These authors contributed equally to this work.
A novel pepper (Capsicum annuum) receptor-like kinase functions as a negative regulator of plant cell death via accumulation of superoxide anions
Article first published online: 1 DEC 2009
© The Authors (2009). Journal compilation © New Phytologist (2009)
Volume 185, Issue 3, pages 701–715, February 2010
How to Cite
Yi, S. Y., Lee, D. J., Yeom, S.-I., Yoon, J., Kim, Y.-H., Kwon, S.-Y. and Choi, D. (2010), A novel pepper (Capsicum annuum) receptor-like kinase functions as a negative regulator of plant cell death via accumulation of superoxide anions. New Phytologist, 185: 701–715. doi: 10.1111/j.1469-8137.2009.03095.x
GenBank accession number of CaRLK1: EF397556.
- Issue published online: 15 JAN 2010
- Article first published online: 1 DEC 2009
- Received: 16 July 2009, Accepted: 22 September 2009
Vol. 190, Issue 3, 808, Article first published online: 25 MAR 2011
- Capsicum annuum receptor-like kinase;
- cell death;
- ectopic expression;
- lesion stimulating disease gene;
- NADPH oxidase;
- receptor-like kinase;
- superoxide anion
- Top of page
- Materials and Methods
- Supporting Information
- •Plant receptor-like kinases belong to a large gene family. The Capsicum annuum receptor-like kinase 1 (CaRLK1) gene encodes a transmembrane protein with a cytoplasmic kinase domain and an extracellular domain.
- •The CaRLK1 extracellular domain (ECD)–green fluorescent protein (GFP) fusion protein was targeted to the plasma membrane, and the kinase domain of the CaRLK1 protein exhibited autophosphorylation activity. CaRLK1 transcripts were more strongly induced in treatment with Xag8ra than in treatment with Xag8-13. Furthermore, infection with incompatible Xanthomonas campestris pv. vesicatoria race 3 induced expression of CaRLK1 more strongly than in the compatible interaction.
- •Cell death caused by both a disease-forming and an HR-inducing pathogen was delayed in the CaRLK1-transgenic plants. Ectopic expression of CaRLK1 also induced transcripts of the lesion stimulating disease (LSD) gene, a negative regulator of cell death. Respiratory burst oxidase homolog (RBOH) genes were up-regulated in the transgenic plants compared with the wild type, as the concentration of the superoxide anion was increased. In contrast, the concentration of H2O2 did not differ between the transgenic and wild-type plants.
- •These results support the theory that the suppression of plant cell death by CaRLK1 is associated with consistent production of the superoxide anion and induction of the RBOH genes and the LSD gene, but not with the concentration of H2O2. Thus, CaRLK1 may be a receptor of an as yet unidentified pathogen molecular pattern and may function as a negative regulator of plant cell death.
- Top of page
- Materials and Methods
- Supporting Information
The plant receptor-like protein kinase (RLK) genes constitute a large gene family in plants, with more than 600 and 1000 genes in the Arabidopsis thaliana and rice (Oryza sativa) genomes, respectively (Shiu et al., 2004). However, in animals there are only a few receptor tyrosine kinases, which are similar to the plant RLK genes. Recently, Oh et al. (2009) reported that recombinant cytoplasmic domains of brassinosteroid insensitive 1 (BRI1) and its co-receptor BRI1-associated kinase 1 (BAK1) autophosphorylated on tyrosine residues, and a specific tyrosine residue has been shown to play an important role in vivo in plant receptor kinase function. In plants, RLKs play important roles in growth (Clark et al., 1997), development (Matsubayashi et al., 2002), cell wall biosynthesis (Mizuno et al., 2007) and defense responses (Gomez-Gomez & Boller, 2000; Heese et al., 2007). They are defined by the presence of a signal sequence, an amino-terminal domain with a transmembrane region, and a carboxyl-terminal kinase domain (Torii, 2000; Shiu et al., 2004). The kinase domains of different RLKs share a high degree of homology (more than 45% identity at the amino acid level), whereas their extracellular domains are much more divergent. Although the functions of most plant RLKs have not been determined, several members of the RLK family have been implicated in controlling disease resistance or in interacting with proteins of microbial origin (Zipfel et al., 2006). These genes, containing membrane-bounded and cytoplasmic protein kinase domains, include rice Xa21, which confers resistance to Xanthomonas oryzae pv. oryzae race 6 (Song et al., 1995), wheat (Triticum aestivum) LRK10, a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat (Feuillet et al., 1997), A. thaliana somatic embryogenesis receptor kinase 3 (SERK3)/brassinosteroid-associated kinase 1 (BAK1) (Heese et al., 2007) and A. thalianaFLAGELLIN-SENSITIVE 2 (FLS2) (Gomez-Gomez & Boller, 2000). For example, SERK3/BAK1 rapidly enters an elicitor–dept complex with FLS2 and its peptide derivative flg22 (a peptide spanning a single stretch of 22 amino acid residues of the most conserved part in the N terminus of flagellin) in A. thaliana (Heese et al., 2007).
