Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity
2010; Springer Nature; Volume: 29; Issue: 6 Linguagem: Inglês
10.1038/emboj.2010.1
ISSN1460-2075
AutoresNina V. Chichkova, Jane Shaw, Raisa A. Galiullina, Georgina E. Drury, Alexander I. Tuzhikov, Sang Hyon Kim, Markus Kalkum, Teresa Hong, Elena N. Gorshkova, L. Torrance, Andrey B. Vartapetian, Michael Taliansky,
Tópico(s)Plant Pathogenic Bacteria Studies
ResumoArticle28 January 2010free access Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity Nina V Chichkova Nina V Chichkova A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Jane Shaw Jane Shaw Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Raisa A Galiullina Raisa A Galiullina A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Georgina E Drury Georgina E Drury Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Alexander I Tuzhikov Alexander I Tuzhikov A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Sang Hyon Kim Sang Hyon Kim Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Division of Biosciences and Bioinformatics, Myongji University, Yongin, Kyeongki-do, Korea Search for more papers by this author Markus Kalkum Markus Kalkum Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA, USA Search for more papers by this author Teresa B Hong Teresa B Hong Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA, USA Search for more papers by this author Elena N Gorshkova Elena N Gorshkova A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Lesley Torrance Lesley Torrance Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Andrey B Vartapetian Corresponding Author Andrey B Vartapetian A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Michael Taliansky Corresponding Author Michael Taliansky Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Nina V Chichkova Nina V Chichkova A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Jane Shaw Jane Shaw Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Raisa A Galiullina Raisa A Galiullina A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Georgina E Drury Georgina E Drury Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Alexander I Tuzhikov Alexander I Tuzhikov A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Sang Hyon Kim Sang Hyon Kim Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Division of Biosciences and Bioinformatics, Myongji University, Yongin, Kyeongki-do, Korea Search for more papers by this author Markus Kalkum Markus Kalkum Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA, USA Search for more papers by this author Teresa B Hong Teresa B Hong Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA, USA Search for more papers by this author Elena N Gorshkova Elena N Gorshkova A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Lesley Torrance Lesley Torrance Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Andrey B Vartapetian Corresponding Author Andrey B Vartapetian A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia Search for more papers by this author Michael Taliansky Corresponding Author Michael Taliansky Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK Search for more papers by this author Author Information Nina V Chichkova1, Jane Shaw2, Raisa A Galiullina1, Georgina E Drury2, Alexander I Tuzhikov1, Sang Hyon Kim2,3, Markus Kalkum4, Teresa B Hong4, Elena N Gorshkova1, Lesley Torrance2, Andrey B Vartapetian 1 and Michael Taliansky 2 1A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia 2Plant Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee, UK 3Division of Biosciences and Bioinformatics, Myongji University, Yongin, Kyeongki-do, Korea 4Department of Immunology, Beckman Research Institute of the City of Hope, Duarte, CA, USA *Corresponding authors: A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119992, Russia. Tel.: +7 495 9394125; Fax: +7 495 9393181; E-mail: [email protected] Pathology Programme, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. Tel.: +44 1382 562731; Fax: +44 1382 562426; E-mail: [email protected] The EMBO Journal (2010)29:1149-1161https://doi.org/10.1038/emboj.2010.1 There is a Have you seen ...? (March 2010) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Caspases are cysteine-dependent proteases and are important components of animal apoptosis. They introduce specific breaks after aspartate residues in a number of cellular proteins mediating programmed cell death (PCD). Plants encode only distant homologues of caspases, the metacaspases that are involved in PCD, but do not possess caspase-specific proteolytic activity. Nevertheless, plants do display caspase-like activities indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases. Here, we