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First published online December 5, 2003; 10.1105/tpc.017889 American Society of Plant Biologists A Plant Caspase-Like Protease Activated during the Hypersensitive Response
a Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia 2 To whom correspondence should be addressed. E-mail mtalia{at}scri.sari.ac.uk; fax 44-1382-562426; or e-mail varta{at}genebee.msu.su; fax 7-095-939-3181
To test the hypothesis that caspase-like proteases exist and are critically involved in the implementation of programmed cell death (PCD) in plants, a search was undertaken for plant caspases activated during the N gene–mediated hypersensitive response (HR; a form of pathogen-induced PCD in plants) in tobacco plants infected with Tobacco mosaic virus (TMV). For detection, characterization, and partial purification of a tobacco caspase, the Agrobacterium tumefaciens VirD2 protein, shown here to be cleaved specifically at two sites (TATD and GEQD) by human caspase-3, was used as a target. In tobacco leaves, specific proteolytic processing of the ectopically produced VirD2 derivatives at these sites was found to occur early in the course of the HR triggered by TMV. A proteolytic activity capable of specifically cleaving the model substrate at TATD was partially purified from these leaves. A tetrapeptide aldehyde designed and synthesized on the basis of the elucidated plant caspase cleavage site prevented fragmentation of the substrate protein by plant and human caspases in vitro and counteracted TMV-triggered HR in vivo. Therefore, our data provide a characterization of caspase-specific protein fragmentation in apoptotic plant cells, with implications for the importance of such activity in the implementation of plant PCD.
Programmed cell death (PCD), or apoptosis, is a basic genetically controlled process that functions in the development of multicellular organisms and in their responses to biotic and abiotic stresses. PCD provides a means to eliminate redundant, damaged, or infected cells. In mammals, apoptosis is recognized as a ubiquitous phenomenon with distinct morphological hallmarks, including condensation of the nucleus and the cytoplasm, cleavage of DNA into short 180-bp fragments (DNA laddering), and surface marking of the dying cells that are ingested by phagocytes (reviewed by Kerr et al., 1972  ). At the molecular level, there are two canonical pathways to apoptotic cell death. One involves the interaction of a death receptor with its ligand, and the second depends on the participation of mitochondria. The release of a number of factors—most notably cytochrome c—from the mitochondrial intermembrane space is regulated by proapoptotic and antiapoptotic members of the Bcl-2 family. Crosstalk between the two pathways exists (reviewed by Green, 1998; Adrain and Martin, 2001; Kaufmann and Hengartner, 2001). The end result of either pathway is the activation of caspases, a family of Cys proteases conserved evolutionarily from nematodes to humans (Thornberry and Lazebnik, 1998; Wolf and Green, 1999). Caspases are activated from dormant precursors in the course of apoptosis and introduce breaks after specific Asp (D) residues (hence their name) in a limited set of key cellular protein substrates. Caspases are among the most specific proteases known, and usually no more than one or two breaks per substrate protein molecule are introduced. Caspase-mediated protein fragmentation eventually leads to cell dismantling. The most prevalent executioner caspase in animal cells is caspase-3, which ultimately is responsible for the majority of the effects orchestrating cell death. In accordance with the pivotal role of caspases in PCD, caspase-specific peptide inhibitors based on the sequences cleaved by caspases and caspase gene knockouts are known to counteract apoptosis in animals (Ekert et al., 1999; Zheng et al., 1999).
In plants, several tissues or whole organs undergo cell death as part of their normal development, such as during senescence, or in response to pathogens and environmental stresses (reviewed by Greenberg, 1996
The case for the existence of caspases in plants is controversial. Although plant Cys proteolytic enzymes are known to be associated with PCD (Minami and Fukuda, 1995
In Arabidopsis suspension cultures, nitric oxide– and H2O2-induced cell death was inhibited by the caspase-1 inhibitor (Clarke et al., 2000 These observations suggest the existence of plant proteases (referred to here and below as plant caspases) with properties and cleavage specificities similar to those of animal caspases but perhaps being structurally different from them. However, for direct detection and analysis of such plant enzymes, it was crucial to identify a genuine plant protein substrate(s) that was (were) unknown.