A rapid increase in reactive oxygen species (ROS) is a hallmark of the early events in interactions between plants and pathogens. Plasma membrane-bound NADPH oxidases play a significant role in the generation of ROS, particularly the superoxide anion (O2−) (Simon-Plas et al., 2002), which is responsible for a one-electron reduction of oxygen molecules. The NADPH oxidase gene was identified in phagocytic cells (neutrophils and macrophages), which are responsible for controlling invasive pathogens (Vulcano et al., 2004). Plant NADPH oxidases consist of one component of c. 105 kDa. NADPH oxidase homologs comprise multigene families in rice, tomato (Solanum lycopersicum), and A. thaliana. The first identified plant NADPH oxidase genes were the A. thaliana respiratory burst oxidase homolog (Atrboh) genes (Torres et al., 1998). Arabidopsis thaliana has 10 Atrboh genes (Torres & Dangl, 2005). AtrbohD and AtrbohF are known to produce superoxide following pathogen invasion (Torres et al., 2002). Recently, Miller et al. (2009) reported that O2− generated by AtRBOHD plays an important role in mediating cell-to-cell communication (rapid systemic signaling) over long distances in the plant. NbrbohA and NbrbohB in Nicotiana benthamianaare also required for H2O2 accumulation and for resistance to Phytophthora (Yoshioka et al., 2003).
Superoxide dismutase (SOD) catalyzes the dismutation of O2− to H2O2. There are three classes of SOD in plants: FeSOD (FSD), MnSOD (MSD), and CuZnSOD (CSD) (Conklin & Last, 1995). The lesion stimulating disease 1 (LSD1) gene is a part of a signaling pathway for the induction of the CSD protein (Kliebenstein et al., 1999). Glutathione S-transferases (GSTs) (EC 184.108.40.206) are a family of multifunctional enzymes that catalyze the conjugation of reduced glutathione to a range of toxic compounds to detoxify xenobiotics and protect plant tissues against oxidative damage (Conklin & Last, 1995). Thus, their expression is induced by a range of stimuli, such as pathogen attack and oxidative stress. The expression of A. thaliana GST1 was found to be strongly induced by ozone treatment (Sharma & Davis, 1997).
In this study, we have identified and functionally characterized the Capsicum annuum (pepper) receptor-like kinase 1 (CaRLK1) gene. The CaRLK1 gene was induced by treatment with pathogen, H2O2, and salicylic acid (SA). We determined the role of CaRLK1 as a critical component of the regulation of O2− production through induction of the RBOH genes.
Materials and Methods
- Top of page
- Materials and Methods
- Supporting Information
Plant growth and bacterial infection
Plants were grown under standard greenhouse, long-day conditions with a relative air humidity of 50–60% at 25°C. For pepper inoculation, 2 × 108 colony-forming units (cfu) ml−1 solutions of Xanthomonas axonopodis pv. glycines (Xag) and Xanthomonas campestris pv. vesicatoria race 3 (Xcv) were pressure-infiltrated into leaf tissues using a needleless syringe (Lee et al., 2002; Kim & Kim, 2009). Bacterial populations were measured as described in Thilmony et al. (1995) and Thara et al. (1999). Three leaf discs of area 0.785 cm2 per plant were collected after inoculation by syringe infiltration of a serially diluted suspension. Bacterial populations were counted after leaf discs had been ground in 10 mM MgCl2 and serial dilutions of bacterial solution were plated on LB agar plates containing 100 mg l−1 rifampicin. The average number of bacterial cells is presented as cfu cm−2 of inoculated leaves.
RNA gel blots analysis and RT-PCR
For northern blot analyses, total RNA was separated on formaldehyde-containing agarose gels and blotted onto nylon membranes following standard procedures (Sambrook et al., 1989). Blots were hybridized with [α-32P] dCTP-labeled probes. A CaRLK1-specific probe was generated via PCR amplification with the gene-specific primers CaRLK1proLP and CaRLK1proRP. The pepper cDNA probe for pathogenesis-related 4 (PR4) was isolated previously from pepper (Lee et al., 2002). For RT-PCR, first-strand cDNA was synthesized from 2 μg of total RNA and used for amplification by PCR. Oligonucleotide primers were made according to the published primer sequences. The nucleotide sequences of other primers are listed in Supporting Information Table S1.
Subcellular localization of CaRLK1
Two constructs, CaRLK1-extracellular domain (ECD):smGFP and CaRLK1-ECD-SP:smGFP, were made for CaRLK1 expression in pepper protoplasts. Two primers, RLK(ECD)LP and RLK(ECD)RP, were used to amplify CaRLK1-ECD:smGFP (amino acids 1-219), and RLK(ECD-SP)LP and RLK(ECD)RP primers were used to amplify CaRLK1-ECD-SP:smGFP (amino acids 19-219, without the putative signal peptide (SP)). Each construct was fused in-frame to the N-terminal region of the green fluorescent protein (GFP) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and introduced into pepper protoplasts prepared from young leaves by polyethylene glycol-mediated transformation (Lee et al., 2001). A DNA construct encoding H+-ATPase:RFP, in which H+-ATPase was fused to the C-terminus of RFP (red fluorescence protein), under the control of the CaMV 35S promoter was simultaneously introduced into protoplasts. Expression of the fusion proteins was monitored at 40 h after transformation by fluorescence microscopy using a Zeiss Axioplan fluorescence microscope (Jena, Carl Zeiss, Berlin, Germany), and images were captured with a cooled charge-coupled device camera. The filter sets were used as described previously (Lee et al., 2001). The data were processed using Adobe Photoshop software (Mountain View, CA, USA) and presented in pseudocolor format. The experiments were repeated independently three times with similar results.
