Plant and Cell Physiology Advance Access originally published online on February 8, 2007
Plant and Cell Physiology 2007 48(3):498-510; doi:10.1093/pcp/pcm021
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MAP Kinases Function Downstream of HSP90 and Upstream of Mitochondria in TMV Resistance Gene N-Mediated Hypersensitive Cell Death
1National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602 Japan
2National Agricultural Research Center, Tsukuba Ibaraki, 305-8666 Japan
*Corresponding author: E-mail, mituhara{at}affrc.go.jp; Fax, + 81-29-8387469.
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Abstract |
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Although the involvement of heat shock protein 90 (HSP90), mitogen-activated protein kinase (MAPK) cascades and organelle dysfunction in plant hypersensitive cell death has been suggested, the mutual relationship among them has not been elucidated. Here, we show the molecular network of HSP90, the wound-induced protein kinase (WIPK)/salicylic acid-induced protein kinase (SIPK)-mediated MAPK cascade and mitochondrial dysfunction in tobacco mosaic virus (TMV) resistance gene N-dependent cell death. p50, the Avr component for N, NtMEK2DD, a constitutively active form of a MAPK kinase of WIPK/SIPK, and a mammalian pro-apoptotic factor Bax were used for cell death induction. Suppression of HSP90 and treatment with geldanamycin, a specific inhibitor of HSP90, compromised p50- but not NtMEK2DD- or Bax-mediated cell death accompanying the reduction of NtMEK2, WIPK and SIPK activation. In WIPK/SIPK-double knockdown plants, p50- and NtMEK2DD- but not Bax-mediated cell death was suppressed. All three types of cell death induced mitochondrial dysfunction, but they were similarly suppressed by Bcl-xL, which is a mammalian anti-apoptotic factor, and prevents mitochondrial dysfunction in plants as it does in animals in the cell death signal pathway. Taken together with the expression profile of hypersensitive reaction marker genes, it was indicated that the MAPK cascade functions downstream of HSP90 and transduces the cell death signal to mitochondria for N gene-dependent cell death. Furthermore, we found that WIPK and SIPK are functionally redundant in cell death signaling using WIPK/SIPK single or double knockdown plants.
Keywords: Heat shock protein 90 - Mitochondria - Mitogen-activated protein kinase - Programmed cell death - Signal transduction - Transient expression
Abbreviations: GDA, geldanamycin; HR, hypersensitive reaction; HSP, heat shock protein; MAPK, mitogen-activated protein kinase; MAPKK, MAPK kinase; MAPKKK, MAPKK kinase; MBP, myelin basic protein; PR, pathogenesis-related; Rh123, rhodamine123; RNAi, RNA interference; SIPK, salicylic acid-induced protein kinase; TMV, tobacco mosaic virus; VIGS, virus-induced gene silencing; WIPK, wound-induced protein kinase
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Introduction |
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The hypersensitive reaction (HR) is a plant-specific defense system against pathogen attack. HR is a resistance response accompanied by programmed cell death (Ross 1961a
, Ross 1961b, Goodman and Novacky 1994). Upon HR, local lesions are formed as the result of autonomous death of infected cells to enclose an avirulent pathogen in the infection site. To investigate the mechanism of programmed cell death in plants, HR cell death is an attractive and useful system.
To study the cell death signal pathway, we have isolated many genes by differential screening such as WIPK (Seo et al. 1995), DS9 (Seo et al. 2000), WRK (Takabatake et al. 2006a) and HSP90 (unpublished) whose expression was rapidly changed during tobacco mosaic virus (TMV) resistance gene N-mediated HR.
Recent studies have emphasized that cytosolic HSP90, which is an essential and abundant molecular chaperone in bacteria and eukaryotes, is required to confer R gene-mediated resistance and cell death. In HSP90-suppressed Nicotiana benthamiana, N gene-mediated and potato virus X resistance gene Rx-mediated resistance were suppressed (Liu et al. 2004, Lu et al. 2003). Pseudomonas syringae resistance gene RPS2- and RPM1-mediated resistance and cell death were suppressed in HSP90-disrupted Arabidopisis (Hubert et al. 2003, Takahashi et al. 2003b).
