BWMK1, a rice mitogen-activated protein kinase, locates in the nucleus and mediates pathogenesis-related gene expression by activation of a transcription factor

Abstract

Mitogen-activated protein kinase (MAPK) cascades are known to transduce plant defense signals, but the downstream components of the MAPK have as yet not been elucidated. Here, we report an MAPK from rice (Oryza sativa), BWMK1, and a transcription factor, OsEREBP1, phosphorylated by the kinase. The MAPK carries a TDY phosphorylation motif instead of the more common TEY motif in its kinase domain and has an unusually extended C-terminal domain that is essential to its kinase activity and translocation to the nucleus. The MAPK phosphorylates OsEREBP1 that binds to the GCC box element (AGCCGCC) of the several basic pathogenesis-related gene promoters, which in turn enhances DNA-binding activity of the factor to the cis element in vitro. Transient co-expression of the BWMK1 and OsEREBP1 in Arabidopsis protoplasts elevates the expression of the beta-glucuronidase reporter gene driven by the GCC box element. Furthermore, transgenic tobacco (Nicotiana tabacum) plants overexpressing BWMK1 expressed many pathogenesis-related genes at higher levels than wild-type plants with an enhanced resistance to pathogens. These findings suggest that MAPKs contribute to plant defense signal transduction by phosphorylating one or more transcription factors.

Figure 1.

BWMK1 belongs to an MAPK family in plants. Phylogenetic tree of plant MAPKs. Plant MAPKs included were alfalfa MMK1-4 (Jonak et al., 1995, 1996) and TDY1 (Schoenbeck et al., 1999); Arabidopsis AtMPK1-7 (Mizoguchi et al., 1993), AtMPK8 (accession no. BAA92222), and AtMPK9 (accession no. BAA92223); parsley ERMK (Ligterink et al., 1997); tobacco NtF3, 4, and 6 (Wilson et al., 1995), WIPK (Seo et al., 1995), and SIPK (Zhang and Klessig, 1997); and rice BWMK1 (He et al., 1999). The phylogenic tree was created using ClustalW program (Thompson et al., 1994).

Figure 2.

The CD of BWMK1 is essential for both kinase activity and nuclear localization. A, Schematic diagram of the GST fusion constructs of BWMK1. Amino acid numbers of domain boundaries are indicated. B, Role of the C-terminal domain in autophosphorylation and myelin basic protein (MBP) kinase activity. +, With MBP; –, without MBP. The arrows indicate the positions where GST-BWMK1 and MBP migrated. The apparent molecular masses (kilodaltons) are indicated at the left. C, Subcellular localization of BWMK1. Arabidopsis protoplasts were cotransfected with three sets of smGFP and RFP constructs, that is, smGFP and RFP (a, d, g, and j), smGFP-fused BWMK1 and RFP-fused NLS (b, e, h, and k), and smGFP-fused KD and RFP-fused NLS (c, f, i, and l). Bars = 20 μm.

Figure 3.

Specificity of Ab-pNBWMK1 antibody. A, SDS-PAGE analysis of GST fusion recombinant proteins of BWMK1. Purified proteins were separated on a 10% (w/v) SDS-PAGE and stained with Coomassie Brilliant Blue R-250. The apparent molecular masses (kilodaltons) are indicated at the left. B, Immunoblot analysis using the recombinant protein. Ab-pNBWMK1 specially recognized GST-BWMK1 and GST-BWMK1 KD. C, Immunoblot analysis using plant extracts from rice. Protein (50 μg) was separated by SDS-PAGE, blotted, and probed with Ab-pNBWMK1 (1:3,000 [v/v] dilution). After incubation with a horseradish peroxidase-conjugated secondary antibody, the complex was visualized using enhanced chemiluminescence.

Figure 4.