Recombinant protein and in vitro phosphorylation assay
The coding region of truncated CaRLK1 was cloned into pRSET-A (Invitrogen, Carlsbad, CA, USA) and expressed in Escherichia coli BL21 cells (Amersham Pharmacia Biotech, Uppsala, Sweden). Two primers (RLK-KD LP and RLK-KD RP) were used to amplify the CaRLK1 kinase domain (CaRLK1-KD; amino acids 306-598). The His-tag fusion protein CaRLK1-KD was purified using Ni-NTA agarose resin, according to the manufacturer’s instructions (Invitrogen). Phosphorylation was performed in a 25-μl assay buffer (50 mM Tris-HCl, pH 7.6, 50 mM KCl, 2 mM DTT and 10% (v/v) glycerol) containing 0.5–1 μg of purified CaRLK1-KD in the presence of 5 mM MnCl2. Phosphorylation was initiated by adding 25 μCi of [γ-32P] ATP (30 Ci mmol−1), and the reaction was incubated at 22°C for 60 min and terminated by the addition of EDTA to a final concentration of 10 mM. The phosphorylated proteins were subjected to 10% SDS-PAGE and transferred onto PVDF membranes (Amersham Pharmacia Biotech). The incorporated phosphate was visualized by autoradiography. Coomassie blue staining of the same membrane was also performed to verify protein loading. The stability of the incorporated phosphate was determined by treating the membranes with water, 1 M HCl or 3 M NaOH for 2 h at room temperature. The treated membranes were then subjected to autoradiography. The experiments were repeated independently three times with similar results.
Transformation of N. benthamiana
The pMBP-1:CaRLK1 construct was introduced into Agrobacterium tumefaciens (GV2260) for plant transformation according to Lee & Zeevaart (2005). Leaf discs were incubated with A. tumefaciens for 10 min and then transferred to Murashige and Skoog (MS) medium containing 1 mg l−1 6-benzylaminopurine (BA) and 0.1 mg l−1 1-naphthaleneacetic acid (NAA) in darkness. After co-culture for 3 d, leaf discs were transferred to selection medium (MS, 1 mg l−1 BA, 0.1 mg l−1 NAA, 100 mg l−1 kanamycin, and 500 mg l−1 carbenicillin). More than 30 individual shoots were cut and transferred to root-inducing medium (MS, 0.1 mg l−1 NAA, 100 mg l−1 kanamycin, and 500 mg l−1 carbenicillin). Plantlets (T1) were planted in soil and grown in a greenhouse. The T1 generation was self-pollinated to obtain the T2 generation. Independent transgenic lines were selected on half-strength MS containing 50 mg l−1 kanamycin. All T1 lines showing 3 : 1 segregation (resistant:sensitive) were selected to obtain homozygous T3 plants. Line 3 from these homozygous lines was chosen as a representative line.
Histochemical detection of cell death
Plant cell death was detected by modified lactophenol-trypan blue staining as described by Koch & Slusarenko (1990). The stock solution of lactophenol-trypan blue contained 10 ml of lactic acid, 10 ml of glycerol, 10 g of phenol, and 10 mg of trypan blue dissolved in 10 ml of deionized distilled water. The working solution was prepared by diluting the stock solution with ethanol (1 : 2 v/v). Leaf discs (1 cm in diameter) were stained with lactophenol-trypan blue working solution and were placed in a heated water bath and boiled for 4 min. The tissues were left overnight, and then destained with chlorohydrate solution (2.5 g of chloral hydrate dissolved in 1 ml of distilled water). They were mounted in 50% glycerol and photographed under a light microscope (Siwon Optical Technology, Suwon, Republic of Korea). The experiments were repeated independently three times with similar results.
Measurement of ion leakage
Cell death was also assayed by measuring ion leakage from leaf discs (Liu et al., 2007). Two days after inoculation with Pseudomonas syringae pv. tabaci, six leaf discs (1 cm diameter) per plant were floated on 6 ml of distilled water for 1 h at room temperature. After incubation, treated tubes were measured for electrical conductivity (Model 455C; Istek, Seoul, Republic of Korea). The experiments were repeated independently three times with similar results.
Detection of O2− and H2O2
Nitroblue tetrazolium (NBT) staining was performed on fully expanded leaves by vacuum-infiltrating NBT solution, containing 1 mM NBT plus 10 mM sodium azide in 50 mM sodium phosphate buffer (pH 7.6) (Achard et al., 2008). Leaves were placed in boiling ethanol for 10 min to remove the chlorophyll and to visualize reduced NBT dye. Use of the luminol-like compound L-012 (8-amino-5-chloro-7-phenylpyridol[3,4-d]pyridazine-1,4(2H,3H)dione) to induce luminescence of reactions is a highly sensitive means of measuring superoxide and nitric oxide (NO) production (Imada et al., 1999; Dyke et al., 2007). An L-012 solution containing 100 μM L-012 in 10 mM sodium phosphate buffer (pH 7.5) was infiltrated into leaves using a needleless syringe. Diphenylene iodonium (DPI) solution (100 μM) in 50 mM sodium phosphate (pH 7.6) was infiltrated 1 h before injection of L-012 (Achard et al., 2008). Intact leaves were removed 30 min after injection and photographed using the MultiDoc-it Digital Imaging System (UVP, Upland, CA, USA).
We stained peroxides in situ in fully expanded leaves or leaf discs (1 cm diameter) of 6-wk-old plants with 3,3′-diaminobenzidine tetrahydrochloride (DAB). Fully expanded leaves or leaf discs were vacuum-infiltrated with DAB solution (0.1 mg ml−1 DAB in 50 mM Tris acetate buffer, pH 5.0) for 10 min and then placed overnight in darkness at 25°C. Green pigments were cleared in 80% (v/v) ethanol for 10 min at 70°C. Control staining was performed in the presence of 5 mM ascorbic acid. The experiments were repeated independently three times with similar results.