Mitogen-activated protein kinase (MAPK) cascades are major pathways for signal transduction from the cell surface to the nucleus in eukaryotic cells. In plants, several MAPKs play pivotal roles in stress and defense responses. Wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK) are stress- and defense-related MAPKs. Both WIPK and SIPK are rapidly activated by pathogens and elicitors (Zhang and Klessig 1998, Romeis et al. 1999, Seo et al. 2001, Zhang and Klessig 2001). Transient expression of NtMEK2DD, a constitutively active form of a tobacco MAPK kinase upstream of WIPK and SIPK, induces HR-like cell death in tobacco (Yang et al. 2001). WIPK, SIPK and NtMEK2 are required for N gene-dependent TMV resistance (Jin et al. 2003).
Although the involvement of HSP90 and the WIPK/SIPK-mediated MAPK cascade in cell death signaling is indicated, their relationship and upstream or downstream components are not clear.
In both plants and animals, programmed cell death has similar physiological roles; elimination of diseased cells and of unwanted cells during development. There are several indications that common pathways are used for controlling programmed cell death in both kingdoms. The Bcl-2 family is a large family of proteins acting as either activators (e.g. Bax and Bak) or suppressors (e.g. Bcl-2 and Bak) of animal programmed cell death by means of mitochondrial dysfunction (Gross et al. 1999). Mammalian Bcl-xL could suppress cell death in tobacco (Mitsuhara et al. 1999, Qiao et al. 2002), and Bax could trigger HR-like cell death in plants (Lacomme and Cruz 1999), while database searches have revealed that no obvious homologs of Bcl-2 family genes are present in plants. Bax inhibitor (BI)-1, an animal programmed cell death suppressor (Xu and Reed 1998), is conserved in plants, and plant BI-1 could suppress Bax- and elicitor-mediated cell death in plants (Kawai-Yamada et al. 2001, Matsumura et al. 2003). Mitochondria are also considered to be a target for Bax and Bcl-xL in plants (Qiao et al. 2002, Yoshinaga et al. 2005), suggesting the involvement of mitochondrial dysfunction in plant cell death signaling. Mitochondrial dysfunction is the commitment step of animal programmed cell death upstream of activation of caspases, but the physiological role and upstream or downstream components of mitochondrial dysfunction in plant programmed cell death have not been clearly identified yet.
In this report, we show that HSP90 functions upstream of the NtMEK2–WIPK/SIPK cascade in N gene-dependent cell death signaling. Moreover, we suggest that mitochondrial dysfunction is induced by the activation of these MAPKs and is a prerequisite for HR cell death execution. From these results, we propose that HSP90, the MAPK cascade and mitochondria in turn function in the R gene-mediated cell death signaling network.
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Results |
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Geldanamycin inhibits N gene-dependent cell death and HR marker gene induction
To determine whether HSP90 is required for N gene-dependent cell death, we studied the effect of geldanamycin (GDA), a specific inhibitor of HSP90, on cell death. GDA is known to affect the ATPase activity of HSP90 by binding to the N-terminal conserved domain (Picard 2002
), and has been reported to be available in plants (Queitsch et al. 2002, Takahashi et al. 2003b). Geldampicin (GDM), a structural analog of GDA, has no effect on HSP90 (Ochel et al. 2003). Using a synchronous HR-inducing system based on a temperature shift from 30°C, a non-permissive temperature for the N gene, to 20°C, a permissive temperature (Takabatake et al. 2006b), the effect of GDA on TMV-induced HR was studied. Necrotic lesions began to be visible at 8–12 h after the temperature shift (Fig. 1A), and the level of electrolyte leakage, a marker of cell death, increased and reached a maximum at 12 h in both control and GDM-treated leaves. However, in GDA-treated leaves, the level of ion leakage was suppressed considerably and necrotic lesion formation was also inhibited (Fig. 1B, C).
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We examined the effect of GDA on the cell death mediated by p50, which is the 50 kDa domain of TMV helicase and the avirulent component for N (Erickson et al. 1999
), NtMEK2DD and Bax by a transient expression system using Agrobacterium infiltration. Necrotic lesion formation was observed at 36–40 h in the area in which p50, NtMEK2DD and Bax were expressed following infiltration (Fig. 1A). As shown in Fig. 1D, p50-mediated cell death was inhibited by GDA treatment, while NtMEK2DD- and Bax-mediated cell death was not. The levels of p50, NtMEK2DD and Bax protein accumulation were elevated at 30 and 35 h after Agrobacterium infiltration, but were not affected by GDA treatment (Fig. 1E). Subsequently, the profiles of HR marker gene expression and the effect of GDA on the expression during p50-, NtMEK2DD- and Bax-mediated cell death was examined. All HR marker genes used here, acidic PR-1 (aPR1) (Mitsuhara et al. 1999), Hsr203J (Marco et al. 1990) and Hin1 (Gopalan et al. 1996), were induced by p50 expression, and it was notable that their expression was completely suppressed by GDA treatment. On the other hand, Hsr203J and Hin1 but not aPR1 were induced by NtMEK2DD, and Bax expression only induced Hin1. The induction of the marker genes by NtMEK2DD and Bax was not inhibited by GDA treatment (Fig. 1F). Similar results were obtained from independent quantitative reverse transcription–PCR (RT–PCR) analysis (data not shown). The effect of GDA on the HR marker genes was coincident with necrotic lesion formation (Fig. 1D). These results indicate that HSP90 is required for N gene-mediated but not NtMEK2DD- or Bax-mediated cell death, and for the induction of HR marker genes.
