Tobacco MAPKKs and their mutants with a Flag-epitope at their N termini were inserted into the LBA4404 carrying different constructs was grown overnight in AB medium (28) containing 100 g/ml streptomycin, 50 g/ml kanamycin, and 100 M acetosyringone. activated by pathogens and pathogen-derived elicitors as well (10, 11, 20). Its ortholog in parsley, and evidence. Expression of cv. Xanthi nc). The reverse transcriptionCPCR products were cloned into pGEM-T vector (Promega). Four unique clones with homology to MAPKKs were identified, which were used to screen a tobacco ZapExpress cDNA library. Positive clones containing the longest insert were sequenced. Preparation of Recombinant Proteins. An cv. Xanthi nc [NN] and cv. Xanthi nc [NN]/NahG transgenic) were grown at 22C in a growth room programmed for a 14-h light cycle. Seven to eight-week-old tobacco plants were used for experiments. Tobacco MAPKKs and their mutants with a Flag-epitope at their N termini were inserted into the LBA4404 carrying different constructs was grown overnight in AB medium (28) containing 100 g/ml streptomycin, 50 g/ml kanamycin, and 100 M acetosyringone. Cells were collected by centrifugation (4,000 AtMEK4 of unknown function. Open in a separate window Figure 1 Alignment of tobacco NtMEK2 with AtMEK4 (“type”:”entrez-protein”,”attrs”:”text”:”BAA28830″,”term_id”:”3219271″,”term_text”:”BAA28830″BAA28830) and human MEK1 (“type”:”entrez-protein”,”attrs”:”text”:”Q02750″,”term_id”:”400274″,”term_text”:”Q02750″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, AT-1001 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. In yeast and animals, MAPKKs are activated through the phosphorylation of two Ser/Thr residues in a conserved S/TxxxS/T motif by MAPKK kinases (17). When these two Ser/Thr residues are replaced with Glu (E) or Asp (D), the mutant MAPKK becomes constitutively active (26, 27). To generate such a mutant of NtMEK2 for gain-of-function studies, we mutated several Ser/Thr residues between the kinase subdomains VII and VIII to D as single, double, or triple mutants. The activities of the His-tagged recombinant proteins were determined by an autophosphorylation assay. Fig. ?Fig.22 shows that HisNtMEK2DD, where the conserved Thr-227 and Ser-233 were replaced by Asp, has much higher kinase activity than the AT-1001 wild-type protein. Mutagenesis of other tobacco MAPKKs at corresponding positions also leads to increases in kinase activity (data not shown). These results suggest that plant MAPKKs have an activation motif with two Ser/Thr residues separated by five amino acids instead of the three in MAPKKs from yeast and animals (Fig. ?(Fig.1). 1). Open in a separate window Figure 2 Constitutively active NtMEK2 mutant phosphorylates and activates SIPK and WIPK. (spp. (20, 23). As shown in Fig. ?Fig.33(20), indicating that NtMEK2 is the upstream kinase of SIPK and WIPK in the same pathway. The truncated NtMEK2, against which the antibody was raised, but not the corresponding regions of NtMEK1 and NtMEK7, could compete for the binding of the Ab-NtMEK2, demonstrating the specificity of the immune complexCkinase assay. The N-terminal portion of NtMEK8, NtMEK8, is insoluble. As a result, it cannot be used as a competitor in the immune complexCkinase assay. Nonetheless, immunoblot analysis indicated that Ab-NtMEK2 does not crossreact with NtMEK8 (Fig. ?(Fig.33cells carrying Flag-tagged and correlated with high MAPKK activity of Flag-NtMEK2DD as demonstrated by immune complexCkinase assay by using HisSIPKR and HisWIPKR as substrates (Fig. ?(Fig.4A 4and (Fig. ?