Activation of a mitogen-activated protein kinase pathway is involved in disease resistance in tobacco

Abstract

Hypersensitive response (HR), a form of programmed cell death, is frequently associated with plant disease resistance. It has been proposed that mitogen-activated protein kinase (MAPK) cascades regulate HR cell death based on pharmacological studies by using kinase inhibitors. However, direct evidence is lacking. Here, we demonstrate that NtMEK2, a MAPK kinase, is upstream of salicylic acid-induced protein kinase (SIPK) and wounding-induced protein kinase (WIPK), two tobacco MAPKs that are activated by various pathogens or pathogen-derived elicitors. Expression of a constitutively active mutant of NtMEK2 induces HR-like cell death in tobacco, which is preceded by the activation of endogenous SIPK and WIPK. In addition, NtMEK2-SIPK/WIPK cascade appears to control the expression of 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and l-phenylalanine ammonia lyase (PAL), two defense genes encoding key enzymes in the phytoalexin and salicylic acid biosynthesis pathways. These results demonstrate that a plant MAPK cascade controls multiple defense responses against pathogen invasion.

Figure 1

Alignment of tobacco NtMEK2 with Arabidopsis AtMEK4 (BAA28830) and human MEK1 (Q02750). Roman numerals indicate the 11 conserved subdomains of the kinase catalytic domain. Numbers in parentheses indicate the percentage of amino acid sequence identity to the NtMEK2. The conserved Ser/Thr residues (Thr-227, Ser-233, and Thr-237) between subdomains VII and VIII for MAPKKs are marked with asterisks underneath. The conserved Lys-111 that is important for the ATP binding is marked with a dot.

Figure 2

Constitutively active NtMEK2 mutant phosphorylates and activates SIPK and WIPK. (A) NtMEK2^DD, the T227D/S233D double mutant of NtMEK2, has elevated kinase activity. Autophosphorylation activities of 0.5 μg of His-tagged wild-type (WT) NtMEK2 and its mutants were determined as described in Materials and Methods. (B) NtMEK2^DD preferentially phosphorylates SIPK and WIPK. Phosphorylation activities of HisNtMEK2^WT and HisNtMEK2^DD (0.1 μg) were determined by using inactive mutant MAPKs (HisMAPK^R, 1 μg) as substrates. Reactions in the absence (−) of either MAPK or MAPKK were used as controls. (C) Phosphorylation by NtMEK2^DD activates SIPK and WIPK. Wild-type MAPKs (HisMAPK, 1 μg) were incubated in the absence (−) or with 0.1 μg of HisNtMEK2^WT or HisNtMEK2^DD in the presence of 50 μM unlabeled ATP. MBP (final concentration of 0.25 μg/μl) and [γ-³²P]ATP (1 μCi per reaction) were then added. Phosphorylated MBP (P-MBP) that reflects the activity of MAPK was visualized by autoradiography after SDS/PAGE.

Figure 3

Activation of NtMEK2 during HR-like cell death initiated by fungal elicitin. (A) Cryptogein (Cry), a fungal elicitin from Phytophthora cryptogea, induces HR-like cell death in tobacco suspension cells. Cells were treated with cryptogein at a final concentration of 25 nM, and cell viability was monitored by the fluorescein diacetate method (20) at the indicated times. Data represent the mean of three replicates ±SE. (B) Ab-NtMEK2 specifically recognized NtMEK2 protein. Two nanograms each of the purified recombinant NtMEK1, NtMEK2, NtMEK7, and NtMEK8 were subjected to immunoblot analysis with Ab-NtMEK2. (C) Activation of NtMEK2 in cells treated with cryptogein. Protein extracts (75 μg) from cryptogein-treated cells were immunoprecipitated with purified Ab-NtMEK2 (4 μg) in the absence (−) or the presence of the N-terminal portion of NtMEK2 (ΔNtMEK2), NtMEK7 (ΔNtMEK7), or NtMEK1 (ΔNtMEK1) as competitor. Kinase activity of the immune complex was subsequently assayed with HisSIPK^R or HisWIPK^R as a substrate.

Figure 4

Expression of NtMEK2^DD activates SIPK and WIPK in tobacco. (A) Induction of NtMEK2 and its mutants by steroid in Agrobacterium-mediated transient transformation. Tobacco leaves were infiltrated with Agrobacterium carrying pTA7002 constructs. DEX (30 μM) was infiltrated 40 h later, and samples were taken at indicated times. The expression of transgenes was monitored by immunoblot analysis by using anti-Flag antibody (Top). The kinase activities of Flag-tagged NtMEK2 and its mutants were determined by immune complex (IC)-kinase assay with HisSIPK^R (Middle) or HisWIPK^R (Bottom) as a substrate. (B) Induction of NtMEK2^DD expression activates SIPK and WIPK in vivo. The MAPK activities in cells after DEX treatment were determined by an in-gel kinase assay with MBP substrate (Top). The identities of the two kinases were confirmed by immune complex–kinase assays by using SIPK-specific (Ab-p48N; Middle) or WIPK-specific (Ab-p44N; Bottom) antibody. (C) Expression of NtMEK7^DD does not activate SIPK or WIPK. The levels of NtMEK7 transgene expression were determined by immunoblot analysis (Upper). Endogenous SIPK and WIPK activities were detected by an in-gel kinase assay with MBP as a substrate (Lower).

Figure 5

Induction of NtMEK2^DD expression leads to HR-like cell death. Different sections of a tobacco leaf were infiltrated with Agrobacterium carrying indicated constructs or vector control, and DEX (30 μM) was applied 40 h later. Solvent (0.1% of ethanol) was used in the control leaf (−DEX). Photo was taken 30 h after application of DEX. In the table, HR denotes the development of an HR-like necrosis in the leaf section; — indicates no visible phenotype.

Figure 6

Induction of NtMEK2^DD expression leads to the activation of a subset of defense genes. Total RNA was isolated from samples collected at the same time as those for protein analyses (Fig. 4). Equal amounts of RNA (10 μg) were electrophoresed on a 1.2% formaldehyde-agarose gel and transferred to Zeta-Probe membrane (Bio-Rad). The transcript levels of HMGR, PAL, and LOX were determined by sequential probing with [α-³²P]CTP random primer-labeled cDNA inserts as previously described (23). An ethidium bromide-stained gel was used to show equal loading of samples.