HSP90 is required for p50- but not NtMEK2DD- or Bax-mediated cell death in Nicotiana benthamiana
We have isolated a cytosolic tobacco HSP90 gene (GenBank accession No. AB264546
[GenBank]
) as one of the genes whose transcripts changed during N gene-dependent synchronized HR cell death (Seo et al. 1995). To study the roles of HSP90 in cell death, we first tried to generate stable HSP90-silenced transgenic tobacco by antisense or RNA interference (RNAi) methods using conserved regions of HSP90 genes, but no severely suppressed plants were obtained.
Then, we prepared HSP90-silenced plants by virus-induced gene silencing (VIGS) (Ratcliff et al. 2001). Nicotiana benthamiana plants were independently silenced by two DNA fragments (VIGS1 and VIGS2) from the NbHSP90 coding regions (Fig. 2A). NbHSP90 protein was detected easily in two representative independent control lines, but the protein levels were severely reduced in plants silenced by VIGS1 or VIGS2 (Fig. 2C). Severe knockdown of HSP90 genes might cause lethality in plants because it has been shown that HSP90 is an essential protein in several eukaryotic cells (Aligue et al. 1994, Cutforth and Rubin 1994). In fact, the VIGS-mediated HSP90-silenced N. benthamiana began to stunt severely at 4 weeks after the introduction of VIGS vectors and finally died, as previously reported by Liu et al. (2004) (data not shown). Therefore, we used HSP90-silenced N. benthamiana at 3 weeks after introduction, at which time the plants looked healthy but HSP90 protein had already been reduced. In two representative independent VIGS1 and VIGS2 plants, p50- but not NtMEK2DD- or Bax-mediated cell death was clearly suppressed, similar to the GDA treatment (Fig. 2B).
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HSP90 is required for N gene-dependent NtMEK2, WIPK and SIPK activation
To study the mechanism of the inhibition of TMV- and p50-mediated cell death by GDA, we examined the effect of GDA on the activation of WIPK and SIPK, which are considered to function downstream of N gene-mediated signaling (Jin et al. 2003
). Both WIPK and SIPK activities reached a maximum at 6–8 h after the temperature shift, but the activation was markedly suppressed by GDA treatment (Fig. 3A). To study the effect of GDA on the activation of WIPK and SIPK during the other types of cell death, p50-, NtMEK2DD- and Bax-mediated cell death were examined. The accumulation of p50, NtMEK2DD and Bax proteins was detected at 30 h but not at 20 h after Agrobacterium infiltration, and the levels increased until necrotic lesions were visible (Fig. 1E). By transient expression of p50 and NtMEK2DD, the activities of both WIPK and SIPK were elevated at 30 and 35 h after the infiltration (Fig. 3B, C), and the activation by p50 but not NtMEK2 DD was clearly inhibited by GDA treatment (Fig. 3B, C). These results show that TMV- and p50-induced activation of WIPK and SIPK is carried out in an HSP90-dependent manner, while NtMEK2DD-induced activation of the MAPKs is done in an HSP90-independent manner. No clear activation of these MAPKs was detected during Bax-mediated cell death (data not shown).
