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The LCB2 subunit of the sphingolip biosynthesis enzyme serine palmitoyltransferase can function as an attenuator of the hypersensitive response and Bax-induced cell death Blackwell Publishing Ltd

Yunzhe Gan, Lisha Zhang, Zhengguang Zhang, Suomeng Dong, Jun Li, Yuanchao Wang and Xiaobo Zheng Department of Plant Pathology, Nanjing Agricultural University, Nanjing 210095, China

Summary Authors for correspondence: Zhengguang Zhang & Yuanchao Wang Tel: +86 25 84396972 Fax: +86 25 84395325 Email: [email protected]; [email protected] Received: 5 June 2008 Accepted: 15 August 2008 New Phytologist (2009) 181: 127–146 doi: 10.1111/j.1469-8137.2008.02642.x Key words: Agrobacterium-mediated transient expression, elicitor, hypersensitive response (HR), programmed cell death (PCD), virus-induced gene silencing (VIGS).

• Previous results showed that expression of the gene encoding the LONG-CHAIN BASE2 (LCB2) subunit of serine palmitoyltransferase (SPT), designated BcLCB2, from nonheading Chinese cabbage (Brassica campestris ssp. chinensis) was up-regulated during hypersensitive cell death (HCD) induced by the Phytophthora boehmeriae elicitor PB90. • Overexpression of BcLCB2 in Nicotiana tabacum leaves suppressed the HCD normally initiated by elicitors and PB90-triggered H2O2 accumulation. BcLCB2 also functioned as a suppressor of mouse Bcl-2 associated X (Bax) protein-mediated HCD and cell death caused by Ralstonia solanacearum. BcLCB2 overexpression suppressed Bax- and oxidant stress-triggered yeast cell death. Reactive oxygen species (ROS) accumulation induced by Bax was compromised in BcLCB2-overexpressing yeast cells. • The findings that NbLCB2 silencing in Nicotiana benthamiana enhanced elicitortriggered HCD, combined with the fact that myriocin, a potent inhibitor of SPT, had no effect on Bax-induced programmed cell death, suggested that suppression of cell death was not involved in the dominant-negative effect that resulted from BcLCB2 overexpression. A BcLCB2 mutant assay showed that the suppression was not involved in SPT activity. • The results suggest that plant HCD and stress-induced yeast cell death might share a common signal transduction pathway involving LCB2, and that LCB2 protects against cell death by inhibiting ROS accumulation, this inhibition being independent of SPT activity.

Introduction The hypersensitive response (HR), a defense response involving rapid localized cell death, allows plants to resist pathogen invasion and growth (Dangl & Jones, 2001; de Jong et al., 2004; Greenberg & Yao, 2004). Cell death associated with the HR is a genetically controlled and regulated process and is an example of programmed cell death (PCD) in plants (Greenberg, 1997). PCD is a hallmark of HR-based immunity in plants, and cell death phenotypes are often used in laboratory experiments to discover and dissect plant immune responses. Diverse chemical elicitors, including proteins, peptides, glycoproteins, lipids and oligosaccharides, trigger defense

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responses that are part of the basal (nonhost) resistance of plants, and are accompanied by hypersensitive cell death (HCD) (Mittler et al., 1997; Nürnberger, 1999). A number of key players involved in the HR have been identified (del Pozo et al., 2004; Gabriëls et al., 2006), and show that mammalian and plant cell death mechanisms share common morphological and biochemical features, including cytoplasm shrinkage, nuclear condensation, DNA laddering, and the release of cytochrome c from mitochondria (Sun et al., 1999; Sasabe et al., 2000; Kim et al., 2003; Ji et al., 2005). However, it is still unclear how signaling pathways lead to local HCD but not whole-plant cell death, and how death occurs.

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Phytopathogens contain a wide range of elicitors that are secreted into the cell wall, including polypeptides, proteins, and oligosaccharides, such as elicitins (Ponchet et al., 1999; Baillieul et al., 2003; Qutob et al., 2003), harpins (Wei et al., 1992), transglutaminases (Brunner et al., 2002), necrosis and ethylene-inducing peptide 1 (Nep1) (Gijzen & Nürnberger, 2006; Qutob et al., 2006), other proteins (Nürnberger et al., 1994; Villaba Mateos et al., 1997; Fellbrich et al., 2002; Torto et al., 2003), and hepta-β-glucoside (Umemoto et al., 1997; Mithöfer et al., 2000; Fliegmann et al., 2004). Most of these elicitors trigger plant defense responses in host and nonhost plants. Phytophthora boehmeriae, a phytopathogenic oomycete that causes cotton blight, does not infect the nonhost plants tobacco (Nicotiana tabacum) and nonheading Chinese cabbage (Brassica campestris ssp. chinensis). We previously used a P. boehmeriae culture filtrate to purify a proteinaceous elicitor, PB90, which has a molecular mass of 90 kDa (Wang et al., 2003). Immunogold labeling revealed that this elicitor localizes to the cell walls of the mycelium and encysting zoospores (Wang et al., 2003). Infiltration of nanomolar concentrations of PB90 into tobacco results in HCD and the activation of systemic acquired resistance (SAR) (Wang et al., 2003). The HR of plants shows similar morphological characteristics to mammalian apoptosis, including rapid chromatin condensation, margination, DNA laddering, and the specific breakage of the 3′-OH ends of DNA (Ji et al., 2005). Our studies strongly suggested that, in tobacco plants treated with PB90, salicylic acid (SA) mediates SAR, but not HR (Zhang et al., 2004). Significantly, PB90 can induce HCD and defense responses in leaves of the nonheading Chinese cabbage, an important vegetable in China, and a PB90-induced H2O2 burst triggers HCD and defense responses (Li et al., 2006). We used a suppression subtractive library-based approach to study HCD-associated signaling and to identify genes involved in HCD and defense responses. We isolated 70 expressed sequence tags (ESTs), classified into nine categories of genes up-regulated in the early induction/execution of the HR of nonheading Chinese cabbage induced by PB90 (Gan et al., 2008). Here, we further identified and characterized the BcLCB2 gene (encoding the LCB2 subunit), which is involved in the regulation of HCD during both immune and susceptible plant–pathogen interactions. Evidence is presented that BcLCB2 functions as a general cell death attenuator in both plants and yeast.

Materials and Methods Plant material, elicitor preparation, and strains Nicotiana tabacum L., Nicotiana benthamiana L. and nonheading Chinese cabbage (Brassica campestris ssp. chinensis Makino) plants were grown in environmentally controlled growth cabinets under a 16-h photoperiod at 25°C according to Li et al.

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(2006). All infiltration treatments with elicitor for N. tabacum and nonheading Chinese cabbage were performed at 10 wk after sowing as described previously (Zhang et al., 2004). PB90, a hypersensitive response extracellular protein elicitor with a molecular mass of 90 kDa, was purified from culture filtrates of P. boehmeriae JX-9 as described by Wang et al. (2003). Recombinant Magnaporthe grisea necrosis and ethyleneinducing peptide (MgNep) was produced in Escherichia coli strain BL21(DE3) by transformation with pET32a:MgNep as described previously (Qutob et al., 2002). Recombinant elicitin gene of Phytophthora infestans (INF1) was produced in E. coli strain BL21(DE3) as described previously (Kamoun et al., 1997, 1998). The bacterial expression of MgNep and protein concentrations were determined using the Bradford reagent (Qutob et al., 2006), and concentrated stock solutions were prepared. Recombinant harpinXoo (HrpXoo) was prepared as described previously (Lee et al., 2001). The amplification concentrations of MgNep1, INF1 and HrpXoo were 1 µM, 100 nM and 1 µM, respectively. Agrobacterium tumefaciens strain GV3101 was used for virus-induced gene silencing (VIGS) in N. benthamiana. GV2260 was used for Agrobacteriummediated transient expression in leaves. Ralstonia solanacearum was used for pathogen assays in N. benthamiana. Cloning of BcLCB2 and sequence analysis An EST clone (GA89) highly represented in a PB90-treated nonheading Chinese cabbage cDNA library shared high homology with the gene encoding the Arabidopsis thaliana LCB2 subunit of serine palmitoyltransferase (SPT), designated BcLCB2. The full-length BcLCB2 open reading frame (ORF) was obtained by 5′ rapid amplification of cDNA ends (Invitrogen, Beijing, China). Amplified PCR fragments were subcloned into the pMD19 T-vector (TaKaRa Biotechnology Co., Dalian, China), and sequenced using the dideoxy method of Sanger with a kit from TaKaRa Biotechnology Co. Amino acid sequence alignment was performed with the bioeditor software (San Diego Supercomputer Center, San Diego, CA, USA). phylip (Phylogeny Inference Package; http://evolution.genetics.washington.edu/phylip.html) was used to analyze protein phylogeny using the neighbor-joining method. The phylogenetic tree was prepared with mega3.0 (The Biodesign Institute, Tempe, AZ, USA). Plasmid constructs and Agrobacterium-mediated transient expression Agrobacterium-mediated transient expression was performed as described in Bos et al. (2006). The 35S:BcLCB2 construct was generated by inserting a fragment encompassing the complete BcLCB2 ORF, amplified by PCR from pMD19:BcLCB2 using the primers FL371 (5′-atgaattcATGATTACGATCCCATACC-3′; EcoRI site underlined) and FL565 (5′gccgaattcTTAATCCAATTTGATGCCAT-3′; EcoRI site

