First published online June 23, 2006; 10.1104/pp.106.080697
Plant Physiology 141:1482-1493 (2006)
© 2006 American Society of Plant Biologists
PLANTS INTERACTING WITH OTHER ORGANISMS
Activation of Ntf4, a Tobacco Mitogen-Activated Protein Kinase, during Plant Defense Response and Its Involvement in Hypersensitive Response-Like Cell Death1
Dongtao Ren2,
Kwang-Yeol Yang2,3,
Guo-Jing Li2,4,
Yidong Liu and
Shuqun Zhang*
State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, China (D.R.); and Department of Biochemistry, University of Missouri, Columbia, Missouri 65211 (D.R., K.-Y.Y., G.-J.L., Y.L., S.Z.)
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ABSTRACT
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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.
The active defense of plants against invading pathogens often includes rapid programmed cell death, known as the hypersensitive response (HR), the generation of reactive oxygen species, the activation of a complex array of defense genes, and the production of antimicrobial phytoalexins (Dangl and Jones, 2001 ; Dixon, 2001; Lam et al., 2001; Martin et al., 2003; Greenberg and Yao, 2004; Torres and Dangl, 2005). In addition to these local responses, the uninfected portions of the plant usually develop systemic acquired resistance, which is manifested as enhanced resistance to a subsequent challenge by pathogens (Durrant and Dong, 2004; Bostock, 2005). The induction of these defense responses is regulated by a complex signaling network initiated after the plant recognition of pathogens, which is mediated either by gene-for-gene interactions between plant resistance (R) genes and pathogen avirulence (Avr) genes, or by the binding of nonhost-specific elicitors, also known as pathogen-associated molecular patterns, to their receptors (Staskawicz et al., 1995; Baker et al., 1997; Dangl and Jones, 2001; Martin et al., 2003; Innes, 2004; Ausubel, 2005; Boller, 2005).
Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules in eukaryotes. They function downstream of sensors/receptors and convert signals generated by the sensors/receptors to cellular responses (Herskowitz, 1995; Mizoguchi et al., 1997; Widmann et al., 1999; Davis, 2000; Chang and Karin, 2001; Innes, 2001; Tena et al., 2001; Zhang and Klessig, 2001; Nakagami et al., 2005; Pedley and Martin, 2005). Biochemical studies from a number of laboratories demonstrated that SIPK and WIPK, two tobacco (Nicotiana spp.) MAPKs, and their orthologs in other plant species, including MPK6 and MPK3 in Arabidopsis (Arabidopsis thaliana), SIMK and SAMK in alfalfa (Medicago sativa), LeMPK1/2 (LeSIPK) and LeMPK3 (LeWIPK) in tomato (Lycopersicon esculentum), and PcMPK6 and PcMPK3 in parsley (Petroselinum crispum), are activated in cultured cells or plants treated with elicitors or after pathogen infection (Stratmann and Ryan, 1997; Zhang and Klessig, 1998a; Zhang et al., 1998, 2000; Romeis et al., 1999; Cardinale et al., 2000; Nühse et al., 2000; Desikan et al., 2001; Lee et al., 2001, 2004; Yuasa et al., 2001; Asai et al., 2002; Link et al., 2002; Ekengren et al., 2003; Holley et al., 2003; Kroj et al., 2003; del Pozo et al., 2004; Pedley and Martin, 2004). In tobacco, SIPK and WIPK share a common upstream MAPK kinase (MAPKK), NtMEK2 (Yang et al., 2001). There are two NtMEK2 orthologs in Arabidopsis, MKK4 and MKK5, which are likely to arise from a relatively recent gene duplication (Asai et al., 2002; MAPK Group, 2002; Ren et al., 2002). The MAPKK kinases (MAPKKKs) upstream of NtMEK2/MKK4/MKK5 include MEKK1 and MAPKKK (Asai et al., 2002; del Pozo et al., 2004).
Pathogen/elicitor-induced activation of SIPK in tobacco or its orthologs in other plant species occurs within 1 to 5 min, representing one of the earliest responses in plants after the perception of invading pathogens. The rapid activation of these MAPKs potentially allows them to regulate a variety of other early, intermediate, and late defense responses. Depending on the nature of the pathogens/elicitors, SIPK activation can either be transient or longer lasting. For instance, pathogens or elicitors that cause HR cell death induce long-lasting activation of SIPK (Zhang and Klessig, 1998a; Zhang et al., 1998, 2000; Suzuki et al., 1999). In contrast, elicitors that do not induce cell death, such as fungal cell wall elicitors, only activate SIPK transiently (Zhang et al., 1998). Based on evidence from correlative biochemical analyses and inhibitor studies, it was concluded that SIPK and WIPK are involved in regulating HR cell death in tobacco disease resistance (Zhang and Klessig, 1998a; Zhang et al., 1998, 2000; Suzuki et al., 1999). This conclusion is further supported by the gain-of-function genetic evidence. Conditional expression of an active form of NtMEK2, which activates SIPK and WIPK, leads to HR-like cell death (Yang et al., 2001; Yoshioka et al., 2003). The same phenotype was observed in other plant species, including Arabidopsis and tomato (Ren et al., 2002; del Pozo et al., 2004; Pedley and Martin, 2004). Later, it was shown that activation of SIPK alone is sufficient to induce cell death (Zhang and Liu, 2001; Samuel and Ellis, 2002), and WIPK accelerates the cell death process (Liu et al., 2003). In mammals, the activation kinetics and amplitude of stress-activated protein kinase/c-Jun N-terminal kinase and p38 kinase also influence the fate of cells under stress (Kyriakis and Avruch, 1996; Widmann et al., 1999; Davis, 2000). Transient activation of these MAPKs induces various defense responses and allows the cells to adapt to adverse environments. In contrast, persistent activation leads to apoptosis (Xia et al., 1995; Chen et al., 1996).