Here, we used an approach based on the exploitation of the Agrobacterium tumefaciens–encoded VirD2 protein, which was predicted recently to be a possible substrate for caspases in our computer-assisted search for novel nuclear proteins subject to caspase fragmentation (N.V. Chichkova and A.B. Vartapetian, unpublished data). A. tumefaciens is a soil-borne plant pathogen capable of transferring DNA to the genome of higher plants, resulting in tumor formation. To initiate the process, VirD2 cleaves the agrobacterial tumor-inducing (Ti) plasmid DNA at a strictly defined sequence and becomes covalently attached to the newly formed 5' DNA terminus (Yanofsky et al., 1986
In Vitro Fragmentation of VirD2 by Human Caspase-3 To test the ability of caspase-3, the principal human executioner caspase, to cleave the VirD2 protein, VirD2 containing an N-terminal six-His [(His)6] tag (VirD2-His) was expressed in Escherichia coli and purified further by nickel–nitrilotriacetic acid (Ni-NTA) agarose affinity chromatography. The purified preparation of the protein gave a single band in SDS-PAGE (Figure 1A, lane 1). Treatment of the VirD2-His protein with human caspase-3 resulted in the formation of two closely spaced bands with higher electrophoretic mobilities (Figure 1A, lane 2), indicating the terminal truncation of VirD2 at two different sites. Of note, the higher molecular mass VirD2 fragment was formed more efficiently (major component) than the lower molecular mass fragment (minor component). The caspase-3–specific inhibitor Ac-DEVD-CHO precluded the fragmentation of VirD2 (Figure 1A, lane 3). Both of the caspase-3 cleavage products could be retarded by the Ni-NTA agarose resin (Figure 1A, lane 4), indicating that they preserved the N-terminal (His)6 tag. Thus, both caspase-3 cleavage sites are located close to the C terminus of the VirD2 molecule.
To construct a convenient protein substrate for further studies, the C-terminal 86–amino acid region of VirD2 (VirD2Ct), probably containing both of the caspase-3 cleavage sites, was fused to the C terminus of GFP and tagged with (His)6. The resulting GFP-VirD2Ct protein (Figure 2A, w.t.) then was confirmed to contain both of the sites recognized and cleaved by human caspase-3 in the entire VirD2 molecule (Figure 1B, lane 2).
Within the C-terminal region of the VirD2 protein fused to the GFP are several D residues that could represent human caspase-3 cleavage sites (Figure 2A). To identify the two that are in fact involved in caspase-3 cleavage, a set of VirD2Ct mutants lacking one or more D residues were generated, fused to GFP, and tagged with (His)6 (Figure 2A). The recombinant proteins were produced in E. coli, purified, treated individually with caspase-3, and analyzed by SDS-PAGE (Figure 2B). The single D10A and D39A mutations resulted in the elimination of the minor, faster migrating product (Figure 2B, lane 6) and the major, slower migrating product (Figure 2B, lane 8), respectively, whereas the double mutant (D10,39A) had acquired complete resistance to caspase-3 hydrolysis (Figure 2B, lane 10). Elimination of all other downstream D residues had no effect on the cleavage pattern of GFP-VirD2Ct (Figure 2B, lane 4). Therefore, our results indicate that D39 and D10 represent the major and minor (suboptimal) caspase-3 cleavage sites, respectively, in VirD2. To further support this conclusion, we inverted the order of the GFP and VirD2Ct moieties in the protein substrate to produce VirD2Ct-GFP (Figure 2A). Efficient caspase-3 cleavage of the VirD2Ct-GFP substrate at the proximal D39 site was observed (Figure 2C, lane 2), thus masking the cleavage at the distal D10 position. Substrate fragmentation at the suboptimal D10 site became evident only after mutating the D39 residue to A (Figure 2C, lane 4). As expected, the double mutant (D10,39A) was resistant to caspase-3 treatment (Figure 2C, lane 6). Thus, regardless of the order of the VirD2Ct and GFP moieties, human caspase-3 appears to cleave VirD2Ct preferentially at D39 and less efficiently at D10 (equivalent to D400 and D371, respectively, in the full-length VirD2). To verify that the cleavage actually takes place at the expected residues, mass spectrometric (MS) characterization of caspase-3 cleavage sites in GFP-VirD2Ct was performed. Gel-purified GFP-VirD2Ct and the major and minor cleavage products generated by caspase-3 were subjected to enzymatic proteolysis using endoproteinase LysC. Digest peptides were captured on reversed-phase beads, desalted, and subjected to MS analysis on a linear-mode matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) MS system. The C-terminal peptide at