Multiple levels of tobacco WIPK activation during the induction of cell death by fungal elicitins

Summary

Three protein kinases of 48, 44 and 40 kDa are activated at different stages in tobacco cells treated with fungal elicitins. Previously we demonstrated that the rapidly activated 48 kDa protein kinase is encoded by SIPK. Here we report that the elicitin-activated 44 kDa kinase is encoded by WIPK. Activation of this kinase occurred 2–4 h after elicitin treatment and was preceded by dramatic increases in WIPK mRNA and protein levels. Studies using actinomycin D and cycloheximide demonstrated that de novo transcription and translation were required for this activation of the kinase activity. Strikingly, the kinetics of WIPK activation following elicitin treatment correlated with the onset of hypersensitive response (HR)-like cell death. Moreover, staurosporine and K-252a, two Ser/Thr protein kinase inhibitors that blocked WIPK activation, suppressed cell death. The timing for elicitin-treated cells to commit to a death program correlated with the appearance of high levels of WIPK activity. These correlative data suggest that WIPK may play a role during HR development in tobacco. Interestingly, a fungal cell-wall elicitor that does not cause cell death induced WIPK mRNA and protein to similar levels as those observed with the elicitins. However, no corresponding increase in WIPK activity was detected. Thus WIPK appears to be controlled at multiple levels.

Keywords: MAP kinases, WIPK, SIPK, elicitin, transcription, translation.

Introduction

The active defenses of plants against infection by invading pathogens, such as fungi, bacteria, and viruses, often include a hypersensitive response (HR) in which rapid and localized cell death occurs around the sites of infection. Plant defense responses are initiated by the recognition of potential pathogens. This can be mediated either by a gene-for-gene interaction or by the binding of a non-host-specific elicitor to a putative receptor. The signals generated by such an interaction are transduced into cellular responses via several interlinked pathways ( Martin, 1999; Scheel, 1998; Yang et al. 1997 ). Plant cells respond to elicitors, including crude fungal cell-wall fragments, elicitins and the Avr9 factor, with a battery of cellular changes ( Hammond-Kosack & Jones, 1996; Ricci, 1997; Yang et al. 1997 ; Yu, 1995). Some of these responses, such as changes in ion fluxes and the generation of reactive oxygen species, occur very rapidly and may involve events that occur primarily at the post-translational level ( Jabs et al. 1996 ; Levine et al. 1994 ; Mehdy, 1994; Viard et al. 1994 ; Zimmermann et al. 1997 ). Other responses, such as the accumulation of phytoalexins and the synthesis of chitinases, glucanases, and other pathogenesis-related (PR) proteins, involve induction of gene expression ( Dixon & Lamb, 1990; Yang et al. 1997 ).

Several lines of evidence have implicated the involvement of protein phosphorylation/dephosphorylation in regulating elicitor-stimulated defense responses ( Grant & Mansfield, 1999; Martin, 1999; Viard et al. 1994 ). Treatment of plant cells with pathogen-derived elicitors has been shown to cause rapid changes in phosphoprotein profiles ( Dietrich et al. 1990 ; Felix et al. 1991 ; Felix et al. 1994 ; Viard et al. 1994 ). The inhibition of protein phosphorylation by K-252a and staurosporine correlates with the blockage of (1) medium alkalization; (2) reactive oxygen species generation; (3) defense gene activation, including that of phenylalanine ammonia lyase (PAL); and (4) HR-like cell death ( Felix et al. 1991 ; Grosskopf et al. 1990 ; Suzuki et al. 1995 ; Suzuki et al. 1999 ; Viard et al. 1994 ). Conversely, protein phosphatase inhibitors can mimic the effects of elicitors, presumably by promoting protein phosphorylation ( Felix et al. 1994 ).