Ntf4 is a tobacco MAPK that shares high homology with SIPK, both of which belong to the A2 subgroup of the plant MAPK family (MAPK Group, 2002). In the fully sequenced Arabidopsis genome, there is only one MAPK that belongs to the A2 subgroup of plant MAPK family, MPK6. Based on the genome analysis, we rule out the possibility that Ntf4 and SIPK are orthologs from the two parental lines of the amphidiploid common tobacco. Ntf4 is highly induced during pollen hydration and is reported to function in pollen germination (Wilson et al., 1997; Voronin et al., 2001). In this report, we provided evidence that Ntf4 is also expressed in tobacco leaves and suspension-cultured cells. We further demonstrate that Ntf4 shares the same upstream MAPKK, NtMEK2, with SIPK and WIPK and is activated by pathogen-derived elicitor, indicating that Ntf4 functions in signaling plant defense response as well. Conditional gain-of-function analysis revealed that the activation of Ntf4 leads to HR-like cell death. These results demonstrate that Ntf4 plays multiple functions in tobacco and is involved in signaling both development and disease resistance in tobacco.
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RESULTS
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Ntf4 and SIPK Are Two Highly Homologous MAPKs in Tobacco
Since the purification and cloning of SIPK, it has become one of the best-studied MAPKs in plants (Zhang and Klessig, 1997). In tobacco, there is another highly homologous MAPK, Ntf4, which shares 93.6% identity with SIPK at the amino acid level and 88.0% identity at the nucleotide level (Fig. 1
). Ntf4 was reported to be expressed specifically in tobacco pollen grains, developing embryos, and seeds (Wilson et al., 1997; Voronin et al., 2001). In the process of cloning additional MAPKs from tobacco, we identified a large number of Ntf4 clones from a tobacco leaf cDNA library, suggesting that Ntf4 is also expressed in tobacco leaf tissues. Ntf4 was cloned initially from Petit Havana SR1 cultivar of common tobacco (Wilson et al., 1995). In our library constructed using Xanthi NC (Guo et al., 2000), two Ntf4 genes were identified. They were named Ntf4-1 (DQ229077) and Ntf4-2 (DQ229078), which are 97.5% and 98.5% identical to Ntf4 at the amino acid level, respectively. At the nucleotide level, Ntf4-1 and Ntf4-2 share 93.6% and 96.3% identity with Ntf4, respectively. If we only consider the sequence in the open reading frame, both are approximately 98% identical to Ntf4 (Fig. 1).
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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.
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Common tobacco (Nicotiana tabacum) is an amphidiploid organism that carries two sets of diploid chromosomes, one from Nicotiana sylvestris and the other from Nicotiana tomentosiformis (Gerstel, 1976). To determine if SIPK and Ntf4 are orthologs from the two parental lines of common tobacco, we used gene-specific primers to PCR amplify the genomic DNA preparations from N. tabacum, N. sylvestris, and N. tomentosiformis (Fig. 2
). If Ntf4 and SIPK are orthologs, we would expect each of the two parental lines to have only one of the two MAPKs. Four pairs of primers, with two targeting each gene, were selected from the divergent regions of Ntf4 and SIPK. The specificity of the primer pairs was confirmed using cDNA clones in pBluescript vector (pBS-Ntf4 and pBS-SIPK) as templates. As shown in Figure 2, the two SIPK-specific primer pairs only amplified the SIPK cDNA plasmid, whereas the two Ntf4-specific primer pairs only amplified the Ntf4 cDNA plasmid. When the genomic DNAs from N. tabacum, N. sylvestris, and N. tomentosiformis were used as template, each pair of primers amplified a fragment of similar size from all three tobacco species (Fig. 2). This result demonstrated that SIPK and Ntf4 are not orthologs from the two parental lines, but rather are paralogs from a gene duplication event. The use of two sets of specific primer pairs that target different regions of SIPK and Ntf4 strengthens this conclusion.
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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.
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No length polymorphism was observed when the F1/B1 primer pairs, which target the N termini of SIPK and Ntf4, were used in PCR (Fig. 2, top). There was a polymorphism in SIPK between N. sylvestris and N. tomentosiformis when the SIPK-F2/SIPK-B2 primer pair was used. Both bands were present in N. tabacum, indicating that the polymorphism developed before the generation of N. tabacum (Fig. 2, bottom). There also was a polymorphism in Ntf4 between N. sylvestris and N. tomentosiformis when the Ntf4-F2/Ntf4-B2 primer pair was used. The length of the PCR fragment from N. tabacum was the same as that from N. sylvestris, suggesting that this polymorphism generated after the formation of N. tabacum.
SIPK and Ntf4 have different intron structure based on the lengths of PCR fragments, suggesting that they are the result of relatively ancient gene duplication. Potato (Solanum tuberosum) and tomato, two other solanaceous species, also have two highly similar MAPKs that fall into the A2 subgroup of plant MAPKs (Fig. 1; Holley et al., 2003). Two separate lineages are maintained during the course of evolution. LeMPK1 and StMPK1 are more closely related to SIPK, and LeMPK2 and StMPK2 are more closely related to Ntf4. It is unknown whether other plants in the Solanaceae family also have two MAPKs in the A2 subgroup.
Ntf4 Is Expressed in Tobacco Leaves
Leaves and suspension-cultured cells were used in a number of studies that probe into the functions of SIPK in tobacco defense signaling (Lebrun-Garcia et al., 1998; Zhang and Klessig, 1998a, 1998b; Zhang et al., 1998, 2000; Romeis et al., 1999; Suzuki et al., 1999; Lee et al., 2001; Yang et al., 2001; Samuel and Ellis, 2002). We would like to know whether Ntf4 is expressed in these tissue/cell types, and whether Ntf4 is involved in defense signaling as well. To avoid potential cross-hybridization in the RNA gel-blot analysis, we used gene-specific primers to determine the expression of SIPK and Ntf4 by reverse transcription-PCR. Poly(A+) RNA was isolated from tobacco pollen grains, leaves, and suspension-cultured cells. After reverse transcription, cDNA was used as template for PCR analysis. As shown in Figure 3
, both SIPK and Ntf4 are expressed in the tissue/cell types that we examined. Primer pairs that target different regions of Ntf4 and SIPK gave identical results. The specificity of the PCR reactions was ensured by running control reactions with Ntf4 and SIPK cDNA clones as templates. As shown in Figure 3, Ntf4-specific primer pairs only amplified pBS-Ntf4, whereas SIPK-specific primer pairs only amplified pBS-SIPK.
