. Studies using chimeras of SIPK and WIPK demonstrated that the C terminus of SIPK contains the molecular determinant for its activation, which is rather surprising because the N termini of SIPK and WIPK are more divergent. SIPK has been implicated previously in the regulation of both plant defense gene activation and hypersensitive response-like cell death based on evidence from pharmacological studies using kinase inhibitors. This gain-of-function study provided more direct evidence for its role in the signaling of multiple defense responses in tobacco.

Figure 1.

Expression of SIPK and Its Inactive Mutant under the Control of a Steroid-Inducible Promoter. (A) A simplified map of MAPK constructs in pTA7002 vector. SIPK or its mutant was inserted into the XhoI–SpeI sites of the steroid-inducible pTA7002 binary vector. The 5′ untranslated region of SIPK was replaced with the Ω sequence from Tobacco mosaic virus. To facilitate the detection of transgene expression, a Flag tag was added to the N terminus of SIPK. (B) Induction of transgene expression. Tobacco leaves were infiltrated with Agrobacterium carrying SIPK or its inactive mutant with K90 replaced by R (SIPK^R). DEX (30 μM) was infiltrated 48 hr later, and samples were taken at the times indicated. The expression of transgenes was monitored by immunoblot (IB) analysis using anti-Flag antibody. (C) Relative levels of transgene expression in leaves under induced conditions. The levels of SIPK protein in leaves were determined by immunoblot analysis using anti-SIPK antibody. The relative amount of Flag-tagged SIPK (closed circles) or Flag-tagged SIPK^R (open circles) versus endogenous SIPK was quantified using NIH Image software (National Institutes of Health, Bethesda, MD). The endogenous SIPK level before induction was normalized to 1.

Figure 2.

Expression of SIPK under the Inducible Promoter Increases SIPK Activity in Tobacco Leaves. (A) The increase of SIPK activity in leaves under induced conditions. Kinase activities in the same protein extracts shown in Figure 1 were determined by in-gel kinase assay using MBP as a substrate. (B) Newly synthesized Flag-tagged SIPK is active, as demonstrated by immune complex (IC) kinase assay. Protein extracts (50 μg) were immunoprecipitated with anti-Flag antibody (2 μg). Kinase activity of the immune complex was assayed subsequently as described in Methods, and the phosphorylated MBP was visualized by autoradiography. (C) Only SIPK is activated after the application of DEX. Protein extracts (50 μg) from leaves transformed with Flag-tagged SIPK were immunoprecipitated with either SIPK-specific (anti-p48N) or WIPK-specific (anti-p44N) antibody (2.5 μg). Kinase activity of the immune complex was assayed subsequently as described in Methods, and the phosphorylated MBP was visualized by autoradiography.

Figure 3.

The Increase of SIPK Activity Requires the Phosphorylation of Flag-Tagged SIPK by Its Endogenous Upstream MAPKK. (A) K252a, a kinase inhibitor known to inhibit SIPK activation (Zhang et al., 1998, 2000), prevents the increase of SIPK activity after the application of DEX. K-252a was infiltrated at various concentrations into leaves together with DEX. Samples were taken 4 hr later. SIPK activities were determined by in-gel kinase assay with MBP as a substrate (top), and transgene expression was monitored by immunoblot (IB) analysis with anti-Flag antibody (bottom). (B) Flag-tagged SIPK activation requires phosphorylation on both Thr and Tyr residues. SIPK protein from transgene expression was immunoprecipitated with anti-Flag antibody. Flag-tagged SIPK in the immune complex was then treated with either a Thr/Ser-specific protein phosphatase, PP-2A₁ (0.25 units in 30 μL), or a Tyr-specific protein phosphatase, YOP (2 units in 30 μL), for 20 min at 30°C in the presence or absence of a phosphatase inhibitor. The PP-2A₁ inhibitor, okadaic acid, and the YOP inhibitor, Na₃VO₄, were used at concentrations of 5 μM and 5 mM, respectively. After the phosphatase treatment, kinase activity was detected using MBP as a substrate. P-MBP, phosphorylated MBP. (C) Activation of Flag-tagged SIPK requires the TEY motif. The Thr-218 and Tyr-220 residues in the TEY motif of SIPK were substituted with Ala (A) and Phe (F), respectively. SIPK^AF, the SIPK(T218A/Y220F) mutant, was transformed into tobacco leaves as SIPK. Transgene expression was monitored by immunoblot analysis with anti-Flag antibody (top), and kinase activity was determined by in-gel kinase assay with MBP as a substrate (bottom).

Figure 4.

