The tobacco wounding-activated mitogen-activated protein kinase is encoded by SIPK

Abstract

It has been demonstrated that both salicylic acid and fungal elicitors activate a 48-kDa mitogen-activated protein kinase termed salicylic acid-induced protein kinase (SIPK) in tobacco suspension cells. Here, we show that infiltration of these agents into tobacco leaves also activates SIPK. Of particular interest, infiltration of water alone activated a kinase of the same size, possibly because of wounding and/or osmotic stresses. The kinetics of kinase activation, however, differ for these different treatments. Various mechanical stresses, including cutting and wounding by abrasion, also activated a 48-kDa kinase. By using an immune-complex kinase assay with antibodies specific for SIPK or wounding-induced protein kinase, we demonstrate that this wounding-activated 48-kDa kinase is SIPK, rather than wounding-induced protein kinase, as reported [Seo, S., Okamoto, M., Seto, H., Ishizuka, K., Sano, H. & Ohashi, Y. (1995) Science 270, 1988-1992]. Activation of SIPK after wounding was associated with tyrosine phosphorylation but not with increases in SIPK mRNA or protein levels. Thus, the same mitogen-activated protein kinase, SIPK, appears to facilitate signaling for two distinct pathways that lead to disease resistance responses and wounding responses.

Figure 1

Infiltration of SA, fungal cell wall elicitor, and water activate a 48-kDa kinase in tobacco leaves. (A) Tobacco leaves were infiltrated with either SA (1 mM) or water, and samples were taken at the indicated times. Kinase activity was determined with an in-gel kinase activity assay by using MBP as the substrate. The size of the activated kinase is given in kilodaltons (kDa). (B) The 48-kDa kinase activities detected in SA-infiltrated (•) and water-infiltrated (○) leaves were quantitated by using a PhosphorImager (Molecular Dynamics), and the relative activities were plotted against time. Kinase activities were normalized to the level present at the zero time point, which was given a value of 1. (C) Tobacco leaves were infiltrated with either a cell wall elicitor (Eli) from P. parasitica (100 μg of glucose equivalent per milliliter) or water, and samples were taken at the indicated times and analyzed as in A. (D) The 48-kDa kinase activities in fungal cell wall elicitor-infiltrated (•) and water-infiltrated (○) leaves were quantitated by using a PhosphorImager, and the relative activities were plotted against time as in B. The difference between elicitor- and water-infiltrated leaves, which presumably represents the elicitor effect, is depicted as a line with triangular symbol (▴).

Figure 2

The 48-kDa kinase activated by SA and fungal cell wall elicitor corresponds to SIPK. (A) Protein extracts (50 μg) from water-infiltrated (0 and 180 min) and SA-infiltrated (0 and 180 min) leaves were immunoprecipitated with the SIPK-specific antibody Ab-p48N in the absence or presence of a competitor peptide (either p48N or p44N). Kinase activity of the immune-complex subsequently was determined with an in-solution kinase assay by using MBP as the substrate, and the phosphorylated MBP was visualized by autoradiography after SDS/PAGE. (B) Protein extracts (50 μg) from water-infiltrated (0 and 30 min) and fungal cell wall elicitor (Eli)-infiltrated (0 and 30 min) leaves were immunoprecipitated with antibody Ab-p48N in the absence or presence of a competitor peptide. Kinase activity of the immune-complex subsequently was determined as described in A.

Figure 3

Wounding by cutting or abrasion also activates a 48-kDa kinase. (A) Leaf discs were punched out of fully expanded tobacco leaves and floated on Hepes buffer as described by Usami et al. (13). At the indicated times, leaf discs were harvested, and protein extracts were prepared. Kinase activity was determined by an in-gel kinase activity assay. (B) Fully expanded tobacco leaves were wounded by rubbing with carborundum as described by Seo et al. (12) and analyzed as in A.

Figure 4

Specificity of antibody Ab-p44N raised against a peptide (p44N) corresponding to the N terminus of WIPK. One nanogram each of HisSIPK, HisNtf4, HisWIPK, and HisNtMPK6, equal mixture of HisSIPK and HisWIPK, or 20 μg of protein extracts from fully expanded tobacco leaf were separated in 10% SDS/polyacrylamide gel. Duplicated blots were subjected to immunoblot analysis by using the antibodies listed below: (A) Ab-p44N (0.5 μg/ml); (B) Ab-p44N preadsorbed with HisWIPK; (C) Ab-p44N preadsorbed with HisSIPK; (D) Ab-p44N (0.5 μg/ml) plus Ab-p48N (0.5 μg/ml); (E) Ab-p48N (0.5 μg/ml). The bands corresponding to HisWIPK and HisSIPK are indicated with asterisks (∗) and dots (⋅), respectively. Recombinant proteins migrate ≈3.6-kDa slower than their endogenous counterparts because of the His-tag; thus, HisWIPK and SIPK migrate similarly as seen in D.

Figure 5

The 48-kDa kinase activated by water infiltration and wounding is encoded by SIPK rather than WIPK. Protein extracts (50 μg) from water-infiltrated, cutting- or abrasion-wounded leaves were immunoprecipitated with either the SIPK-specific antibody Ab-p48N (Upper) or the WIPK-specific antibody Ab-p44N (Lower). Kinase activity of the resultant immune-complexes subsequently was determined as described in the text.

Figure 6

Activation of SIPK by water infiltration or wounding is regulated at the post-translational level by phosphorylation. (A) Total RNA was extracted from water-infiltrated or wounded leaves and was subjected to RNA blot analysis. Blots were hybridized sequentially with the 5′ untranslated region and then the full-length SIPK cDNA. Because both probes yielded the same result, only the autoradiogram produced with the full length cDNA probe is shown. (B) Protein extracts (20 μg) were subjected to immunoblot analysis by using the SIPK-specific antibody Ab-p48N. (C) Protein extracts (50 μg) from water-infiltrated or wounded leaves (0 and 10 min post-treatment) were immunoprecipitated with Ab-p48N in the absence or the presence of the competitor peptides p48N or p44N. Phosphotyrosine residues in the immune-complex were detected by reaction with the phosphotyrosine-specific monoclonal antibody 4G10. The upper band corresponds to the heavy chain of rabbit IgG (IgGHC). (D) The kinase activity in the immune-complex from (C) was determined by an in-gel kinase assay by using MBP as the substrate.

Figure 7

Water infiltration and wounding induce transient increases in WIPK mRNA levels but little or no increases in WIPK protein level. (A) Total RNA was extracted at the indicated times from water-infiltrated or wounded leaves and was subjected to RNA blot analysis. Blots were hybridized sequentially with the 3′ untranslated region and then the full length WIPK cDNA. Both probes yielded the same result; thus, only the autoradiogram produced with the full-length cDNA probe is shown. (B) Protein extracts (20 μg) were subjected to immunoblot analysis with the WIPK-specific antibody Ab-p44N.