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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.
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In addition to the transcript level, we also examined Ntf4 expression at the protein level. The difference between Ntf4 and SIPK is mostly in their N termini, which allowed us to raise a specific antibody against SIPK, Ab-p48N. The C termini of SIPK and Ntf4 are almost identical. As a result, Ab-p48C, the antibody generated against the peptide corresponding to the C-terminal 18 amino acids of SIPK, recognizes SIPK and Ntf4 equally well (Zhang and Klessig, 1998a, 1998b; Zhang et al., 1998). Immunoblot analysis using Ab-p48N demonstrated that SIPK is expressed in all the tissues/organs that we examined, including leaf, stem, root, pollen, anther, pistil, ovary, and flower petal (Fig. 4A
, top). Immunoblot analysis using Ab-p48C, which recognizes both SIPK and Ntf4, revealed an identical pattern at the 48-kD position (Fig. 4A, bottom). This result indicates that the relative levels of SIPK and Ntf4 in combination are similar to those of SIPK in these tissues/organs. Otherwise, we should have observed different patterns when Ab-p48N and Ab-p48C antibodies were used.
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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.
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No Ntf4-specific antibody is available since the reported Ntf4-specific antibody was later found to cross-react with SIPK (Wilson et al., 1997; Voronin et al., 2001). To provide more direct evidence to support the presence of Ntf4 in leaves, we performed immune-depletion analysis. SIPK protein in the total leaf extract was first depleted by immunoprecipitation using different amounts of SIPK-specific antibody Ab-p48N. As shown in Figure 4B (top), no SIPK protein could be detected in the supernatant after immune depletion with Ab-p48N, demonstrating that SIPK was effectively removed from the leaf protein extract. However, Ab-p48C, which recognizes SIPK and Ntf4, still detected a 48-kD protein in the SIPK-depleted leaf protein extract (Fig. 4B, bottom), which confirms the presence of Ntf4 protein in tobacco leaf tissues. Based on the fact that Ab-p48C antibody detected relatively high levels of 48-kD protein (Ntf4) in the SIPK-depleted leaf extracts (Fig. 3B, bottom, lanes 2, 3, and 4) in comparison to the total protein extract (Fig. 3B, bottom, lane 1), which contains both Ntf4 and SIPK, we conclude that Ntf4 is not a minor MAPK in the leaf tissue. Because of the limited amount of Ab-p48N antibody available, we could not perform immune-depletion experiments on all the cell/tissue types. However, based on similar levels of Ntf4 transcript (Fig. 3), we assume that the Ntf4 protein is present in these cell/tissue types as well.
NtMEK2 Is the Upstream MAPK Kinase of Ntf4
To determine whether Ntf4 shares the same upstream MAPKK with SIPK and WIPK, we performed in vitro phosphorylation and activation assays. As shown in Figure 5A
, His-tagged Ntf4 has low basal level activity in phosphorylating myelin basic protein (MBP) and can autophosphorylate (Fig. 5A). The basal activity of Ntf4 is comparable with that of SIPK, and both are lower than that of WIPK (Zhang and Liu, 2001). Mutation of the catalytic essential Lys (K88) to Arg in the kinase domain renders Ntf4 inactive (data not shown). This inactive Ntf4KR mutant protein was used as a substrate to determine if Ntf4 can be phosphorylated by NtMEK2. As shown in Figure 5B, wild-type NtMEK2 protein had little activity in phosphorylating Ntf4KR. In contrast, NtMEK2DD, the active mutant of NtMEK2, phosphorylated Ntf4KR protein strongly, and the level of phosphorylation was similar to that when SIPKKR and WIPKKR were used as substrates (Fig. 5B). NtMEK2DD, which migrates in between the Ntf4/SIPK and WIPK proteins, has higher autophosphorylation activity and autophosphorylated NtMEK2DD is visible in the autoradiogram (Fig. 5B; Yang et al., 2001).
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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, NtMEK2DD phosphorylates Ntf4, SIPK, and WIPK with equal efficiency. Phosphorylation activities of HisNtMEK2WT and HisNtMEK2DD (0.1 µg) were determined using inactive mutant MAPKs (Ntf4KR, SIPKKR, and WIPKKR, 1 µg) as substrates. The image is from an x-ray film with 1-h exposure. C, Activation of Ntf4 by NtMEK2DD phosphorylation. Wild-type recombinant MAPKs (Ntf4, SIPK, and WIPK, 0.25 µg) were incubated with 0.025 µg of HisNtMEK2WT or HisNtMEK2DD in the presence of 50 µM unlabeled ATP. MBP (final concentration 0.25 µg/µL) and  -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.
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To determine if the phosphorylation of Ntf4 by NtMEK2 activates Ntf4, we incubated wild-type Ntf4 recombinant protein with either NtMEK2 or NtMEK2DD in the presence of unlabeled ATP to allow the phosphorylation of Ntf4. MBP was then added together with -32P-ATP to determine the activity of Ntf4. As shown in Figure 5C, phosphorylation of Ntf4 by NtMEK2DD resulted in high kinase activity of Ntf4. In contrast, incubation of Ntf4 with wild-type NtMEK2 protein did not lead to Ntf4 activation because of the much lower MAPKK activity of the wild-type NtMEK2 protein (Fig. 5C; Yang et al., 2001). We estimated that the activity of Ntf4 increases by several hundred-fold after NtMEK2DD phosphorylation. Figure 5A, which shows the basal kinase activity of Ntf4, is from an overnight exposed x-ray film with 1 µg of recombinant Ntf4. In contrast, only 0.25 µg of Ntf4 was used in Figure 5C and the film was exposed for only 10 min.