The mitogen-activated protein kinase (MAPK) cascade is one of the major pathways by which extracellular stimuli are transduced into intracellular responses in yeast and mammalian cells ( Herskowitz, 1995; Kyriakis & Avruch, 1996; Seger & Krebs, 1995). The basic assembly of a MAPK cascade is a three-kinase module conserved in all eukaryotes. MAPK, the last kinase in this cascade, is activated by dual phosphorylation of threonine and tyrosine residues in a TXY motif located between subdomains VII and VIII of the kinase catalytic domain by MAPK kinase (MAPKK). MAPKK is, in turn, activated by MAP kinase kinase kinase (MAPKKK). In mammals, three of the five subgroups of the MAP kinase family, the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK), the p38 kinase, and ERK5, are activated in response to various stress signals, including UV and ionizing radiation, hyperosmolarity, oxidative stress, and cytokines such as tumor necrosis factor and interleukin-1 ( Kyriakis & Avruch, 1996; Widmann et al. 1999 ). The ability of these three MAPKs to transduce various stimuli into different responses is determined to a large extent by the kinetics and/or magnitude of their activation ( Chen et al. 1996 ; Marshall, 1995; Widmann et al. 1999 ). For instance, rapid and transient activation of the SAPK/JNK kinase by a variety of stress stimuli leads to responses that allow the cell to adapt to its environment ( Kyriakis & Avruch, 1996). In contrast, prolonged activation of this kinase initiates apoptosis ( Chen et al. 1996 ).

An increasing body of evidence suggests that MAPKs play similar roles in plants responding to various stresses ( Hirt, 1997; Mizoguchi et al. 1997 ; Zhang & Klessig, 2000). For example, a fungal cell-wall elicitor rapidly activates a 47 kDa MAPK-like kinase in tobacco suspension cells ( Suzuki & Shinshi, 1995). This kinase preferentially phosphorylates myelin basic protein (MBP) and is itself phosphorylated on tyrosine residues upon activation. Similarly, the Pep25 elicitor, derived from the Phytophthora sojae glycoprotein elicitor, activates a 45 kDa MBP kinase in parsley, thought to be encoded by ERMK ( Ligterink et al. 1997 ). In addition, cold, drought and mechanical stresses have been shown to induce the expression of MMK4 in alfalfa ( Bögre et al. 1997 ; Jonak et al. 1996 ). Wounding/cutting of tobacco leaves has also been shown to activate a 46 kDa kinase ( Seo et al. 1995 ; Usami et al. 1995 ) and induce the accumulation of transcripts for WIPK. Based on these results, WIPK was hypothesized to encode the wounding-activated 46 kDa kinase ( Seo et al. 1995 ). However, subsequent studies revealed that the major kinase activated by wounding is encoded by another tobacco MAPK gene, SIPK ( Zhang & Klessig, 1998a). SIPK (salicylic acid (SA)-induced protein kinase) was initially identified as a 48 kDa MAPK that is rapidly activated by SA, an endogenous signaling molecule involved in defense responses ( Zhang & Klessig, 1997). Both SIPK and WIPK are activated in a gene-for-gene-specific manner in tobacco plants resisting infection by TMV or responding to the Cladosporium fulvum-encoded elicitor Avr9 ( Romeis et al. 1999 ; Zhang & Klessig, 1998b). Interestingly, however, the activation pathways for these kinases differ; SIPK is activated only post-translationally while WIPK activation in TMV-infected tobacco is preceded by increases in both mRNA and protein levels ( Zhang & Klessig, 1998b).

Previously we reported that the elicitins parasiticein and cryptogein, small proteins synthesized by phytopathogenic Phytophthora spp. ( Ricci, 1997), activate three kinases with molecular mass of 48, 44 and 40 kDa in tobacco cells. The 48 kDa kinase was shown to be SIPK ( Zhang et al. 1998 ). In this paper we demonstrate that the elicitin-activated 44 kDa kinase is encoded by WIPK, and present data suggesting that this kinase may be involved in regulating HR-like cell death. Further data are presented showing that this MAPK is regulated at multiple levels.