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NtMEK2 has been reported to act as an upstream mitogen-activated protein kinase (MAPKK) for WIPK and SIPK (Yang et al. 2001
). However, endogenous NtMEK2 activity has rarely been confirmed in the stress and defense responses that are accompanied by the activation of WIPK and SIPK, including N gene-dependent HR. We tried to detect the activation of endogenous NtMEK2 during N gene-dependent cell death and analyze the effect of GDA treatment on the activation. Antibodies were raised against a synthetic peptide corresponding to residues 25–38 (PPPPSRNRPRRRTD) of the N-terminal amino acid regions of NtMEK2, and the specificity of the antibodies was confirmed (data not shown). First, we examined an immune complex-kinase assay of protein extracts from TMV-inoculated and temperature-shifted leaves by using the NtMEK2-specific antibodies and recombinant SIPK (rSIPK) as a substrate. In the system, the amount of radiolabeled rSIPK corresponds to the NtMEK2 activity. However, we could not detect significant SIPK phosphorylation activities of NtMEK2 even at 8 h after the temperature shift (data not shown), at which time SIPK was clearly activated (Fig. 3A). We then tried to detect immunoprecipitated NtMEK2-mediated rSIPK activation using myelin basic protein (MBP) as an artificial substrate, and found that rSIPK was activated considerably by endogenous NtMEK2 at 6 and 8 h after the temperature shift (Fig. 3E). No clear activation was detected without NtMEK2 antibodies or rSIPK at 8 h after the temperature shift, eliminating the possible effect of endogenous SIPK or any other kinases on MBP phosphorylation (Fig. 3F). The activation by endogenous NtMEK2 was severely inhibited by GDA treatment (Fig. 3E), suggesting that HSP90 functions upstream of NtMEK2 in cell death signaling.
Either WIPK or SIPK is required for p50- and NtMEK2DD- but not Bax-mediated cell death
The data shown in Fig. 3 suggest that the activation of WIPK and SIPK most probably triggers p50- and NtMEK2DD-mediated cell death execution. HR cell death or HR-like cell death often accompanies the activation of MAPKs. Overexpression of MAPKKK
which is an HR- and disease resistance-related MAPKK kinase (MAPKKK), induces cell death, and requires SIPK but not WIPK (del Pozo et al. 2004). INF1, the major elicitin secreted by Phytophtora infestans, induces HR cell death in several Nicotiana species, and activates WIPK and SIPK in tobacco (Kamoun et al. 1998). However, INF1-mediated cell death was not influenced in both WIPK- and SIPK-silenced N. benthamiana (Sharma et al. 2003). These results indicate that there is no general conclusion that WIPK or SIPK, or both, are actually required for HR cell death. To determine whether p50- and NtMEK2DD-mediated cell death requires WIPK or SIPK, or both, we generated transgenic tobacco plants in which the expression of each MAPK gene, or both, was suppressed by the RNAi method. The 422 and 472 bp gene-specific regions including the 3'-untranslated regions of WIPK or SIPK are used, respectively. For double silenced plants, the gene-specific regions were aligned in tandem (Fig. 4A). Three independent lines for each transgenic plant were selected and used as the representatives for further analysis. The transcript levels of WIPK or SIPK, or both, were reduced approximately 90% compared with that of the control plants (Fig. 4B). When p50, NtMEK2DD and Bax were transiently expressed by Agrobacterium infiltration, normal necrotic lesions developed in WIPK silenced plants (IR-W) or SIPK silenced plants (IR-S), and no clear differences were observed in the timing or the extent of the lesions in three independent transgenic lines compared with those of control lines (Fig. 4C). In contrast, in the double silenced plants (IR-WS), p50- and NtMEK2DD-mediated cell death was not observed (Fig. 4C). Bax-mediated necrosis was observed even in IR-WS plants, but the extent was somewhat attenuated. These results indicate that either WIPK or SIPK is required for p50- and NtMEK2DD-mediated cell death and they have functionally redundant roles in N gene-dependent cell death.
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Mitochondrial dysfunction likely precedes p50- and NtMEK2DD-mediated cell death
Bax is known to target mitochondria in both animals and plants (Gross et al. 1999
, Yoshinaga et al. 2005). In animal programmed cell death, Bcl-xL, which localizes to the mitochondrial outer membrane, blocks Bax- and Bak-induced membrane potential disruption (Gross et al. 1999, Kaufmann et al. 2003). We reported previously that Bcl-xL also inhibits plant cell death and is considered to work mainly at mitochondria (Qiao et al. 2002); mitochondrial membrane potential could be maintained for a longer time under a high salt stress condition compared with control cells in Bcl-xL-expressing tobacco suspension-cultured cells, and in which the accumulation of Bcl-xL protein was abundant in the mitochondrial fraction. To study whether mitochondrial dysfunction is a prerequisite for cell death, two independent Bcl-xL transformants M65-4 and M65-23-4 which constitutively accumulate Bcl-xL protein were used for Agrobacterium infiltration (Mitsuhara et al. 1999) (Fig. 5B). p50-, NtMEK2DD- and Bax-mediated cell death was suppressed in both Bcl-xL-expressing lines (Fig. 5A). Three days after Agrobacterium infiltration, necrotic regions were visible in control plants although no clear necrotic regions were detected in either M65-4 or M65-23-4. About 4–5 d after infiltration, necrosis originated by to p50 and NtMEK2DD expression began to appear even in Bcl-xL-expressing lines (data not shown), while Bax-mediated cell death was suppressed throughout the period of observation. These results indicate that Bcl-xL abolishes Bax-mediated cell death and considerably suppresses p50- and NtMEK2DD-mediated cell death.