Expression of WIPK under the Inducible Promoter Does Not Result in an Increase of Its Activity. (A) Expression of WIPK under the same conditions used for SIPK fails to result in an increase of its activity. Tobacco leaves were infiltrated with Agrobacterium carrying WIPK or its inactive K73R mutant (WIPK^R). DEX (30 μM) was infiltrated 48 hr later, and samples were taken at the times indicated. Expression of transgenes was monitored by immunoblot (IB) analysis using anti-Flag antibody (top), and kinase activities in the leaf tissues were determined by in-gel kinase assay using MBP as a substrate (bottom). (B) Recombinant WIPK is active in both autophosphorylation and cross-phosphorylation of MBP. Autophosphorylation activities of 0.5 μg of His-tagged wild-type SIPK and WIPK and their mutants were determined as described in Methods. Phosphorylated SIPK (P-SIPK) and WIPK (P-WIPK) were visualized by autoradiography after SDS-PAGE (top). Cross-phosphorylation (Cross-P) activities of SIPK and WIPK (0.1 μg) were determined using MBP as a substrate. Phosphorylated MBP (P-MBP) was visualized by autoradiography after SDS-PAGE (bottom).

Figure 5.

The C Terminus of SIPK Plays an Important Role in Its Activation. (A) Construction of SIPK and WIPK chimeras. A PstI site was introduced into the WIPK gene at the position corresponding to that of SIPK by site-directed mutagenesis. Two chimeric proteins were constructed by swapping the C termini of SIPK and WIPK. (B) The (W/S)IPK, but not the (S/W)IPK, chimera is activated when expressed in tobacco leaves. Tobacco leaves were infiltrated with Agrobacterium carrying (S/W)IPK or (W/S)IPK in pTA7002 vector. DEX (30 μM) was infiltrated 48 hr later, and samples were taken at the times indicated. Expression of transgenes was monitored by immunoblot (IB) analysis using anti-Flag antibody (top), and kinase activities in the leaf tissues were determined by in-gel kinase assay using MBP as a substrate (bottom).

Figure 6.

Mutation of the Two Asp (D) Residues in the MAPK CD Domain Does Not Enhance the Activation of SIPK or WIPK. (A) The CD domain is evolutionarily conserved in plants, animals, and yeast. (B) Mutation of the conserved Asp residues in the CD domain to Asn does not enhance SIPK activation. D353 alone, or both D350 and D353, in the CD domain of SIPK were replaced with Asn (N). SIPK^N, the SIPK(D353N) mutant, and SIPK^NN, the SIPK(D350N/D353N) double mutant, were transformed into tobacco leaves. Transgene expression was monitored by immunoblot (IB) analysis with anti-Flag antibody (top), and MAPK activity was determined by in-gel kinase assay with MBP as a substrate (bottom). (C) Substitution of the conserved Asp residues in the CD domain of WIPK fails to result in its activation. D337 alone, or both D334 and D337, in the CD domain of WIPK were replaced with Asn (N). WIPK^N, the WIPK(D337N) mutant, and WIPK^NN, the WIPK(D334N/D337N) double mutant, were transformed into tobacco leaves. Transgene expression was monitored by immunoblot analysis with anti-Flag antibody (top), and MAPK activity was determined by in-gel kinase assay with MBP as a substrate (bottom).

Figure 7.

Induction of the Expression of SIPK and the (W/S)IPK Chimera Leads to HR-Like Cell Death. Different sections of a tobacco leaf were infiltrated with Agrobacterium carrying the constructs indicated, and DEX (30 μM) was applied 48 hr later. The photograph was taken 30 hr after the application of DEX. HR indicates the development of HR-like necrosis in the leaf section, and dashes indicate no visible phenotype.

Figure 8.

The Increase of SIPK Activity Induces the Expression of the HMGR Gene. Tobacco leaves were transformed transiently with SIPK. SIPK^R, an inactive mutant of SIPK, was used as a control. Total RNA was isolated from samples collected at the times indicated after the application of DEX. Equal amounts of RNA (10 μg) were electrophoresed on a 1.2% formaldehyde-agarose gel and transferred to a Zeta-Probe membrane (Bio-Rad). The transcript levels of HMGR were determined by probing with an α-³²P-CTP random primer–labeled cDNA insert, as described in Methods. An ethidium bromide (EtBr)-stained gel was used to show equal loading of samples.

Figure 9.

Model of the Possible Mechanism of SIPK Activation in Cells under Induced Conditions. In unstimulated cells, the actions of the upstream kinase of SIPK, possibly NtMEK2, which activates SIPK, and the unknown MAPK phosphatase (MPase), which inactivates SIPK, reach equilibrium to give a low basal activity of SIPK. A sudden increase of Flag-tagged SIPK protein (F-SIPK) from transgene expression after DEX application leads to the increase of SIPK activity in the cells, and there is not enough MAPK phosphatase to inactivate them. The increase of total SIPK activity leads to the phosphorylation of its substrate(s), which in turn evokes tobacco defense responses such as defense gene activation and HR-like cell death. BPs, binding proteins; P, phosphorylated.