Results

The 44 kDa protein kinase activated by elicitins is encoded by WIPK

Treating tobacco suspension cells with cryptogein or parasiticein was previously shown to induce rapid activation of SIPK and delayed activation of a 44 and a 40 kDa protein kinases ( Fig. 1a; Zhang et al. 1998 ). The size and substrate preference of this 44 kDa kinase are similar to those exhibited by the TMV-induced 44 kDa WIPK. To determine whether the elicitin-induced 44 kDa kinase is encoded by WIPK, a WIPK-specific antibody, Ab-p44N, was used in an immune-complex kinase assay ( Fig. 1b). As anticipated, a kinase activity induced by cryptogein was immunoprecipitated by Ab-p44N. Addition to the immunoprecipitation reaction of p44N, the peptide to which Ab-p44N was raised, abolished precipitation of this kinase activity. In contrast, addition of a control peptide corresponding to the N-terminus of SIPK, p48N, had no effect. Thus the immune-complex kinase assay is specific for WIPK. A similar result was obtained with parasiticein-induced kinases (data not shown). Therefore, we conclude that both elicitins activate WIPK.

Figure 1. Activation of WIPK at multiple levels in tobacco cells treated with cryptogein, an elicitin from Phytophthora cryptogea.

Tobacco cells were treated with 25 n m cryptogein and two sets of cell samples were harvested at the times indicated. One set was used to prepare protein extracts.

(a) Kinase activity was determined with an in-gel kinase assay using MBP as a substrate.

(b) To confirm that the 44 kDa kinase is WIPK, an immune-complex kinase assay was used. Selected protein extracts were immuno- precipitated with Ab-p44N, a WIPK-specific antibody in the absence (–) or presence of competitor peptides p48N or p44N, which correspond to the N-termini of SIPK and WIPK, respectively. Kinase activity of the immune complex was assayed using MBP as the substrate. The phosphorylated MBP was visualized by autoradiography.

(c) Total RNA was isolated from the other set of cell samples and WIPK mRNA levels were determined by RNA blot analysis.

(d) WIPK protein level was determined by immunoblot analysis using the WIPK-specific antibody Ab-p44N.

Activation of WIPK is dependent on transcription and translation

A dramatic increase in the levels of WIPK mRNA and protein preceded the appearance of the 44 kDa WIPK enzyme activity ( Fig. 1c,d). The basal steady-state level of WIPK mRNA was very low, but within 30 min of cryptogein treatment, WIPK mRNA levels started to increase and peaked at 2 h post-treatment (hpt) ( Fig. 1c). The basal level of WIPK protein was also very low; it was estimated to be approximately 10% that of SIPK ( Zhang & Klessig, 1998a). Within 4 h of cryptogein treatment, WIPK protein levels increased markedly and peaked at 8 hpt. Long exposures of the in-gel kinase assay revealed a small increase in WIPK activity by 15 min. Since this slight increase preceded rises in WIPK mRNA and protein levels, it presumably reflects post-translational activation of the basal level of WIPK by the WIPK kinase.

To study whether increases in WIPK gene expression and protein synthesis were required for the high levels of WIPK activity seen at 4–8 h post-elicitin treatment, actinomycin D (actD), a transcription inhibitor, and cycloheximide (CHX), a translation inhibitor, were employed. Pretreatment of cells with either actD or CHX suppressed the strong activation of WIPK induced by cryptogein ( Fig. 2a). The low level of WIPK activity remaining in the presence of these inhibitors was probably due to activation of the pre-existing WIPK protein ( Fig. 2b). As shown in Fig. 2(c,b), respectively, actD blocked cryptogein-induced increases in WIPK mRNA levels while CHX inhibited rises in the corresponding protein levels. Thus, the marked increase in WIPK activity following cryptogein treatment required both transcription and de novo protein synthesis. This is in marked contrast to SIPK ( Fig. 2a) and all yeast and animal MAPKs, which are activated exclusively by post-translational phosphorylation ( Zhang & Klessig, 1998a; Zhang & Klessig, 1998b).

Figure 2. Strong induction of WIPK activity is dependent on transcription and translation.

Tobacco cells were pretreated with actinomycin D (ActD, 50 μg ml⁻¹) or cycloheximide (CHX, 10 μg ml⁻¹) for 15 min before the addition of 25 n m cryptogein (Cry). Aliquots of cells were harvested at 4 and 8 hpt for kinase activity (a), protein (b) and RNA (c) analyses. Kinase activity, WIPK protein and WIPK mRNA levels were determined by an in-gel kinase assay, immunoblot analysis, and RNA blot analysis, respectively.