report the identification and characterisation of a novel PCD-related subtilisin-like protease from tobacco and rice named phytaspase (plant aspartate-specific protease) that possesses caspase specificity distinct from that of other known caspase-like proteases. We provide evidence that phytaspase is synthesised as a proenzyme, which is autocatalytically processed to generate the mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to tobacco mosaic virus and abiotic stresses. Phytaspase is constitutively secreted into the apoplast before PCD, but unexpectedly is re-imported into the cell during PCD providing insights into how phytaspase operates. Introduction Programmed cell death (PCD, or apoptosis) is a basic process for elimination of redundant and damaged cells in multicellular organisms, which operates in the course of their development and in response to various stress-inducing stimuli. In animals, an important component of the PCD machinery is a family of apoptotic proteases termed as caspases (for cysteine-dependent aspartate-specific proteases). These proteolytic enzymes become activated in the course of apoptosis and introduce specific breaks after aspartate (D) residues in a limited number of cellular proteins thus mediating PCD (Thornberry and Lazebnik, 1998; Wolf and Green, 1999). Two types of caspases exist: initiator and effector caspases (Boatright and Salvesen, 2003). Initiator caspases (e.g. caspases-2, -8, -9 and -10) cleave inactive pro-forms of effector caspases, thereby activating them. Effector caspases (e.g. caspases-3, -6 and -7) in turn cleave other protein substrates within the cell, to trigger the apoptotic process. The initiation of this cascade reaction is regulated by caspase inhibitors (Ekert et al, 1999). Caspase inactivation is known to suppress cell death (Zheng et al, 1999). PCD in the plant and animal kingdoms share a number of morphological and biochemical features, including condensation and shrinkage of the nucleus and cytoplasm, DNA laddering and cytochrome c release from mitochondria (Danon et al, 2000; Balk and Leaver, 2001; Lam et al, 2001; Hoeberichts and Woltering, 2003). However, direct structural homologues of animal caspases with an analogous cleavage specificity and function have not been shown to exist in plants (reviewed by Bonneau et al, 2008). Plants encode only distant homologues of caspases, metacaspases, that may be involved in PCD, but do not possess caspase-specific proteolytic activity (Bozhkov et al, 2005; Watanabe and Lam, 2005; Vercammen et al, 2006; He et al, 2007; Sundström et al, 2009). However, some specific peptide inhibitors of animal caspases have been shown to affect the development of plant PCD. One of the examples of PCD in plants is the hypersensitive response (HR) that results from incompatible plant–pathogen interactions, which serves to prevent the spread of pathogens from the infection site (Greenberg, 1997; Heath, 2000). For example, specific inhibitors of animal caspase-1 and -3 [N-acetyl-YVAD-chloromethylketone (Ac-YVAD-CMK) and Ac-DEVD-aldehyde (Ac-DEVD-CHO), respectively] could attenuate bacteria- and tobacco mosaic virus (TMV)-induced HR in tobacco leaves (del Pozo and Lam, 1998). The baculovirus anti-apoptotic proteins p35 and IAP (inhibitor of apoptosis), which are known to inhibit animal caspases, were also efficient in preventing plant PCD induced by bacterial, fungal and viral infections (Hansen, 2000; Dickman et al, 2001; del Pozo and Lam, 2003). Furthermore, caspase-like activities have been detected in plants using synthetic fluorogenic caspase substrates (reviewed by del Pozo and Lam, 1998; Bonneau et al, 2008) indicating that enzymes structurally distinct from classical caspases may operate as caspase-like proteases in plants. Indeed, it has recently been shown that the plant protease vacuolar-processing enzyme (VPE) is required for PCD induced by TMV (Hatsugai et al, 2004) and fumonisin (Kuroyanagi et al, 2005) as well as for developmental cell death in seed integuments (Nakaune et al, 2005) and displays a caspase-1 (YVADase) activity (Hatsugai et al, 2004; Rojo et al, 2004). However, VPE is a legumain, a cysteine protease, that is structurally different from classical caspases. It has also been found that caspase inhibitors prevent ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) proteolysis during victorin-induced PCD, and the use of the pan-caspase substrate benzyloxycarbonyl-VAD-7-amino-4-trifluoromethylcoumarin (z-VAD-AFC) allowed the detection of caspase-like activity in extracts from victorin-treated Avena sativa leaves (Coffeen and Wolpert, 2004). The purified active protein contained amino-acid sequences homologous