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underlined), into the EcoRI site in the pCJ-p19 binary vector under the control of the constitutive 35S CaMV promoter. The 35S:Bax construct was generated by inserting a fragment encompassing the complete Bax ORF, amplified by PCR from pGilda:Bax using the primers baxF (5′-ccatcgatATGGACGGGTCCGGGGAG-3′; ClaI site underlined) and baxR (5′-cggtcgacTCAGCCCATCTTCTTCCAG-3′; SalI site underlined), between the ClaI and SalI sites in the potato virus X (PVX) binary vector under control of the constitutive 35S CaMV promoter. After verification by sequencing, the constructs were introduced into A. tumefaciens strain GV2260. Overnight cultures were harvested by centrifugation, and the cells were resuspended in 10 mM MgCl2, 10 mM 2-(Nmorpholino) ethanesulfonic acid (MES), pH 5.6, and 150 µM acetosyringone to an optical density (OD600) of 1.0, incubated for 2 h at room temperature, and then infiltrated into N. benthamiana leaves using a 1-ml needleless syringe (Romeis et al., 2001). For controls and to test responses in the absence of BcLCB2 or Bax, A. tumefaciens strains carrying the appropriate empty vector were used. Cell death measurements in tobacco leaves Cell death was evaluated by (a) histochemical analysis using trypan blue and (b) measurement of ion leakage from leaf discs according to Li et al. (2006). For trypan blue staining, samples were covered with an alcoholic lactophenol trypan blue mixture (30 ml of ethanol, 10 g of phenol, 10 ml of water, 10 ml of glycerol, 10 ml of lactic acid, and 10 mg of trypan blue), placed in a boiling water bath for 2–3 min, left at room temperature for 1 h, transferred into a chloral hydrate solution (2.5 g ml–1), and boiled for 20 min to destain. After multiple changes of chloral hydrate solution to reduce the background, samples were equilibrated with 50% (w/v) glycerol, and photographed with a digital camera. For ion leakage measurement, five leaf discs (7 mm diameter) were floated abaxial side up on 5 ml of distilled water for 24 h at room temperature. After incubation, the conductivity of the bathing solution was measured with a conductivity meter (CyberScan 510; Eutech, Ayer Rajah Crescent, Singapore). DAB staining Histochemical detection of hydrogen peroxide in tobacco leaves was performed using 3,3′-diaminobenzidine·4HCl (DAB) staining with a procedure adapted from those of OrozcoCárdenas & Ryan (1999) and Thordal-Christensen et al. (1997). Briefly, leaves were harvested and immediately vacuum-infiltrated for 20 min with phosphate-buffered saline (PBS; pH 7.4) containing 0.5% (w/v) DAB. The leaves were placed under light for 10 h and then boiled for 20 min in 80% ethanol. The intensity and patterns of DAB staining were assessed visually.

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PVX construct and PVX-mediated gene silencing in N. benthamiana Total RNA from N. benthamiana leaves was reverse-transcribed using an oligo(dT) 20 primer (TaKaRa Biotechnology Co.). Using this cDNA as a template, a cDNA containing the entire NbLCB2 ORF was amplified by PCR with forward (NbLCB2ORFU: 5′-ATGATCACCATTCCATATTTGAC-3′) and reverse (NbLCB2-ORFR: 5′-TCATTCCAGCTTGACTCTGTTTT-3′) primers designed from the sequence of an LCB2 cDNA from N. tabacum (GenBank accession number AB264104). The PCR-amplified product (1470 bp) was inserted into the pMD19-T vector (TaKaRa Biotechnology Co.) to generate pMD19:NbLCB2. The NbLCB2 sequence was submitted to GenBank (accession number EU117119) after verification of its sequence. A portion of the NbLCB2 (positions 899–1202) cDNA amplified by reverse transcriptase–polymerase chain reaction (RT-PCR) with FL1106 (5′-gcggtcgacGCTGATGTGGACATTATGAT-3′; SalI site underlined) and FL1107 (5′gcggcggccgcATCACAGGAGAATCATTGTC-3′; NotI site underlined) using pMD19:NbLCB2 as the template was fused between the SalI and NotI sites of the PVX vector pgR106 in the antisense direction, generating PVX:NbLCB2. This construct was transferred into A. tumefaciens strain GV3101 by the freeze– thaw method. A single colony of A. tumefaciens GV3101 containing PVX:NbLCB2 in Luria-Bertani (LB) was incubated overnight at 28°C. The culture was centrifuged and resuspended in 10 mM MgCl2. Bacterial suspensions were infiltrated into the undersides of 3-wk-old N. benthamiana leaves using a 1-ml needleless syringe, with A. tumefaciens GV3101 harboring an empty binary vector serving as a negative control. RT-PCR For gene expression analysis, RT-PCR was carried out using gene-specific primer sets to amplify cDNA fragments, as described in Zhang et al. (2004): BcLCB2 (5′-CTTCTTCTTACCTAGGAGTG-3′; 5′-TCTCGCCAGTGCTTGAAGC-3′), NbLCB2 (5′-ATGATCACCATTCCATATTTGACC-3′; 5′-TCATTCCAGCTTGACTCTGTTTT-3′), BcActin (5′CAAGTCCTTCCTGATATCCAC-3′; 5′-ACCGAGAGATTCAGGTGCCC-3′), and N. benthamiana elongation factor 1 alpha (NbEF1α; 5′-AGACCACCAAGTACTACTGCAC-3′; 5′-CCACCAATCTTGTACACATCC-3′). Yeast cell death assays Saccharomyces cerevisiae strain W303 (ade2-1, can1-100, his3-11.15, leu2-3.112, trp1-1, ura3-1; Harris et al., 2000) containing the HIS3-marker plasmid pGilda:Bax (Kampranis et al., 2000), which expresses the mouse Bax under the control of the galactokinase 1 (GAL1) promoter when induced by galactose, was used. The expression vector was amplified by PCR using the BcLCB2 forward and reverse primers FL371

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and FL565, which introduce an EcoRI restriction site. The amplified product was digested and inserted into the EcoRI site of the URA3-marker vector pYES2 (Invitrogen), giving rise to pYES2:BcLCB2, which expresses BcLCB2 under the control of the GAL1 promoter. Plasmid DNA transformations were performed using the lithium acetate method (Schiestl & Gietz, 1989). Transformants harboring pGilda:Bax/pYES2 or pGilda:Bax/pYES2:BcLCB2 were then streaked on SDHis-Ura (glucose) plates or SG-His-Ura (galactose) plates, and the plates were incubated at 30°C for 3 d. Saccharomyces cerevisiae strain W303 was obtained from Clontech (Palo Alto, CA, USA), and the growth, transformation, and gene expression analysis in this strain were essentially as described by Kampranis et al. (2000). W303 cells were grown in yeast extract-peptone-dextrose (YPD) medium containing 1% yeast extract, 2% Difco peptone, and 2% glucose. BcLCB2 was inserted behind a galactose-inducible promoter in the yeast expression vector pYES2, and the plasmid was transformed into W303 after verification by DNA sequencing. Cells were grown in synthetic dropout (SD) medium with 2% glucose lacking uracil (SD-Ura) to select for the presence of the plasmid. W303 cells containing BcLCB2 were grown overnight in SDUra. The cells were pelleted, washed, and resuspended in SD medium containing 2% galactose and 1% raffinose as carbon sources (SD/gal/raff/-Ura) to induce the expression of the fusion protein from the GAL1 promoter. After 10 h of induction, the cells were diluted to OD600 = 0.05 and treated in one of the following ways. For chemical treatments, H2O2 or tertbutylhydroperoxide (t-BHP) was added to the medium at selected final concentrations and the cultures were incubated at 30°C with vigorous shaking for 6 h. For heat stress, the cells were incubated at 37°C for 30 min with vigorous shaking, transferred to a water bath at 51°C for 25 min, and then incubated at 30°C with vigorous shaking for 6 h. Following these treatments, viability was determined by plate counting. Treated and untreated cells were sampled and spread onto YPD medium solidified with 2% agar, and then incubated at 30°C for 48 h. The number of colony-forming units (CFU) from treated cells (both W303 and W303 carrying BcLCB2) was compared with that from untreated cells. All experiments were repeated in triplicate. BcLCB2 expression assay in yeast cells and SPT assay The BcLCB2 protein expression assay was carried out in the W303 yeast host as described previously (Li et al., 2007). To determine whether or not BcLCB2 was expressed in yeast, the yeast pYES2:BcLCB2 transformant and control yeast pYES2 transformant were grown in liquid SD-Ura medium containing 2% glucose for 24 h at 30°C, and cells were precipitated, washed, and grown in SD-Ura medium containing 2% galactose and 1% raffinose overnight in a 30°C shaker (225 rpm). To extract proteins, cells were collected, suspended in lysis buffer (PBS, pH 7.4, 1% Nonidet P-40, and a cocktail of protease inhibitors; Roche, Indianapolis, IN, USA), mixed with an