Suppression of elicitin-induced WIPK activation correlates with inhibition of cell death

Under our assay conditions, treatment of tobacco suspension cells with 25–50 n m of either elicitin led to almost 100% cell death within 24 hpt ( Zhang et al. 1998 ). To study the possible involvement of WIPK in elicitin-induced cell death, the Ser/Thr protein kinase inhibitors staurosporine and K-252a were used ( Fig. 3). Cells were pretreated with either K-252a or staurosporine for 5 min, then either cryptogein or parasiticein was added. Cell death was determined at 3 and 8 hpt by a fluorescein diacetate assay ( Widholm, 1972). Pretreating the cells with either kinase inhibitor almost completely prevented cell death at 3 hpt, and reduced cell death by approximately 65–85% at 8 hpt. The lack of a more complete inhibition at 8 hpt appears to be due to depletion of the kinase inhibitors through either cellular metabolism or their inherent instability. When a second dose of staurosporine was added 3 h after the initial treatment, cell death at 8 hpt was suppressed approximately 95%.

Figure 3. Inhibition of elicitin-induced HR-like cell death by kinase inhibitors.

Cells were pretreated with the kinase inhibitors K-252a or staurosporine (Stauro) for 5 min. For the control, an equal volume of DMSO, which was the solvent used to prepare the inhibitor stock solutions, was added. Either cryptogein or parasiticein (Par) was then added at zero time to a final concentration of 25 n m, and cell death was determined at 4 and 8 hpt as described in Experimental procedures. +/+ denotes a second addition of staurosporine 3 h after the initial inhibitor treatment.

To assess how effectively K252a and staurosporine reduced the elicitin-induced activation of WIPK, as well as that of SIPK and the 40 kDa kinase, in-gel kinase assays were performed ( Fig. 4). At 3 hpt, activation of WIPK and the 40 kDa kinase was almost completely blocked by either inhibitor, corresponding with the very low levels of cell death observed at this time ( Fig. 3). SIPK activity was also greatly reduced in the presence of either inhibitor at 3 hpt ( Fig. 4). By 8 hpt, when the inhibitors appear to have lost much of their effectiveness, the activities of WIPK and the 40 kDa kinase reappeared, correlating with the increased level of cell death ( Figs 3 and 4). SIPK activity also increased at this time, returning to the levels exhibited by control cells not treated with either inhibitor ( Fig. 4). A second addition of staurosporine, which facilitated continued suppression of cell death ( Fig. 3) was able to suppress WIPK activity effectively throughout the time course; however, it only maintained SIPK activity at a reduced level. Thus, WIPK activity levels appear to correlate best with the amount of cell death detected at both 3 and 8 hpt.

Figure 4. Inhibition of cell death by kinase inhibitors correlates with suppression of WIPK activation.

Protein extracts were prepared from cells harvested in the experiment described in Fig. 3. Kinase activity was determined by an in-gel kinase assay with MBP as a substrate. 2× Stauro denotes a second addition of staurosporine at 3 h after the initial inhibitor treatment of Cry-induced cells.

The correlation between cell death and the appearance of WIPK activity following elicitin treatment was further tested by adding staurosporine at various times relative to the addition of cryptogein at zero time. Addition of the kinase inhibitor at various times up to 2 h after cryptogein was observed to block cell death effectively at 3 and 8 hpt; however, addition at 3 h was too late to prevent cell death at 8 hpt ( Fig. 5a). Adding staurosporine up to 2 h after elicitin treatment also effectively suppressed WIPK activation at 3 and 8 hpt, and SIPK activation at 3 hpt. In contrast, staurosporine addition 3 h after elicitin treatment no longer repressed the activation of either WIPK or SIPK. Taken together, these results suggest that the cells were not committed to a death program until between 2 and 3 h after elicitin treatment, and that this commitment involves either the activation of a kinase between 2 and 3 h after addition of cryptogein or the prolonged activation of a kinase during the first 2 hpt. The window of time during which staurosporine could effectively block cryptogein-induced cell death was similar to that during which WIPK and SIPK activities could be inhibited. This correlation therefore suggests that WIPK, and possibly SIPK, are involved in the elicitin-mediated induction of cell death. In these experiments activation of the 40 kDa kinase by cryptogein at 3 and 8 h was quite variable, with staurosporine either having no effect or even enhancing the activation. Thus, 40 kDa kinase activity did not correlate with cell death.