Superoxide anion-generating activity in leaf discs
Leaf discs of 4 mm diameter were washed thoroughly in 50 mM Tris buffer (pH 7.4) for 1 h, blotted dry, and incubated at 25°C for 1 h in 50 mM Tris buffer (pH 7.4). The assay solution consisted of 0.3 mM XTT (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt) and 2 mM NADPH in 50 mM Tris buffer (pH 7.4), containing 5 μM DPI (Achard et al., 2008). The A450 was measured and expressed in micromoles of superoxide generated per minute using the molar extinction coefficient for the XTT formazan product of 21 600 M−1 cm−1 (Johnson et al., 2003; Achard et al., 2008). The experiments were repeated independently three times with similar results.
Enzyme assay for GST and quantification of protein
Mature leaves were harvested, ground with a pestle and mortar in liquid nitrogen and suspended in grinding buffer (50 mM sodium phosphate buffer, pH 7.0, and 0.5% Triton X-100) at 4°C and centrifuged at 13 000g for 5 min. The aqueous phase was used as an enzyme solution. Spectrophotometric measurements at 340 nm were performed at 25°C in 50 mM potassium phosphate buffer (pH 6.5), containing 5 mM reduced glutathione (GSH) plus 1 mM 1-chloro-2,4-dinitrobenzene (CDNB). The specific activity of GST was calculated with an extinction coefficient of 9.6 mM−1 cm−1 (Ricci et al., 1984; Ali et al., 2005). Protein assays were carried out using Quick StartTM Bradford Dye Reagent (Bio-Rad, Hercules, CA, USA). The experiments were repeated independently three times with similar results.
- Top of page
- Materials and Methods
- Supporting Information
Isolation of CaRLK1 cDNA
To isolate full-length cDNA, a partial cDNA fragment obtained from differential display RT-PCR with sequence homology to the serine/threonine kinase protein was used as a probe to screen a cDNA library previously constructed from C. annuum (Yi et al., 2006). Twenty-two positive clones were isolated and further analyzed by restriction enzyme digestion and DNA sequencing, resulting in the identification of six clones with > 2.5-kb cDNA inserts. Five of these clones were predicted to encode full-length proteins with an open reading frame of 627 amino acids and a calculated molecular mass of 69 kDa (Fig. 1a). (Pfam is designed to be a comprehensive and accurate collection of protein domains and families.) Analyses of the structural properties of the predicted protein using the Pfam database (Bateman et al., 2004) indicated that this cDNA encodes a putative receptor-like kinase (CaRLK1) with four distinctive regions: an N-terminal hydrophobic signal peptide (SP), an extracellular domain (ECD), a transmembrane domain (TM), and a cytosolic kinase domain (KD) (Fig. 1b).
CaRLK1 is a putative novel receptor-like kinase
Plant RLKs are classified into multiple subfamilies based on their internal kinase domain (IKD) and ECDs. A large number of RLKs do not have any established sequence motifs in their ECDs (Shiu & Bleecker, 2001; Shiu et al., 2004). The ECD of CaRLK1 has low homology to those of other known plant RLKs (Braun & Walker, 1996; Shiu et al., 2004) or any other proteins in the protein databases. CaRLK1 contains 27 leucine residues in its ECD (Fig. 1) which cannot be organized as a leucine-rich repeat consensus as defined by Kajava (1998), indicating that it contains a new type of ECD. Two hypothetical proteins from Vitis vinifera (CAN81685 and CAV65965) show the highest homology to the ECD of CaRLK1 (c. 27% identity and 45% similarity), and one of four wall-associated kinases in the LRK10L-1a subfamily, At1g25390, also shows 45% similarity (Wagner & Kohorn, 2001; Shiu et al., 2004; Lehti-Shiu et al., 2009). Thus, the CaRLK1 gene belongs to the LRK10L-1a subfamily (Shiu et al., 2004; Lehti-Shiu et al., 2009). Two hypothetical proteins from V. vinifera (CAN69599 and CAN76446) show 70% identity with the IKD (amino acids 250-627) of CaRLK1. Interestingly, the IKDs of the putative rust resistance kinase Lr10 (BAB17345) from O. sativa and of an RLK from A. thaliana (At1g70250) show 75% similarity to the IKD of CaRLK1 (Fig. S2).
Expression of CaRLK1 is induced by pathogen
We examined whether expression of CaRLK1 mRNA is induced upon pathogen attack in pepper (C. annuum) cv. Bukang. Bukang is a commercial cultivar, and was used to produce C. annuum expressed sequence tags (http://genepool.kribb.re.kr/new/index.php). Inoculation with the nonhost pathogen Xag8ra can induce the leaf hypersensitive response (HR) in hot pepper, but inoculation with Xag8-13 can not. The expression of CaRLK1 was induced at 4 h post inoculation (hpi) and sustained in both Xag8ra-treated and Xag8-13-treated leaves (Fig. 2a). However, the expression of CaRLK1 was much stronger and of longer duration in both Xag8ra-treated and Xag8-13-treated leaves (Fig. 2a). As a positive control, the expression of PR4 (Park et al., 2001) was not induced in a mock treatment. However, it was first induced at 4 hpi with Xag8ra or Xag8-13 and strongly induced at 8–12 hpi.
To evaluate the specificity of CaRLK1 expression, leaves of pepper cultivars ECW-20R (Bs2/Bs2) and ECW (bs2/bs2) were syringe-infiltrated with the pepper bacterial spot pathogen Xanthomonas campestris pv. vesicatoria (Xcv) race3, which expresses the avrBs2 gene. The Bs2 gene specifically confers resistance to Xcv that contain the avirulence gene, avrBs2. The compatible host (cv. ECW) did not exhibit any visible responses until 24 hpi. In contrast, the incompatible host (cv. ECW-20R) developed the HR within 24 hpi (data not shown). In incompatible interactions, the amount of CaRLK mRNA detected was significantly increased at 4 hpi and remained at a high level for 36 h, whereas in compatible interactions the increase in the amount of CaRLK transcript was less and was not sustained to the same extent from 4 to 24 hpi (Fig. 2b). As expected, the PR4 transcript was strongly up-regulated in ECW-20R (BS2/BS2) but not in ECW (bs2/bs2) (Fig. 2b).