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To confirm the change of mitochondrial membrane potential during cell death in tobacco leaves, the active membrane potential was visualized with rhodamine 123 (Rh123), which stains mitochondria green depending on the active membrane potential. Rh123 solution (20 µM) was infiltrated into the region into which Agrobacterium with p50, NtMEK2DD or empty vector had been infiltrated. At 32–33 h after Agrobacterium infiltration, at which time cell death has not been observed, the phenotypes of mesophyll cells were optically sectioned at 0.4 µm and observed using a confocal laser scanning microscope. In control cells into which Agrobacterium containing empty vector was introduced, small green fluorescent signals, which indicate the mitochondria with normal membrane potential, were found (Fig. 5E, a), and these green signals disappeared by the action of the uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) which leads to mitochondrial dysfunction (data not shown). In p50- or NtMEK2DD-expressing cells, green signals were rarely found (Fig. 5E, b and c), indicating that activation of WIPK and/or SIPK caused loss of mitochondrial membrane potential. However, in Bcl-xL-expressing lines, the signals were clearly detected even in p50-expressing cells (Fig. 5E, d). Similar results were obtained from NtMEK2DD- and Bax-expressing cells (data not shown), indicating that mitochondrial membrane potential was retained in the Bcl-xL-expressing plants. At 32–33 h after the Agrobacterium infiltration, at which time mitochondrial membrane potential was already lost, necrotic regions were not visible (Fig. 1A) and cell death was not detected in p50- or NtMEK2DD-expressing tissues by trypan blue staining although the infiltrated tissues were well stained at 40 h after the infiltration (Fig. 5D). The distribution profile of red signals, which are due to autofluorescence from chlorophylls, was not different between p50-expressing control and Bcl-xL plants (Fig. 5E. b and d). The round shape of mesophyll cells was still maintained even in p50- or NtMEK2DD-expressing control cells (Fig. 5E, b or c). These results suggest that the loss of function of mitochondria preceded p50-, NtMEK2DD- and Bax-mediated cell death execution, and Bcl-xL inhibited the cell death through the prevention of mitochondrial dysfunction.
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Discussion |
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We demonstrate here that the NtMEK2–WIPK/SIPK cascade functions downstream of HSP90 preceding mitochondrial dysfunction in N gene-dependent cell death signaling (Fig. 6). Further, the function of WIPK and SIPK is shown to be redundant for cell death. The evidence provides a detailed understanding of the mechanism of HR cell death.
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Both WIPK and SIPK function downstream of HSP90 in N gene-dependent cell death
Kanzaki et al. (2003
) reported that the WIPK/SIPK-mediated MAPK cascade plays a role in hypersensitive cell death signaling downstream or independent of HSP90. In this report, it was concluded that both WIPK and SIPK function downstream of HSP90 in the N gene-dependent cell death signaling pathway. The accumulation of several R proteins such as Rx, RPM1 and RPS5 was reduced in HSP90-suppressed plants (Lu et al. 2003, Holt et al. 2005). Since N protein was reported to associate directly with HSP90 (Liu et al. 2004), inhibition or reduction of HSP90 would prohibit N protein abundance, leading to the suppression of N-mediated cell death. Interestingly, wound-induced activation of WIPK and SIPK was also inhibited by GDA treatment (Fig. 3D), suggesting that HSP90 could affect WIPK and SIPK activation not only by N protein accumulation but also by unknown mechanism(s). GDA inhibits cell death signaling at least upstream of NtMEK2 activation (Fig. 3E). Raf-1, a well known mammalian MAPKKK, associates with HSP90 and is destabilized by GDA treatment. Raf-1-mediated MAPK signaling was inhibited by GDA in mammalian cells (Schulte et al. 1996, 1997). One possibility is that GDA would affect the predicted MAPKKK(s) that functions upstream of NtMEK2 in both wound and HR signaling.
