Activation of Ntf4, a tobacco mitogen-activated protein kinase, during plant defense response and its involvement in hypersensitive response-like cell death

Abstract

Mitogen-activated protein kinase (MAPK) cascades are important signaling modules in eukaryotic cells. They function downstream of sensors/receptors and regulate cellular responses to external and endogenous stimuli. Recent studies demonstrated that SIPK and WIPK, two tobacco (Nicotiana spp.) MAPKs, are involved in signaling plant defense responses to various pathogens. Ntf4, another tobacco MAPK that shares 93.6% and 72.3% identity with SIPK and WIPK, respectively, was reported to be developmentally regulated and function in pollen germination. We found that Ntf4 is also expressed in leaves and suspension-cultured cells. Genomic analysis excluded the possibility that Ntf4 and SIPK are orthologs from the two parental lines of the amphidiploid common tobacco. In vitro and in vivo phosphorylation and activation assays revealed that Ntf4 shares the same upstream MAPK kinase, NtMEK2, with SIPK and WIPK. Similar to SIPK and WIPK, Ntf4 is also stress responsive and can be activated by cryptogein, a proteinaceous elicitin from oomycetic pathogen Phytophthora cryptogea. Tobacco recognition of cryptogein induces rapid hypersensitive response (HR) cell death in tobacco. Transgenic Ntf4 plants with elevated levels of Ntf4 protein showed accelerated HR cell death when treated with cryptogein. In addition, conditional overexpression of Ntf4, which results in high cellular Ntf4 activity, is sufficient to induce HR-like cell death. Based on these results, we concluded that Ntf4 is multifunctional. In addition to its role in pollen germination, Ntf4 is also a component downstream of NtMEK2 in the MAPK cascade that regulates pathogen-induced HR cell death in tobacco.

Figure 1.

Phylogenetic analysis of A2 subgroup of MAPKs from solanaceous species and Arabidopsis. The phylogenetic tree was created by using the Clustal method (MegaAlign program of DNAStar). Only DNA sequences in the open reading frames were used for alignment. Tobacco WIPK, which belongs to the A1 subgroup, was used to anchor the A2 subgroup. The GenBank accession numbers are (in parentheses) Ntf4 (X83880), Ntf4-1 (DQ229077), Ntf4-2 (DQ229078), LeMPK2 (AY261513), StMPK2 (AB062139), LeMPK1 (AY261512), StMPK1 (AB062138), SIPK (U94192), MPK6 (D21842), and WIPK (D61377). The percentage of sequence identity to Ntf4 is listed on the right in parentheses.

Figure 2.

Ntf4 and SIPK coexist in the two parental lines of the amphidiploid common tobacco. Genomic DNA was isolated from common tobacco (N. tabacum) and its parental lines, N. sylvestris and N. tomentosiformis. Four pairs of primers, two specific to Ntf4 and two specific to SIPK, were selected from the divergent regions of Ntf4 and SIPK, and were used to amplify the genomic fragments of Ntf4 and SIPK. The top section shows the PCR products using SIPK-F1/SIPK-B1 and Ntf4-F1/Ntf4-B1 primer pairs (F1/B1), and the bottom section shows the PCR products using SIPK-F2/SIPK-B2 and Ntf4-F2/Ntf4-B2 primer pairs (F2/B2). The specificity of the primer pairs was confirmed using cDNAs (pBS-Ntf4 and pBS-SIPK) as templates. Two-log DNA size marker (NEB) was used to determine the sizes of amplified fragments.

Figure 3.

Expression of Ntf4 and SIPK in tobacco pollen grains, leaves, and cultured suspension cells. Poly(A⁺)-RNA samples from tobacco pollen grains, leaves, and cultured suspension cells were reverse transcribed to produce cDNAs. The presence of Ntf4 and SIPK transcripts was determined by PCR using the gene-specific primers. The top section shows the PCR products using SIPK-F1/SIPK-B1 and Ntf4-F1/Ntf4-B1 primer pairs (F1/B1), and the bottom section shows the PCR products using SIPK-F2/SIPK-B2 and Ntf4-F2/Ntf4-B2 primer pairs (F2/B2). The specificity of the PCR reactions was monitored using cloned SIPK and Ntf4 plasmids (pBS-Ntf4 and pBS-SIPK) as templates. Two-log DNA size marker (NEB) was used to confirm the sizes of amplified fragments.

Figure 4.

Expression of SIPK and Ntf4 in various tobacco tissues/organs. A, Protein extracts (10 μg) from various tissues/organs were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membrane. Duplicate blots were prepared. One was probed with Ab-p48N, which specifically recognizes SIPK, and the other probed with Ab-p48C, which recognizes both SIPK and Ntf4. B, Immune-depletion analysis of tobacco leaf protein extract. SIPK in the total leaf protein extract (75 μg) was immune depleted with different amounts (0, 2, 5, and 10 μg) of Ab-p48N. The supernatants were then subjected to immunoblot analysis using Ab-p48N and Ab-p48C. Asterisks indicate the rabbit IgG heavy chain of the residual Ab-p48N in the supernatant that was recognized by secondary antibody in the immunoblot analysis.