Above experiments revealed that the active NtMEK2 can phosphorylate and activate Ntf4 in vitro, qualifying NtMEK2 as the upstream kinase of Ntf4. To provide in vivo evidence, we transiently expressed Ntf4 in the steroid-inducible promoter:NtMEK2DD or NtMEK2KR transgenic tobacco (Jin et al., 2003; Kim and Zhang, 2004). In this experiment, the Ntf4 transgene was under the control of the cauliflower mosaic virus (CaMV) 35S promoter and had a Flag-epitope tag to allow the detection of Ntf4 activity by the immune-complex kinase assay. Flag-Ntf4, Flag-Ntf4KR, and empty vector were transiently transformed into the stable NtMEK2DD or NtMEK2KR transgenic plants. Two days later, NtMEK2 was induced by the application of dexamethasone (DEX), and the activity of Ntf4 was determined by the immune-complex kinase assay using anti-Flag antibody. As shown in Figure 6
, the induction of NtMEK2DD after DEX application elevated the Flag-tagged Ntf4 activity, providing in vivo evidence that NtMEK2 functions upstream of Ntf4. No MBP-phosphorylating activity was detected in vector control or Ntf4KR transgene control. In the negative control NtMEK2KR transgenic background, no activation of Ntf4 was observed after DEX application, demonstrating that the activation of Ntf4 in NtMEK2DD transgenic background is a result of NtMEK2DD expression. In this experiment, both NtMEK2DD and Ntf4 were tagged with the same Flag epitope. However, since NtMEK2DD does not phosphorylate MBP (Yang et al., 2001), the phosphorylation activity detected in Figure 6 should be from Ntf4. Consistent with this conclusion, no MBP-phosphorylating activity was detected in the vector or Ntf4KR controls in the NtMEK2DD transgenic background. Previously, we reported that the expression of NtMEK1DD, NtMEK7DD, and NtMEK8DD in tobacco leaves failed to induce any detectable MAPK activation (Yang et al., 2001). Since the in-gel kinase assay used in the report allows the detection of Ntf4 activity, a negative result suggests that these MAPKKs are unable to activate Ntf4, demonstrating the specificity of NtMEK2DD activation of Ntf4 shown in Figure 6. Consistent with the in vivo results, recombinant NtMEK1DD, NtMEK7DD, and NtMEK8DD failed to phosphorylate and activate Ntf4 (data not shown).
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Figure 6. In vivo activation of Ntf4 by NtMEK2 in tobacco. Stable steroid-inducible promoter:NtMEK2DD and NtMEK2KR transgenic plants were transiently transformed with Flag-tagged Ntf4 or its inactive mutant Ntf4KR 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."
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To this end, we provided both in vitro and in vivo evidence that NtMEK2 functions upstream of Ntf4 in addition to SIPK and WIPK.
Activation of Ntf4 by Cryptogein, a Proteinaceous Elicitor from the Oomycetic Pathogen Phytophthora cryptogea
After we found that Ntf4 is expressed in tobacco leaf tissue and shares the same upstream kinase with SIPK and WIPK, we tested whether Ntf4 can be activated by pathogen-derived elicitors. Because no member-specific Ntf4 antibody is available, we used tobacco plants transformed with a Flag-tagged version of Ntf4 for the experiments. Leaves of 6-week-old wild-type tobacco plants were infiltrated with Agrobacterium carrying Flag-Ntf4 or Flag-Ntf4KR control construct. We also included the empty vector as another negative control. Two days later, the leaf tissue was subjected to stress treatment, and the activation of Ntf4 was determined by in-gel kinase assay and immune-complex kinase assay.
Cryptogein, a proteinaceous elicitor from P. cryptogea, induces prolonged activation of SIPK and WIPK (Lebrun-Garcia et al., 1998; Zhang et al., 1998). As shown in Figure 7A
(top), cryptogein induced a long-lasting activation of two MAPK bands in the vector control plants as determined by in-gel kinase assay. The top 48-kD activity contains SIPK and the smaller 44-kD activity corresponds to WIPK based on previous studies (Zhang et al., 1998, 2000; Lebrun-Garcia et al., 2002). In leaves transformed with Flag-Ntf4, in-gel kinase assay revealed a broader band at the 48-kD position, consistent with the activation of Flag-tagged Ntf4. With a Flag tag, Flag-Ntf4 is larger and therefore migrates a little slower than the endogenous SIPK and Ntf4. In contrast, leaves transformed with the inactive Flag-Ntf4KR or vector control did not have this broad kinase band. This result suggests that Ntf4 can be activated by cryptogein and the 48-kD band is likely a mixture of SIPK and Ntf4.
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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 Ntf4KR 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 Ntf4KR from transgene (top); one was probed with Ab-p48C, which recognizes Flag-tagged Ntf4 and Ntf4KR from the transgenes and the endogenous Ntf4 and SIPK (middle); and the third was probed with Ab-p48N, which recognizes SIPK only (bottom).
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Immunoblot analysis using anti-Flag antibody demonstrated that Flag-Ntf4 and Flag-Ntf4KR transgenes were expressed at similar levels (Fig. 7B, top). To confirm that the broader 48-kD kinase band in Flag-Ntf4-transformed leaves is due to the activation of Flag-Ntf4, we performed further analysis using the immune-complex kinase assay. Flag-tagged Ntf4 or Ntf4KR was first immunoprecipitated from protein extracts using anti-Flag affinity gel, and the kinase activity in the immune complex was determined in solution in the presence of -32P-ATP and MBP. As shown in Figure 7A (middle), higher kinase activation was detected in Flag-Ntf4-transformed, but not in Flag-Ntf4KR-transformed or empty-vector control leaf tissues after cryptogein treatment. Since the expression levels of Flag-Ntf4 were similar throughout the time course (Fig. 7B, top), we concluded that the increase in kinase activity is a result of kinase activation.