Effect of exogenous H2O2 on expression of CaRLK1
In an effort to elucidate the relationship between oxidative stress signals and the in vivo function of CaRLK1, hot pepper plants were treated with H2O2 at various time intervals and at various concentrations. CaRLK1 expression was induced 2 h after treatment with 0.25–2 mM H2O2 (Fig. 3a). In addition, expression of CaRLK1 was rapidly induced as early as 1 h after treatment and remained at a high level until 12 h after treatment with 1 mM H2O2 (Fig. 3b), indicating that the expression of CaRLK1 is regulated by H2O2, and that CaRLK1 expression may be involved in oxidative stress signaling or defense signaling.
Subcellular localization of CaRLK1
In order to determine the subcellular localization of CaRLK1 in vivo, two CaRLK1 fusion constructs with GFP were generated and transiently expressed under the control of the 35S CaMV promoter in pepper protoplasts (Fig. 4a). After 40 h of incubation, the very thin green fluorescent signal of CaRLK1-ECD:smGFP (amino acids 1-219) completely overlapped with the thin red fluorescent signal in the plasma membrane, indicating the co-localization of CaRLK1-ECD:smGFP and H+-ATPase:RFP in the plasma membrane, although cytoplasm is thick enough to cover the several chloroplasts (Fig. 4a). An H+-ATPase:RFP construct was used as a positive control for plasma membrane localization (Kim et al., 2001). The green fluorescent signals were localized in the cytoplasm when the truncated CaRLK1 construct without the putative signal peptide (for ECD-SP:smGFP; amino acids 19-219) was used as a positive control (Fig. 4a). This observation suggests that the putative signal peptide acts as an essential component for plasma membrane targeting of the CaRLK1 protein, and that deletion of the putative signal peptide results in misplacement of the CaRLK1 protein.
CaRLK1 is a functional protein kinase with Mn2+ as a cofactor
To determine whether CaRLK1 encodes a functional protein kinase, we performed a kinase assay with recombinant CaRLK1 proteins expressed in E. coli. When the recombinant CaRLK1-KD (amino acids 306-598) was incubated with [γ-32P]ATP, 32P was specifically incorporated into the protein when Mn2+ was used as a cofactor. A lower level of incorporation was observed in the presence of Ca2+ or Mg2+. Some plant protein kinases, including tobacco protein kinase, NPK5 (Muranaka et al., 1994), CBL-interacting protein kinase 1 (CIPK1) (Shi et al., 1999) and RLK5 (Horn & Walker, 1994), are more active when Mn2+ is used as a cofactor. Sodium pyrophosphate, an inhibitor of protein kinase, abolished the kinase activity (Fig. 4b). These data suggest that CaRLK1-KD encodes a functional protein kinase.
Ectopic expression of CaRLK1 in N. benthamiana
To determine whether ectopic expression of CaRLK1 affects the plant defense response and cell death in another species, we introduced the 35S::CaRLK1 cDNA construct into N. benthamiana by Agrobacterium tumefaciens-mediated transformation (Lee & Zeevaart, 2005). Eight homozygous lines from more than 30 lines expressing CaRLK1 were finally selected for resistance against kanamycin (50 mg l−1), and did not show any morphological difference in phenotype (data not shown). To confirm the presence of the CaRLK1 gene in the transgenic plants, we performed genomic PCR amplification with the primer set CaRLK1LP1354 and CaRLK1RP1883 (Table S1). The PCR products (0.5 kb) were only detected without any size of other PCR products in all transgenic lines (data not shown). Transgenic line 3 (CaRLK1-3) was the main line chosen for further characterization.
Effect of ectopic expression of CaRLK1 on plant cell death
To investigate the possible role of CaRLK1 in inhibiting host cell death, we challenged at least three fully grown leaves per 5- to 6-wk-old plant with a virulent pathogen (P. syringae pv. tabaci strain 11528; As shown in Fig 5a, we used two kinds of concentration of cell, OD600 = 0.01 and 0.005), and detected cell death using trypan blue exclusion (Fig. 5). Strikingly, at least four of six inoculated spots were turned black and finally died in wild-type plants and GFP overexpression plants as a transgenic control, but only one of six inoculated spots were black to death in CaRLK1 overexpression plants (Fig. 5a). When challenged with P. syringae pv. tomato T1 (OD600 = 0.01 and 0.005), WT and GFP overexpression plants also displayed the normal cell death phenotype, but CaRLK1 overexpression plants showed only a weak cell death phenotype (Fig. 5b).
Cell death in wild-type leaves was characterized by intense staining with lactophenol-trypan blue (Fig. 5c). Host cell death in the CaRLK1 overexpression line was found to a much lesser extent than in wild-type leaves at both 24 and 48 h post inoculation of P. syringae pv. tabaci, indicating that inhibition of cell death is attributable to the specific expression of CaRLK1 (Fig. 5c, Fig. S3).
LSD1 is well known to be an important factor in the regulation of programmed cell death (PCD) in plants (Dietrich et al., 1997). The lsd1 mutant fails to restrict spread of cell death under various conditions, such as pathogen infection, strong light, and treatment with SA (Dietrich et al., 1997; Rustérucci et al., 2001). Expression of NbLSD1 transcripts was increased in the transgenic plants compared with wild type (Fig. 5d), indicating that constitutive expression of LSD under the control of CaRLK1 could suppress the spread of cell death in the transgenic plant. The putative amino acid sequences of two genes (BoLSD1 and BoLSD2) from Brassica oleracea show 85% similarity with that of NbLSD1 (data not shown) (Coupe et al., 2004). Interestingly, the AtLSD1 protein shows 75% similarity with NbLSD1 (data not shown).