to plant subtilisin-like serine proteases and was named saspase (serine-dependent aspartate-specific protease). Saspase has also been shown to be indirectly involved in victorin-induced cleavage of Rubisco during PCD in oats (Coffeen and Wolpert, 2004). However, the function of saspase in PCD execution remains unknown. Recently, we have identified a caspase-like activity in tobacco that seems to represent a functional analogue of animal caspases (Chichkova et al, 2004). This protease activity displayed an exceptional selectivity introducing a single break in the Agrobacterium tumefaciens VirD2 protein, after the D400 residue within the TATD motif, in a caspase-like manner. The tobacco enzyme, like animal caspases, was inactive in healthy tissues and became active in the course of TMV-induced HR in tobacco. Furthermore, suppression of enzyme activity by a peptide aldehyde matching its cleavage site inhibited PCD mediated by TMV in tobacco leaves (Chichkova et al, 2004). Fragmentation of the A. tumefaciens VirD2 protein by tobacco caspase-like protease results in the detachment of the C-terminal nuclear localisation signal (NLS)-containing peptide of VirD2. The NLS in the VirD2 protein is essential for nuclear uptake of foreign DNA within the plant cell during bacterial infection and plant transformation (Steck et al, 1990; Shurvinton et al, 1992). Therefore, inactivation of VirD2 mediated by tobacco caspase-like protease may represent a protective mechanism aimed at limiting delivery and expression of foreign genes in plants. Indeed, substitution of the wild-type VirD2 protein with its mutant form, which is resistant to tobacco caspase-like protease, was shown to markedly improve A. tumefaciens-mediated foreign gene delivery (Reavy et al, 2007). Proteases with specificity and biochemical properties similar to those of the tobacco enzyme have been shown to be ubiquitous in mono- and dicotyledonous plants (Chichkova et al, 2008). Here, we report the isolation and identification of this protease from tobacco and rice and propose to name it phytaspase (plant aspartate-specific protease). Mutational analysis of the recombinant enzyme showing serine dependence confirmed the designation of phytaspase as a subtilisin-like protease predicted from the amino-acid sequence. Being synthesised as a proenzyme, phytaspase is autocatalytically processed to generate a mature enzyme. Overexpression and silencing of the phytaspase gene showed that phytaspase is essential for PCD-related responses to biotic (TMV) and abiotic stresses. The substrate specificity of phytaspase is distinct from that of other known caspase-like proteases. Another intriguing peculiarity of phytaspase is that it is constitutively secreted into the apoplast before PCD, but unexpectedly is partially re-imported into the cell during PCD. Results and discussion Identification of phytaspase Using affinity chromatography with biotinylated TATD aldehyde (bio-TATD-CHO) on avidin resin, phytaspase was purified from Nicotiana tabacum leaves to homogeneity producing a single major ∼80 kDa protein (Figure 1A, left panel; Supplementary Figure S1). Mass spectrometric analysis of the protein allowed the identification of an N. tabacum EST encoding a central portion of the enzyme and by amplification of a cDNA, the complete ORF of the protein was obtained and sequenced (these sequence data have been submitted to the GenBank databases under accession No. GQ249168). The deduced complete amino-acid sequence of tobacco phytaspase indicated that it corresponds to a putative subtilisin-like protease of the S8 family predicted to possess a 24 amino-acid N-terminal signal peptide (for extracellular targeting) and a prodomain (Figure 1B; Supplementary Figure S2). Indeed, the mature tobacco phytaspase was found to lack both the signal sequence and the prodomain (Supplementary Figure S2). A rice phytaspase was similarly identified (accession GI 32977156) and it displayed 53% amino-acid sequence identity with the tobacco phytaspase (Figure 1A, right panel; Supplementary Figure S2). Figure 1.Identification and molecular characterisation of tobacco phytaspase. (A) Affinity chromatography purification of tobacco (left panel) and rice (right panel) phytaspases with their inhibitor, bio-TATD-CHO on avidin resin; bio-DEVD-CHO, which does not inhibit phytaspase, was used as a control. Protein samples were fractionated by SDS–gel electrophoresis and stained with Coomassie Blue or zinc-imidazole. Positions of MW protein markers (M) are indicated on the left. (B) Schematic representation of phytaspase domains. (C) Recombinant tobacco phytaspase (rec) displays the same ability to cleave GFP-VirD2Ct protein as natural tobacco enzyme (nat). The bio-TATD-CHO was used at 100 μM for enzyme inhibition. Coomassie Blue-stained gel is shown. The D39A mutation (D39A mut) (Chichkova et al, 2004) preventing cleavage in the GFP-VirD2Ct protein corresponds to the D400 residue in full-length VirD2. (D) The S537A mutation prevents phytaspase-mediated cleavage of GFP-VirD2Ct in vitro (Coomassie Blue staining). C540A, a mutation of a nearby residue, only marginally affects proteolytic activity. Recombinant enzymes were produced as in (C). 