WIPK and SIPK are functionally redundant in cell death signaling
WIPK and SIPK have been considered to have individual physiological role(s). There are several reports showing that WIPK and SIPK have individual specific substrate(s), including transcriptional factors (Katou et al. 2005, Menke et al. 2005, Yap et al. 2005). MPK6, which is an Arabidopsis ortholog of SIPK, was reported to be involved in ethylene production through phosphorylation and activation of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS), the rate-limiting enzyme for ethylene biosynthesis. However, MPK3, an Arabidopsis ortholog of WIPK, seems not to have an important role in ethylene production (Liu and Zhang 2004).
The level of WIPK or SIPK suppression was not significantly different between IR-W and IR-WS plants, or between IR-S and IR-WS plants, respectively. However, p50- and NtMEK2DD-mediated cell death was clearly suppressed only in IR-WS plants, but not in IR-W or IR-S plants. These results indicate that WIPK and SIPK are redundant in N gene-dependent cell death signaling. This is not surprising in view of the fact that both WIPK and SIPK are activated by a common MAPKK (NtMEK2), and activated by the same stimulus and with the same timing including N gene-dependent HR. However, we could not rule out the possibility that WIPK or SIPK were independently essential components for N gene-dependent cell death because we used RNAi knockdown plants but not knockout null mutants of WIPK and SIPK, suggesting that the loss of function of each MAPK gene was severe but not complete.
How do WIPK and SIPK work synergistically in cell death signaling? WIPK and SIPK might share a common substrate(s) that plays a positive role in plant cell death and would lead to mitochondrial dysfunction, although possible candidates have not yet been identified.
Individual expression profile of HR marker genes upon p50-, NtMEK2DD- and Bax-mediated cell death
We used here three HR marker genes, aPR1, Hsr203J and Hin1, which were all induced by p50. The induction of aPR1 was inhibited by GDA, indicating that the expression of aPR1 is dependent on HSP90 (Fig. 1F). In contrast, NtMEK2DD induced Hsr203J but not aPR1, indicating that the expression of Hsr203J but not aPR1 is dependent on the WIPK/SIPK-mediated MAPK cascade. Furthermore, Bax induced Hin1 but not aPR1 and Hsr203J, suggesting that the expression of Hin1 but not aPR1 and Hsr203J is induced after mitochondrial dysfunction. The expression profile of these genes is consistent with our working model (Fig. 6).
In the model, the induction machinery of aPR1 is upstream of the MAPK cascade and mitochondrial dysfunction, expecting that Bcl-xL does not affect the induction of aPR1 by p50. We actually confirmed that the induction level was not significantly different between control and Bcl-xL-expressing lines (Fig. 5C). On the other hand, we have previously reported that the protein accumulation of aPR1 with TMV-mediated HR was restricted in Bcl-xL-expressing tobacco, suggesting that Bcl-xL suppressed aPR1 induction (Mitsuhara et al. 1999). The reason for this discrepancy is not clear at present. The difference in the cell death induction system could be the possible cause. In particular, it should be noted that the area in which TMV-mediated HR is induced is composed of TMV-infected living and dying cells, and uninfected healthy cells (Fig. 1C), whereas the whole p50-expressed area is clearly dead (Figs. 1D, 2B, 4C, 5A). We previously showed that aPR1 is strongly induced in living cells around the HR lesion (Ohshima et al. 1990). Bcl-xL might suppress such a local induction of aPR1.
N-mediated cell death signaling is transduced via mitochondrial dysfunction
There are several reports that imply HSP90, the NtMEK2–WIPK/SIPK cascade and the loss of function of mitochondria are involved in plant programmed cell death (del Pozo et al. 1994, Lacomme et al. 1999, Mitsuhara et al. 1999, Jin et al. 2003, Yoshinaga et al. 2005). However, the mutual relationship between these three components has not been established. p50- but not NtMEK2DD-mediated cell death was suppressed by GDA, and p50- and NtMEK2DD-mediated cell death was suppressed by Bcl-xL, indicating that the MAPK cascade functions downstream of HSP90 and upstream of mitochondria in N gene-dependent cell death signaling. That is to say, the cell death signaling is in turn transduced via HSP90, the WIPK/SIPK-mediated MAPK cascade and then mitochondria. All of our results demonstrated here support this hypothesis, except for the finding that Bax-mediated cell death occurred but was somewhat attenuated in IR-WS plants. If Bax triggered the cell death signaling downstream of the MAPK cascade, Bax-mediated cell death would not be attenuated even in IR-WS plants. We wondered if mitochondrial dysfunction might feedback positively to the MAPK cascade. In fact, CCCP treatment induces WIPK activation and WIPK expression (Takahashi et al. 2003a).