average mass-to-charge ratio (m/z) 6697.5 ± 0.8 was identified from the major (slower migrating) cleavage product (Figure 2D). The N terminus of this peptide h, expected at m/z 6697.3, is attributed to LysC cleavage within the GFP part of the substrate at K226, whereas its C terminus represents the caspase cleavage site at TATD292 (corresponding to TATD39 of the VirD2Ct part). The faster migrating band (Figure 1B, thin arrow) gave rise to the characteristic signal at m/z 3887.1 ± 0.8. The corresponding C-terminal peptide g (expected at m/z 3887.3) ends with the caspase-3 cleavage site at GEQD263 (corresponding to GEQD10 of the VirD2Ct part). None of these peptides was detected in LysC mapping experiments with intact GFP-VirD2Ct, although signals of several LysC-specific digest products (a to f) verified the identity of the protein (Figure 2D). Our analysis lacks the detection, from the intact GFP-VirD2Ct, of the expected LysC digest product at m/z 8531.4 (residues 227 to 309) that contains both caspase cleavage sites. Incomplete LysC cleavage in this C-terminal portion of the intact GFP-VirD2Ct or insufficient recovery of this big peptide might be responsible. Therefore, we conclude that TATD39 and GEQD10 are the caspase-3 cleavage sites in GFP-VirD2Ct. It is noteworthy that both of the identified caspase-3 cleavage sites precede the bipartite NLS of VirD2, and as result, each cleavage of GFP-VirD2Ct produces a GFP-containing fragment devoid of the NLS. By contrast, the GFP-encompassing products of fragmentation of the alternative substrate, VirD2Ct-GFP, retain the NLS with either cleavage (for structures, see Figure 2A). This outcome was the key feature of our strategy to detect plant caspase activity in vivo.
Detection of a Plant Caspase-Like Activity in Tobacco Plants Two types of host plants were used for inoculation with the recombinant viruses: tobacco cv Samsun (with the nn genotype exhibiting systemic TMV infection [no HR] at both 24 and 33°C) and tobacco cv Samsun NN (with the NN genotype exhibiting temperature-dependent TMV-mediated HR as described above). The intracellular localization at the developing infectious sites of GFP-VirD2Ct and VirD2Ct-GFP was examined by confocal laser scanning microscopy and fluorescence microscopy. As expected, nuclear fluorescence was observed exclusively in the cells infected with TMV(GFP-VirD2Ct) or TMV(VirD2Ct-GFP) in the absence of the HR—that is, in Samsun NN plants at 33°C (Figure 3A) and in Samsun plants at either 33 or 24°C (data not shown). In a control experiment, infection with TMV expressing free GFP [TMV(GFP)] lacking the VirD2 sequence resulted in the distribution of GFP fluorescence throughout the cell (Figure 3A). This result indicated that no cleavage of the VirD2 NLS from GFP occurred in the absence of the HR. However, when the HR was induced by transferring Samsun NN plants from 33 to 24°C, rapid relocalization of fluorescence to the cytoplasm occurred in the case of TMV(GFP-VirD2Ct) (Figure 3B), presumably as a result of cleavage of the NLS from GFP.
Cytoplasmic fluorescence became visible as early as 1 h after transfer, and at 4 h after transfer, it had spread throughout the entire patch of infected cells. By contrast, fluorescence in cells infected with TMV(VirD2Ct-GFP) was localized to the nucleus even in plants expressing the HR (Figure 3B). Interestingly, examination of a group of infected cells located at different distances from the center of the necrotic lesion revealed that, for TMV(GFP-VirD2Ct), the initial nuclear fluorescence of freshly infected cells farthest from the necrotic center was replaced by nuclear/cytoplasmic fluorescence in cells close to the necrotic area (Figure 3C), whereas for TMV(VirD2Ct-GFP), it remained confined to the nucleus (Figure 3C). These results suggested that the observed relocalization of the GFP-VirD2Ct fluorescent protein might be associated with its cleavage rather than with some general alteration in nuclear-cytoplasmic trafficking in the course of the HR. Considering the fact that human caspase-3 could cleave off the NLS sequences from the GFP moiety in GFP-VirD2Ct but not in VirD2Ct-GFP (see above), we suggest that a putative plant caspase with a similar specificity to human caspase-3 is activated during the HR in Samsun NN plants. To further test this idea, the effects of the single D10A and D39A mutations and the double D10,39A mutation, corresponding to the cleavage sites of caspase-3 (Figures 2A and 2B), on the intracellular localization of GFP-VirD2Ct were assessed. These mutations were introduced individually into TMV(GFP-VirD2Ct) to give TMV(D10A), TMV(D39A), and