1 and 0.2 indicate relative amounts of recombinant enzymes taken to assess their hydrolytic activity, as verified by western blotting with anti-GST antibody. Control sample (control) obtained in an identical manner from vector-only agroinfiltrated leaves. (E) Phytaspase mutants with impaired processing. Western blot analysis of the crude extracts from leaves agroinfiltrated with constructs expressing wt and mutated phytaspase forms fused to GFP with anti-GFP antibody. In contrast to the purified enzyme (A) showing only one band (mature enzyme), extracts containing wt phytaspase display two bands (proenzyme and mature enzyme) apparently because in the latter case, we used immediate denaturation of samples (boiling in SDS). Both the S537A (catalytic residue) and D117A (the prodomain/catalytic domain junction) mutations impair proenzyme processing in N. benthamiana leaves. Control, vector-only agroinfiltrated leaves. Download figure Download PowerPoint The tobacco phytaspase gene was expressed in N. benthamiana leaves as phytaspase-glutathione-S-transferase (GST) fusion using an Agrobacterium-mediated transient expression system and purified by glutathione Sepharose chromatography. The recombinant phytaspase displayed the same ability to cleave a substrate protein GFP-VirD2Ct (Chichkova et al, 2004) at the TATD motif in vitro, as the native enzyme (Figure 1C). S8 subtilisin-like proteases are serine proteases. Accordingly, mutation of the predicted catalytic S537 residue (Rawlings et al, 2008; Rawlings and Barrett, 2009) of tobacco phytaspase abolished its proteolytic activity (Figure 1D). To determine whether processing of the prodomain occurs autocatalytically, green fluorescent protein (GFP) was fused to the C-terminus of the full-length tobacco phytaspase. Western blots of total protein extracted from these leaves were reacted with anti-GFP antibody, and two protein bands (Figure 1E) were detected corresponding to the partially processed enzyme-containing QSETYVIHM at its N-terminus (proenzyme, lacking the predicted 24-amino-acid signal peptide) and completely processed protein with the TTHTSQFL as the N-terminal amino-acid sequence (lacking the signal peptide and prodomain), respectively, as was shown by Edman degradation sequencing (Supplementary Figure S2). Of note, the inactive S537A mutant displayed only one protein band with an MW similar to the wild-type proenzyme (Figure 1E) and its N-terminal sequence determined by Edman degradation sequencing as QSETYVIHM corresponded to the amino-terminus of the proenzyme lacking the signal peptide (Supplementary Figure S2). This indicates that no cleavage of the prodomain had occurred in the catalytically inactive mutant and shows that phytaspase seems to be a self-processing enzyme—a property shared by subtilases of various origin including plant subtilisin-like proteases (Cedzich et al, 2009). Consistent with this conclusion, the D residues in both tobacco and rice phytaspase precursors immediately precede the TTHT motif, which is the N-terminus of the mature enzyme (Figure 1B; Supplementary Figure S2). Accordingly, mutation of the D117 to A in tobacco phytaspase impaired proenzyme processing (Figure 1E). Substrate specificity of phytaspase To determine the substrate specificities of the tobacco and rice phytaspases, we used a panel of peptide aldehyde inhibitors of animal caspases in the in vitro GFP-VirD2Ct cleavage assay. Their inhibitory potential was markedly distinct, with Ac-VEID-CHO showing the strongest inhibition, whereas DEVD-CHO produced no effect (Figure 2A and B). Employment of a range of peptide-based fluorogenic substrates of animal caspases produced complementary results: Ac-VEID-AFC (7-amino-4-trifluoromethylcoumarin) was found to be the optimal substrate of phytaspases, whereas Ac-DEVD-AFC remained uncleaved (Figure 2C). Phytaspase inactivation by peptide aldehyde inhibitors was found to be reversible (Figure 2D). These results describing VEID as a preferred motif for phytaspase cleavage differentiate tobacco and rice phytaspases from the saspase obtained from A. sativa, which does not cleave VEID (nor VDVAD or WEHD) at all (Coffeen