Relatively high basal kinase activity was detected in samples without Flag-tagged protein (vector control) or with Flag-tagged inactive kinase (Flag-Ntf4KR), suggesting nonspecific binding and/or cross-linking of another kinase(s) to the affinity beads. Tobacco is rich in poly-phenol and other secondary metabolites, which tend to cause nonspecific cross-linking during incubation. To confirm this speculation, we performed in-gel kinase assay after anti-Flag immunoprecipitation. As shown in Figure 7A (bottom), Flag-Ntf4-transformed leaves showed kinase activity at the expected position (48-kD Ntf4 plus 1-kD Flag epitope) with an induction kinetics consistent with the broader bands in the in-gel kinase assay (Fig. 7A, top). In contrast, no kinase band was present at the 49-kD position in vector- or Flag-Ntf4KR-transformed leaves. This result confirmed that the activity detected in vector- or Flag-Ntf4KR-transformed leaves by immune-complex kinase assay is due to kinase(s) other than Flag-Ntf4 from nonspecific binding or cross-linking to the protein A-agarose beads. We also performed immunoblot analysis using Ab-p48C and Ab-p48N antibodies. As shown in Figure 7B (middle and bottom), the Ab-p48C antibody recognized the endogenous SIPK and Ntf4, as well as transgene products Flag-Ntf4 and Flag-Ntf4KR. In contrast, the Ab-p48N antibody only detected SIPK, which shows constant level of expression.
Long-Lasting Activation of Ntf4 Leads to HR-Like Cell Death in Tobacco
We observed faster HR cell death in Flag-Ntf4-transformed tobacco leaves treated with cryptogein, suggesting that Ntf4 might be involved in regulating this process. To obtain additional supporting evidence, we attempted conditional overexpression of Ntf4 in tobacco leaves using the steroid-inducible pTA7002 binary vector (Aoyama and Chua, 1997). Previously, we found that overexpression of SIPK, but not WIPK, under such condition leads to the activation of newly synthesized SIPK protein (Zhang and Liu, 2001). Agrobacterium cells carrying the Flag-tagged Ntf4 or its mutants in pTA7002 vector (Fig. 8A
) were infiltrated into newly fully expanded leaves of 4-week-old tobacco plants. Transgene expression was induced by the application of DEX (30 µM) 2 d later. Immunoblot analysis with anti-Flag antibody revealed that the Flag-tagged Ntf4 from transgene expression started to accumulate within 3 h (Fig. 8B, top). Between 6 and 12 h, the level of Ntf4 protein peaked and stayed at a similar level up to 24 h. The increase in Flag-tagged Ntf4 protein was accompanied by an increase in Ntf4 activity, as demonstrated by the in-gel kinase activity assay (Fig. 8B, bottom).
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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  sequence from tobacco mosaic virus. Ntf4 or its mutant was inserted into the XhoI-SpeI sites of the steroid-inducible pTA7002 binary vector. Ntf4KR, the Ntf4(K88R) mutant, loses the kinase activity completely because it is not able to bind ATP. Ntf4AF, the Ntf4(T217A/Y219F) mutant, cannot be activated by upstream MAPKK, but should still retain the basal kinase activity. Ntf42N, the Ntf4(D352N) mutant, and Ntf4NN, 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).
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In leaves transformed with Flag-Ntf4KR, the inactive mutant of Ntf4, little activation of MAPK was detected, although the induction of Ntf4KR protein was similar (Fig. 8B). This result indicates that the activity detected by the in-gel kinase assay is Flag-Ntf4 from the transgene expression, and the endogenous MAPKs, including Ntf4, SIPK, and WIPK, were not activated in the process. Similar to what we observed with SIPK, expressing Ntf4 under the control of the constitutive CaMV 35S promoter failed to result in the increase in the activity of Ntf4 transgene product (Fig. 7A, 0-h time point). We speculated that the activation of newly synthesized MAPK after DEX induction is because of the loss of balance between the MAPK-activating activity (MAPKK) and the MAPK-inactivating activity (MAPK phosphatase) after a sudden influx of newly synthesized MAPK (Zhang and Liu, 2001).
To determine if the activation of newly synthesized Ntf4 requires dual phosphorylation of the TEY activation motif by its upstream MAPKK, NtMEK2, we constructed Ntf4AF, an Ntf4 mutant with the Thr-217 and Tyr-219 residues in the TEY activation motif mutated to Ala (A) and Phe (F), respectively. As shown in Figure 8B, this mutation eliminated the activation of Ntf4. The Ntf4AF mutant protein was expressed at a similar level to Ntf4. These results demonstrated that the dual phosphorylation of the TEY motif in Ntf4 by its upstream MAPKK is required for Ntf4 activation in the transient transformation experiments.
Ntf4 also has the conserved common docking domain, which was first identified in MAPKs from yeast and mammals and is conserved in plant MAPKs (Zhang and Liu, 2001). This domain contains two invariable acidic amino acid residues and is involved in the interaction of MAPKs with their upstream kinases, downstream substrates, and their negative regulators, MAPK phosphatases (Tanoue et al., 2000). Similar to SIPK, mutation of either the second Asp (D349) or both Asp (D349 and D352) residues in the common docking domain to Asn (N) did not alter the activation of Ntf4 significantly. As shown in Figure 8, both Ntf42N, the Ntf4(D352N) mutant, and Ntf4NN, the Ntf4(D349N/D352N) mutant, proteins were expressed at similar levels as wild-type Ntf4. However, there was no enhancement of Ntf4 activity. In contrast, kinase activation decreased slightly, suggesting that the conserved Asp residues play a different role in plants and might be involved in Ntf4 activation. These observations are similar to what we found with SIPK (Zhang and Liu, 2001).