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equal volume of acid-washed glass beads (diameter 400 µm; Sigma, St Louis, MO, USA), and homogenized in a Fast-Prep cell disrupter (Bio101, Carlsbad, CA, USA) four times for 40 s at 4°C. Homogenized samples were centrifuged at 12 000 g for 30 min at 4°C, and supernatants were recovered. Proteins were then concentrated through a centrifugal filter (Millipore, Bedford, MA, USA), measured using a BCA protein assay kit (Pierce, Rockford, IL, USA), and verified by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blotting using the anti-His antibody (Invitrogen). SPT was assayed using yeast microsomal protein as described previously (Gable et al., 2000). Background incorporation of L-[γ-3H]Ser was measured without the addition of palmitoylCoA and subtracted. BcLCB2 conserved motif deletion mutant and site-directed mutagenesis To mutate the putative BcLCB2 pyridoxal phosphate binding site, we constructed the plasmid pYES2:BcLCB2ΔK311E (containing the BcLCB2ΔK311E mutation) via overlap PCR. The PCR product amplified with primers FL371 and FL716 (5′-GCCACCACAAGATCCGAAAGATTCGGTAAAAGTCCCCATCATAAT-3′) was mixed at an equal molecular ratio with the fragment amplified with primers FL715 (5′ATTATGATGGGGACTTTTACCGAATCTTTCGGATC TTGTGGTGGC-3′) and FL565. Overlapping PCR with this mixture as template was used to amplify the BcLCB2 fragment containing the expected point mutation of Lys(AAA) to Glu(GAA) with primers FL371 and FL565. All PCR reactions were performed with the Pfu DNA polymerase (TaKaRa Biotechnology Co.). PCR products amplified by FL371 and FL565 were digested with EcoRI and cloned into the EcoRI site of vector pYES2 to generate pYES2: BcLCB2ΔK311E. This vector was sequenced to confirm the point mutation and direction of BcLCB2ΔK311E. To generate mutant BcLCB2 with a deletion of residues 307–314 (BcLCB2Δ307–314), the same strategy as used for the site-directed mutagenesis of BcLCB2ΔK311E was adopted to construct plasmid pYES2:BcLCB2Δ307–314 via overlap PCR using primers FL714 (5′-GGCAATATAGCCACCACAAGACATCATAATGTCCACATCAGC-3′)/FL371 and FL713 (5′-GCTGATGTGGACATTATGATGTCTTGTGGTGGCTATATTGCC-3′)/FL565. The overlap PCR product was amplified with primers FL371 and FL565, cloned into the vector pYES2, generating the expression construct pYES2:BcLCB2Δ307–314. This vector was sequenced to confirm the deleted mutation and direction of BcLCB2Δ307–314. Reaction oxygen species (ROS) detection in yeast cells Transformed yeast cells were harvested from the early to middle exponential growth phases using a brief centrifugation,

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washed three times with distilled water, resuspended in either SD-His-Ura (glucose) or SD-His-Ura (galactose) liquid medium, and cultured at 30°C for 6 h. The cell suspensions were washed briefly and diluted to 1 ml at OD600 = 1.0. To test for the presence of ROS, yeast cells were washed three times in ddH2O, incubated with 5 g ml–1 dihydrorhodamine (DHR; Sigma) for 2 h at 25°C on a rotary shaker (150 rpm), and then rinsed three times with fresh suspension buffer (10 mM Tris-HCl, pH 7.4) to remove excess fluorophore, according to Fahrenkrog et al. (2004). Cells were observed under a Leica DM R microscope (Leica Microsystems, Wetzlar, Germany). TUNEL assay The terminal transferase dUTP nick end-labeling (TUNEL) assay was performed according to Gavrieli et al. (1992) using the in situ cell death detection fluorescein kit (1684795; Roche Diagnostics, Basel, Switzerland). One million cells were washed twice with 1 ml PBS, fixed in 4% (w/v) paraformaldehyde for 30 min on ice, washed twice more, resuspended in 100 µl of permeabilization solution (0.1% Triton X-100 (Sigma) in 0.1% trisodium citrate dihydrate (Sigma)), incubated on ice for 2 min, and then washed twice with PBS. After addition of the TUNEL mixture (terminal deoxy nucleotidyl transferase (TdT):fluorescein-deoxy uridine triphosphate (fluoresceindUTP) = 1 : 9), the cells in suspension were incubated for 60 min at 37°C, washed three times in PBS, and finally suspended in mounting medium containing 4′,6′-diaminido2-phenylindole (DAPI) (Quesney et al., 2001; Marcon & Boissonneault, 2004).

Results Cloning and sequence analysis of BcLCB2 Using a suppression subtractive library-based approach, we identified 70 ESTs representing genes (GenBank accession numbers EB041701–EB041723 and EB041725–EB041771) up-regulated in nonheading Chinese cabbage exhibiting an early response to the elicitor PB90. Of these, the sequence of the EST GA89 (GenBank accession number EB041714) was very similar to the sequence encoding the C-terminal domain of the LCB2 subunit of A. thaliana, AtLCB2, a subunit of SPT (Gan et al., 2008). The sequence of the full-length LCB2 cDNA was obtained using a SMART rapid amplification of cDNA ends (RACE) amplification kit (BD Bioscience-Clontech, Palo Alto, CA, USA). Three clones of the full-length cDNA, named BcLCB2, were sequenced to confirm that the correct gene had been cloned. DNA sequencing revealed that all three clones contained a complete ORF within a 1470-bp cDNA (GenBank accession number EU117118) that encodes a 53.83-kDa protein of 489 amino acid residues with a predicted isoelectric point (pI) of 8.77. The predicted BcLCB2

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protein contains a highly conserved motif, 307GTFTKSFG314 (Fig. 1a), which suggests its involvement in pyridoxal phosphate binding (Chen et al., 2006). Analysis of the deduced amino acid sequence of BcLCB2 indicated that the protein shares a high level of similarity with other LCB2 proteins, as well as with human LCB2 (Fig. 1b). For example, BcLCB2 shares 90% and 86% identity with AtLCB2 and NtLCB2, respectively, and is 43–50% identical to HsLCB2 and ScLCB2 (Fig. 1b). Phylogenetic analysis revealed that the plant sequences are distant from animal and yeast sequences. The plant sequences were grouped together and subdivided into monocotyledonous and dicotyledonous species, with the latter further divided according to family (Solanaceae and Brassicaceae). This phylogenetic tree thus closely mirrors accepted evolutionary classifications. The presence of the BcLCB2 transcript over a 60-h period during the interaction between plants and PB90 was examined. RT-PCR revealed that BcLCB2 transcription was rapidly and strongly induced in leaf tissues treated with PB90, with a peak at 24 h (Fig. 2a), as compared with controls treated with water. BcLCB2 expression was detected by RT-PCR in all nonheading Chinese cabbage organs: young leaves, stems, roots, and flowers (Fig. 2b). Consistent with our results for BcLCB2, AtLCB1 (At4g36840) (Chen et al., 2006) and AtLCB2 (At5g23670) (Tamura et al., 2001; Zimmermann et al., 2004) were also found to be expressed ubiquitously in all organs of A. thaliana examined. However, the transcript level was higher in flowers and stems than in leaves and roots (Fig. 2b). BcLCB2 suppresses the elicitor- and Bax-induced HR in N. tabacum leaves and cell death during compatible plant–pathogen interactions Some plant proteins, such as Bax inhibitor 1 (BI-1) (Kawai et al., 1999; Kawai-Yamada et al., 2001, 2004; Bolduc et al., 2003; Matsumura et al., 2003; Watanabe & Lam, 2006), HEAT SHOCK PROTEIN 90 (HSP90) (Kanzaki et al., 2003; Lu et al., 2003), and ethylene-responsive element binding protein (AtEBP) (Pan et al., 2001; Ogawa et al., 2005), can inhibit Bax- or abiotic stress-induced PCD in yeast and/or plants. We found that the expression of BcLCB2, which encodes a subunit of SPT, was up-regulated in nonheading Chinese cabbage in the early response to the elicitor PB90 (Fig. 2a). SPT catalyzes the condensation of serine with palmitoylCoA to form 3-ketosphinganine in the first step of de novo biosynthesis of sphingolipid, which is involved in apoptosis and defense (Hannun & Luberto, 2000; Perry et al., 2000; Ng et al., 2001). These results suggest that BcLCB2 is involved in the PB90-induced HR. To test this hypothesis, we infiltrated A. tumefaciens carrying the plasmid p19:BcLCB2 or p19 (empty-vector control) into N. tabacum leaves to overexpress BcLCB2 using Agrobacterium-mediated transient assays (agroinfiltration; van de Ackerveken et al., 1996). After 6 h, the sites were infiltrated with 10 nM PB90, and cell death