Figure 5.

NtMEK2 phosphorylates and activates Ntf4 in vitro. A, Basal kinase activity of recombinant Ntf4. Phosphorylation and autophosphorylation activities of His-tagged Ntf4, SIPK, and WIPK (1 μg) were determined as described in “Materials and Methods.” The top band in each lane is the autophosphorylated MAPKs, and the lower one is the phosphorylated MBP (P-MBP). The image is from an overnight-exposed x-ray film. B, NtMEK2^DD phosphorylates Ntf4, SIPK, and WIPK with equal efficiency. Phosphorylation activities of HisNtMEK2^WT and HisNtMEK2^DD (0.1 μg) were determined using inactive mutant MAPKs (Ntf4^KR, SIPK^KR, and WIPK^KR, 1 μg) as substrates. The image is from an x-ray film with 1-h exposure. C, Activation of Ntf4 by NtMEK2^DD phosphorylation. Wild-type recombinant MAPKs (Ntf4, SIPK, and WIPK, 0.25 μg) were incubated with 0.025 μg of HisNtMEK2^WT or HisNtMEK2^DD in the presence of 50 μm unlabeled ATP. MBP (final concentration 0.25 μg/μL) and γ-³²P-ATP (1 μCi per reaction) were then added. Phosphorylated MBP (P-MBP), which reflects the activity of MAPK, was visualized by autoradiography after SDS-PAGE. The image is from an x-ray film exposed for 10 min.

Figure 6.

In vivo activation of Ntf4 by NtMEK2 in tobacco. Stable steroid-inducible promoter:NtMEK2^DD and NtMEK2^KR transgenic plants were transiently transformed with Flag-tagged Ntf4 or its inactive mutant Ntf4^KR under control of the CaMV 35S promoter. As another control, empty vector was also included. Two days later, DEX (30 μm) was applied to induce the expression of NtMEK2, and samples were collected 6 h later. Ntf4 activity from Flag-tagged transgene was determined by anti-Flag immune-complex kinase assay following the procedure described in “Materials and Methods.”

Figure 7.

Activation of Ntf4 by cryptogein, a proteinaceous elicitor from oomycetic pathogen P. cryptogea. A, Wild-type tobacco plants were transiently transformed with Flag-tagged Ntf4 (F-Ntf4) or its inactive mutant Ntf4^KR by Agrobacterium-mediated transformation. Empty vector was included as a negative control. Two days after Agrobacterium infiltration, cryptogein (50 nm) was infiltrated and leaf discs were collected at the indicated times. Three different kinase assays were performed: in-gel kinase assay using total extract (top); immunoprecipitation using anti-Flag antibody followed by in-solution assay, so-called immune-complex kinase assay (middle); and immunoprecipitation using anti-Flag antibody followed by in-gel kinase assay (bottom). B, Expression levels of Ntf4 in protein extracts subjected to kinase assays. Protein extracts (10 μg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membrane. Triplicate blots were prepared. One was probed with anti-Flag, which specifically recognizes the Ntf4 and Ntf4^KR from transgene (top); one was probed with Ab-p48C, which recognizes Flag-tagged Ntf4 and Ntf4^KR from the transgenes and the endogenous Ntf4 and SIPK (middle); and the third was probed with Ab-p48N, which recognizes SIPK only (bottom).

Figure 8.

Expression of Ntf4 under the control of a steroid-inducible promoter increases Ntf4 activity in tobacco leaves. A, A diagram illustrates the Ntf4 constructs in pTA7002 vector with various mutants marked. To facilitate the detection of transgene expression, a Flag tag was added to the N terminus of Ntf4. The 5′-untranslated region of Ntf4 was replaced with the Ω sequence from tobacco mosaic virus. Ntf4 or its mutant was inserted into the XhoI-SpeI sites of the steroid-inducible pTA7002 binary vector. Ntf4^KR, the Ntf4(K88R) mutant, loses the kinase activity completely because it is not able to bind ATP. Ntf4^AF, the Ntf4(T217A/Y219F) mutant, cannot be activated by upstream MAPKK, but should still retain the basal kinase activity. Ntf4^2N, the Ntf4(D352N) mutant, and Ntf4^NN, the Ntf4(D349N/D352N) mutant, have mutations in the conserved MAPK common docking domain. B, Induction of Ntf4 expression under the control of steroid-inducible promoter leads to an increase in Ntf4 activity in tobacco leaves. Tobacco leaves were infiltrated with Agrobacterium carrying Ntf4 or its mutants. DEX (30 μm) was infiltrated 2 d later, and samples were taken at the times indicated. The expression of transgenes was monitored by immunoblot (IB) analysis using anti-Flag antibody (top). Kinase activities in the same protein extracts were determined by in-gel kinase assay using MBP as a substrate (bottom).

Figure 9.

Induction of Ntf4 and the mutants that do not interfere with the kinase activity/activation 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 h later. The photograph was taken 30 h after the application of DEX. HR indicates the development of HR-like necrosis in the leaf section, and dashes indicate no visible phenotype.