(Fig.44carrying pTA7002 constructs. DEX.Interestingly, we noticed consistently that the leaves collapse a few hours earlier in the plants (data not shown). In addition to the HR-like cell death, expression of and expression with SIPK activation by the use of kinase inhibitor (23). and wounding-induced protein kinase (WIPK), two tobacco MAPKs, to HR cell death induced by fungal elicitins (20). SIPK was first identified as SA-induced protein kinase (21). It is also activated by various biotic and abiotic stresses, much like mammalian JNK/SAPK (12, 16). WIPK, which was first cloned as a gene whose transcript is induced by wounding (22), is activated by pathogens and pathogen-derived elicitors as well (10, 11, 20). Its ortholog in parsley, and evidence. Expression of cv. Xanthi nc). The reverse transcriptionCPCR products were AT-1001 cloned into pGEM-T vector (Promega). Four unique clones with homology to MAPKKs were identified, which were used to screen a tobacco ZapExpress cDNA library. Positive clones containing the longest insert were sequenced. Preparation of Recombinant Proteins. An cv. Xanthi nc [NN] and cv. Xanthi nc [NN]/NahG transgenic) were grown at 22C in a growth room programmed for a 14-h light cycle. Seven to eight-week-old tobacco plants were used for experiments. Tobacco MAPKKs and their mutants with a Flag-epitope at their N termini were inserted into the LBA4404 carrying different constructs was grown overnight in AB medium (28) containing 100 g/ml streptomycin, 50 g/ml kanamycin, and 100 M acetosyringone. Cells were collected by centrifugation (4,000 AtMEK4 of unknown function. Open in a separate window Figure 1 Alignment of tobacco NtMEK2 with AtMEK4 (“type”:”entrez-protein”,”attrs”:”text”:”BAA28830″,”term_id”:”3219271″,”term_text”:”BAA28830″BAA28830) and human MEK1 (“type”:”entrez-protein”,”attrs”:”text”:”Q02750″,”term_id”:”400274″,”term_text”:”Q02750″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. In yeast and animals, MAPKKs are activated through the phosphorylation of two Ser/Thr residues in a conserved S/TxxxS/T motif by MAPKK kinases (17). When these two Ser/Thr residues are replaced with Glu (E) or Asp (D), the mutant MAPKK becomes constitutively active (26, 27). To generate such a mutant of NtMEK2 for gain-of-function studies, we mutated several Ser/Thr residues between the kinase subdomains VII and VIII to D as single, double, or triple mutants. The activities of the His-tagged recombinant proteins were determined by an autophosphorylation assay. Fig. ?Fig.22 shows that HisNtMEK2DD, where the conserved Thr-227 and Ser-233 were replaced by Asp, has much higher kinase activity than the wild-type protein. Mutagenesis of other tobacco MAPKKs at corresponding positions also leads to increases in kinase activity (data not shown). These results suggest that plant MAPKKs have an activation motif with two Ser/Thr residues separated by five amino acids instead of the three in MAPKKs from yeast and animals (Fig. ?(Fig.1). 1). Open in a separate window Figure 2 Constitutively active NtMEK2 mutant phosphorylates and activates SIPK and WIPK. (spp. (20, 23). As shown in Fig. ?Fig.33(20), indicating that NtMEK2 is the upstream kinase of SIPK and WIPK in the same pathway. The truncated NtMEK2, against which the antibody was raised, but not the corresponding regions of NtMEK1 and NtMEK7, could compete for the binding of the Ab-NtMEK2, demonstrating the specificity of the immune complexCkinase assay. The N-terminal portion of NtMEK8, NtMEK8, is insoluble. As a result, it cannot be used as a competitor in the immune complexCkinase assay. Nonetheless, immunoblot analysis indicated that Ab-NtMEK2 does not crossreact with NtMEK8 (Fig. ?(Fig.33cells carrying Flag-tagged and correlated with high MAPKK activity of Flag-NtMEK2DD as demonstrated by immune complexCkinase assay by using HisSIPKR and HisWIPKR as substrates (Fig. ?(Fig.4A 4and (Fig. ?(Fig.44carrying pTA7002 constructs. DEX (30 M) was infiltrated 40 h later, and samples were taken at indicated times. The expression of transgenes was monitored by TIMP2 immunoblot analysis by using anti-Flag antibody (transgene expression were determined by immunoblot analysis (and data not shown). Induction of carrying carrying indicated constructs or vector control, and DEX (30 M) was applied 40 h later..