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Fig. 1 Brassica campestris BcLCB2 amino acid sequence phylogeny and properties. (a) Comparison of deduced amino acid sequences of LONG-CHAIN BASE 2 subunit (LCB2) from tobacco (Nicotiana tabacum; NtLCB2; accession number BAE96963), rice (Oryza sativa; OsLCB2; EAY81086), Arabidopsis thaliana (AtLCB2; NP 197756), yeast (ScLCB2; CAA98880), and humans (HsLCB2, BAA25452). Numbers on the left indicate amino acid positions. Residues identical to those in BcLCB2 are shaded in black. Hyphens indicate gaps introduced to maximize the alignment. The motif of a putative pyridoxal 5′-phosphate binding site (GTFTKSFG) is underlined, and an asterisk indicates the lysine residue that forms a Schiff base with pyridoxal 5′-phosphate. (b) Phylogenetic tree of sequences of LCB2 proteins from various organisms, constructed using the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) method using MEGA3.0. Sequences were obtained from GenBank. The scale below the figure represents amino acid substitutions, and branches lengths are drawn to scale.

symptoms were observed and scored 1–5 d later. Surprisingly, BcLCB2 suppressed the HR induced by PB90, whereas the vector construct or MgCl2 solution did not. The cell death suppression was stable and was observed for up to a week after infiltration with PB90 (Fig. 3a). Cell death was further investigated in situ using trypan blue, which accumulates in dead cells. The application of 10 nM PB90 induced blue staining that was localized to treated tissues, whereas BcLCB2pretreated tissue infiltrated with PB90 remained unstained, with a negligible number of blue spots (Fig. 3b). No difference was observed between tissues pretreated with the empty vector and those pretreated with the MgCl2 solution. Clear electrolyte leakage was also observed in leaves infiltrated

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with PB90 (PB90) and A. tumefaciens containing the empty vector (PB90 + p19), but was not observed in leaves infiltrated with PB90 and A. tumefaciens containing p19:BcLCB2 (PB90 + BcLCB2) (Fig. 3c). Tobacco leaves infiltrated with A. tumefaciens containing the empty vector (p19) or p19:BcLCB2 alone did not show clear ion leakage (data not shown). Together, these data effectively demonstrate that the overexpression of BcLCB2 in tobacco leaves suppressed cell death triggered by PB90. We further tested whether BcLCB2 can suppress cell death by using INF1 from Phytophthora infestans, MgNep1 from Magnaporthe grisea (MGG_08454.5), and harpinXoo from Xanthomonas oryzae pv. oryzicola (GenBank accession number

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Fig. 2 Expression of Brassica campestris BcLCB2 in nonheading Chinese cabbage (Brassica campestris). (a) Up-regulation of BcLCB2 in nonheading Chinese cabbage treated with PB90. Reverse transcriptase–polymerase chain reaction (RT-PCR) was performed with RNA isolated from PB90- and water-treated leaves, respectively. (b) RT-PCR analysis of BcLCB2 expression in leaves, stems, flowers, and roots. Primers used were specific to BcLCB2, and, as an endogenous control using the same total RNAs, to the actin gene in nonheading Chinese cabbage. PCR products (33 cycles for BcLCB2 and 28 cycles for actin) were separated on an agarose gel and stained with ethidium bromide. Three independent replicates provided the same results. CK, control water treatment.

AY875714) (Zou et al., 2006). INF1 is an elicitor of the HR in N. benthamiana (Kamoun et al., 1997, 1998), and Nep1 can trigger HRs in many dicots (Gijzen & Nürnberger, 2006; Qutob et al., 2006). Escherichia coli expressing MgNep1 can trigger the HR in tobacco, soybean (Glycine max), cotton (Gossypium hirsutum), A. thaliana, and cucumber (Cucumis sativus) (W. Li et al., unpublished), and harpinXoo can induce the HR in tobacco (Zou et al., 2006). We overexpressed BcLCB2 in N. tabacum leaves 6 h before infiltrating elicitors into the agroinfiltration sites. As expected, the HR induced by infiltrating these elicitors into the BcLCB2 transient overexpression area was suppressed, even after 36 h (Fig. 4a). However, these elicitors did trigger HCD upon agroinfiltration with empty vector controls. The mouse protein Bax is a member of the B cell lymphoma/ leukemia-2 (Bcl-2) family of pro-apoptotic proteins and triggers PCD by disrupting mitochondria and causing the release of cytochrome c and other pro-apoptotic factors (Jürgensmeier et al., 1998). Expression of the Bax protein initiates cell death in yeast and rapid cell death in plants in a process closely resembling the HR (Lacomme & Cruz, 1999;

Fig. 3 Suppression of the PB90-triggered hypersensitive response (HR) in tobacco (Nicotiana tabacum cv. Xanthi nc.) leaves overexpressing BcLCB2. Transient overexpression of BcLCB2 via agroinfiltration suppressed the PB90-induced HR in N. tabacum leaves. The Agrobacterium tumefaciens strain GV2260 containing p19:BcLCB2 was infiltrated into leaves 6 h before treatment with PB90. (a) The PB90-induced HR was observed from 36 h post-infiltration in infiltration sites expressing the empty vector but was suppressed in BcLCB2-overexpressing sites. The fraction underneath each white word indicates the number of times the results shown were obtained over the number of times the experiments was performed. (b) Leaves were sampled and analyzed for cell death using trypan blue staining 12 h after infiltration with PB90. The experiments were performed in triplicate. (c) Overexpression of BcLCB2 in tobacco leaves suppressed PB90-induced ion leakage. The experiments were performed in six independent replicates. Error bars show the standard deviation of the mean for six replicates. Asterisks indicate a significant difference between conductivity in control leaves (treated with PB90 and empty vector) and that in BcLCB2-overexpressing leaves treated with PB90 at P = 0.01 according to Duncan’s range test.

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Fig. 4 Suppression of the hypersensitive response (HR) in tobacco induced by various elicitors and Bcl-2 associated X protein (Bax). (a) Transient overexpression of BcLCB2 using agroinfiltration suppressed the HR induced by various elicitors (including elicitin of Phytophthora infestans (INF1), Magnaporthe grisea necrosis and ethylene-inducing peptide 1 (MgNep1) and harpinXoo). Leaves were photographed 36 h postinfiltration with elicitors. The fraction in white indicates the number of times the results shown were obtained over the number of times the experiment was performed. (b) BcLCB2 suppresses cell death initiated by Bax. Agrobacterium tumefaciens strain GV2260 carrying binary vectors encoding Bax or BcLCB2 was co-infiltrated into Nicotiana benthamiana leaves. Leaves were photographed after 7 d. The experiment was performed at least five times, and representative photographs are shown. (c) BcLCB2 plays a role in the development of bacterial disease symptoms. Nicotiana tabacum plants overexpressing BcLCB2 or containing only the empty vector were infiltrated with 5 × 105 colony-forming units (CFU) ml–1 of Ralstonia solanacearum at 10 d after infection. Disease symptoms in the infiltrated area pretreated with A. tumefaciens containing p19 are shown in 1 and 2, and disease symptoms that only occurred in the infiltrated area pretreated with A. tumefaciens containing p19:BcLCB2 are shown in 3 and 4. The leaf on the right is the same leaf as shown in the left panel after bleaching in ethanol. The experiment was performed with three plants, with two leaves inoculated per plant. The dark colour indicates the infiltrated area.

Kawai-Yamada et al., 2001), suggesting that a common PCDactivating mechanism exists across kingdoms. Here, transient overexpression of the mouse Bax under control of the CaMV 35S promoter triggered the HR-like response in N. tabacum. However, infiltration of BcLCB2 into N. tabacum leaves for 6 h, using Agrobacterium-mediated transient expression, before infiltration of A. tumefaciens harboring 35S:Bax into the agroinfiltration regions suppressed the Bax-triggered HR-like response (Fig. 4b). To test the hypothesis that BcLCB2 also plays a role in disease-associated cell death, we overexpressed BcLCB2 in N. tabacum and inoculated the leaves with a disease-causing titer (5 × 105 CFU ml–1) of the virulent pathogen R. solanacearum by infiltration using a 1-ml needleless syringe (Fig. 4c). No disease symptoms were observed in any BcLCB2overexpressing sites at 10 d after R. solanacearum inoculation, whereas disease-associated cell death developed in all nonBcLCB2-overexpressing sites (Fig. 4c). The ability of BcLCB2 to broadly suppress PCD triggered by various elicitors from fungi, oomycetes, and bacteria, as well as the pro-apoptotic mouse protein Bax, suggests that BcLCB2 functions as a general attenuator of PCD in tobacco.