Figure 5. Elicitin-induced cell death com- mitment correlates with the appearance of WIPK activity.

(a) Cryptogein was added to tobacco cells at the zero time and staurosprine was added either 5 min before (−5 min) or 30, 60, 120, or 180 min afterwards. Cell death was determined at 3 and 8 h after the cryptogein treatment.

(b) In parallel, protein extracts were prepared from the treated cells and kinase activity was determined by an in-gel kinase assay with MBP as the substrate.

A fungal elicitor that fails to induce cell death only marginally activates WIPK

Treating tobacco cells with a carbohydrate elicitor derived from the cell walls of Phytophthora parasitica was previously shown to induce SIPK activation in the absence of cell death ( Zhang et al. 1998 ). To determine whether this elicitor induces WIPK gene expression and/or enzyme activation, these phenomena were monitored in elicitor-treated tobacco cells. WIPK transcripts began to accumulate 30 min after elicitor treatment, and by 2 h they had peaked ( Fig. 6a). Significant levels of WIPK protein were first detected at 2 hpt and plateaued at 4 hpt ( Fig. 6b). However, no increase in WIPK activity accompanied this rise in protein level ( Fig. 6c). A longer exposure of the in-gel kinase activity gel shown in Fig. 6(c) revealed an early, weak activation of WIPK (data not shown) which was similar to that previously observed with cryptogein ( Fig. 1a,b). This weak activation, which was most evident at 1 hpt, preceded the rise in WIPK protein which began at 2 hpt ( Fig. 6b). Post-translational activation of the basal level WIPK protein detected by immunoblot analysis ( Fig. 6b) therefore appears to be responsible for the early weak activation of WIPK. In contrast, this elicitor strongly induced SIPK activation ( Fig. 6c; Zhang et al. 1998 ), suggesting that either the activation of SIPK is not sufficient to induced cell death or SIPK is not involved in cell death signaling.

Figure 6. A fungal cell wall elicitor induces the accumulation WIPK transcripts and protein but no subsequent increase in WIPK activity.

Tobacco cells were treated with a fungal cell wall elicitor (50 μg glucose equivalents per ml), and two sets of cell samples were taken at the indicated times.

(a) Total RNA was isolated from one set of cells and WIPK mRNA level was determined by RNA blot analysis. The other set of cells was used to prepare protein extracts.

(b) WIPK protein level was determined by immunoblot analysis using the WIPK-specific antibody Ab-p44N.

(c) Kinase activity was determined with an in-gel kinase assay using MBP as a substrate.

Discussion

Multiple levels of WIPK regulation

In this study we demonstrate that the Phytophthora elicitin-activated 44 kDa protein kinase is encoded by WIPK. Moreover, using actD and CHX, we demonstrate that increases in WIPK mRNA and protein levels are required for elicitin-mediated activation of this kinase. Increases in WIPK mRNA and protein levels have previously been shown to precede WIPK activation in tobacco resisting TMV infection ( Zhang & Klessig, 1998b). Thus, in both systems, activation of WIPK appears to be regulated at the transcriptional level. The ability of a carbohydrate cell wall-derived elicitor from P. parasitica to induce large increases in WIPK mRNA and protein levels, but no subsequent rise in kinase activity, suggests that WIPK is also regulated at the post-translational level. This is consistent with the previous demonstration that WIPK activity is regulated post-translationally in wild-type tobacco resisting TMV infection ( Zhang & Klessig, 1998b) and in Cf-9-expressing transgenic tobacco plants and cells treated with Avr9 ( Romeis et al. 1999 ). WIPK activation may also be regulated at the translational level; wounding wild-type tobacco enhances WIPK transcript levels without a corresponding increase in protein levels or kinase activity ( Zhang & Klessig, 1998a), and Avr9 treatment of Cf-9-expressing tobacco induces a delayed accumulation of WIPK mRNAs but no corresponding increase in the basal level of protein ( Romeis et al. 1999 ).

