The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling

Abstract

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H(2)O(2), but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.

Figure 1.

Analysis of ProMKK3:GUS, mkk3-1, and MKK3-Overexpressing Lines. (A) and (B) Histochemical analysis of GUS activity in ProMKK3:GUS plants in response to Pst DC3000 infiltration. Mock-infiltrated seedlings (A) and seedlings infiltrated with 10⁶ colony-forming units (cfu)/mL Pst DC3000 (B) are shown. (C) Effect of treatments on GUS activity of short-day-grown, 16-d-old transgenic Arabidopsis seedlings carrying the –1997 ProMKK3:GUS construct. CONT, untreated control; Pst, infiltrated with 10⁶ cfu/mL Pst DC3000; Cold, 4°C in dark; Salt, 400 mM NaCl; Drought, lid of the Petri dishes removed. The duration of all treatments was 20 h. Similar results were obtained with five independent transgenic lines. Specific GUS activity is expressed as pmol 4-methylumbelliferyl-β-d-glucuronide·mg⁻¹ protein·min⁻¹. Values obtained with untransformed Arabidopsis seedlings were subtracted. Error bars show se. GUS staining and flourimetric assays were repeated three times with similar results. (D) Genomic organization of the MKK3 gene and the position of the T-DNA insertion within mkk3-1 as determined by PCR and sequencing of the flanking regions. Bar = 100 bp. (E) Analysis of MKK3:myc expression and MKK3 transcript levels from 16-d-old seedlings. MKK3 protein was detected using anti-myc antibody, and the large subunit (LSU) of ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco) is shown as a loading control for equal protein amounts in wild-type Col-0 and plants overexpressing wild-type or constitutively active MKK3-EE. The mkk3 null line (mkk3-1), wild-type Col-0, and MKK3-overxpressing plants were analyzed by RT-PCR. MKK3 was amplified using gene-specific primers, and the ACT3 actin gene was used as a control for equal cDNA amounts. Samples were analyzed from three plants per line with similar results.

Figure 2.

Characterization of MKK3 in Pathogen Response. (A) Analysis of PR gene expression in long-day-grown mkk3-1, Col-0, and MKK3-EE–overexpressing plants by quantitative RT-PCR. PR genes were amplified using gene-specific primers, and the UBQ4 gene was used as an internal control. The experiment was repeated two times with similar results, and the averages of the two independent biological repeats are shown. Error bars represent se. (B) Growth of Pst DC3000 in mkk3-1, wild-type Col-0, and MKK3-overxpressing plants (MKK3-WT and MKK3-EE) after infection by dipping. Growth curves represent the bacterial titers of three pooled leaf discs in six replicates at different times after infection on a logarithmic scale. The experiment was repeated three times with similar results, and the averages of all experiments are shown. Different letters at the 48- or 72-h data points indicate significant differences (P < 0.05) calculated with data from all replicates at each time point with one-way analysis of variance and Tukey's honestly significant difference test. Error bars represent se.

Figure 3.

Identification of Downstream MAPKs of MKK3 by Directed Two-Hybrid Analysis. (A) Quantitative yeast two-hybrid analysis of pBTM116-MKK3 with 14 different pGAD424-MPKs, representing all MAPK groups in the Arabidopsis genome. (B) to (D) Quantitative yeast two-hybrid analysis of the MKK3-interacting MPKs with all 10 MKKs of the Arabidopsis genome. (B) pGAD424-MPK1 with pBTM116-MKKs. (C) pGAD424-MPK2 with pBTM116-MKKs. (D) pGAD424-MPK7 with pBTM116-MKKs. Interaction is expressed as specific β-galactosidase activity in units per milligram of protein. For each plasmid combination, the background values of both constructs measured with the corresponding empty vector were subtracted. The error bars represent the sd from three technical repeats. Two biological repeats were performed, with similar results.

Figure 4.

Activation of Downstream MAPKs by MKK3 and Complex Formation of MPK7 and MKK3 in Planta. (A) Activation of different MPKs by MKK3. MPKs were transiently transformed into Arabidopsis protoplasts. Activation of MPKs was tested by coexpression of the MPKs with either wild-type MKK3-WT or constitutively active MKK3-EE. The kinase activity of immunoprecipitated MPKs was determined with MBP as an artificial substrate and autoradiography after SDS-PAGE. Expression of the MPKs and MKK3 was detected by protein gel blot analysis with anti-HA (MPKs) or anti-myc (MKK3) antibody. (B) In vitro phosphorylation of MPK7 by MKK3. Wild-type (wt) and constitutively active (EE) myc epitope–tagged MKK3 were immunoprecipitated from Arabidopsis protoplasts and subsequently used for the phosphorylation of recombinant kinase-inactive GST-MPK6 and GST-MPK7. Phosphorylation of MPKs was analyzed by autoradiography after SDS-PAGE. MKK3 protein was detected using anti-myc antibody; a Coomassie blue stain of the MPK substrates is shown in the lower panel. (C) MKK3 coimmunoprecipitates with MPK7 from plant extracts. Protein extracts from seedlings of wild-type Col-0 (Col-0) and myc-tagged MKK3-overexpressing (MKK3:myc) plants were immunoprecipitated (IP) either with anti-MPK7 antibody or without antibody addition. Protein gel blot analysis shows the presence of MKK3:myc in the crude protein extracts (input) or in the immunoprecipitated protein complexes. (D) MPK7 coimmunoprecipitates with MKK3 from protoplast extracts. Protein extracts from protoplasts transiently transformed with HA-tagged MPK7 and myc-tagged MKK3 were immunoprecipitated either with anti-myc antibody or without antibody addition. Protein gel blot analysis shows the presence of MPK7:HA in the crude protein extract (input) or in the immunoprecipitated protein complexes. The MPK activation experiments were repeated five times and the coimmunoprecipitation experiments were repeated three times, with similar results.

Figure 5.

MKK3-Mediated Activation of MPK7 and MPK6 by H₂O₂ and flg22. (A) to (D) Arabidopsis protoplasts were transiently transformed with MPK7 ([A] and [B]) or with MPK6 ([C] and [D]) alone or in the presence of MKK3 or MKK4. Protoplasts were treated with either H₂O₂ ([A] and [C]) or flg22 ([B] and [D]) for the indicated periods. The kinase activity of immunoprecipitated MPKs was determined with MBP as an artificial substrate and by autoradiography after SDS-PAGE. Expression of MPK7 and MPK6 and of MKK3 and MKK4 was detected by protein gel blot analysis with anti-HA (MPKs) or anti-myc (MKKs) antibody. These activation assays were repeated three times for MPK7 and twice for MPK6. (E) Stabilization of MKK3 by MG115. Wild type (wt) and constitutively active (EE) myc epitope–tagged MKK3 from nontreated and MG115-treated protoplasts was detected by protein gel blot analysis with anti-myc antibody. This experiment was repeated twice.

Figure 6.

Induction of the PR1 Promoter by the MKK3-MPK7 Module. Arabidopsis protoplasts were transformed with either a ProPR1:GUS fusion construct alone or in combination with different MAPKs and constitutively active MAPK kinases. GUS activity is expressed as pmol 4-methylumbelliferyl-β-d-glucuronide·mg⁻¹ protein·min⁻¹. The error bars represent se from three measurements. Values obtained from untransformed Arabidopsis protoplasts were subtracted.