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NbLCB2-silenced plants are more sensitive to elicitors The activity of the wild-type gene disrupted by overexpression of mutant polypeptides often leads to dominant-negative effects (Herskowitz, 1987; Veitia, 2007). If the overexpression of BcLCB2 causes the dominant-negative effect, the elicitortriggered HRs should be suppressed in NbLCB2-silenced N. benthamiana plants. Here, we used VIGS to determine whether the effect was involved in the suppression of HR. A cDNA encoding NbLCB2 (GenBank accession number EU117119) was isolated from N. benthamiana according to N. tabacum LCB2 cDNA (GenBank accession number AB264104). A partial cDNA fragment was inserted into a PVX vector (PVX:NbLCB2) in the antisense direction and used for VIGS of NbLCB2. As expected, VIGS of NbLCB2, verified by RT-PCR 4 wk post pretreatment with PVX:NbLCB2 by agroinfiltration (Fig. 5a), enhanced several elicitor-triggered HRs in N. benthamiana leaves pretreated with PVX:NbLCB2, but neither the empty vector PVX nor MgCl2 produced any difference in the elicitor-triggered HR of plants (Fig. 5b,c). Ethanol bleaching has been used as a convenient assay to enhance the visualization of the HR in

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Fig. 5 The elicitor-triggered hypersensitive response (HR) is accelerated by virus-induced gene silencing (VIGS) in NbLCB2-silenced plants. (a) Reverse transcriptase–polymerase chain reaction (RT-PCR) results show that the expression of NbLCB2 is silenced in Nicotiana benthamiana plants 3 wk following inoculation with an Agrobacterium tumefaciens culture harboring PVX:NbLCB2. Total RNA was isolated from the third leaves above the inoculation site 4 wk post-agroinfitration and used for RT-PCR as described by Zhang et al. (2004). Primers used were specific to NtLCB2, and, as an endogenous control using the same total RNAs, to elongation factor 1α (EF1α). PCR products (35 cycles for NbLCB2 and 28 cycles for EF1α) were separated on an agarose gel and stained with ethidium bromide. Equal input of cDNA template for PCR was demonstrated by amplification of the constitutively expressed EF1α gene. (b) After the establishment of NbLCB2 silencing, leaves of control (ck, first row; PVX-inoculated, second row) and NbLCB2-silenced (PVX:NbLCB2-inoculated, third row) plants were infiltrated with the Magnaporthe grisea elicitor ethylene-inducing peptide (MgNep; left), elicitin of Phytophthora infestans (INF1) (middle), or Xanthomonas oryzae pv. oryzae harpin (right). In PVX-infected and PVX:NbLCB2-infected leaves, HR developed 48 h following treatment with elicitors. This elicitor-triggered HR was stronger in Nicotiana benthamiana plants with VIGS caused by NbLCB2 than in plants infected with PVX. (c) Leaves were removed from various plants 48 h after treatment and bleached in ethanol. The bottom panels show the leaves in the top panels following bleaching in ethanol. The dashed black circle indicates the inoculated region, which is more transparent and flattened than the rest of the leaf. This experiment was performed four times. Representative results from one of these experiments are shown in (a) to (c).

leaves responding to an avirulent pathogen (Schornack et al., 2004; Weber et al., 2005). The HR was observed earlier in NbLCB2-silenced leaves treated with elicitors than in nonsilenced NbLCB2 leaves (in a different set of plants), as observed after ethanol bleaching (Fig. 5c). Southern blotting showed that NbLCB2 may be a single copy gene in the N. benthamiana genome (see Supporting Information Fig. S1a,b). Also, NbLCB2 silencing did not result in any alterations in growth and development in plants (data not shown). Collectively, these results suggest that the silencing of NbLCB2 accelerates elicitor-triggered cell death and the dominant-negative effect is not involved in suppression of cell death by overexpression of BcLCB2. Overexpression of BcLCB2 inhibits the elicitor-triggered H2O2 burst in tobacco leaves The accumulation of ROS is characteristic of the HR in plant tissues and functions as a second signal mediating plant PCD during HR (Lamb & Dixon, 1997; Alvarez et al., 1998; Gechev & Hille, 2005; Li et al., 2006). We analyzed PB90-

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triggered H2O2 burst in leaves overexpressing BcLCB2. On DAB staining, numerous irregular dark-brown patches were visible after pre-infiltration of plants with MgCl2 or the empty vector p19 6 h after infiltration with PB90. However, less dark-brown precipitate was visible after pre-infiltration of plants with p19:BcLCB2, suggesting that the overexpression of BcLCB2 significantly suppressed the accumulation of H2O2 induced by PB90 (Fig. 6a,b). A similar observation was made when INF1 was infiltrated into leaves after pre-infiltration with p19:BcLCB2 (data not shown). The data strongly suggest that the overexpression of BcLCB2 suppressed the elicitortriggered H2O2 burst in leaves. BcLCB2 suppresses PCD in yeast In S. cerevisiae, PCD induced by mammalian pro-apoptotic factors, such as Bax, exhibits many of the hallmarks of metazoan apoptosis, including cytochrome c release, DNA fragmentation, and chromatin condensation (Madeo et al., 2002). To test whether BcLCB2 could rescue yeast from Bax-induced PCD, Bax-expressing yeast cells were transformed

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Fig. 6 Transient overexpression of BcLCB2 inhibits H2O2 accumulation in tobacco (Nicotiana tabacum) cv. Xanthi nc. triggered by PB90. The Agrobacterium tumefaciens strain GV2260 containing p19:BcLCB2 was infiltrated into leaves before a 6-h treatment with PB90. (a) Leaves were sampled and analyzed for H2O2 accumulation by 3,3′-diaminobenzidine·4HCl (DAB) staining at 6 h after infiltration with PB90. The stained leaves were then boiled for 20 min in 80% ethanol to remove chlorophyll, and H2O2 production was observed with a light microscope. (b) magnified images of (a). The experiment was performed six times, and representative photographs are presented.

with pYES2 expressing BcLCB2 under the control of the strong galactose-responsive GAL1 promoter. As a control, yeast was transformed with the empty vector pYES2. Overnight cultures of three independent transformants of each strain were serially diluted and spotted on galactose (SG-His-Ura) or control glucose (SD-His-Ura) plates. Although all strains grew on the glucose plates, yeast pYES2:BcLCB2 transformants grew well but vector-transformed yeast failed to grow on the SG-His-Ura plates (Fig. 7a). We further investigated whether BcLCB2 could be expressed stably in wild-type W303 S. cerevisiae cells. Immunoblot analysis revealed that the BcLCB2 protein was stably expressed in yeast cells and that it could be detected with anti-His antibody to the yeast His6-BcLCB2 fusion protein (Fig. 7b). The results exclude the possibility that the degradation of BcLCB2 contributes to the suppression of Bax-induced PCD in yeast. To further explore whether BcLCB2 has anti-apoptotic activity, Bax-expressing and Bax/BcLCB2-co-expressing yeast cells were analyzed for apoptotic hallmarks, such as chromatin condensation and fragmentation, DNA strand breaks, and the accumulation of ROS. Cells were grown to mid-log phase, and apoptosis was induced by incubation in galactose medium for 6 h. The TUNEL assay and DAPI staining were then used to visualize DNA fragmentation and chromatin condensation, respectively. The DNA in Bax-expressing cells was strongly condensed and TUNEL-positive within the nucleus, similar to the DNA in DNase I-treated cells, whereas in Bax/BcLCB2-co-expressing cells, the DNA was evenly distributed within the nucleus and had a weak TUNELnegative phenotype (Fig. 8). To rule out the dominant-negative effect, we used myriocin, a potent inhibitor of SPT (Hanada et al., 2000b; He et al., 2004), to assess the contribution of SPT activity to Bax-induced yeast apoptosis. Myriocin had no effect on Bax-induced cell death (Fig. 9), suggesting that SPT activity was not involved

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in the Bax-induced apoptosis and overexpression of BcLCB2 in yeast cells, and did not cause a ‘dominant-negative effect’. We further examined the protective capacity of BcLCB2 by examining cell death initiated by oxidative, salt, and heat stresses, according to the method of Abramovitch et al. (2003). BcLCB2 was expressed in the yeast strain W303 and the yeast cells were treated with the oxidants t-BHP (0.75 mM) and H2O2 (15 mM). The yeast cells expressing BcLCB2 were markedly resistant to cell death induced by H2O2 and t-BHP (Fig. 10), with > 80% and > 50% of the survival rates of yeast harboring the pYES2 empty vector, respectively (Fig. 10b). However, BcLCB2 did not suppress salt- or heat shock-induced cell death (data not shown). The capacity of BcLCB2 to suppress cell death in plants and protect yeast from oxidative-triggered cell death clearly establishes BcLCB2 as a eukaryotic cell death inhibitor. BcLCB2 suppresses Bax-triggered ROS accumulation The accumulation of ROS is a critical step in many types of apoptosis (Zha et al., 1996; Gechev & Hille, 2005), although, in certain systems, apoptosis may occur in the absence of ROS (Glazener et al., 1996; Tada et al., 2004). In yeast, ROS generation plays a key role in Bax-induced apoptosis (Mange et al., 2002). We therefore investigated whether the ectopic expression of BcLCB2 interfered with the PCD process before or after ROS generation. Yeast cells can be tested for ROS production by incubation with dihydrorhodamine 123 (DHR), which, in the presence of ROS, is oxidized to become fluorochrome rhodamine 123 (Schulz et al., 1996). Yeast cells were transformed with Bax alone or with Bax and BcLCB2, grown in glucose-based medium to mid-log phase, and then transferred to galactose-based medium. ROS accumulation was examined 6 and 12 h after changing the medium. Most yeast cells co-expressing Bax and BcLCB2 showed no fluorescence, whereas > 70% of the yeast cells expressing Bax