To confirm that cell death was suppressed in transgenic plants, we quantified electrolyte leakage. The conductivity of the wild type was always higher than that of CaRLK1 overexpression plants in the absence of pathogen application, indicating that the transgenic plant may always be preparing for self-defense (Fig. 6a). This result was consistent with observations of reduced electrolyte leakage after pathogen inoculation; the conductivity of CaRLK1-overexpressing plants only changed moderately following infection with P. syringae pv. tabaci. However, that of the wild type increased threefold (Fig. 6b). This result also suggests that CaRLK1 is involved in negative regulation of plant cell death, as shown by lactophenol-trypan blue staining (Fig. 5c).
Interestingly, pathogen colonization was two or three times higher in the wild type than in the CaRLK1 overexpression plants at 2–3 d post inoculation (dpi) (Fig. 6c), although the difference was less than we expected. This result suggests that the transgenic plants have more tolerance to P. syringae pv. tabaci than wild-type plants because they delay pathogen growth. In addition, expressions of pathogenesis-related genes (PR1s), systemic acquired resistance 8.2 (SAR8.2) and nonexpressor of pathogenesis-related genes (NPR) was also induced in the transgenic plants (Fig. S1c). This suggests that CaRLK1 may be involved in plant basal resistance.
Effect of CaRLK1 on accumulation of the superoxide anion
It was of interest to determine whether the expression of CaRLK1 is related to ROS, because CaRLK1 expression was induced c. 4 h after infiltration with pathogens (incompatible or compatible interaction) (Fig. 2a,b) and was quickly induced 1 h after treatment with H2O2 (Fig. 3b). NBT, a well-known scavenger of the superoxide anion, has generally been used to show the production of ROS, especially O2−. As shown in Fig. 7a,b, and Fig. S4, CaRLK1-transgenic leaves showed higher levels of superoxide production than those of wild type, after infiltration of NBT solution plus sodium azide into intact leaves, because NBT staining was more intense in the CaRLK1-overexpressing line than in the wild type. Microscopy showed that the dark blue-colored formazan precipitants were located in vascular systems rather than in the chloroplasts (Fig. 7b). The difference in staining intensity can be assumed to be specific to NADPH oxidase because treatment with sodium azide, an inhibitor of peroxidase, prevents production of ROS by H2O2-generating peroxidases such as French bean peroxidase type 1 (FBP1) (Bindschedler et al., 2006). This result suggests that the superoxide anion is generated at the cell surface rather than inside the cell.
The luminal derivative L-012 can be used for measurement of peroxynitrite (OONO−), a product of the superoxide anion (O2−) and NO, at high sensitivity (Imada et al., 1999; Dyke et al., 2007). Superoxide-triggered chemiluminescence was found to be much higher in the transgenic plants than in the wild-type plants as the L-012 solution was infiltrated into the leaves (Fig. 7c). Diphenylene iodonium (DPI), an irreversible inhibitor of NADPH oxidase, blocked this increase in chemiluminescence in the transgenic plants (data not shown), although it did not block basal chemiluminescence, which might be generated by DPI-insensitive, azide-insensitive ROS sources (Bindschedler et al., 2006). This result strongly suggests that the induction of RBOH genes contributes to the production of the superoxide anion and peroxynitrite in the transgenic plants.
In higher plants, production of H2O2 is thought to be driven by increases in the concentrations of superoxide anions. However, a slightly lower level of DAB staining was observed in transgenic leaves compared with wild-type leaves (Fig. 7d). This implies that superoxide anions produced in transgenic plants may not be converted to H2O2 or that H2O2 may quickly disappear in transgenic plants. Thus, we confirmed that superoxide anions are the main ROS in the transgenic plants.
Effect of CaRLK1 on the expression of NbRBOH genes
To elucidate which genes are involved in the production of the superoxide anion, we compared the relative expression of RBOH genes. RBOH genes, encoding NADPH oxidase, are known to produce the superoxide anion in the extracellular region after pathogen attack or other stresses (Torres et al., 2005). These NADPH oxidases are the primary candidates suggested to be responsible for ROS accumulation, which may be regulated by expression of the CaRLK1 gene. The function of NbRBOHA and NbRBOHB was already determined to share superoxide anion production, H2O2 generation and resistance to bacterial pathogene (Yoshioka et al., 2003). Partial cDNA sequences of NbRBOHD genes were isolated from an expressed sequence tag (EST) database (http://www.pdrc.re.kr). A phylogenetic tree constructed using the clustal w program with PIR as the output format (http://align.genome.jp/) to compare the amino acid sequences of 10 AtRBOH genes and three NbRBOH genes showed that the NbRBOHD gene has relatively high sequence homology to AtRBOHD (Fig. S6). The expression of the NbRBOHA, NbRBOHB, and NbRBOHD genes was higher in the transgenic plants than in the wild type, suggesting that the expression of the CaRLK1 gene plays a role in ROS accumulation at the extracellular matrix through the production of superoxide anions (Fig. 8a).