Increase in MAPK activity after the induction of Ntf4, Ntf42N, and Ntf4NN protein led to HR-like cell death. Within 12 to 16 h after the induction of Ntf4 expression, cell death in small areas started to appear. By 30 h, areas infiltrated with Agrobacterium carrying Ntf4 collapsed (Fig. 9
). The cell death phenotype is identical to that induced after SIPK expression (Zhang and Liu, 2001). The induction of Ntf4KR, an inactive mutant, or Ntf4AF, a mutant that cannot be activated, did not result in the cell death phenotype, suggesting that Ntf4 activity is required (Fig. 9). The phenotype induced by Ntf42N or Ntf4NN was weaker and delayed, which correlates with their reduced activation (Fig. 8B, bottom). These results demonstrate that the activation of Ntf4 alone is sufficient to induce HR-like cell death, providing gain-of-function evidence supporting a role of Ntf4 in regulating HR-like cell death in tobacco.
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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.
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DISCUSSION
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Tobacco has two MAPKs, SIPK and Ntf4, in the A2 subgroup of plant MAPKs (MAPK Group, 2002). SIPK was implicated in signaling various plant defense responses. One specific role of SIPK is to regulate the ethylene production in plants under stress (Kim et al., 2003; Liu and Zhang, 2004). SIPK was shown to be expressed in at least leaf tissues and suspension-cultured cells, and studies of SIPK were mostly performed using these two organ/cell types (Lebrun-Garcia et al., 1998; Zhang and Klessig, 1998a, 1998b; Zhang et al., 1998, 2000; Romeis et al., 1999; Suzuki et al., 1999; Lee et al., 2001; Yang et al., 2001; Samuel and Ellis, 2002). In contrast, Ntf4 was reported to be expressed specifically in pollen grains, developing embryos, and seeds (Wilson et al., 1997; Voronin et al., 2001). In this report, we demonstrate that SIPK is universally expressed in all tissues/organs examined, including leaf, stem, root, pollen, anther, pistil, ovary, and flower petal (Fig. 4A, top). More importantly, we found that Ntf4 is also expressed in tobacco leaves and suspension-cultured cells. In addition, Ntf4 and SIPK share the same upstream MAPKK, NtMEK2. Cryptogein, a proteinaceous elicitor from P. cryptogea, induces long-lasting activation of Ntf4, which correlates to a rapid HR-like cell death. Conditional gain-of-function analysis revealed that the activation of Ntf4 alone is sufficient to lead to HR-like cell death. Based on these data, we conclude that Ntf4 is also involved in signaling tobacco defense response.
Ntf4 and SIPK in Tobacco Stress Response and Pollen Germination
SIPK was initially purified and cloned as salicylic acid (SA)-induced protein kinase, and was so named (Zhang and Klessig, 1997). This conclusion is based on the fact that only the SIPK gene contains all the tryptic peptides that we identified from the purified kinase using microsequencing. Based on the findings in this report, it is likely that the purified SIPK sample in our previous report was actually a mixture of SIPK and Ntf4. The masses and pIs of SIPK and Ntf4 are almost identical, which would prevent their separation during the purification by column chromatography. Later, it was found that SIPK can be activated by a number of stress stimuli, including wounding, ozone, elicitors, and pathogen infection (Zhang and Klessig, 1998a, 1998b; Zhang et al., 1998, 2000; Lee et al., 2001; Samuel and Ellis, 2002). These stress stimuli activate SIPK independently of SA because the activation of SIPK by these stresses occurs within a few minutes, preceding the accumulation of SA. In addition, stress stimuli that do not induce SA, such as wounding, also activate SIPK strongly. As a result, we concluded that the activation of SIPK by stress stimuli is independent of SA (Zhang et al., 1998). Later, we suggested viewing SIPK as the acronym of stress-induced protein kinase to avoid confusion (Zhang and Liu, 2001). Recently, it was shown that SA is an uncoupler and inhibitor of mitochondrial electron transport (Norman et al., 2004), which could be sensed as an intracellular stress by the cells and lead to SIPK activation.
Although our research, as well as that of a number of others, is focused on the function of SIPK in tobacco stress signaling, we cannot rule out the involvement of Ntf4 in the process. However, this question was set aside because it was reported that Ntf4 is not expressed in the leaf tissues (Wilson et al., 1997; Voronin et al., 2001). Here, we show that Ntf4 has a similar expression pattern as SIPK and is also expressed in leaves and cultured suspension cells. SIPK and Ntf4 cannot be separated in the in-gel kinase assay and show as one kinase band. Immune-complex kinase assay using a SIPK-specific antibody can only demonstrate the presence of SIPK, but cannot rule out the presence of Ntf4. Since no Ntf4-specific antibody is available, there is no previous report to implicate Ntf4 in signaling tobacco stress responses. Using a tagged version of Ntf4, we demonstrated the activation of Ntf4 by pathogen-derived elicitor (Fig. 7) and wounding (data not shown). Consistent with our finding, both LeMPK1, which is more closely related to SIPK, and LeMPK2, which is more closely related to Ntf4, were reported to be expressed in tomato cell cultures and leaves and to be responsive to stress stimuli (Holley et al., 2003).
Recently, it was reported that SIPK is also expressed in pollen, consistent with our data shown in Figure 4. In addition, transient delivery of a loss-of-function NtMEK2 mutant gene to pollen grains by particle bombardment inhibits pollen germination, indicating that NtMEK2 and its downstream MAPKs, Ntf4 and SIPK, are involved in pollen germination (Voronin et al., 2004). Together with data shown in this report, it can be concluded that this MAPKK-MAPK module plays important roles in signaling both developmental processes and responses to pathogen invasion. There are ample precedents in animal systems that a single MAPK can play diverse roles in an organism (Widmann et al., 1999; Kolch, 2005). More research is needed to determine the underlying molecular mechanisms by which Ntf4 and/or SIPK carry out distinct functions in both pollen germination and plant response to environmental stimuli.
Is the Suppression of SIPK Alone Sufficient to Give the Loss-of-Function Phenotype?