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Fig. 7 BcLCB2 inhibits the programmed cell death (PCD) initiated by Bcl-2 associated X protein (Bax) in yeast (Saccharomyces cerevisiae). (a) The yeast strain W303 expressing Bax under the control of a galactose-inducible promoter (in the vector pGilda) was transformed with the empty pYES2 vector, the pYES2:BcLCB2 construct encoding BcLCB2, or the pYES2 construct encoding the indicated deletion. Serial dilutions of cultures of three independent transformants (a–c) were grown overnight in SD-His-Ura liquid medium and then diluted to equal densities. Six-fold serial dilutions of each culture were spotted (left to right) onto SD-His-Ura (glucose) or SG-His-Ura (galactose) plates, grown at 30°C for 4 d, and photographed. The triangles above the image respresent the decreases in cell numbers for cells plated in each spot. The experiment was performed at least five times, and representative photographs are presented. (b) Western blot analysis of BcLCB2 protein in yeast cells. Transformant only containing the pYES2:BcLCB2 construct was grown to log phase in SD-Ura liquid medium and was then transferred to SG-Ura medium (containing 2% galactose and 1% raffinose) for 18 h to induce BcLCB2 expression. Equal amounts of protein extract were subjected to western blotting using anti-His antibody. Lanes 303, pYES2 and BcLCB2 show an extract from yeast strain W303, one transformant containing the empty vector pYES2 and one transformant carrying the pYES2:BcLCB2 construct, respectively. The molecular size marker is in kilodaltons.

or harboring only the pYES2 empty vector showed intense intracellular fluorescence, as determined by DHR staining and integration of the signal intensity of the digital image within individual cells (Fig. 11). This suggests that the BcLCB2-suppressed Bax-induced ROS accumulation resulted in inhibition of the cell death initiated by Bax. BcLCB2 does not require the conserved motif and SPT activity to suppress Bax-induced cell death The BcLCB2 sequence contains a highly conserved motif, 307GTFTKSFG314, which is associated with pyridoxal phosphate binding (Tamura et al., 2001). To evaluate the involvement of this motif in anti-cell death activities, we generated a mutant BcLCB2Δ307–314 in which the motif was deleted and another BcLCB2ΔK311E containing a directed mutation in the Lys codon of the pyridoxal phosphate binding site. The constructs pYES2:BcLCB2Δ307–314 and pYES2:BcLCB2ΔK311E were transformed into yeast possessing galactose-induced mammalian

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Bax for expression of the BcLCB2 mutant proteins. Like the full-length BcLCB2, both of the above BcLCB2 mutants rescued Bax-initiated cell death in yeast (Figs 8 and 12). We further evaluated the involvement of the pyridoxal phosphate binding motif in the suppression of ROS accumulation. Both BcLCB2Δ307–314 and BcLCB2ΔK311E still suppressed the Bax-induced ROS burst (Fig. 11). Similarly, overexpression of the BcLCB2 mutants also suppressed elicitor-triggered PCD (data not shown). SPT functions as a heteromeric enzyme that requires both LCB1 and LCB2 activity in yeast (Gable et al., 2000), Arabidopsis thaliana (Chen et al., 2006) and mammalian cells (Hanada et al., 2000a,b). However, AtLCB2 (accession number AB025633) did not rescue lcb2Δ mutant S. cerevisiae cells, although it had low levels of SPT activity when expressed in yeast (Tamura et al., 2001). We wondered whether the BcLCB2 subunit may homodimerize to form a functional SPT enzyme, resulting in suppression of yeast PCD. To this end, a functional assay based on yeast complementation and measurement of SPT activity was used. A pYES2:BcLCB2 construct carrying full-length BcLCB2 was transformed into lcb2ΔKAN mutant S. cerevisiae cells (6028, mating type alpha (Matα), His3, Leu2, Ura3, Lys2). Consistent with the expression of BcLCB2 in the wild-type yeast stain W303 (Fig. 7b), immunoblot analysis revealed that BcLCB2 protein was stably expressed and that it could be detected with polyclonal antiHis antibody to the yeast BcLCB2-His fusion protein in lcb2ΔKAN mutant S. cerevisiae cells (data not shown). Yeast cells lacking LCB2 require exogenous long-chain bases (such as phytosphingosine) for growth. However, BcLCB2 did not efficiently rescue the lcb2ΔKAN mutant S. cerevisiae cells, which were unable to grow without exogenous phytosphingosine (Fig. 13), consistent with the results of other workers who found that AtLCB2 cDNA did not complement the lcb2Δ mutant phenotype of S. cerevisiae cells (Nagice et al., 1996; Tamura et al., 2001), indicating that it did not restore SPT activity. We further measured SPT activity in isolated microsomes from the lcb2Δ mutant S. cerevisiae cells to determine whether BcLCB2 alone had SPT activity. Similar to the results of other workers (Tamura et al., 2001; Chen et al., 2006), the BcLCB2 expressed in the lcb2ΔKAN mutant S. cerevisiae cells had very low SPT activity of c. 7.34 ± 0.58 pmol min–1 mg–1 protein, which was significantly lower than that of the wild-type strain (DHY4a, Ura3, His3, Leu2, Met15, Lys2; Chen et al., 2006) (138 ± 4.58 pmol min–1 mg–1 protein). Both BcLCB2Δ307–314 and BcLCB2ΔK311E mutative proteins can also be stably expressed in W303, but the BcLCB2 mutant protein expressed in the lcb2Δ mutant had no detectable SPT activity. Because the yeast, mammalian and A. thaliana SPT enzymes are heterodimers of the LCB1 and LCB2 subunits (Hanada & Nishijima, 2003; Chen et al., 2006; Diretrich et al., 2008), we further investigated whether co-expression of BcLCB2 and BcLCB1 would rescue the SPT activity of the yeast mutants to confirm whether BcLCB2 encodes a bona

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Fig. 8 Overexpression of BcLCB2 and BcLCB2 mutant genes prevents apoptosis-like cell death by in situ detection of DNA cleavage. The top panels are images obtained by normal light microscopy, and coincident fluorescence images (middle and bottom panels) are shown. In the middle panels, the terminal transferase dUTP nick end-labeling (TUNEL) procedure was used to detect DNA fragmentation. In the bottom panels, 4′,6′diaminido-2-phenylindole (DAPI) was used to stain the DNA. Bar, 20 µm. DNase I: positive control (cells treated with DNase at 25°C for 10 min). The yeast cells in panels 2–6 contained GAL1-Bax. 3, 4, 5 and 6 indicate yeast lines expressing GAL1-Bax that were transformed with the empty pYES2 vector; the pYES2:BcLCB2 plasmid, which encodes BcLCB2; the pYES2:BcLCB2Δ307–314 vector, which encodes BcLCB2 with deletion of residues 307–314; and the pYES2:BcLCB2ΔK311E vector, which encodes BcLCB2 with a directed mutation in the pyridoxal phosphate-binding site (K311E), respectively. The experiments were performed five times.

Fig. 9 Bcl-2 associated X protein (Bax)induced yeast (Saccharomyces cerevisiae) cell death was not affected by the serine palmitoyltransferase (SPT) activity inhibitor myriocin. Yeast cells expressing Bax were cultured on synthetic dropout (SD) medium containing 2% glucose (Glc) or galactose (Gal) with or without 1 µM myriocin.

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Fig. 10 BcLCB2 rescues yeast from oxidative stress-induced cell death. (a) BcLCB2 protects Saccharomyces cerevisiae strain W303 from programmed cell death (PCD) induced by 0.75 mM tertbutylhydroperoxide (t-BHP). The agar plates show the increased survival of yeast cells expressing BcLCB2 as compared with the wild type after treatment with 0.75 mM t-BHP. (b) BcLCB2 protects yeast from cell death triggered by 0.75 mM t-BHP or 15 mM H2O2. Treated and untreated cells were sampled and spread onto yeast extractpeptone-dextrose (YPD) agar, incubated at 30°C for 48 h, and then counted and photographed. Error bars show the standard deviation of the mean for three experiments. Asterisks indicate a significant difference between cell survival in control W303 (or W303 containing empty vector pYES2) and that in BcLCB2-overexpressing W303 treated with oxidant at P = 0.01 according to Duncan’s range test.