and Wolpert, 2004). By the same substrate specificity criterion, phytaspase is more similar to animal caspase-6 and to an unidentified protease involved in PCD during embryogenesis in Norway spruce (Bozhkov et al, 2004). Figure 2.Substrate specificities of tobacco and rice phytaspases. (A) Peptide aldehyde inhibitors of animal caspases at 100 μM, with the exception of DEVD-CHO, impair tobacco (upper panel) and rice (lower panel) phytaspase-mediated fragmentation of GFP-VirD2Ct substrate protein. (B) The use of the same set of inhibitors at 10 μM shows their markedly distinct inhibitory potential and minor differences between tobacco and rice phytaspase specificities. Note that in both cases, Ac-VEID-CHO is the most potent phytaspase inhibitor. (C) Fluorogenic peptide substrates of animal caspases are hydrolysed by tobacco (left panel) and rice (right panel) phytaspases. Relative rates of peptide-AFC (20 μM) hydrolysis expressed as relative fluorescence units per hour (RFU/h, mean values from three samples) are slightly different for rice and tobacco enzymes. Note that the naturally occurring phytaspase cleavage site, STATD, in the VirD2 protein is one of the poorest cleavage sites at the peptide level. (D) Inhibition of phytaspase with peptide aldehyde inhibitors is reversible. Phytaspase was pre-incubated with the indicated inhibitors (at 100 μM), samples were diluted five-fold and fluorogenic substrate Ac-VAD-AFC was added up to 20 μM directly to the mixture to determine relative rates of substrate hydrolysis in the presence of the inhibitors (grey bars). Alternatively, before addition of the fluorogenic substrate, free inhibitors were eliminated by spin gel filtration followed by 1 h incubation at room temperature to allow possible reversion of inhibition (open bars). Data (means from three experiments) are given for tobacco phytaspase. Rice phytaspase behaves similarly (data not shown). Download figure Download PowerPoint Overexpression and silencing of phytaspase gene in tobacco transgenic plants To address the possible function of phytaspase in the HR, we generated transgenic N. tabacum Samsun NN plants, which either overproduced tobacco phytaspase or possessed markedly decreased levels of phytaspase activity because of RNAi silencing induced by transgenic expression of three independent hairpin RNA constructs designed to avoid ‘off-target’ silencing. Using the siRNA scan website (http://bioinfo2.noble.org/RNAiScan.htm) (Xu et al, 2006), sequences of these three independent fragments of the phytaspase ORF were screened against datasets from tobacco, tomato and Arabidopsis to seek 21 nt stretches of homology with other genes and thus the potential for ‘off-target’ silencing. For fragment 1 (nt 7–237), no matches were identified to any sequences in all the datasets searched. For the other two fragments, fragment 2 (nt 1096–1446) and fragment 3 (nt 1888–2295) matches were made to sequences of only one tomato gene (SGN-U329832) annotated as a gene for subtilisin-like protease, which may or may not be a homologue of tobacco phytaspase. However, taking into account the unavailability of full-genome sequence of tobacco, we performed a functional complementation assay to ascertain that the phenotypic effects observed on RNAi were due to specific silencing of the phytaspase gene, as described below. Tests using qRT–PCR revealed >90% reduction in transcript level in leaf tissues of T1 seedlings in each of five to seven lines of phytaspase-silenced (knocked down, KD) tobacco plants generated with each of the hairpin RNAi constructs. Typical results characteristic of all the KD transgenic lines for each construct are shown in Figure 3A. In all the phytaspase overexpressing (OE) lines, exemplified by three of them in Figure 3B, phytaspase gene expression was increased about seven- to eight-fold compared with wild-type tobacco. Measurements of phytaspase enzymatic activity confirmed the significant increase (approximately four-fold) and reduction (approximately eight-fold) of phytaspase-specific activity in overproducing and silenced transgenic plants, respectively (Figure 3C and D). In spite of these differences, neither overproduction of phytaspase nor its silencing showed any discernible altered phenotype. It is possible that phytaspase overexpression could be neutralised by export of the enzyme out of the cell (see below), whereas (as suggested by the silencing experiments) phytaspase is possibly not involved in plant growth and development (at least under the optimal glasshouse conditions). Alternatively, functional redundancy may exist and another enzyme may compensate for the low level of phytaspase during plant growth