With the demonstration that Ntf4 is a stress-responsive MAPK and is likely to be functionally redundant with SIPK in the signaling pathway, it becomes a question whether suppression of only SIPK is sufficient to generate a loss-of-function phenotype. In our earlier report, we found that suppression of SIPK by virus-induced gene silencing compromises the N-gene-mediated tobacco mosaic virus resistance (Jin et al., 2003). Silencing of SIPK also resulted in the loss of resistance to an incompatible bacterial pathogen, Pseudomonas cichorii (Sharma et al., 2003). The region that we targeted to silence SIPK by virus-induced gene silencing shares high homology with Ntf4. As a result, both SIPK and Ntf4 were likely to be suppressed. Similarly, the region of SIPK targeted by Sharma et al. (2003) is almost identical between SIPK and Ntf4. In light of the findings reported here, it is likely that the loss-of-resistance phenotype is a result of silencing of both SIPK and Ntf4. However, in neither of these two reports was Ntf4 expression examined.
Suppression of SIPK by RNA interference increases the sensitivity of tobacco to ozone stress (Samuel and Ellis, 2002). It was concluded in the paper that the silencing of SIPK is specific, citing that Ntf4 gene was still expressed in the SIPK RNAi lines. Since no in-gel kinase assay result was shown for the total extracts from SIPK RNAi plants after ozone treatment, it is unknown whether Ntf4 can be activated by ozone in the SIPK-silenced plants. If ozone can activate Ntf4, some of the conclusions might have to be reevaluated. Alternatively, it is possible that both SIPK and Ntf4 can be activated by ozone, and the phenotype is a result of dose-dependent event, i.e. loss of SIPK alone or loss of SIPK together with partial loss of Ntf4 is sufficient to generate the phenotype.
Why Do Solanaceous Species Need Two MAPKs in the A2 Subgroup?
In addition to tobacco, two other solanaceous species, tomato and potato, in which an extensive search for MAPKs and expressed sequence tag sequencing has been carried out, also have two MAPK members in the A2 subgroup (Holley et al., 2003; Fig. 1). Furthermore, two distinct lineages can be identified in the phylogenetic tree (Fig. 1), suggesting that the gene-duplication event occurred before these three different species arose, but after Solanaceae and Cruciferae diverged in the course of evolution. At present, it is unknown if other species in the Solanaceae family have two MAPKs in the A2 subgroup as well. The significance of having two MAPKs in the A2 subgroup in these species is also unknown. Although our analyses reveal identical properties and functions for Ntf4 and SIPK in this report, it is still possible that these two MAPKs have subtle differences in their functions that remain to be identified. However, since other plant species, including Arabidopsis, only have one MAPK in the A2 subgroup, it is unlikely that Ntf4 plays an essential function that cannot be replaced by SIPK, and vice versa. On the other hand, the conservation of having two members in the A2 MAPK subgroup over the course of evolution in tobacco, tomato, potato, and, potentially, other solanaceous species does suggest the importance of having both MAPKs in these plants. One possibility for the importance of having two members in these species could be simply a gene-dosage effect. In our mutant analysis using Arabidopsis, we identify distinct phenotypes in heterozygous MAPK mutant plants under certain conditions, suggesting that the copy number of MAPK genes could be important for their function (Y. Liu, N. Ngwenyama, and S. Zhang, unpublished data). The additional copy of MAPK may give certain unique properties to this family of plants, including how they respond to pathogen invasion and other environmental stress stimuli. Duplications of genes encoding other subgroups of plant MAPKs have also occurred during the course of evolution. Recent genomic comparison of MAPKs and MAPKKs in Arabidopsis, rice (Oryza sativa), and poplar (Populus spp.) revealed extensive gene duplications that are unique to different species (Hamel et al., 2006). Although direct evidence is lacking, it is possible that gene copy numbers may affect the strength of signaling processes, which in turn plays a role in determining the specific forms (growth and development pattern/program) of plants and their response to environment.
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MATERIALS AND METHODS
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Plant Growth and Treatments
Wild-type tobacco plants [Nicotiana tabacum cv Xanthi NC (NN), Nicotiana sylvestris, and Nicotiana tomentosiformis] and steroid-inducible promoter:NtMEK2DD or NtMEK2KR transgenic plants in the Xanthi background (Jin et al., 2003) were grown at 22°C in a growth room programmed for a 14-h light cycle. Different organs/tissues were collected from flowering plants. Fully expanded leaves of 4- or 6-week-old tobacco plants were used for Agrobacterium-mediated transient transformation experiments. Suspension-cultured cells were maintained and collected as described previously (Zhang and Klessig, 1997; Zhang et al., 1998).
Samples for protein and RNA preparations were collected without treatment or at the indicated times after treatment, quick-frozen in liquid nitrogen, and stored at –80°C until use.
Cloning of the Tobacco Ntf4 Gene and Mutagenesis
SIPK cDNA insert was labeled with -32P-dCTP and used as a probe to screen a ZapExpress tobacco leaf cDNA library (Guo et al., 2000) under low stringency to identify additional tobacco MAPK genes. Positive clones were grouped based on restriction enzyme mapping. Clones with the longest inserts from each group were fully sequenced. Eleven positive clones that belong to the Ntf4-1 and Ntf4-2 groups were obtained by screening approximately 1 x 106 pfu.
Various mutants of Ntf4 were generated by QuickChange site-directed mutagenesis (Stratagene) and confirmed by sequencing.
Genomic DNA Isolation and PCR Analysis
Tobacco genomic DNA was isolated using Nucleon Phytopure plant DNA extraction kit (Amersham Life Science). Ntf4 and SIPK gene-specific primers were selected from the divergent regions between Ntf4 and SIPK, and the specificity of the primer pairs was confirmed by using SIPK and Ntf4 cDNA clones as templates.
The following primers were used: Ntf4-F1, 5'-TTCCCCCAACAACACGCACAT-3'; Ntf4-F2, 5'-CTAATCAGGGATTATCAGAAGAACAT-3'; Ntf4-B1, 5'-TGAATGCTTCTCTTTGAGGCG-3'; Ntf4-B2, 5'-TTACATGTGGAAATTTTTCGACAAAA-3'; SIPK-F1, 5'-GACATACTACGGCCCTTCTTCC-3'; SIPK-F2, 5'-AGGGTTTATCTGAGGAGCAC-3'; SIPK-B1, 5'-AAAGGCCTCTCTCTGTGGTG-3'; and SIPK-B2, 5'-GTACATGTGGAAACTTTTCAGTGAAT-3'.