To determine whether the higher expression levels of RBOH genes contribute much amount of superoxide anion, O2−-generating activity was measured using the leaf disc method (Sagi & Fluhr, 2001; Achard et al., 2008). Leaf discs from the transgenic plants showed higher O2−-generating activity than those from wild-type plants (Fig. 8b). Treatment with DPI decreased the induction of O2−-generating activity in the transgenic plants, although it did not decrease the basal O2−-generating activity in wild-type plants (Fig. 8c), as it blocked the increase in chemiluminescence in the transgenic plants after treatment with L-012 (Fig. 7c). These results strongly suggest that CaRLK1 may control O2− accumulation through induction of the RBOH genes.
Effect of CaRLK1 on expression of antioxidant-related genes
The expression of two (NbCSD2 and NbCSD3) of the three CuZnSOD (CSD) genes was slightly increased in the transgenic plants as compared with the wild type (Fig. 9a), but there was no increase in the expression of the two NbFeSOD or the two NbMnSOD genes (data not shown). Although two CSD genes are thought to be targeted to the chloroplast but superoxide anion is believed to be extracellular, signal changes in the redox cycle at the extracellular matrix may be transduced to the chloroplast, a major ROS-producing organelle in plants, to protect the chloroplast.
It is known that an ascorbate-glutathione cycle is required to protect plants from oxidative stress (Wojtaszek, 1997). Expression of three GST genes (NbGST11, NbGST12 and NbGST27) was also increased in the transgenic plants (Fig. 9a). In addition, the specific activities of total GST were 1.5- to 2-fold higher in the transgenic plants than in the wild-type plants (Fig. 9b), suggesting that an increase in both GST expression and GST activity is necessary to detoxify xenobiotic molecules, which might be produced by increasing the concentration of the superoxide anion in the transgenic plants. However, none of five putative ascorbate peroxidase genes (NbAPX) tested was expressed at a higher level in the transgenic plants than in the wild-type plants. Total ascorbate peroxidase activity did not differ significantly between the wild-type and transgenic plants (data not shown).
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Superoxide production and cell death mechanisms
At one time ROS were considered to function merely as cytotoxic molecules inducing oxidative damage and aging, but it is now becoming clear that their functions are more diverse. In the mammalian system, mature human monocyte-derived dendritic cells were found to express NADPH oxidase and to release enough superoxide anions to kill pathogens such as E. coli and Salmonella (Vulcano et al., 2004; Craig & Slauch, 2009). They are also involved in disease resistance, adaptation of oxidative stress, development and the production of secondary messenger. Torres et al. (2005) showed that reactive oxygen intermediates (ROIs) generated by AtRBOH proteins can antagonize salicylic acid-dependent pro-death signals and that the oxidative burst can suppress cell death in cells surrounding sites of NADPH oxidase activation. Using NBT and L-012, we observed that ectopic expression of CaRLK1 gave rise to strong superoxide anion signals (Fig. 7), and also up-regulated the expression of NbRBOH genes (Fig. 8a), indicating that the transgenic plants consistently produce more superoxide anions than wild-type plants, as shown in Fig. 8b,c.
Host cell death caused by P. syringae pv. tabaci and P. syringae pv. tomato T1 was delayed in the CaRLK1-overexpressing lines (Fig. 5), although induced extracellular ROS in plants have been proposed to drive PCD correlated with disease resistance (the HR) (Torres et al., 2005). There are proposed to be at least two putative mechanisms for postponing cell death under the control of CaRLK1. The first involves regulation of the concentrations of superoxide and NO. ROS can function as both positive and negative regulators of PCD, depending on the conditions and requirements (Jabs et al., 1996; Torres et al., 2002; Wrzaczek et al., 2009). Superoxide anions in the CaRLK1-overexpressing lines reacted with NO to generate peroxynitrite (ONOO−) (Fig. 7c). It was previously reported that peroxynitrite is not a mediator of hypersensitive cell death and that the HR is triggered only by balanced production of NO and ROIs (Groves, 1999; Dyke et al., 2007).
According to the ‘balance model’ for O2−, H2O2, and NO (Delledonne et al., 2001), when the NO/O2− balance is in favor of O2−, there is no available NO left for interaction with H2O2, and then cell death is delayed. Moreover, hypersensitive cell death is activated after interaction of NO with H2O2, but not with O2−. The results shown in Fig. 7 strongly support this model: constitutive generation of the superoxide anion and peroxynitrite result in the depletion of NO and a delay in host cell death. Taken together, the results suggest that the constitutive expression of NbRBOH genes in the transgenic plants could be necessary to protect plant cells from the cell death signal. Thus, we concluded that the inhibition of cell death in the CaRLK1-overexpressing plants was not attributable to an indirect or pleiotropic effect as a consequence of transformation, but instead to the function of the CaRLK1 gene.
According to the ‘speculative model’ of Beers & McDowell (2001), PCD, such as the HR, is triggered by H2O2 rather than O2− because the HR is inhibited by expression of SOD, and antioxidant enzymes are strongly induced by overproduction of O2−, but not H2O2. Thus, it is very important to elucidate whether the high concentration of O2− in the transgenic plants induced a high or low concentration of H2O2. Less DAB staining was observed in the transgenic leaves than in wild-type leaves (Fig. 7d). Treatment with ascorbic acid and DAB together completely suppressed this DAB staining (Fig. S5). These results suggest that the delay of cell death in the transgenic plants is associated with a low concentration of H2O2.
Both of these models strongly suggest that constitutive generation of O2− and peroxynitrite and a low concentration of H2O2 lead to the collapse of the NO/O2− balance and a less reaction of NO with H2O2, and finally delay cell death in the transgenic plants. Thus, O2− generated in the transgenic plants seems to be a survival signal.