TMV(D10,39A), respectively. Confocal laser scanning microscopy of infected sites on the Samsun NN plants inoculated with these viruses showed that all of the mutant GFP-VirD2Ct proteins accumulated efficiently in the nucleus at 33°C, as did the wild-type protein (data not shown). However, after the temperature was shifted to 24°C, both mutant proteins carrying single mutations (either D10A or D39A) became distributed throughout the cell (Figure 3D), whereas the protein carrying the double mutation (D10,39A) still retained its nuclear localization (Figure 3D, right column), indicating a lack of cleavage. This finding suggests that GFP-VirD2Ct is cleaved by a plant enzyme activated during TMV-mediated HR in vivo, at the same sites as those identified for caspase-3 in in vitro experiments (see above). In parallel experiments, protein gel blot analysis using polyclonal antibody prepared against GFP showed that in the absence of the HR, either in Samsun plants at both 33 and 24°C (for 24°C, see Figure 4A) or in Samsun NN plants infected at 33°C (Figure 4A), the nonmutated recombinant proteins (VirD2Ct-GFP and GFP-VirD2Ct) were intact. A difference in size between VirD2Ct-GFP and GFP-VirD2Ct was caused by the presence of a short additional vector-derived sequence in VirD2Ct-GFP. However, when Samsun NN plants were shifted to 24°C to trigger the HR, fragmentation of these substrate fusion proteins at two distinct sites was evident (Figure 4B, lanes 2 and 7). Furthermore, under the same conditions, the single D10A or D39A mutation in GFP-VirD2Ct resulted in the elimination of the faster or the slower migrating product, respectively (Figure 4B, lanes 8 and 9). The relative size of the fragments formed was in agreement with the proposed pattern of proteolytic processing of VirD2. Most notably, the double mutation D10,39A prevented GFP-VirD2Ct from being cleaved even during the HR (at 24°C; Figure 4B, lane 10).
The results obtained are fully consistent with the notion of the activation, in the course of the HR, of a plant caspase(s) with the specificity of human caspase-3. In the wild-type protein substrate GFP-VirD2Ct, proteolytic cleavage at either D10 or D39 removes the NLS from the fluorescent protein, thus triggering its relocalization from the nucleus. Accordingly, the single D/A mutations do not rescue the nuclear accumulation of the cleavage product. However, when both of the cleavage sites are mutated simultaneously, the substrate protein acquires resistance to cleavage by a putative plant caspase and preserves the nuclear localization.
Partial Purification and Properties of a Plant Caspase
The D39A mutation in GFP-VirD2Ct abrogated substrate fragmentation by the plant enzyme (Figure 5, lane 4). Furthermore, mass spectrometric analysis of the gel-purified and LysC-digested GFP-VirD2Ct cleavage product generated by plant caspase permitted the identification of the same C-terminal peptide at average m/z 6698.1 ± 0.8 (Figure 2D, bottom) as the peptide originating from the caspase-3 major cleavage product. Thus, plant caspase cleaves GFP-VirD2Ct at TATD39, indicating that the substrate specificity of the plant caspase is similar in part to that of the human caspase. However, the partially purified plant caspase reproducibly failed to hydrolyze the substrate at GEQD10, which is the suboptimal cleavage site for human caspase-3. Therefore, we suggest that yet another caspase-like enzyme may become activated in plant cells that is directly responsible for the D10 cleavage and the relocalization in vivo of GFP-VirD2Ct containing the mutation D39A. To determine whether the plant caspase-like enzyme under study is likely to be a Cys proteinase, as animal caspases are, inhibition of GFP-VirD2Ct cleavage with various protease inhibitors was attempted. Ser proteinase [4-(2-aminoethyl)benzenesulfonyl fluoride and chymostatin], aspartyl proteinase (pepstatin), and metallo proteinase (EDTA) inhibitors were ineffective at inactivating the plant enzyme (Figure 6A). Of the Cys protease inhibitors tested, mercuric chloride abolished the substrate fragmentation by both plant and human enzymes, N-ethylmaleimide inhibited human enzyme but not the plant enzyme, and neither of the enzymes was inhibited by E-64 (Figures 6A and 6B). Notably, mercuric chloride under the conditions used failed to inhibit thermolysin and pepsin, a metallo protease and an aspartyl protease, respectively (Figure 6A, lanes 10 to 13). These results indicate the specificity of the inhibitory action of mercuric chloride and support the notion that the plant caspase-like enzyme possesses a Cys residue essential for its catalytic activity.