and development. The results presented below were typical of all transgenic lines; most tests were performed using silenced line KD1-1 generated with the RNAi fragment 1 and OE line OE-1. Figure 3.Relative expression of phytaspase ORF in transgenic N. tabacum Samsun NN plants with silenced (knocked down, KD) or overexpressed (OE) phytaspase ORF. (A, B) Phytaspase ORF expression was measured by qRT–PCR in wild-type (wt) plants and in three independent phytaspase-silenced transgenic lines KD1-1, KD2-1 and KD3-1 generated using different RNAi constructs (A) and three independent lines overexpressing phytaspase (B, OE-1, OE-2 and OE-3). The results presented in (A) and (B) were typical of all transgenic lines produced. Data are means±s.d. from six independent plants. The levels of ubiquitin mRNA used as a constitutively expressed internal control were similar in all the samples analysed above (data not shown). (C, D) Transgenic tobacco plants with overexpressed (OE) and silenced (KD) phytaspase display altered phytaspase enzymatic activity relative to wild-type (wt) plants. (C) Crude leaf extracts from three independent OE and KD lines and from wt plants were tested for in vitro cleavage of GFP-VirD2Ct substrate protein. Reaction mixtures were fractionated by 12% SDS–gel electrophoresis. An unknown protein band marked with an asterisk comes from extracts and indicates similar sample loading. Lane M, substrate protein cleaved with purified tobacco phytaspase. (D) Serial dilutions of arbitrary chosen extracts of each type allow quantification of phytaspase enzymatic activity: approximately four-fold increase in OE plants and approximately eight-fold decrease in KD plants, relative to wt plants. Download figure Download PowerPoint Phytaspase is involved in the hypersensitive cell death response and resistance to TMV N. tabacum plants carrying the N gene (e.g. Samsun NN) are resistant to TMV and exhibit an HR at temperatures <27°C: TMV is localised to the vicinity of the necrotic lesions formed at the late stages of the HR (Kassanis, 1952). At higher temperatures (30°C), the resistance response is inoperative and TMV is able to multiply and spread, but after decreasing the temperature, the HR is synchronously induced in all of the infected cells (Mittler et al, 1995). To assess a potential function of phytaspase in the TMV-mediated HR, transgenic plants overproducing or silencing phytaspase were infected with TMV at 30°C and examined after induction of the HR by temperature shift to 24°C. TMV-mediated HR was suppressed in phytaspase-silenced (KD) plants, resulting in the formation of less severe amorphous lesions some of which continued to grow reaching up to 1 cm in size compared with wild-type plants, which under these conditions formed necrotic lesions of ∼2–3 mm in diameter (Figure 4A). In contrast, the HR in plants overproducing phytaspase (OE lines) resulted in the formation of more severe and sharply defined necrotic lesions compared with those in wild-type plants, although their sizes did not differ significantly (2–3 mm) (Figure 4A). These data indicate that deficiency of phytaspase markedly suppressed TMV-mediated HR, whereas its overproduction facilitated HR. The same conclusion was drawn from evaluation of expression of a plant gene, HSR203J that is an early marker of HR (Pontier et al, 1994) (Figure 4B). To ascertain whether the phenotypic effects observed on RNAi were due to specific silencing of the phytaspase gene, we performed a functional complementation assay: Agrobacterium-mediated expression of the heterologous wild-type rice phytaspase gene in the RNAi transgenic leaves 24 h before induction of HR by temperature shift (30–24°C) of leaves pre-infected with TMV at 30°C led to remarkable strengthening of HR (necrotisation of lesions and HSR203J expression) (Figure 4A and B). In contrast, expression of the inactive rice phytaspase (S535A mutant; Supplementary Figure 2) did not affect the HR phenotype of phytaspase-silenced plants, indicating specificity of RNA silencing (Figure 4A and B). Of note, no 21 nt regions of homology were found between any of the three RNAi constructs used for generation of transgenic plants and the rice phytaspase ORF. Figure 4.Effects of transgenic phytaspase deficiency or overproduction on PCD in tobacco (Samsun NN) plants induced by TMV. (A) TMV-mediated HR is suppressed in phytaspase-silenced (KD; line KD1-1) plants compared with control (wt) plants, resulting in the formation of less severe amorphous lesions some of which (shown by arrows) continue to grow. The HR in plants overproducing (OE; OE-1 line) phytaspase results in th
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