In Bcl-xL-expressing tobacco suspension-cultured cells, high salt stress-induced vacuolar disruption was suppressed (Qiao et al. 2002). Bax-mediated mitochondrial dysfunction was followed by vacuolar rupture in Arabidopsis (Yoshinaga et al. 2005), suggesting that mitochondrial dysfunction finally leads to vacuolar disruption. Vacuolar collapse has been considered to play the primary role in both HR and developmental programmed cell death in plants (Jones 2001). Hatsugai et al. (2004) showed that vacuolar processing enzyme (VPE), a cysteine proteinase responsible for the maturation of vacuolar proteins, is essential for plant programmed cell death. In further analysis, vacuoles and other organelles would be positioned in the cell death signaling network.
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Materials and Methods |
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Plant materials
Tobacco plants (Nicotiana tabacum cv. Samsun NN) were grown in growth chambers at 24–26°C with a 16 h light/8 h dark cycle, and fully expanded upper leaves were used for the experiments. Nicotiana benthamiana plants were transformed with the genomic clone of the N gene using Agrobacterium tumefaciens LBA4404, and the homozygous plants were used for VIGS experiments.
Inhibitor treatments
GDA was purchased from Sigma. GDM was provided by the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (USA). GDA and GDM were diluted from stock solutions in dimethylsulfoxide (DMSO). GDA (10 µM) was used for the infiltration studies, and 10 p.p.m. (
17.8 µM) of GDA and GDM were used for the measurement of electrolyte ion leakage. For the control experiments, an equivalent concentration of DMSO was applied.
Molecular cloning and plasmid constructions
The cDNAs of NtHSP90 (GenBank accession No. AB264546
[GenBank]
) and NtMEK2 (GenBank accession No. AB264547
[GenBank]
) were isolated from tobacco (Samsun NN) using a SMARTTM RACE cDNA Amplification Kit (Clontech, Palo, Alto, CA, USA).
For Agrobacterium-mediated transient expression, the FLAG tag sequence, corresponding to 5'-GACTACAAAGACGATGATGACAAG-3', was translationally fused to the C-terminus of p50, and an HA tag sequence, corresponding to 5'-TATCCATACGATGTTCCAGATTATGCT-3', was fused to the N-terminus of NtMEK2DD. The mouse Bax cDNA was purchased from Upstate Biotechnology. p50-FLAG, HA- NtMEK2DD and Bax were cloned into pEl2
-MCS vector (Ohtsubo et al. 1999), and expressed under the control of the highly efficient promoter El2 (Mitsuhara et al. 1996). For the WIPK and SIPK inverted repeat constructs, a 422 bp region corresponding to positions 979–1,400 of WIPK, and a 472 bp region corresponding to positions 1,049–1,520 of SIPK, were used (see Fig. 4A). For WIPK- and SIPK-double silenced constructs, the DNA fragments for WIPK and SIPK were inserted in tandem. The DNA sequence of ß-glucuronidase (989 bp corresponding to positions 821–1,809) was used as a linker between the gene-specific fragments in the antisense and sense orientations for WIPK and SIPK constructs. These constructs were finally inserted into the pEl2 -MCS vector.
Determination of ion leakage
Electrolyte leakage from tobacco leaf discs was measured as described previously by Mitsuhara et al. (1999) using a model CDD-6A conductivity detector (Shimadzu, Kyoto, Japan), and expressed as µS per gram of fresh leaf per hour.
Tobacco transformation
For stable transformations, binary plasmids containing various constructs were introduced into A. tumefaciens LBA4404 by electroporation, and tobacco plants were transformed by the leaf disc co-cultivation method (Horsh et al. 1985). Agrobacterium-mediated transient transformation was conducted as described by Takabatake et al. (2006b).
Antibody preparation and immunoblot analysis
NtHSP90 and NtMEK2 antibodies were raised in rabbits against the purified full-length recombinant protein of HSP90 and a synthetic peptide corresponding to residues 5–38 (PPPPSRNRPRRRTD) of the N-terminal region of NtMEK2, respectively. Total protein extracts (20 µg) were electrophoresed on SDS–polyacrylamide gels (10–15%), blotted onto PVDF membrane (Immobilon, Millipore) and then treated with anti-FLAG M2 (Sigma, St. Louis, MO, USA), anti-HA 3F10 (Roche, Nutley, NJ, USA), anti-Bax (Sigma), anti-Hsp90 or anti-Bcl-xL (Mitsuhara et al. 1999) antibodies. After washing the membranes, the antibody–antigen complexes were detected with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Kirkegaard & Perry Laboratories).