Activation of BWMK1 by defense signals. A, Kinase activities of BWMK1. Protein extracts (100 μg) from the suspension cell cultures treated with above stresses for various times were immunoprecipitated with anti-pNBWMK1 antibody and the performed the kinase assay as described in “Materials and Methods.” B, Protein levels of BWMK1. Fifty micrograms of protein extracts used for kinase assay was analyzed for BWMK1 protein levels by immunoblot analysis using the anti-pNBWMK1 antibody. C, Transcription levels of BWMK1. Total RNAs (20 μg) isolated from rice suspension cells treated by stresses were separated by 1.5% (w/v) formaldehydeagarose gel, transferred onto a membrane, and then hybridized with ³²p-labeled BWMK1 cDNA. Fungal elicitor, Crude extract (50 μg mL^–1) prepared from the rice blast Magnaporthe grisea; H₂O₂, 1 mm H₂O₂; SA, 1 mm SA; JA, 0.1 mm JA; ethephon, 5 mm ethaphon; H₂O/DMSO, water or dimethyl sulfoxide control.

Figure 5.

Overexpression of BWMK1 in transgenic tobacco plant induces HR-like cell death and elevates SAR gene expression. A, Development of HR-like cell death in transgenic tobacco plants. Lesions formed in 6-week-old representative transgenic BWMK1 tobacco plants (4-11 and 6-2) and wild-type (WT) plant. B, Accumulation of autofluorescent materials in HR-like cell death lesions of transgenic plants. Fully expanded leaves from 6-week-old plants were used for microscopic analysis after clearing with lactophenol. Magnification is ×50. White, Lactophenol-cleaned leaves examined under a light microscope. UV, UV-stimulated autofluorescence in lactophenol-cleared tobacco leaves. C, Constitutive expression of PR genes in transgenic plants.

Figure 6.

Enhanced disease resistance of transgenic tobacco plants that constitutively overexpressed BWMK1. A, Disease responses to the virulent oomycete pathogen, P. parasitica var nicotianae, at 7 d after inoculation. B, In planta bacterial growth. Pst was inoculated into leaves of mature wild-type plants (WT) and two independent transgenic plant lines (4-11 and 6-2) at 10⁵ colony-forming units (cfu) mL^–1, and in planta bacterial growth was monitored over 5 d. Values represent average and sds of cfu extracted from leaf discs in three independent samplings.

Figure 7.

BWMK1 phosphorylates OsEREBP1 and the phosphorylation enhances the ability of the transcription factor to bind to the cis-acting element. A, Alignment of the OsEREBP1 amino acid sequence with the EREBPs, AtERF1-5 (Fujimoto et al., 2000). Overlined sequences, Conserved EREBP/AP2 domain. Underlined sequences, N-terminal acidic domain. Identical residues are indicated with dots. A putative MAPK phosphorylation site is indicated by an asterisk. B, Yeast two-hybrid interaction of BWMK1 with OsEREBP1. Yeast strain pJ69-4A transformed with the constructs indicated in the left plant were grown on synthetic complete (SC) medium minus Trp and Leu (+Ade) or in SC medium minus Trp, Leu, and Ade (–Ade). β-Galactosidase activity in the colonies grown in +Ade medium (LacZ) was determined by filter-lift assay. BD, Fusion to a plasmid containing the GAL4 DNA-binding domain; AD, Fusion to a GAL4 transcriptional activation domain. C, BWMK1 phosphorylates OsEREBP1 in vitro. Arrows indicate the positions of autophosphorylated GST-BWMK1 and phosphorylated GST-OsEREBP1. The apparent molecular masses (kilodaltons) are indicated at the left. D, The DNA-binding activity of OsEREBP1 to the GCC box motif (AGCCGCC) is enhanced by BWMK1-mediated phosphorylation. The arrowheads mark the position of the protein-DNA complex and free probe.

Figure 8.

Transactivation of the GCC-box-m35S-GUS fusion gene by BWMK1 and OsEREBP1 in transient assay using Arabidopsis protoplasts. A, Schematic diagram of the effector and reporter plasmids used in cotransfection experiments. B, BWMK1 enhances GCC box-driven gene expression by activating OsEREBP1. Bars = se of three replicates. The numbers show the fold of expression compared with the value obtained with the vector control.