The discovery that WIPK activation is regulated at multiple levels distinguishes this kinase from all other MAPKs identified thus far. In mammals and yeast, MAPK activation is mediated solely by post-translational phosphorylation ( Widmann et al. 1999 ). The same post-translational mechanism is also used to regulate SIPK activity in tobacco ( Zhang & Klessig, 1997; Zhang & Klessig, 1998a; Zhang & Klessig, 1998b). Accumulation mRNAs of several other plant MAPKs, including ERMK ( Ligterink et al. 1997 ), AtMPK3 ( Mizoguchi et al. 1996 ) and MMK4 ( Jonak et al. 1996 ) is activated by stresses. However, as mRNAs for these MAPKs accumulate relatively slowly while the corresponding kinases are activated very rapidly, it is unclear whether activation of these kinases is regulated at the transcriptional level. In comparison, increases in WIPK transcription, as well as translation, both precede and are required for high levels of its kinase activity.

Protein kinases and cell death

In mammalian cells, activation of the SAPK/JNK and p38 subgroups of MAPKs plays a role in stress-induced apoptosis ( Chen et al. 1996 ; Kyriakis & Avruch, 1996). Similarly, our analyses suggest that the level of activated WIPK correlates with the amount of cell death observed in elicitin-treated tobacco cells. Initial experiments ( Zhang et al. 1998 ) also suggested a correlation between the activity of the 40 kDa kinase and cell death. While a role for this kinase cannot be ruled out, subsequent experiments revealed that the activity of this kinase was quite variable, irrespective of cell death levels. The identity of the 40 kDa kinase is currently unknown; it may correspond to the 40–42 kDa kinase induced by osmotic stress ( Hoyos & Zhang, 2000; Mikoajczyk et al. 2000 ). During a pathogen- or elicitor-induced HR, plant cells undergo cytoplasmic condensation and a loss of turgor pressure; this process is similar to plasmolysis. Possibly, HR-associated changes are sensed as osmotic stress by the cells, which leads to the activation of the 40 kDa kinase. Activation of this kinase by elicitins might therefore be a secondary effect of the defense response to pathogen infection.

In addition to WIPK, it is also possible that SIPK plays a role in cell death. The window of time during which staurosporine inhibited cryptogein-induced cell death matched that for inhibition of both SIPK and WIPK activation. However, several stimuli that activate SIPK fail to induce cell death, including the carbohydrate cell wall-derived elicitor ( Fig. 6; Zhang et al. 1998 ) and SA ( Zhang & Klessig, 1997). If this kinase participates in cell death, these stimuli either induce SIPK activation to a magnitude and/or for a duration of time that is inappropriate for initiation of a cell-death program or they fail to activate other factor(s) required for cell death (perhaps WIPK).

In contrast to the delayed activation of WIPK in our elicitin-treated cells, both WIPK and SIPK activities increased rapidly and transiently in Cf-9 -transgenic tobacco treated with Avr9 ( Romeis et al. 1999 ). Lebrun-Garcia et al. (1998) also demonstrated rapid, coordinated and transient activation of a 50 and a 46 kDa MAPK-like kinase in tobacco cells treated with cryptogein. These two kinases were recently shown to be SIPK and WIPK, respectively (A. Lebrun and A. Pugin, unpublished results). The difference in WIPK activation kinetics between their studies and ours may be due to differences in culturing protocols. We treated the cells with elicitins in the original culture flask, whereas they washed and incubated their cells in a different buffer for several hours before the addition of elicitin or Avr9 ( Lebrun-Garcia et al. 1998 ; Romeis et al. 1999 ). In our hands, such pretreatment results in strong activation of SIPK and elevated levels of WIPK protein (E.M. Hoyos, Y. Liu and S. Zhang, unpublished results). These researchers detected similarly elevated levels of the 46 kDa protein (WIPK) in their untreated and cryptogein- or Avr9-treated cells ( Lebrun-Garcia et al. 1998 ; Romeis et al. 1999 ). In their experiments, addition of cryptogein or Avr9 probably served as a second stimulus that rapidly activated already elevated levels of WIPK.