RNA Isolation and Reverse Transcription-PCR Analysis
Total RNA from pollen grains, leaves, and cultured suspension cells was extracted using Trizol reagent (Invitrogen) according to the manufacturer's instructions. Poly(A+) RNA was purified from total RNA using the Qiagen Oligotex mRNA mini kit. After reverse transcription, Ntf4 and SIPK were amplified using the same primer pairs listed above. The specificity of the PCR was monitored using cloned SIPK and Ntf4 plasmids as templates.
Expression and Purification of Recombinant Tobacco Ntf4
Recombinant Ntf4 and Ntf4KR were prepared as before (Zhang et al., 1998). A BamHI site was introduced in front of the ATG start codon of Ntf4 by PCR and then ligated in frame into the pET-28a(+) vector (Novagen). BL21(DE3) cells transformed with pET-28a(+) constructs were induced with 0.5 mM isopropylthio- -galactoside for 3 h. His-tagged proteins were purified using nickel-affinity columns (Amersham Pharmacia Biotech), concentrated, and desalted using Centricon-10 (Millipore).
Agrobacterium-Mediated Transient Transformation
Agrobacterium tumefaciens-mediated transient transformation experiments were performed as described previously (Yang et al., 2001). Ntf4 and its mutants with a Flag epitope at their N termini were inserted into the XhoI-SpeI sites of the steroid-inducible pTA7002 binary vector (Aoyama and Chua, 1997) or a modified pBI121 binary vector for constitutive expression as described previously (Zhang and Liu, 2001; Liu and Zhang, 2004). Four- or 6-week-old tobacco plants were used for experiments. Two days after Agrobacterium infiltration, leaf tissues were used for experiments. For constructs with transgene under the control of steroid-inducible promoter, DEX (30 µM) was applied by spraying or infiltration to induce the transgene expression. Elicitin (cryptogein, 50 nM) was delivered by infiltration.
Protein Extraction and Immunoblot Analysis
Protein was extracted from leaf tissue and stored at –80°C (Zhang and Klessig, 1998b). The concentration of protein extracts was determined using the Bio-Rad protein assay kit with bovine serum albumin as standard. Immunoblot detection of Flag-tagged proteins and endogenous MAPKs was performed as described previously (Zhang and Klessig, 1997; Yang et al., 2001).
Immune-Depletion Analysis
Total leaf protein extract (75 µg) was incubated with different amounts (0, 2, 5, and 10 µg) of Ab-p48N in the immunoprecipitation buffer at 4°C for 2 h (Zhang et al., 1998b). Washed protein A-agarose beads (10 µL packed volume) were then added, and the incubation continued for another 4 h at 4°C on a rotating mixer. After centrifugation to remove the immune-complex-protein A beads, the supernatants were subjected to immunoblot analysis using Ab-p48N or Ab-p48C, which recognize SIPK or SIPK plus Ntf4. The absence of signals in Ab-p48N immunoblot analysis indicates the effective removal of SIPK from the leaf extract.
Phosphorylation Assays
Autophosphorylation and phosphorylation activities of recombinant MAPKs and their mutants were determined by incubating equal amounts (1 µg) of purified recombinant proteins in reaction buffer (25 mM Tris, pH 7.5, 10 mM MnCl2, 1 mM EGTA, and 1 mM DTT) in the presence of 0.25 µg/µL MBP and 25 µM of -32P-ATP (1 µCi per reaction) at 30°C for 30 min. The reaction was stopped by the addition of SDS-loading buffer, and kinase activities were detected by autoradiography after SDS-PAGE.
Phosphorylation activities of HisNtMEK2 and HisNtMEK2DD were determined by using inactive mutant MAPKs (HisMAPKKR, 1 µg) as substrates under the same conditions as the autophosphorylation assay, except 0.1 µg of HisNtMEK2 or HisNtMEK2DD was added to each reaction.
MAPK activation assay was carried out by first incubating HisMAPKs (0.25 µg) with 0.025 µg of HisNtMEK2 or HisNtMEK2DD in the presence of 50 µM unlabeled ATP at 30°C for 15 min. MBP (final concentration of 0.25 µg/µL) and -32P-ATP (1 µCi per reaction) were then added. The reactions were stopped by the addition of SDS-loading buffer after 30 min. The phosphorylated MBP was visualized by autoradiography after being resolved on a 15% SDS-polyacrylamide gel.
Immune-complex kinase assays of Flag-tagged Ntf4 were performed using 75 µg protein extract with anti-Flag affinity gel (10 µL packed volume; Sigma), as described previously (Zhang and Klessig, 1998b).
The in-gel kinase activity assay was performed as described previously (Zhang and Klessig, 1997).
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers DQ229077 (Ntf4-1) and DQ229078 (Ntf4-2).
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ACKNOWLEDGMENTS
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We thank N.-H. Chua for the pTA7002 vector, and Sunjoo Joo and Njabulo Ngwenyama for critical reading of the manuscript.
Received March 21, 2006;
returned for revision June 14, 2006;
accepted June 16, 2006.
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FOOTNOTES
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1 This work was supported by the National Science Foundation (grant nos. MCB–9974796 and IBN–0133220 to S.Z.), the National Natural Science Foundation of China (grant nos. 30421002 and 30370140 to D.R.), and the National Basic Research Program of China (grant no. 2003CB114304 to D.R.). 
2 These authors contributed equally to the paper.
3 Present address: Department of Plant Biotechnology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500–757, Republic of Korea.
4 Present address: College of Bioengineering, Inner Mongolia Agricultural University, Huhhot 010018, China.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Shuqun Zhang (zhangsh{at}missouri.edu).
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.080697.
* Corresponding author; e-mail zhangsh{at}missouri.edu; fax 573–884–9676.
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