To compare in more detail the plant caspase activity with that of human caspase-3, a set of peptide-based inhibitors known to inactivate animal caspases was tested. Surprisingly, neither zVAD-fmk nor biotinyl-DEVD-CHO could inhibit the plant enzyme even at 100 µM (Figure 6C, lanes 4 and 5), whereas human caspase-3 hydrolysis was abolished completely by these inhibitors at 10 µM (Figure 6D, lanes 3 and 4). Based on the sequence of the plant caspase cleavage site in VirD2, TATD39 (identified above; see also Figure 2A), a potential inhibitor of plant caspase, biotinyl-TATD-CHO, was designed and synthesized. This biotinylated peptide aldehyde inhibited plant caspase at inhibitor concentrations of  40 µM (Figure 6C, lanes 6 and 7). It also proved to be a human caspase-3 inhibitor, with hydrolysis being prevented completely at 10 µM (Figure 6D, lane 5).
Therefore, we conclude that the plant caspase under study has greater substrate specificity than does human caspase-3. To further substantiate this conclusion, we tested an unrelated human protein, prothymosin
Effect of Plant Caspase Inhibitor on Cell Death Mediated by TMV Infection
To confirm this suggestion at the molecular level, we examined the effect of biotinyl-TATD-CHO on the expression of a plant gene, HSR203J, an early marker of HR cell death, whose expression is activated several hours before the appearance of necrotic lesions induced by pathogens (Pontier et al., 1994 ). Semiquantitative reverse transcriptase–mediated PCR showed that in the presence of biotinyl-TATD-CHO, HSR203J RNA transcripts accumulated with a delay (they were not detected at 30 h after inoculation; Figure 7C, lanes 3 versus lanes 2) and to significantly lower levels, being reduced by >85% compared with samples inoculated with TMV in the absence of biotinyl-TATD-CHO (Figure 7C, lanes 3 versus lanes 2) at 52 h after inoculation. At later time points (72 h after inoculation), the difference in the expression of HSR203J disappeared (Figure 7C, lanes 3 versus lanes 2). The levels of ubiquitin mRNA used as a constitutively expressed internal control with or without biotinyl-TATD-CHO were similar in all samples (Figure 7C, ubiquitin). Thus, the inhibition of the necrotization of Samsun NN leaves upon TMV infection by biotinyl-TATD-CHO was correlated with a delay in the activation of HSR203J expression. By contrast, the TMV-mediated activation of the PR-1a gene (a common marker for the defense response) was suppressed only slightly by biotinyl-TATD-CHO at 30 h after inoculation and was not affected at all at later stages (Figure 7C, lanes 3 versus lanes 2). All attempts to suppress the formation of necrotic lesions in TMV-infected tobacco Samsun NN leaves by coinoculation of biotinyl-DEVD-CHO, which did not inhibit the plant caspase in vitro (see above), also were unsuccessful (Figure 7B). Nor did biotinyl-DEVD-CHO affect the multiplication and spread of TMV in these plants or TMV-induced HSR203J or PR1-1a gene expression (Figure 7C, lanes 4 versus lanes 2). We conclude that the effect of biotinyl-TATD-CHO on the development of the TMV-mediated HR is highly specific. Although the plant caspase inhibitor biotinyl-TATD-CHO could not completely abolish the TMV-induced HR, it markedly retarded the appearance of the HR cell death. However, this partial inhibition of the HR did not result in the systemic spread of TMV (data not shown).