Cryptogein and Avr9 failed to induce cell death in the pretreated cells of Lebrun-Garcia et al. (1998) and Romeis et al. (1999) . By contrast, cell death was detected in Cf-9-transgenic tobacco plants treated with Avr9, and these plants exhibited the same kinetics of WIPK and SIPK activation as the cells ( Romeis et al. 1999 ). One possible interpretation of these results is that WIPK activation is not required for the induction of cell death. However, we have also observed that culturing conditions, as well as duration of the culturing process, can suppress the ability of elicitins to induce cell death. Thus, we suspect that factor(s) downstream of WIPK, or in a parallel pathway, provide an additional level of regulation in the cell-death pathway. In our experiments two general kinase inhibitors were used, since PD098059 and U0126, two specific inhibitors of MAPK activation in mammalian cells, do not inhibit the activation of either SIPK or WIPK (unpublished data). As staurosporine and K-252a block a variety of kinases including MAPKs, our results are correlative and do not rigorously establish a role for WIPK in tobacco HR-like cell death. Future analyses of mutant or transgenic plants in which WIPK expression/activation is altered should help clarify the role of WIPK in the induction of cell death and other defense responses in pathogen-infected and elicitor-treated tobacco.

Experimental procedures

Treatment of tobacco cell suspension culture

The cell suspension culture was maintained and treated as previously described ( Zhang & Klessig, 1997). Log-phase cells were used 3 days after a 1 : 10 dilution. Treatment with elicitors was done in the original flasks in the dark to avoid any stresses associated with transfer. At various times, 10 ml cells (0.2–0.3 g FW) were harvested by filtration. The cells were quickly frozen in liquid nitrogen and stored at −80°C until analysis. For inhibitor studies, the cells were pretreated for the times indicated by the addition of stock solutions in DMSO.

The fungal cell-wall elicitor was prepared from a heat- released cell-wall fraction of the fungal pathogen Phytophthora parasitica and quantified as previously described ( Zhang et al. 1998 ), and was used at a final concentration of 50 μg glucose equivalents per ml cell suspension culture, or as stated in the figure legend. Parasiticein and cryptogein were generous gifts from Dr Brett Tyler. Both elicitins were used at a final concentration of 25 n m.

Preparation of protein extracts

To prepare extracts from treated cells, cells were mixed with two volumes (w/v) of extraction buffer (100 m m Hepes pH 7.5, 5 m m EDTA, 5 m m EGTA, 10 m m DTT, 10 m m Na₃VO4, 10 m m NaF, 50 m mβ-glycerolphosphate, 1 m m phenylmethylsulfonyl fluoride [PMSF], 5 μg ml⁻¹ antipain, 5 μg ml⁻¹ aprotinin, 5 μg ml⁻¹ leupeptin, 10% glycerol, 7.5% polyvinylpolypyrrolidone) and sonicated with a W-375 Sonicator (Heat System-Ultrasonics Inc., Farmingdale, NY, USA) until all the cells were disrupted. After centrifugation at 20 000 g for 20 min, supernatants were transferred into clean tubes, quickly frozen in liquid nitrogen and stored at −80°C.

Protein kinase activity assay

Kinase activity assays were performed as described previously ( Zhang & Klessig, 1997). Briefly, for the in-gel kinase activity assay extracts containing 10 μg protein were electrophoresed on 10% SDS–polyacrylamide gels embedded with 0.25 mg ml⁻¹ MBP in the separating gel as a substrate for the kinases. After electrophoresis, SDS was removed by washing the gel with washing buffer (25 m m Tris pH 7.5, 0.5 m m DTT, 0.1 m m Na₃VO₄, 5 m m NaF, 0.5 mg ml⁻¹ BSA, 0.1% Triton X-100 [v/v]) three times, each for 30 min at room temperature. The kinases were allowed to renature in 25 m m Tris pH 7.5, 1 m m DTT, 0.1 m m Na₃VO₄, 5 m m NaF at 4°C overnight with three changes of buffer. The gel was then incubated at room temperature in 30 ml reaction buffer (25 m m Tris pH 7.5, 2 m m EGTA, 12 m m MgCl₂, 1 m m DTT, 0.1 m m Na₃VO4) with 200 n m ATP plus 50 μCi γ-³²P-ATP (3000 Ci mmol⁻¹) for 60 min. The reaction was stopped by transfering the gel into 5% trichloroacetic acid (TCA) (w/v)/1% NaPPi (w/v). The unincorporated γ-³²P-ATP was removed by washing in the same solution for at least 6 h with five changes. The gel was dried onto 3MM paper and exposed to Kodak XAR-5 film. Prestained size markers (Bio-Rad Laboratories, Hercules, CA, USA) were used to calculate the size of kinases.