The second is the levels of LSD. The LSD1 protein functions to negatively regulate a pro-death pathway component by regulating ROS concentrations or to activate a repressor of plant cell death (Torres et al., 2005). The lesion stimulating disease 1 (lsd1) mutant has been shown to develop superoxide-dependent necrotic cell death in spreading lesions (Dietrich et al., 1997). These spreading legions in the lsd mutant were found to be induced by the application of superoxide (Jabs et al., 1996). Expression of CaRLK1 was substantially up-regulated by treatment with 0.1–0.5 mM exogenous SA in pepper leaves (Fig. S1b). SA is required for the function of CaRLK1, and SA accumulates in response to pathogen attack. In turn, induction of CaRLK1 may increase expression of the putative CaLSD, as the ectopic expression of CaRLK1 induced NbLSD1 in N. benthamiana (Fig. 5d). LSD1 is part of a signaling pathway for the induction of the CuZnSOD protein to prevent cell death (Kliebenstein et al., 1999), which reminds us that the expression of two CSD genes (NbCSD2 and NbCSD3) was slightly increased in the transgenic plants. Thus, consistent up-regulation of the production of the LSD protein in the CaRLK1 transgenic plants can play a role in inhibiting cell death caused by pathogens or other factors.
How to survive in highly oxidative conditions?
When plants are exposed to stress, survival mechanisms are turned on to reduce damage. The balance between stress and survival signals determines the degree of damage suffered by the plant. Recent studies have demonstrated that ROS function as important signaling molecules involved in the control of processes such as pathogen defense, hormonal signaling, stress responses, plant growth, and development (Torres et al., 2002; Foreman et al., 2003; Kwak et al., 2003). ROS may play different or even opposite roles under different plant growth and developmental conditions. During the HR, they are actively produced to trigger cell death. However, the production of ROS under some abiotic stress conditions is thought to be a byproduct of stress metabolism for survival of the cell (Neill et al., 2002; Mittler et al., 2004). Rustérucci et al. (2001) suggested that the nature of ROIs produced by cells undergoing the HR is different from that of ROIs associated with signaling from those cells, and monitored by LSD1. Interestingly, CaRLK1 has been suggested to play a role in delaying cell death (Fig. 5).
Superoxide production was enhanced by elicitors, mechanical factors, or pathogens. Thus, pathogens such as Xag8ra and Xcv or their elicitors can induce NADPH oxidase expression regulated by CaRLK1 to produce superoxide anions. In turn, GST genes, one component of the ROS scavenging system, were also induced. Expression of GST11, GST12 and GST27 was increased in the transgenic plants (Fig. 9a). There is a interesting report that O2− rather than H2O2 is the primary ROI signal for induction of GST because sodium diethyldithiocarbamate, a SOD inhibitor, substantially up-regulated expression of GST after treatment of soybean (Glycine max) cells with P. syringae pv. glycinea carrying avrA (Delledonne et al., 2001). Thus, the induction of GST11, GST12 and GST27 transcripts and the increase in the specific activity of GST are attributable to superoxide anions accumulated through ectopic expression of CaRLK1. Although their spatial expression was not elucidated in N. benthamiana, these three GST genes can protect transgenic plants from the superoxide anion by removing xenobiotic material or other oxidized material, because GSTs can catalyze the cross-linking of reduced glutathione and xenobiotic material to remove toxic molecules. In addition, increased expression of NbLSD1 may also induce other antioxidant molecules, because LSD1 is required for the SA-dependent induction of antioxidant CuZnSOD or other antioxidant molecules (Kliebenstein et al., 1999).
Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione disulfide (GSSG) to glutathione (GSH) using NADPH as a substrate. This is an essential reaction that maintains the GSH:GSSG ratio in the cytoplasm. Unfortunately, expressions of GR1 and GR2 were not induced in the transgenic plants (data not shown), reflecting that normal expressions of GR1 and GR2 are enough to protect plant from oxidative stress.
Ectopic expression of CaRLK1 did not affect overall phenotype of N. benthamiana (data not shown). The overall phenotypes of CaRLK1-overexpressing plants, including plant height, leaf size, root length, flower shape, fruit size, and flowering time, were not distinguishable from that of the wild type, although all the transgenic plants were under mild oxidative stress conditions at all times (data not shown). This indicates that the concentration of the superoxide anion in the transgenic plants is not high enough to be cytotoxic, whereas in the lsd mutant the accumulation of the superoxide anion is high enough to cause runaway cell death under stress conditions (Jabs et al., 1996; Dietrich et al., 1997).
Further studies on function of CaRLK1 using molecular genetics and biochemical approaches, including the determination of its ligand in pathogen-challenged conditions, are warranted. Research on the downstream target proteins will also provide insights into the role of CaRLK1 in pathogen-induced cell death signaling, as the CaRLK1-KD domain showed autophosphorylation activity in hot pepper (Fig. 4b).
Ectopic expression of CaRLK1 in agricultural plants may be useful, in light of the finding that superoxide anions were mainly produced in the vascular system in the transgenic plants (Fig. 7b). This observation suggests that ectopic expression of CaRLK1 may protect the plant from vascular tissue-invading pathogens such as Ralstonia solanacearum (Park et al., 2007) and Fusarium spp., similarly to the way in which macrophages produce superoxide and kill infecting bacteria in mammalian systems (Vulcano et al., 2004; Craig & Slauch, 2009).
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We would like to thank Dr Inhwan Hwang for the RFP:H+ATPase plasmid (Postech, Pohang, Korea). We also thank Dr Choong-Min Ryu (Kribb, Daejon, Korea) for critical reading of the manuscript. This work was supported financially by grants from CFGC (CG1431) and the 21st Century Frontier Research Program funded by MOST of the Korean Government. This work was also supported in part by a grant from KRF (KRF-2008-313-C00853). SYY was supported by the MOEHRD program of KRF.
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