A large body of circumstantial evidence, based primarily on the use of mammalian caspase inhibitors, suggested that plant caspases might exist and be involved in the implementation of the PCD in various plant systems. However, these proteolytic enzymes remained elusive, mainly because natural protein targets of putative plant caspases were not known, and no reliable way to find them existed. Nor were their cleavage site requirements known, so research was based on analogy with animal caspase cleavage sites. In our work, the VirD2 protein of A. tumefaciens was used for the detection, identification, and partial purification of a tobacco caspase, based on our prediction that this protein might represent a genuine caspase target. Indeed, we demonstrated that VirD2 could be cleaved by human caspase-3 at one major and one suboptimal site within the C-terminal portion of VirD2, upstream of its NLS. Moreover, because VirD2 is active in plant cells, this finding provided grounds for a search for a plant caspase-like activity capable of VirD2 processing at identical sites. To classify the plant enzyme as an analog of animal caspases, we propose four major criteria: (1) this protein should be a protease; (2) its substrate specificity should be similar to that of an animal caspase; (3) it should become activated in the course of plant PCD; and (4) specific inhibitor(s) of this enzyme should counteract plant PCD. The plant enzyme under study meets all four of these criteria. In the first approach, VirD2 was expressed as N- or C-terminal fusion proteins with GFP from a TMV vector. In these experiments, TMV also played the role of inducer of the HR. When the HR was induced, rapid relocalization of the fluorescence to the cytoplasm occurred in TMV(GFP-VirD2Ct), presumably because the NLS became detached from GFP-infected plants, whereas fluorescence in cells infected with TMV(VirD2Ct-GFP) remained localized to the nucleus. Given the fact that human caspase-3 also could cleave off the NLS sequences from the GFP moiety in GFP-VirD2Ct but not in VirD2Ct-GFP, it seemed possible that some putative plant caspase with a similar specificity is activated during the HR in tobacco plants. Mutational analysis of potential cleavage sites has identified two sites at which GFP-VirD2Ct is cleaved by a plant enzyme activated during TMV-mediated HR in vivo (TATD and GEQD) that are identical to those identified for caspase-3 in the experiments in vitro. In the second approach, we used VirD2 as a substrate to test plant caspase activity induced during the HR in vitro. This approach allowed us to partially purify the enzyme and confirm its cleavage specificity. In the third approach, the tetrapeptide aldehyde biotinyl-TATD-CHO, designed and synthesized on the basis of the elucidated plant caspase cleavage site, was shown to prevent the fragmentation of VirD2 by plant and human caspases in vitro and to counteract TMV-triggered HR in vivo. Thus, our findings provide strong in vivo and in vitro evidence that a caspase-like protease is activated in tobacco plants during the N gene–mediated HR triggered by TMV infection. However, because no direct homologs to animal caspases have been found in plants in database searches, the plant caspase identified in this work presumably represents a functional analog of animal caspases.
The tobacco caspase described in this work appears to possess even greater specificity than human caspase-3 because (1) the plant enzyme hydrolyzes only a single peptide bond in the substrate protein, corresponding to the major cleavage site of human caspase; (2) of several caspase-targeted peptide inhibitors tested (zVAD-fmk, Ac-DEVD-CHO, biotinyl-DEVD-CHO, and biotinyl-TATD-CHO), all were efficient in caspase-3 inhibition, whereas the plant enzyme was inhibited only by biotinyl-TATD-CHO synthesized on the basis of the plant caspase cleavage site; and (3) plant caspase failed to process a model protein, prothymosin
The biotinyl-TATD-CHO concentration required to inhibit the plant enzyme was severalfold greater than that required to inhibit human caspase-3. A possible explanation for this difference is that a longer peptide moiety is optimal for recognition by plant caspase protease. Animal caspase-2, for example, requires pentapeptide substrates for efficient recognition (Talanian et al., 1997 Our findings also suggest that in plants, as in animals, a family of caspases may exist, of which the enzyme identified is one of the members. Indeed, to prevent the proteolytic processing and nuclear escape of GFP-VirD2Ct substrate during the HR in tobacco leaves, mutation of both of the sites cleaved by human caspase-3, TATD39 and GEQD10, was required, whereas the plant enzyme under study was strictly specific to the former site. We infer from this observation that another plant caspase with a recognition sequence matching GEQD may become activated in the course of plant PCD. Consistent with this notion, the use of a set of peptide inhibitors targeting different members of the plant caspase family may lead to a more pronounced suppression of the HR in plants than did biotinyl-TATD-CHO alone.
Our observations show that the synthetic peptide inhibitor for caspase, biotinyl-TATD-CHO, dramatically retards the development of necrotic lesions, the macroscopic symptoms of cell death mediated by TMV. The fact that the suppression by biotinyl-TATD-CHO of the induction of TMV-triggered cell death was correlated with a delay in the activation of the HSR203J gene, an early HR-specific molecular marker, indicates that the plant caspase described here regulates an early step in the HR preceding the activation of HSR203J. By contrast, the finding that the expression of the PR-1a gene (a marker for the host defense response during HR) was affected only slightly by biotinyl-TATD-CHO is consistent with the idea that host defense mechanisms and HR cell death may be regulated by different pathways that are expressed coordinately (del Pozo and Lam, 1998
Interestingly, del Pozo and Lam (1998)
The HR is a general response of plants to attack by different pathogens, including Agrobacterium spp. Certain (incompatible) Agrobacterium spp–plant interactions lead to the development of PCD-like HR (Hansen, 2000 The challenge for the future is to purify to high homogeneity and characterize plant caspase and to identify a gene for this enzyme. This may provide new insights into the molecular mechanisms of PCD in plants and could lead to powerful approaches for the modulation of plant growth and development and for protection from pathogen attack and abiotic stresses. Moreover, comparative analysis of plant and animal caspases will illuminate elements of the PCD machinery that are conserved between the plant and animal kingdoms.