Fig. 11 Overexpression of BcLCB2 or a BcLCB2 mutant prevents Bcl-2 associated X protein (Bax)-induced reactive oxygen species (ROS) generation in yeast (Saccharomyces cerevisiae). Yeast cells harboring pGilda:Bax were transformed with the pYES2 vector containing BcLCB2 or BcLCB2 with a deletion of residues 307–314 or BcLCB2 containing the mutation K311E in the pyridoxal phosphate binding site. To determine the effect of the expression of BcLCB2 or the deletion mutant on ROS accumulation in Bax-expressing yeast, transformants cultured in synthetic dropout (SD)-galactose medium for 6 h were treated with dihydrorhodamine 123 and observed under a fluorescence microscope. Normallight (first column of panels) and coincidentfluorescence (second column of panels) images are shown. mBcLCB2 represents BcLCB2 with a deletion of residues 307–314 and BcLCB2 containing the mutation K311E. The experiment was performed at least three times, and representative photographs are presented. Bar, 30 µm.

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Fig. 12 BcLCB2 with a deletion of residues 307–314 or containing a directed mutation in the pyridoxal phosphate binding site, K311E, suppresses Bcl-2 associated X protein (Bax)-induced cell death in yeast (Saccharomyces cerevisiae). Yeast expressing Bax under the control of a galactose-inducible promoter was transformed with the empty pYES2 vector (Vector) or with pYES2 constructs encoding BcLCB2, BcLCB2 carrying a deletion of residues 307–314 (BcLCB2Δ307–314), or BcLCB2 containing the mutation K311E in the pyridoxal phosphate binding site (BcLCB2ΔK311E). Log-phase cells were diluted 10-fold to optical density (OD600) = 1.0, and 5 µl of each dilution was spotted onto synthetic dropout (SD) solid medium containing glucose or galactose. Photographs were taken after 4 d of incubation at 30°C. The triangles above the image represent the decrease in cell numbers for cells plated in each spot. The experiment was performed at least five times, and representative photographs are shown.

fine LCB2 subunit. We firstly designed primers FL2206 (5′ATGGCTTCGAATCTCGTGGAAATG-3′) and FL2207 (5′-TCAGGACTTGAGTAGAAGCTCTG-3′) based on A. thaliana LCB1 (At4g36840) to amplify BcLCB1 full-length cDNA and genomic DNA by RT-PCR and PCR, respectively. BcLCB1 genomic sequencing revealed that it contained nine introns and a complete ORF within a 1449-bp cDNA (GenBank accession numbers EU717909 and EU717910) that encoded a 482-amino acid protein which shared 19.2% identity with BcLCB2. A pGilda:BcLCB1 construct carrying full-length BcLCB1 also did not restore the phenotype of lcb1Δ mutant S. cerevisiae cells. However, co-expression of BcLCB1 and BcLCB2 could complement the lcb1Δ, lcb2Δ and lcb1Δlcb2Δ mutant S. cerevisiae cells, allowing them to grow without exogenous phytosphingosine (Fig. 13b). These findings suggest that BcLCB2 encodes a bona fide LCB2 subunit and the SPT activity in yeast results from co-expression of the BcLCB1 and BcLCB2 subunits. These results suggest that the activity of SPT is not required to suppress Bax-induced cell death and the ROS burst, and that cell death in yeast and cell death in plants share a common pathway involving LCB2.

Discussion SPT, a heterodimer that consists of the polypeptides LCB1 and LCB2, catalyzes the first step in the synthesis of longchain bases, the signature components of all sphingolipids (Sperling & Heinz, 2003). Here, we report for the first time that BcLCB2, an LCB2 subunit of SPT from B. campestris, functions as an attenuator of cell death. Significantly, we found that BcLCB2 also regulates cell death in susceptible New Phytologist (2009) 181: 127–146 www.newphytologist.org

Fig. 13 (a) The phytosphingosine-dependent growth defect of the Saccharomyces cerevisiae lcb2ΔKAN mutant cannot be complemented by expression of BcLCB2. The S. cerevisiae lcb2ΔKAN mutant (a gift from Dr E. B. Cahoon) was transformed with empty vector pYES2, or the construct pYES2:BcLCB2 expressing BcLCB2 under the control of the galactose-inducible galactokinase1 (GAL1) promoter. The wild-type (wt) and the lcb2ΔKAN mutant carrying the plasmid pYES2:BcLCB2 or empty vector were plated in 6-fold serial dilutions on galactose (2%)-containing medium with (left panel) or without (right panel) phytosphingosine and 0.2% NP-40. The plates were incubated for 5 d at 30°C. The triangles above the image respresent the decreases in cell numbers for cells plated in each spot. The experiment was performed three times, and representative photographs are shown. (b) Co-expression of BcLCB1 and BcLCB2 complemented the long-chain auxotrophy of yeast LCB1 and LCB2 single and double mutants. Each yeast strain transformed with plasmid pGilda containing BcLCB1 or pYES2 containing BcLCB2, or cotransformed with BcLCB1 and BcLCB2, was spotted on synthetic dropout (SD) + galactose (SG) medium without phytosphingosine and the indicated amino acid. pGilda and pYES2 represent cells harboring the empty vectors.

leaves undergoing R. solanacearum infection. The overexpression of BcLCB2 can also inhibit Bax- and oxidant-triggered cell death in yeast, and NbLCB2-silenced plants showed accelerated elicitor-triggered HCD. This suggests that LCB2 functions as a general attenuator of cell death across kingdoms, and plant HR and stress-induced cell death share a common signal transduction pathway involving LCB2. LCB2 functions as a general attenuator of cell death in both plants and yeast The first known plant SPT gene was isolated from potato infected with P. infestans, in which the gene was activated and

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highly expressed in the early stages of the HR (Birch et al., 1999). Here, we isolated a full-length LCB2 cDNA from B. campestris (BcLCB2) by RACE. Broccoli (Brassica oleracea var. italica) and A. thaliana SPTs are associated with PCD during harvest-induced senescence (Coupe et al., 2004). Transcripts of broccoli and A. thaliana SPT homologs rapidly accumulate to maximum levels at 96 h after harvest (Ng et al., 2001). In this report, BcLCB2 was rapidly up-regulated by treatment with the PB90 elicitor, with a peak in leaves at 24 h post-infiltration, suggesting that BcLCB2 is involved in the regulation of the HR triggered by PB90. SPT catalyzes the first committed step in the synthesis of sphingolipids, a precursor in the generation of ceramide during apoptosis, and ceramide is involved in apoptosis (Perry et al., 2000). Moreover, sphingolipids and their associated metabolites, such as ceramide, sphingosine, and sphingosine-1-phosphate, which are formed by SPT activity, are regulatory molecules for a variety of cellular processes in plants, including pathogenesis, membrane stability, and the response to drought (Hannun & Luberto, 2000; Ng et al., 2001). Exogenous sphingosine sphingoid bases, such as dihydrosphingosine and phytosphingosine, are potent elicitors of apoptosis (Kagedal et al., 2001; Cheng et al., 2003). However, overexpression of BcLCB2 did not cause cell death in tobacco and yeast, which may have been because the BcLCB2 protein expressed in cells did not yield enough sphingolipids and their associated metabolites to trigger cell death in plants and yeast. This possibility was supported by the observation that BcLCB2 expressed in yeast only had low SPT activity. Partial RNA interference suppression of AtLCB1 expression resulted in much smaller pavement cells (Chen et al., 2006). Mutation of one of the two AtLCB2 genes had no effect on A. thaliana development, but double mutants for both AtLCB2 genes were not viable (Dietrich et al., 2008; Teng et al., 2008). However, NbLCB2 silencing did not result in any alterations in growth and development in plants, and PCD in plant leaves staining by trypan blue (data not shown), although NbLCB2 may be a single copy gene in the N. benthamiana genome (Fig. S1). Loss of function of NbLCB2 through VIGS enhanced the HR. SPT consists of two subunits, LCB1 and LCB2, and Lcb2p is unstable in cells lacking Lcb1p (Gable et al., 2000). However, AtLCB1 and AtLCB2 of A. thaliana could be stably expressed in wild-type, lcb1Δ, lcb2Δ and lcb1Δlcb2Δ yeast cells (Chen et al., 2006). Consistent with AtLCB2 expression in yeast, in this report immunoblot analysis showed that the BcLCB2 protein was stably expressed in yeast cells. Also, overexpression of BcLCB2 could suppress yeast cell death triggered by the pro-apoptotic mouse protein Bax and oxidants, including t-BHP and hydrogen peroxide. However, it did not suppress salt- and heat-induced yeast cell death, suggesting that these stresses triggered cell death in yeast via different pathways. Herskowitz (1987) provided the classical definition of dominant-negative mutations as mutations producing mutant