Cell viability assay

Cell viability was assayed as described by Widholm 1972) . Briefly, a drop of cells was taken at the indicated times and mixed with a drop of fluorescein diacetate solution (0.01% [w/v] in culture medium) on a microscope slide. The living cells, which fluoresce bright green, were viewed under an epifluorescence microscope (Zeiss Axioskop 20; Carl Zeiss Inc., Thornwood, NY, USA). Three fields, each containing over 100 cells without big clumps, were counted. The percentage of dead cells was calculated as an average ±SEM.

RNA blot analysis

RNA was extracted using Trizol reagent (Gibco BRL, Gaithersburg, MD, USA) following the manufacturer's instructions. Fifteen micrograms of total RNA per lane were separated on 1.2% formaldehyde–agarose gels and transferred to Zeta-probe membranes (Bio-Rad Laboratories, Hercules, CA, USA). Blots were hybridized with random primer-labeled WIPK cDNA as previously described ( Zhang et al. 1998 ).

Immunoblot analysis

For immunoblot analysis, 15 μg of total protein per lane were separated on 10% SDS–polyacrylamide gels and the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell Inc., Keene, NH, USA) by semi-dry electroblotting. After blocking for 1 h in TBS buffer (20 m m Tris pH 7.5, 150 m m NaCl, 0.1% Tween 20) with 5% non-fat dried milk (Carnation) at room temperature, the membranes were incubated with Ab-p44N antibody (0.2 μg ml⁻¹ final concentration in TBS buffer) for 1 h. Following four washes in TBS buffer, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody (Sigma, St Louis, MO, USA, 1 : 10 000 dilution), and the complexes visualized using an enhanced chemiluminescence kit (NEN) following the manufacturer's instructions.

Immune complex kinase activity assay

Protein extract (50 μg) with or without peptide competitor (20 μg ml⁻¹ final concentration) was incubated with Ab-p44N (2.5 μg) in immunoprecipitation buffer (20 m m Tris pH 7.5, 150 m m NaCl, 1 m m EDTA, 1 m m EGTA, 1 m m Na₃VO₄, 1 m m NaF, 10 m mβ-glycerophosphate, 2 μg ml⁻¹ antipain, 2 μg ml⁻¹ aprotinin, 2 μg ml⁻¹ leupeptin, 0.5% Triton X-100, 0.5% non-idet P-40) at 4°C for 2 h on a rocker. About 20 μl packed volume of protein A-agarose was added, and the incubation was continued for another 2 h. Agarose bead–protein complexes were pelleted by brief centrifugation and washed three times with immunoprecipitation buffer, then three times with reaction buffer. Kinase activity in the complex was assayed at room temperature for 20 min in a final volume of 25 μl containing 0.1 mg ml⁻¹ MBP, 10 μm ATP with 1 μCi [γ-³²P]-ATP. The reaction was stopped by the addition of SDS–PAGE sample loading buffer. After electrophoresis on a 15% SDS–polyacrylamide gel, the phosphorylated MBP was visualized by autoradiography.

Acknowledgements

We thank Brett Tyler for generously providing cryptogein and parasiticein and D'Maris Dempsey for critically reviewing the manuscript. Jean Wang is gratefully acknowledged for assistance in preparation of the manuscript. This work was supported by Grant no. 9802200 from the US Department of Agriculture, Grant No. MCB-9723952 from the National Science Foundation to D.F.K. and a MU-Monsanto grant to S.Z.