Plasmid Construction The protein-encoding portion of virD2 cDNA in pQE13 vector (Qiagen, Valencia, CA) was obtained from V. Lunin (Institute of Agricultural Biotechnology, Moscow). The virD2 cDNA then was excised from this plasmid as a 100-bp SauIIIA-PstI fragment and a 1240-bp PstI-XbaI DNA fragment and inserted between the BamHI and XbaI sites of pUC19 to produce pUC19/VirD2. Elimination of the Ecl136II-Bsp120I fragment from pUC19/VirD2, filling in with the Klenow fragment, and self-ligation of the rest of the plasmid produced pUC19/VirD2Ct.
Bacterial Expression
Mutations were introduced in the virD2 sequence by PCR on pUC19/VirD2Ct with the mutagenic primers 5'-TCggaTcCACGCGGACGCTTTG-3' (for VirD2Ct
To construct GFP-virD2Ct fusions, a Bsp120I/Klenow-XbaI DNA fragment from pUC19/VirD2 was inserted into pFRED25 The cDNA fragment encoding human caspase-3 was amplified by PCR from a HeLa cDNA library using primers 5'-CATGGAGAACACTGAAAAC-3' and 5'-GGTGATAAAAATAGAGTTCTT-3'. An 830-bp PCR product was inserted into pBluescript II SK+ vector (Stratagene) between the SmaI and XhoI/Klenow sites, excised with NcoI (partial digestion) and XhoI, and inserted into the NcoI-XhoI–digested pET33b(+) vector (Novagen, Madison, WI). The structure of the resulting construct was confirmed by sequencing.
Expression in Plants
Purification of Recombinant Proteins
E. coli BL21(DE3) cells freshly transformed with a caspase-3–encoding plasmid were grown in 80 mL of Luria-Bertani medium containing 25 µg/mL kanamycin at 30°C with shaking. Exponentially growing cells were induced with 0.5 mM isopropyl-
In Vitro Transcription, Inoculation of Plants, Confocal Laser Scanning Microscopy, and Fluorescence Microscopy Infected leaves were viewed using a stereofluorescence microscope. To visualize nuclear DNA, leaf tissues were stained with 4',6-diamidino-2-phenylindole (1 µg/mL). Fluorescent infection sites also were monitored using an MRC 1000 confocal laser scanning microscope (Bio-Rad, Hercules, CA) equipped with a 25-mW krypton/argon laser. For GFP imaging, blue excitation at 488 nm with emission filter 522 DF (32 nm) was used. For experiments with synthetic caspase inhibitors, potential inhibitors, biotinyl-TATD-CHO (custom synthesized by Bachem, Merseyside, UK), or biotinyl-DEVD-CHO (Bachem) was dissolved in DMSO, diluted in 0.01 M phosphate buffer, pH 5.5, to a final concentration of 2 mM, mixed with an equal volume of purified TMV preparation, and inoculated immediately into tobacco cv Samsun NN leaves as described above. The same volume of DMSO was added to control TMV samples. Infected plants were kept at 24°C.
Protein Gel Blot Analysis
Partial Purification of Tobacco Caspase
Caspase Hydrolysis and Inhibition Treatment of 3-µg aliquots of GFP-VirD2Ct with partially purified plant caspase (1.0 µL of the enzyme preparation per sample) was performed in B1 buffer, pH 5.5, in a total volume of 15 µL at 27°C for 1.5 h. For direct comparison of the inactivation of human and plant enzymes with inhibitors, caspase-3 hydrolysis and inhibition, as depicted in Figures 6B and 6D, also were performed in B1 buffer, pH 5.5. Enzymes were preincubated in the same buffer with the indicated concentrations of zVAD-fmk, biotinyl-DEVD-CHO, or biotinyl-TATD-CHO at 27°C for 1 h before their addition to the substrate. Alternatively, enzymes were preincubated for 30 min at 27°C (plant caspase) or 37°C (caspase-3) in B1 buffer, pH 5.5, containing 200 µM DTT in the presence of 1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride, 100 µM chymostatin, 10 µM E-64, 5 mM N-ethylmaleimide, 200 µM HgCl2, 5 mM EDTA, or 1 µM pepstatin. Then, the substrate was added and the samples were incubated at the temperatures indicated for 2 h. Reaction mixtures were fractionated by 12% SDS-PAGE, and proteins were stained with Coomassie Brilliant Blue R 250.