RNA gel blot analysis
Total RNA was isolated by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. For RNA blot hybridization analysis, 15 µg of total RNA per lane was fractionated by electrophoresis in a 1.2% denaturating agarose gel containing formaldehyde, and then blotted onto a Hybond-N+ nylon membrane (Amersham, Piscataway, NJ, USA). The blots were subjected to hybridization with 32P-labeled 3'-non-coding regions of acidic PR-1 or the coding regions of Hsr203J or HinI. Washing of the membranes was performed under high-stringency conditions (Takabatake et al. 2001).
Real-time RT–PCR
Total RNA was isolated as described above. A 2 µg aliquot of RNA was subjected to reverse transcription with a poly(T) primer. Real-time PCR was performed with an iCycler iQ real-time PCR detection system (BIO-RAD, Hercules, CA, USA) using the BIO-RAD iQ SYBR Green Supermix. Results were normalized to the expression of actin mRNA. Primers for WIPK (5'-CCAAGTATCGTCCTCCTATTATG-3' and 5'-TCACGGAGAGTCCTCTTAGC-3'), SIPK (5'-CCACGGTGGCAGGTTCATTC-3' and 5'-CAGAACAAACGATGCCGTAAGC-3') and Actin (5'-GGGTTTGCTGGAGATGATGCT-3' and 5'-GCTTCGTCACCAACATATGCAT-3') were used.
Immune complex kinase assay
Immunoprecipitations were performed using 50 µg of total protein with 2 µg of anti-WIPK or anti-SIPK antibodies. The immunoprecipitates were then subjected to an MBP kinase assay as described previously by Seo et al. (1999). For detecting the activity of endogenous NtMEK2 (eNtMEK2), immunoprecipitation was performed using 100 µg of total protein with 2 µg of anti-NtMEK2 antibody. Immunoprecipitated eNtMEK2 was added to 0.1 µg of recombinant SIPK and incubated at 24°C for 10 min. Then, 3.7 µg of MBP was added as a substrate and incubated at 24°C for 20 min, and the reaction mixtures separated by electrophoresis on a 15% SDS–polyacrylamide gel. MBP phosphorylation was analyzed by autoradiography.
VIGS assay
The TRV vector was described in Ratcliff et al. (2001). pBINTRA6 and pTV00 contain TRV RNA1 and RNA2, respectively. Nicotiana benthamiana plants were grown in pots at 24°C in a growth chamber. For the VIGS assay, pBINTRA6- and pTV00-derived constructs were introduced into Agrobacterium strains C58C1 and GV3101, respectively. Agrobacterium cultures containing pBINTRA6 and TRV derivative plasmids were mixed in a 1 : 1 ratio prior to infiltration. Two expanded leaves of 5- to 6-week-old N. benthamiana plants were infiltrated with the mixture. p50, NtMEK2 DD and Bax were transiently expressed by Agrobacterium infiltration at 3 weeks after the introduction of the TRV vector.
Trypan blue staining
Trypan blue staining was performed as described in Bowling et al. (1997). Leaf discs were submerged in a lactic acid–phenol–trypan blue solution [0.3 mg ml–1 trypan blue, 33% (v/v) lactic acid, 33% (w/v) phenol, 33% (v/v) glycerol and H2O), and heated over boiling water for 5 min, and then destained with a chloral hydrate solution (2.5 g ml–1 of H2O).
Microscopic analysis
Microscopic observation was carried out using a confocal laser scanning microscope (FLUOVIEW, OLYMPUS, Tokyo, Japan). Six- to 7-week-old transgenic tobacco leaves infiltrated with 20 µM Rh123 (Wako, Japan) to visualize active mitochondria were placed on glass slides and examined with an excitation wavelength of 488 nm (Ar laser) for Rh123 and of 543 nm (HeNe laser) for chlorophyll autofluorescence, and with emission signals of green (530 ± 20 nm) and red (>600 nm), respectively.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgmentsReferences |
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We thank D. C. Baulcombe (Sainsbury Laboratory) for providing the TRV vector (pTV00 and pBINTRA6) and Agrobacterium strain GV3101; B. Baker (University of California) for providing the N-genomic binary plasmid; and the Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (USA) for providing geldampicin. We wish to thank Y. Gotoh and M. Teruse for technical assistance. This work was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN).
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Footnotes |
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The nucleotide sequences reported in this paper have been deposited in the GenBank database (accession Nos. AB264546 and AB264547).
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References |
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(Received December 25, 2006; Accepted February 1, 2007)