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polypeptides that disrupt the activity of wild-type genes when overexpressed. According to this definition, if BcLCB2 overexpression cause the dominant-negative effect as a result of the disruption of SPT activity in yeast or plants, the elicitortriggered HR should be suppressed in NbLCB2-silenced tobacco plants and Bax-induced cell death in yeast should be inhibited by myriocin, an inhibitor of SPT activity. However, NbLCB2 silencing by VIGS did not inhibit HR but enhanced HR induced by elicitors, and myriocin had no effect on Bax-induced yeast cell death. The results excluded the possibility that the degradation of BcLCB2 contributes to the suppression of Bax-induced PCD in yeast and suggested that no dominant-negative effect exists. LCB2 protects against cell death by inhibition of ROS accumulation that does not require SPT activity In plants, SPT is a heteromeric enzyme that requires both the LCB1 and LCB2 subunits for activity, because only the expression of both AtLCB1 and AtLCB2 is able to complement S. cerevisiae SPT mutants (Chen et al., 2006; Diretrich et al., 2008). In this study, consistent with the results of other workers who observed that LCB2 cDNA did not complement the lcb2Δ mutant phenotype of S. cerevisiae cells (Nagice et al., 1996; Tamura et al., 2001), the full-length BcLCB2 cDNA did not complement the lcb2Δ mutant phenotype of yeast, and BcLCB2 expressed in yeast only had low SPT activity, indicating that the BcLCB2 protein expressed in yeast cells does not yield significant SPT activity. Moreover, only co-expression of BcLCB1 and BcLCB2 could rescue the mutant phenotype of lcb1Δ, lcb2Δ and lcb2Δ lcb2Δ S. cerevisiae mutants. We also found that both a BcLCB2 mutant in which the highly conserved motif essential for catalysis was deleted and a BcLCB2 mutant containing a directed mutation in the BcLCB2 pyridoxal phosphate binding site could suppress Bax- and oxidant stress-induced cell death in yeast, although the two mutant genes did not complement the lcb2Δ mutant phenotype similarly to the BcLCB2 wild-type gene (data not shown). Also, the mutant protein stably expressed in the lcb2Δ mutant did not yield detectable SPT activity. However, overexpression of these mutated proteins also suppressed the HR triggered by elicitors. These results suggest that the suppression of cell death by LCB2 is not involved in SPT activity. A series of suppressors of PCD have been identified as suppressors of elicitor- or Bax-triggered PCD in plants (tobacco or A. thaliana): HSP90 (Kanzaki et al., 2003; Lu et al., 2003; Gabriëls et al., 2006), HSP70 (Kanzaki et al., 2003), A. thaliana AtBI-1 (Kawai et al., 1999; Sanchez et al., 2000; Kawai-Yamada et al., 2001, 2004), A. thaliana ethyleneresponsive element binding protein (AtEBP) (Ogawa et al., 2005), tobacco leucine-rich repeat (NtLRP1) (Jacques et al., 2006), L19 ribosomal protein (Gabriëls et al., 2006), and A. thaliana BON1-associated protein (BAP1) and BAP2

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(Yang et al., 2007). These proteins possess a diverse variety of biochemical activities and localize to different cellular compartments, suggesting the involvement of many biochemical and cellular processes in regulating or executing PCD. Plants continuously produce ROS, such as H2O2, O2−, and singlet oxygen, as products of normal cellular metabolism when subjected to stressors such as pathogens, elicitors, or ozone. Plants can rapidly metabolize these ROS using antioxidant enzymes or metabolites, but the presence of ROS above threshold levels leads to PCD (Levine et al., 1994; Lamb & Dixon, 1997; Pennell & Lamb, 1997; Dat et al., 2000; Tiwari et al., 2002). H2O2 can act as a signaling molecule that regulates plant development, stress adaptation, and PCD (Gechev & Hille, 2005). We previously reported that the PB90-induced H2O2 burst triggers PCD (Li et al., 2006). In this study, DAB staining revealed that the overexpression of BcLCB2 in tobacco suppresses the elicitor-induced ROS burst and ion leakage, suggesting that BcLCB2 acts to suppress cell death by directly interacting with a component necessary for cell death, or by altering cell physiology to stimulate a cell death-suppressive cellular environment. The mouse Bax protein triggers cell death in animal cells by inserting into the mitochondrial outer membrane, destabilizing the mitochondria and releasing cytochrome c and ROS. The ROS further destabilize the mitochondrial outer membrane, resulting in a positive feedback loop as more ROS are released. Recent evidence suggests that ROS such as H2O2 and nitric oxide (NO) produced by the oxidative burst act together to activate caspases through a mitogen-activated protein kinase (MAPK) pathway (Zha et al., 1996; Madeo et al., 1999). Thus, we used a yeast genetic system to evaluate the function of BcLCB2 in oxidative cell death. Our results indicated that BcLCB2 could suppress the generation of ROS induced by Bax and that BcLCB2 suppresses elicitor- and Bax-induced cell death downstream of ROS generation in PCD. BcLCB2 is different from AtEBP, BAP1 and BAP2 as these proteins could suppress PCD but did not alter the generation of H2O2 during Bax-induced cell death (Ogawa et al., 2005; Yang et al., 2007). As BcLCB2 can suppress cell death in both yeast and plants, it presumably targets a molecule(s) that is common to the PCD signal transduction pathways in both kingdoms, and probably acts on a target far downstream in the HR and PCD signaling processes. It will be interesting to use BcLCB2 as a tool to investigate PCD in plants and yeast. Some evidence suggests that Ca2+ flux from the endoplasmic reticulum (ER) into plant cells occurs both upstream and downstream of ROS generation and regulates cell survival/ death during the plant innate immune response to pathogens (Harding et al., 2002; Torres et al., 2006). Although the localization of BcLCB2 is unknown, green fluorescent protein (GFP)-tagged AtLCB2 has been localized to the ER in tobacco cells (Tamura et al., 2001) and AtLCB1 is an ER-localized polypeptide (Chen et al., 2006). LCB2 will be a useful tool with which to dissect the molecular basis of PCD signaling,

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which is presently poorly understood. Further studies will clarify where LCB2 acts in the signal transduction cascade leading to PCD and whether LCB2 alters Ca2+ homeostasis in the ER, leading to PCD. BcLCB2 is a signaling component that mediates both immunity and disease susceptibility A role for BcLCB2 in cell death underlying both the elicitorinduced HR and disease susceptibility supports the emerging theme that common signaling pathway(s) operate in elicitormediated HR and disease cell death. This idea is reinforced by several recent findings (Yao et al., 2002; Tao et al., 2003; del Pozo & Lam, 2003; del Pozo et al., 2004). BcLCB2 overexpression also suppressed death of diseased cells, suggesting that LCB2 is involved in the signal transduction pathways associated with both plant immunity and disease susceptibility. Our results strongly support the notion that cell death events involved in elicitor-mediated HR and in the death of diseased cells are mechanistically related at the level of the LCB2 switch. Our results also indicate that LCB2 may be a necessary host component for bacterial parasitism, and support the hypothesis that virulent bacterial pathogens such as P. syringae induce host cell death pathways during the late infection stage, possibly as nutrients become scarce (Stone et al., 2000). However, we cannot exclude the possibility that LCB2 might mediate other susceptibility-related pathways in addition to controlling cell death. Confusion of these pathways could also protect against bacterial growth.

Acknowledgements We gratefully acknowledge funding from the National Basic Research Program of China (grant 2006CB101907 to ZGZ), the Innovative Scholar Project of Jiangsu of China (award BK2005421 to ZGZ), the Natural Science Foundations of China (grants 30471123 to ZGZ and 30571206 to XBZ), the China 863 project (grant 2008AA10Z410) and the New Century Excellent Scholar Project of the Ministry of Education of China (grant NCET-07-0442). We thank David Baulcombe (Sainsbury Laboratory, John Innes Centre, Norwich, UK) for the gift of the PVX vector and Agrobacterium strains, Sophien Kamoun (Department of Plant Pathology, Ohio State University, Ohio Agricultural Research and Development Center, Wooster, OH, USA) for the gift of the INF1 gene, Simon Santa Cruz (Department of Virology, Scottish Crop Research Institute, Invergowrie, UK) for the vector pGilda:Bax, E. B. Cahoon (Department of Agricultural-Agricultural Research Service, Plant Genetics Research Unit, Donald Danforth Plant Science Center, St Louis, MO, USA) for wild-type, lcb1Δ, lcb2Δ and lcb1Δlcb2Δ strains of S. cerevisiae, Gongyou Chen (Department of Plant Pathology, Nanjing Agricultural University, Nanjing, China) for the gift of pET30:harpinXoo, Xueping Zhou (College of Agriculture

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and Biotechnology, Zhejiang University, Hangzhou, China) for the gift of Nicotiana benthamiana seeds, and Xilin Hou (College of Horticulture, Nanjing Agricultural University, Nanjing, China) for the gift of nonheading Chinese cabbage seeds. We also thank two anonymous reviewers for important suggestions on a previous version of the manuscript.

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Supporting Information Additional supporting information may be found in the online version of this article. Fig. S1 NbLCB2 is a single copy gene in the Nicotiana benthamiana genome.

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