Recombinant human prothymosin
Analysis of TMV Multiplication
RNA Isolation and Reverse Transcriptase–Mediated PCR Analysis
Mass Spectrometric Analysis of Caspase Cleavage Sites in GFP-VirD2Ct Spectra were acquired on a Voyager-DE STR MALDI mass spectrometer (ABI) in linear mode. Singly charged ions of bovine insulin and ubiquitin served as external calibration standards using average masses of the corresponding singly charged protonated species. The spectra were recalibrated internally on peaks identified as LysC digestion products of GFP-VirD2Ct at m/z 1534.76 and 4732.15. Upon request, materials integral to the findings presented in this publication will be made available in a timely manner to all investigators on similar terms for noncommercial research purposes. To obtain materials, please contact M.E. Taliansky, mtalia{at}scri.sari.ac.uk; or A.B. Vartapetian, varta{at}genebee.msu.su.
Accession Number
We thank V. Lunin for providing VirD2 cDNA, G. Pavlakis for providing vectors encoding a bright version of GFP, A. Evstafieva for providing human prothymosin  , T. Pestova and C. Hellen for their constant support, and S. MacFarlane and B. Harrison for critical reading of the manuscript. This work was supported in part by the Ludwig Institute for Cancer Research (to A.B.V.), by a grant-in-aid from the Scottish Executive Environment and Rural Affairs Department (to M.E.T.), and by grants from the Russian Foundation for Basic Research (to N.V.C. and A.B.V.), the U.S. Civilian Research and Development Foundation for the Independent States of the Former Soviet Union (to A.B.V.), the Russian Frontiers in Genetics Program (to A.B.V.), and the Royal Society (ex-quota visit fellowship to N.O.K.).
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.017889.
1 These authors contributed equally to this work. Received October 1, 2003; accepted October 14, 2003.
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ABSTRACT
INTRODUCTION
30 h after inoculation and became well developed at 42 h after inoculation in the control (diluent-coinoculated) half-leaves (
Naegfphyg (Stauber et al., 1998
-D-thiogalactoside and harvested at 3 h after induction. Cell pellets were washed with TBST buffer (20 mM Tris-HCl, pH 7.5, 140 mM NaCl, and 0.05% Tween 20), resuspended in 1 mL of buffer A (20 mM Tris-HCl, pH 8.0, 50 mM NaCl, and 5% glycerol) supplemented with aprotenin (4 µg/mL), leupeptin (2 µg/mL), and phenylmethylsulfonyl fluoride (PMSF; 1.2 mM), sonicated on ice by four 30-s bursts, and centrifuged at 12,000g for 10 min. The supernatant was discarded, and the pellet was resuspended in 1 mL of buffer B (10 mM Tris-HCl, pH 8.0, 1 M NaCl, and 8 M urea), incubated for 30 min at room temperature, and sonicated on ice by three 30-s bursts. After centrifugation at 12,000g for 10 min, cleared lysates were incubated in batches with nickel–nitrilotriacetic acid (Ni-NTA) agarose (Qiagen) equilibrated with buffer B for 3 h at room temperature with shaking. The resin then was washed with 5 x 1 mL of buffer C (10 mM Tris-HCl, pH 6.3, 100 mM NaCl, 100 mM Na2HPO4, and 8 M urea), and the proteins were eluted with 400 µL of buffer C containing 100 mM EDTA for 1 h at room temperature with shaking and then with two additional 100-µL portions of the same buffer. The eluates were combined and dialyzed overnight against 200 mL of buffer 20/100 (20 mM Hepes, pH 6.8, 100 mM NaCl, 2 mM DTT, and 0.05% Tween 20) or against B1 buffer, pH 5.5 (50 mM Hepes, pH 5.5, 50 mM NaCl, 2 mM DTT, 0.1% Tween 20, and 5% glycerol). 
